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1521-0111/87/2/251–262$25.00 http://dx.doi.org/10.1124/mol.114.094821MOLECULAR PHARMACOLOGY Mol Pharmacol 87:251–262, February 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
Potent and Efficacious Inhibition of CXCR2 Signaling byBiparatopic Nanobodies Combining Two Distinct Modesof Action s
M. E. Bradley, B. Dombrecht, J. Manini, J. Willis, D. Vlerick, S. De Taeye,K. Van den Heede, A. Roobrouck, E. Grot, T. C. Kent, T. Laeremans, S. Steffensen,G. Van Heeke, Z. Brown, S. J. Charlton, and K. D. CromieNovartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom (M.E.B., J.M., J.W., E.G., T.C.K., G.V.H.,Z.B., S.J.C.); Ablynx NV, Zwijnaarde, Belgium (B.D., D.V., S.D.T., K.V.H., A.R., S.S., K.D.C.); and Structural Biology ResearchCenter, Vlaams Instituut voor Biotechnologie, Brussels, Belgium (T.L.)
Received August 27, 2014; accepted December 2, 2014
ABSTRACTChemokines and chemokine receptors are key modulators ininflammatory diseases and malignancies. Here, we describe theidentification and pharmacologic characterization of nanobodiesselectively blocking CXCR2, the most promiscuous of all chemo-kine receptors. Two classes of selective monovalent nanobodieswere identified, and detailed epitope mapping showed that thesebind to distinct, nonoverlapping epitopes on the CXCR2 receptor.TheN-terminal–binding or class 1monovalent nanobodies possessedpotencies in the single-digit nanomolar range but lacked completeefficacy at high agonist concentrations. In contrast, the extracellularloop-binding or class 2 monovalent nanobodies were of lowerpotency but weremore efficacious and competitively inhibited the
CXCR2-mediated functional response in both recombinant andneutrophil in vitro assays. In addition to blocking CXCR2 signalingmediated by CXCL1 (growth-related oncogene a) and CXCL8(interleukin-8), both classes of nanobodies displayed inverse agonistbehavior. Bivalent and biparatopic nanobodies were generated,respectively combining nanobodies from the same or differentclasses via glycine/serine linkers. Interestingly, receptor mutationand competition studies demonstrated that the biparatopic nano-bodies were able to avidly bind epitopes within one or across twoCXCR2 receptor molecules. Most importantly, the biparatopicnanobodies were superior over their monovalent and bivalentcounterparts in terms of potency and efficacy.
IntroductionChemokines provide directional cues for leukocyte migra-
tion and tissue colonization. The chemokine receptor CXCR2is considered a key molecular target for the diagnosis and treat-ment of a variety of acute and chronic inflammatory diseasessuch as chronic obstructive pulmonary disease, asthma, fibrosis,psoriasis, multiple sclerosis, cystic fibrosis, rheumatoid arthritis,inflammatory bowel disease, allograft rejection, and angiogenesisand also in cancer metastasis (Stadtmann and Zarbock, 2012;Hertzer et al., 2013; Sharma et al., 2013). CXCR2 is a G protein–coupled receptor (GPCR) that is expressed on many differentcells and tissues, including neutrophils, mast cells, CD81T cells,epithelial, endothelial, smoothmuscle, and a variety of cell typesin the central nervous system (Chapman et al., 2009). Severalhigh-affinity ligands have been identified, CXCL1 (growth-related oncogene a [GRO-a]), CXCL8 (interleukin-8), andCXCL5 (ENA-78) as well as lower affinity ligands CXCL2
(GRO-b), CXCL3 (GRO-g), CXCL6 (GCP-2), and CXCL7 (NAP-2),making it the most promiscuous of all CXC chemokine receptors.Targeting of the CXCR2 receptor with small-molecule antag-onists has been pursued for pharmacologic treatment of variousdisease states in which cells of myeloid lineage are thought tocontribute to disease pathophysiology (Chapman et al., 2009).GPCRs are one of the most important classes of targets for
small-molecule drug discovery, but many GPCRs of currentinterest are proving intractable to small-molecule discoveryand may be better approached with biotherapeutics. Inhibitionof CXCR2 with small molecules has shown promising thera-peutic benefits in a variety of acute and chronic inflammatorydiseases (Chapman et al., 2009), but many have failed duringclinical trials (Schall and Proudfoot, 2011). Although some ofthese are likely to be due to the target biology, others havefailed due to unacceptable pharmacologic properties, insuffi-cient selectivity, poor pharmacokinetics, or off-target toxicity,and an antibody approach may prove to be better treatment.Nanobodies are a novel class of antibody-derived therapeutic
proteins based on immunoglobulin single-variable domains(Van Bockstaele et al., 2009; Kolkman and Law, 2010). Nano-body is a trademark of Ablynx N.V. (Ghent, Belgium). These
M.E.B. and B.D. contributed equally to this work.dx.doi.org/10.1124/mol.114.094821.s This article has supplemental material available at molpharm.aspetjournals.
org.
ABBREVIATIONS: CHO, Chinese hamster ovary; ECL, extracellular loop; FACS, fluorescence-activated cell sorter; FBS, fetal bovine serum;GPCR, G protein–coupled receptor; HCAb, heavy chain-only antibody; PBS, phosphate-buffered saline; Sch527123, 2-hydroxy-N,N-dimethyl-3-{2-[[(R)-1-(5-methyl-furan-2-yl)-propyl]amino]-3,4-dioxo-cyclobut-1-enylamino}-benzamide; TM, transmembrane.
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domains are derived from the variable domains of heavy chain-only antibodies (HCAb), which are naturally occurring in camelids(Hamers-Casterman et al., 1993). In contrast to the heterotetra-meric conventional antibodies, HCAbs are homodimers consistingof two heavy chains that lack the CH1 domain. To compensate forthe reduced antigen-binding interface (formed by both the heavyand light chain variable domains in conventional Abs), HCAbsand nanobodies derived from these have evolved toward longercomplementary determining regions in their variable domains.This, combined with their small size (12–15 kDa), enablesnanobodies to recognize unique antigenic sites with high affinityand specificity that are normally not recognized by conventionalantibodies and engineered Fab and sc-Fv fragments (Unciti-Broceta et al., 2013). Additional characteristics such as highsolubility, low immunogenicity, and flexible formatting makethem excellent candidates as standalone biotherapeutics inaddition to use for targeted delivery of biologically activecomponents (Muyldermans, 2001; Siontorou, 2013).Therapeutic nanobodies have been generated against cancer-
specific drug targets such as the receptor tyrosine kinasesepidermal growth factor receptor (Omidfar et al., 2004; Rooverset al., 2007), human epidermal growth factor receptor 2(Vaneycken et al., 2011), c-Met (Slørdahl et al., 2013), andvascular endothelial growth factor receptor 2 (Behdani et al.,2012). In addition, there have been studies demonstratingthe successful generation of inhibitory nanobodies directedagainst GPCRs and specifically the chemokine receptors CXCR4(Jähnichen et al., 2010) and CXCR7 (Maussang et al., 2013).Here, we describe the identification and pharmacologic charac-terization of inhibitory nanobodies directed against the CXCR2receptor. Two different classes of monovalent nanobodies wereidentified, based on binding to two distinct nonoverlappingepitopes within the CXCR2 receptor. Bivalent and biparatopicnanobodies were generated, respectively combining monovalentnanobodies from the same or different classes via glycine/serinelinkers. Interestingly, receptor mutation and competition stud-ies demonstrated that the biparatopic nanobodies were able toavidly bind epitopes within one or across two CXCR2 receptormolecules. Most importantly, the biparatopic nanobodies weresuperior over their monovalent and bivalent counterparts interms of potency and efficacy.
Materials and MethodsCell Culture and Membrane Preparation
All cell culture reagents purchased fromLife Technologies (Carlsbad,CA). All cells were grown adherently at 37°C, 5% CO2, and 100%humidity and subcultured when 90% confluent using trypsin/EDTAsolution. All Chinese hamster ovary (CHO) cells were grown in Ham’sF-12mediumwith Glutamax, supplementedwith 10% (v/v) fetal bovineserum (FBS), although the selection agent differed; CHO-CXCR2(Euroscreen, Gosselies, Belgium) included 400 mg/ml geneticin; CHO-CXCR2-W15A, W112A, and D274A included 500 mg/ml hygromycin;CHO-CXCR2-CCR9-ECL2 (extracellular loop 2) included 300 mg/mlZeocin and 10 mg/ml blasticidin; and CHO-W15A-CXCR2:CXCR2-CCR9-ECL2 cells included 500 mg/ml hygromycin, 300 mg/ml Zeocin,and 10 mg/ml blasticidin. L2071-CXCR1 cells (Richard Ye, University ofIllinois) were grown in Dulbecco’s modified Eagle medium withoutsodium pyruvate, with glucose and pyridoxine HCL, supplementedwith 10% (v/v) FBS and 0.3mg/ml geneticin.Membranes were preparedfrom CHO-CXCR2 cells as previously described elsewhere (Salchowet al., 2010), and the protein concentration was determined using themethod described by Bradford (1976).
Immunizations, Phage Display Selection, and Production ofNanobodies
Llamas were immunized essentially as described by Maussang et al.(2013) with CXCR2-expressing cells (four animals) and/or pVAX1-humanCXCR2 DNA (four animals) or pVAX1-human CXCR2 D1–17 DNA (fouranimals), in which the sequence encoding the first 17 N-terminal aminoacids was deleted. After immunization, immune blood and lymph nodesamples were taken, and total RNA was extracted. Nanobody-encodingcDNA was amplified by reverse-transcription polymerase chain reactionand ligated into phagemid vector pAX50 to generate nanobody phagelibraries (size ∼107) where the phage particles express individualnanobodies with C-terminal c-Myc and His6 tags in fusion with theGene-III protein, as previously described elsewhere (Roovers et al., 2007).
Phage display selectionswere performed using either a biotinylatedpeptide consisting of the first 1 to 19 amino acids of the CXCR2receptor (Bachem, Bubendorf, Switzerland), cells expressing CXCR2receptor, or cell membranes prepared from CHO-CXCR2 cells. Aftertwo to three rounds of selection on various combinations of thesetarget formats, the resulting phage outputs were transfected intoEscherichia coli strain TG1, and individual colonies were grown in96-deep-well plates. The expression of monoclonal nanobodies wasinduced by the addition of isopropyl b-D-1-thiogalactopyranoside, andthe periplasmic extract containing the nanobodies was prepared byfreeze-thawing the bacterial pellets in phosphate-buffered saline (PBS)and subsequent centrifugation to remove cell debris.
Small to medium scale productions and purifications of mono-valent, bivalent, and biparatopic c-Myc and His6-tagged nanobodieswere performed essentially as described elsewhere (Maussang et al.,2013). The flexible Gly-Ser linkers used to combine the monovalentbuilding blocks in the bivalent and biparatopic constructs are referredto as 9GS (GGGGSGGGS) or 35GS ([GGGGS]7).
Off-Rate Determinations for Nanobodies Binding toBiotinylated CXCR2 1–19 Peptide
Off-rate analysis of the nanobodies was performed using surfaceplasmon resonance technology, using the ProteOn XPR36 instrument(Bio-Rad Laboratories, Hercules, CA). ProteOn PBS/Tween (phos-phate buffered saline, pH 7.4, 0.005% Tween20) was used as runningbuffer, and the experiments were performed at 25°C. Biotinylatedpeptide representing the N-terminus (CXCR2 1-19) of human CXCR2was captured on a ProteOn NLC Sensor Chip, injected at 50 mM inProteOn PBS/Tween. Periplasmic extracts of the nanobodies werediluted 10 times in ProteOn PBS/Tween and injected for 2 minutes at45 ml/min and allowed to dissociate for 600 seconds. Between differentsamples, the surfaces were regenerated with a 50-second injection ofsodium hydroxide (50 mM) at 30 ml/min. Analysis was performed withProteOn Manager 2.1.0.38, version 2.0.1 (Bio-Rad Laboratories). Datawere double referenced by subtraction of reference ligand lane and a blankbuffer injection. Processed curveswere evaluated via fitting a standard 1:1ligand-binding model (Langmuir model provided by ProteOn software).
CXCL1 Binding Assay
CHO-CXCR2 cells (2 � 105) were incubated with purified anti-CXCR2nanobodies in fluorescence-activated cell sorter (FACS) buffer [PBS, 10%(w/v) FBS] for 30minutes at 4°C. Fluorescencemicrovolume assay technologyBlue-labeled CXCL1 (labeled CXCL1) (Applied Biosystems, Foster City,CA) (3 nM) diluted in FACS buffer was added to the cellmix and incubatedfor a further 30 minutes at 4°C in the dark. The cells were then washed3 times in FACS buffer. Dead cells were stained with propidium iodidebefore the samples were analyzed on the FACSarray (BD Biosciences,San Jose, CA) to detect the amount of labeled CXCL1 bound to the cells.
[35S]GTPgS Binding Assay
[35S]GTPgS binding to CHO-CXCR2 membranes was determinedusing the method described previously elsewhere (Bradley et al., 2009).However, human serum albumin replaced bovine serum albumin in the
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assay buffer. To determine IC50 values for anti-CXCR2 nanobodies,the activity of either recombinant human CXCL1 or CXCL8 (LifeTechnologies) (EC50 concentration) was measured in the presence orabsence of a range of concentrations of purified anti-CXCR2 nano-bodies. For Schild analysis experiments, dilution series were preparedfor CXCL1 and incubated with a range of concentrations of anti-CXCR2nanobody; the rest of the protocol was as previously described. Finally,to investigate the effect of nanobodies on basal levels of [35S]GTPgSbinding, the assay was performed as detailed for IC50 determinationswith the exception that assay buffer was added in the place of agonist.
Nanobody Binding Competition FACS Assay
CHO-CXCR2 (105/well) cells were incubated for 1.5 hours at 4°Cwith a mixture of serially diluted c-Myc–tagged nanobodies and afixed concentration (EC30) of 3x-Flag–tagged nanobody. Detection wasperformed using anti-Flag M2 antibody (Sigma-Aldrich, St. Louis, MO)for 30 minutes at 4°C followed by the addition of phycoerythrin-labeledgoat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) fora further 30 minutes at 4°C. Cells were washed 3 times with FACSbuffer between each incubation, and dead cells were stained withTO-PRO-3 (Molecular Probes/Life Technologies). The amount of anti-FLAG antibody bound to the cells was detected on a FACSArraymachine (BD Biosciences).
CXCR1 Receptor Selectivity Assay
L2071 cells expressing human CXCR1 receptor were seeded in96-well plates and incubated overnight at 37°C. On the day of theexperiment, the cells were loaded with Fluo-4 dye for 30 minutes at37°C followed by 30 minutes of incubation with purified monovalentand biparatopic nanobodies. Finally, the addition of CXCL8 (EC80–100
concentration) was performed using a Fluorometric Imaging PlateReader (Molecular Devices, Sunnyvale, CA) followed by the detectionof a fluorescent signal, corresponding to the release of intracellularcalcium. The Schering Plough low-molecular-weight antagonistSch527123 (2-hydroxy-N,N-dimethyl-3-{2-[[(R)-1-(5-methyl-furan-2-yl)-propyl]amino]-3,4-dioxo-cyclobut-1-enylamino}-benzamide) was usedas a control (Gonsiorek et al., 2007).
Human Neutrophil Whole Blood Shape Change Assay
Human whole blood was collected from healthy volunteers andanticoagulated with 52 mM EDTA. A range of concentrations ofpurified nanobodies were incubated with whole blood for 10 minutesat room temperature. CXCL1 (EC70 concentration) was then added tothe blood and incubated at 37°C for a further 5 minutes with gentleshaking. The tubes were then placed on ice while ice-cold optimizedCellFix solution (BD Biosciences) was added to each tube. The tubeswere incubated for a further 5 minutes with gentle shaking, afterwhich time the ammonium chloride solution was added to each of thetubes to lyse the red blood cells. This was incubated for 20 minutesbefore the samples were analyzed using flow cytometry (FACSCalibur;Becton Dickinson, Franklin Lakes, NJ). Specifically, cells were gated onforward scatter/side scatter (FSC/SSC) parameters, followed by forwardscatter/fluorescence-channel 2 (FSC/FL-2) gating using the granulocytegates from the first analysis; 5000 events were counted per sample.
Human Neutrophil Chemotaxis Assay
The chemotaxis of human neutrophils to CXCL1 (EC80 concentration)after preincubationwith either assay buffer or a range of concentrationsof purified anti-CXCR2 nanobody was determined using the methodpreviously described elsewhere (Bradley et al., 2009).
Epitope Mapping of CXCR2 Nanobody Binding Sites
A CXCR2 receptor mutation library was created by random mutagen-esis using the shotgunmutagenesis platform fromIntegralMolecular (Paeset al., 2009). A total of 714 clones were generatedwith all residuesmutated
twice, with one conservative and one nonconservative mutation includingone alanine substitution. All clones were expressed inmammalian cells,and cell surface expression wasmeasured using a commercially availableanti-CXCR2 polyclonal antibody. Nanobodies bound to CXCR2 mutantswere detected using an anti–c-Myc antibody (Integral Molecular,Philadelphia, PA) followed by a goat anti-mouse–horseradish peroxidaseconjugate (Jackson ImmunoResearch). Residues involved in the nano-body epitope were identified as those that fell within the bindingthresholds (,30% nanobody reactivity and.60% polyclonal anti-CXCR2antibody), and that included an alanine residue substitution and werelocated in the extracellular loops.
Generation of Mutated CXCR2 Receptor Cell Lines
Single-Point Mutations. CXCR2-W15Amutant with anN-terminalFLAG-tag and the CXCR2-W112Amutant with anN-terminal 3xHA-tagwere produced by gene synthesis and inserted into a pMA vector. Thesequences were subcloned into the pcDNA5/FRT vector using restrictiondigestion. The D274Amutant was produced by site-directedmutagenesisof the HA-tagged wild-type CXCR2 using a Quickchange II kit (Agilent,Santa Clara, CA) according to the manufacturer’s protocol. CHO Flp-Incells (Life Technologies) were grown overnight without antibiotics andthe next day were transfected with using Lipofectamine 2000. CXCR2constructs weremixed with pOGG44 vector in a ratio of 1:9 in Opti-Mem.Lipofectamine 2000 was diluted in Opti-Mem (1 in 25 dilution) and thenadded to the DNA/Opti-Mem mix in a 1:1 ratio and incubated at roomtemperature for 20minutes before being added to the cells. The cells werethen incubated overnight at 37°C, and transfected cells were selectedusing 500 mg/ml of hygromycin.
Chimeric CXCR2-CCR9-ECL2. Human CXCR2 cDNA incorpo-rated in a pcDNA3.1(1) vector was mutated whereby the extracellu-lar loop 2 (ECL2) of CXCR2 was replaced with the ECL2 from theCCR9 receptor creating a CXCR2-CCR9 chimeric cDNA sequencewith a triple HA tag engineered at the N-terminus (Missouri S&TcDNA Resource Center, Rolla, MO). This chimeric HA-tagged cDNAwas subsequently subcloned into pcDNA4/TO at HindIII(59) and XhoI(39) sites, and the correct recombinant plasmids were identified byrestriction enzyme analysis. The HA-CXCR2-CCR9-ECL2 cell linewas produced using Nucleofector Kit T (Amaxa Biosystems, Cologne,Germany).We transfected 1� 106 CHOT-Rex cells [this is a tetracycline-regulated expression (T-Rex) cell line] with 5 mg of plasmid DNAaccording to the kit instructions. Transfected cells were treated with300 mg/ml of Zeocin 48 hours after transfection. Positive clones wereselected after 24 hours of incubationwith 2mg/ml of doxycycline to inducereceptor expression and 60 minutes of incubation with 5 mg/ml mouseanti-HA fluorescein isothiocyanate–conjugated antibody (Sigma-Aldrich)before measurement of fluorescence using FACs.
Double Mutant Receptor Cell Line. CHO-CXCR2-W15A cellswere grown overnight in the absence of antibiotics before the additionof CXCR2-CCR9-ECL2 plasmid (16 mg) using an Opti-MEM/Fugene(Promega,Madison,WI)mixture (ratio 33:1), which had been incubatedat room temperature for 20 minutes before being added to the cells.Selection of positive transformants was done with medium containingboth 500 mg/ml of hygromycin and 300 mg/ml of Zeocin. Single-cellsorting was performed using the FACS Aria (Becton Dickinson) mademore efficient with the use of the fluorescein isothiocyanate–conjugatedanti-HA tag antibody, which enabled only those cells expressing theHA-tagged receptor to be selected.
Alexa647–Anti-his Tag Nanobody Binding Assays Using theIN Cell Analyzer
All cells were plated at 3000 cells/well in media in 96-well blackclear-bottom plates (Corning Life Sciences, Corning, NY) and in-cubated overnight at 37°C. Dilution series were prepared for 127D1,163E3, and 127D1-163E3 in blocking buffer [PBS with 20% (v/v) FBS]and added to the cell plate. This plate was further incubated for60 minutes at 37°C before the plate was washed 2 times with washbuffer [PBS, 0.1% (w/v) human serum albumin]. All nanobodies used
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in this assay have a His6-tag, so anti-His Alexa647-conjugated antibody(ABD Serotec, Oxford, UK) diluted (1 in 20) in blocking buffer containing2 mM Hoechst (Molecular Probes/Life Technologies, Carlsbad, CA) wasadded and incubated for 15 minutes at room temperature before theplate was washed again before the measurement of fluorescence on theIN Cell Analyzer 2000 (GE Healthcare, Fairfield, CT).
Data Analysis and Statistical Procedures
All values reported in the text and tables are mean 6 S.E.M. forthree separate experiments, unless indicated otherwise. All experimentswere analyzed by nonlinear regression using Prism 6.0 (GraphPadSoftware, San Diego, CA). For statistical procedures, one-way or two-wayanalysis of variance (stated in table legends) was performed, followed byTukey’s honest significant difference post hoc test.
ResultsIdentification of Monovalent CXCR2 Binding Nano-
bodies. Phage display selections identified 24 distinct nano-body families (derived from different B-cell lineages) bindingspecifically to CXCR2, as determined by inhibition of labeledCXCL1 binding to CXCR2 expressing cells. Eight of these familieswere found to bind the N-terminal of CXCR2 by ELISA (notshown) or off-rate analysis against a peptide constituting residues1–19. The libraries derived from the animals immunized withCXCR2-expressing cells exclusively delivered nanobodies show-ing binding to the N-terminal peptide, whereas libraries derivedfrom the llamas immunizedwithDNAalso delivered nanobodiesthat inhibited CXCL1 binding but did not bind to theN-terminalpeptide.Six nanobodies, representing different families, 2B2, 54B12,
127D1, 97A9, 163E3, and 163D2 were analyzed in more detail.All of these nanobodies showed concentration-dependent in-hibition of labeledCXCL1 binding to CHO-CXCR2 cells (Table 1).Periplasmic extracts of nanobodies 2B2, 54B12, and 127D1showed binding to the biotinylated CXCR2 1–19 peptide with off-rates shown inTable 1. Interestingly, none of these three peptide-binding nanobodies were able to fully inhibit the binding ofCXCL1 to CXCR2. In contrast, nanobodies 97A9, 163E3, and163D2, which did not show binding to the CXCR2 1–19 peptide,were able to fully inhibit the binding of labeled CXCL1 to CXCR2but were significantly less potent than nanobodies 2B2, 54B12,and 127D1 (Table 1).All 24 nanobody families that were identified fell into one of
two distinct classes. Class 1 nanobodies were those that wereable to bind the CXCR2 1–19 peptide but were unable to fullyinhibit the labeled CXCL1 binding to CHO-CXCR2 cells; incontrast, class 2 nanobodies were unable to bind to CXCR2 1–19peptide but were able to fully inhibit labeled CXCL1 binding toCHO-CXCR2 cells. In addition, class 1 nanobodies were morepotent than the class 2 nanobodies at inhibiting labeled CXCL1binding to CHO-CXCR2 cells, despite lack of full efficacy.Pharmacologic Characterization of Monovalent
Nanobodies. Further characterization of these nanobodiesshowed inhibition of CXCR2 agonist-mediated signaling inCHO-CXCR2 membranes and neutrophils (two examplesfrom class 1: 2B2 and 127D1; and three examples from class2: 97A9, 163E3, and 163D2). Table 2 shows the pIC50 valuesand the percentage of inhibition of the maximum agonistresponse for monovalent CXCR2 nanobodies determinedwith CXCL1- and CXCL8-mediated [35S]GTPgS binding toCHO-CXCR2 membranes. Per class, the nanobodies wereable inhibit both CXCL1 and CXCL8 with similar pIC50
values and the percentage of inhibition of maximum response.In addition, all monovalent nanobodies showed inhibition ofwhole blood neutrophil shape change and chemotaxis witha range of potencies, as shown in Table 2. It is of note that theclass 1 nanobodies (2B2 and 127D1) that were unable to fullyinhibit the binding of labeled CXCL1 were also unable to fullyinhibit CXCL1 mediated [35S]GTPgS binding. In whole bloodneutrophil shape change, 2B2 was also unable to fully inhibitthe CXCL1 response.As a result of these observations, further studies were per-
formed, using representatives from each class, to assess themechanism of action of these nanobodies. Concentration effectcurves were generated for each nanobody in the presence ofincreasing concentrations of CXCL1. The influence of agonistconcentration on nanobody function was investigated in twodifferent assays. As shown in Fig. 1A, the ability of 127D1 tofully inhibit [35S]GTPgS binding (to CHO-CXCR2 membranes)is totally dependent on the concentration of CXCL1 used and at150 nM CXCL1 (10 � EC50) 127D1 is almost inactive. Thisobservation was not restricted to experiments performed onrecombinant cells: Fig. 1B shows that in the presence of 20 nMCXCL1 (approximately 20 � EC50) 2B2 is completely inactivein a neutrophil shape change assay. In contrast, as shown inFig. 1C, 163E3 is able to fully inhibit [35S]GTPgS binding at allof the CXCL1 concentrations tested.To further understand the antagonist mechanism of action
of these two classes of CXCR2 nanobodies, we performed aSchild analysis. Figure 2A shows that 30 nM 127D1 canproduce a rightward shift in the CXCL1 concentration effectcurve, but further increasing concentrations of 127D1 haveno additional effect. This pattern of behavior is not consistentwith simple competitive behavior. In contrast, Fig. 2B showsthat 163E3 produces concentration-dependent rightward shiftsin the CXCL1 concentration effect curve indicative of a compet-itive mechanism of action, and the Schild plot (Fig. 2C) yieldeda straight line with a mean slope of 0.97 6 0.04, in line witha competitive mode of action and a mean pKB of 27.78 6 0.11.As the data for 127D1 did not indicate a competitive mode ofaction, a Schild plot to determine a pKB was not generated.Competition experiments using a representative FLAG-
tagged monovalent nanobody from each class (127D1 forclass 1 and 163E3 for class 2) showed that nanobodies within thesame class compete with each other, whereas nanobodies fromdifferent classes do not compete with each other, as shown in
TABLE 1Activity of representative nanobodies: off-rate determinations onbiotinylated CXCR2 1–19 peptide (single experiment using periplasmicextracts) and inhibition of labeled CXCL1 binding to CHO-CXCR2 cells(purified nanobodies)Number is given in parentheses.
Nanobody
1–19 Peptide Binding CXCL1 Binding Assay
kd [1/s] pIC50% MaximalInhibition
2B2 7.4 � 1023 8.87 6 0.14 (6)a 40.3 6 2.4a
127D1 7.7 � 1024 9.40 6 0.11 (4)b 59.8 6 2.0b
54B12 1.4 � 1023 9.14 6 0.25 (2)a,b 35.0 6 5.0a
97A9 No binding 7.85 6 0.06 (8)c 93.2 6 2.4c
163E3 No binding 8.02 6 0.02 (4)c,d 94.0 6 1.4c
163D2 No binding 8.31 6 0.10 (5)d 96.8 61.1c
Values annotated with the same superscript letter(s) are not statistically significantlydifferent, based on comparison of all nanobodies for the CXCL1-binding assay data(P , 0.05; Tukey’s honest significant difference test after one-way analysis of variance).
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Fig. 3. Mean pIC50 values were determined to be 7.33 6 0.04and 7.59 6 0.18, for 163E3 and 163D2 (in competition with163E3) and 9.13 6 0.03 and 8.08 6 0.02 for 127D1 and 2B2 (incompetition with 127D1), respectively. It is therefore possiblefor two monovalent nanobodies from different classes to simulta-neously bind epitopes within one receptor subunit.Finally, in terms of selectivity, all the monovalent nano-
bodies tested were shown to be inactive against the relatedchemokine receptor CXCR1 (Supplemental Fig. 1 shows theresults for 127D1 and 163E3, as representatives from eachclass).Pharmacologic Characterization of Bivalent and
Biparatopic Nanobodies. Next, we engineered a series ofbivalent but also biparatopic nanobody constructs using themonovalent building blocks described previously, linkedtogether with either 9GS or 35GS linkers. A number of trendswere observed from an initial characterization, which com-pared 6 bivalent and 15 biparatopic nanobodies and theirability to inhibit labeled CXCL1 binding to CHO-CXCR2 cells(Supplemental Table 1). First, a combination of two identicalor different class 2 (non 1–19 peptide binding) nanobodies didnot show a strong increase in potency over their monovalentcounterparts, as exemplified by 163E3-35GS-163E3 in Table 2,where at best a fourfold increase in potency was observed forthe inhibition of CXCL1-stimulated [35S]GTPgS binding toCHO-CXCR2 cell membranes. Second, a combination of twoidentical class 1 (1–19 peptide binding) nanobodies showedsignificantly increased potency over their monovalent counter-part, as exemplified by 2B2-9GS-2B2 in Table 2, with a 134-foldincrease in potency in the whole blood neutrophil shape changeassay. However, this bivalent construct was still unable to fullyinhibit CXCL1-stimulated [35S]GTPgS binding to CHO-CXCR2cell membranes; in whole blood neutrophil shape change, at highconcentrations of CXCL1 (20� EC50), it was completely inactive(data not shown). Finally, biparatopic nanobodies that werecomposed of one class 1 nanobody and one class 2 nanobody (e.g.,127D1-35GS-163E3; Fig. 4 and Table 2) showed the greatestincreases in potency when compared with their class 2 mono-valent counterparts, and increased efficacy when compared withtheir class 1 monovalent counterparts, although the gains inpotency were not as large in the labeled CXCL1-binding assayand CXCL1-stimulated [35S]GTPgS binding assay (Table 2) ascomparedwith all the other assay formats. Aswith themonovalentnanobodies, the bivalent and biparatopic nanobodies could inhibitCXCL8-stimulated [35S]GTPgS binding with similar potencyand efficacy as for CXCL1 (Table 2) and were completelyinactive at the CXCR1 receptor (Supplemental Fig. 1).To further characterize the biparatopic nanobody 127D1-
35GS-163E3, we performed a Schild analysis, and the dataare shown in Fig. 5A. Parallel rightward shifts of the CXCL1concentration effect curve were observed, indicative of acompetitive antagonist, and the Schild plot (Fig. 5C) yieldeda straight line with a mean slope of 0.60 6 0.01 and a meanpKB of 29.97 6 0.03. If the slope of the line is equal to 1, asis the case for monovalent 163E3 (Fig. 2C), then this couldbe indicative of a competitive antagonist. However, for thebiparatopic nanobody, the slope is significantly less than 1,suggesting that the mechanism of inhibition of the formattednanobody may not be identical to a competitive antagonist.Finally, we performed a Schild analysis on equimolar mixturesof monovalent components 127D1 and 163E3, which wereadded to the assay at the same time (Fig. 5B). The resultingT
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CXCL1
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pIC50
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pIC50
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pIC50
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2B2
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7.90
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60.07
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8.20
60.11
b94
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9.60
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9.63
60.07
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Schild plot yielded a mean slope of 0.80 6 0.06 and a meanpKB of28.956 0.38 (Fig. 5C). This pKB value is a log order ofmagnitude lower in potency than the biparatopic nanobody,suggesting that when linked, the binding of one monomerforces the second tethered one to stay close to its correspond-ing binding site. This forced proximity favors the bindingand rebinding (once dissociated) of each nanobody to theirdiscreet epitopes and hence leads to the increased potency/affinity that we observe with the biparatopic constructs(Vauquelin and Charlton, 2013).Finally, the biparatopic nanobody showed inverse agonist
effects on basal [35S]GTPgS binding to CHO-CXCR2 mem-branes (Fig. 6) and produced a mean pIC50 value of 9.17 60.10, which is in line with the potency derived in the presenceof the agonist. In addition, Fig. 6 shows that the monovalent
nanobodies 127D1 and 163E3 also exhibited inverse agonisteffects on basal [35S]GTPgS binding to CHO-CXCR2 mem-branes, with mean pIC50 values of 8.48 6 0.61 and 7.89 60.28, respectively. These potency values are in line with thosegenerated in the presence of the agonist, and the percentagedecrease of basal [35S]GTPgS binding is less with 127D1 ascompared with 163E3, correlating with the reduced efficacywe observed with the class 1 nanobodies.Epitope Mapping of Nanobody Binding Sites. To
define the binding sites of 127D1 and 163E3 nanobodies, weapplied the shotgun mutagenesis technology platform from
Fig. 1. Effect of increasing CXCL1 concentration on anti-CXCR2 nanobodyinhibition of CXCL1-stimulated [35S]GTPgS binding assay using CHO-CXCR2 cell membranes (A and C) and of human whole blood neutrophilshape change assay (B). Data presented for (A) and (C) are mean 6 S.E.M.from three separate experiments. Data presented for (B) are representativeof three experiments performed once.
Fig. 2. Schild experiments performed using CXCL1-stimulated [35S]GTPgSbinding assay, with CHO-CXCR2 cell membranes, in the presence of a rangeof concentrations of (A) 127D1 and (B) 163E3 anti-CXCR2 nanobodies. (C)Schild plot for 163E3 derived from data shown in (B). Data are presented asthe mean 6 S.E.M. of three separate experiments.
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Integral Molecular (Philadelphia, PA), in addition to gener-ating both single-point mutations and a chimeric CXCR2/CCR9 receptor. The shotgun mutagenesis analysis indicatedthat 127D1 and 163E3 bind to distinct, nonoverlapping bindingsites on the CXCR2 receptor as shown in Fig. 7. As expected,the class 1 nanobody 127D1mapped to theN-terminal region ofthe receptor, specifically to residues F11, F14, and W15; whenmutated to alanine residues, these resulted in less than 30%binding of 127D1, when compared with wild-type CXCR2(Supplemental Table 2). The close proximity of the criticalresidues (F11, F14, andW15) suggests that the epitope is linearin nature, hence the ability of this class of nanobodies to bindto the CXCR2 1–19 peptide. The CXCR2 receptor with the
single-point mutation W15A in the N-terminus was expressedon the surface of CHO Flp-In cells, and fluorescence imageanalysis showed that the binding of 127D1 was lost in thepresence of this mutation (Fig. 8, Fig. 9B, and Table 3) whilebinding of 163E3 was retained, thus confirming the shotgunmutagenesis data.In contrast to the linear epitope for the class 1 nanobody,
the class 2 nanobody 163E3 appears to bind to a conforma-tionally complex epitope formed primarily by extracellularloops 1 and 3 (ECL1 and ECL3). When mutated to alanine,residues W112, F114, G115, D274, I282, T285, and D293 allexhibited loss of 163E3 binding (#30% binding when comparedwith wild-type CXCR2) (Supplemental Table 2). The epitopealso appears to be highly sensitive to the conformational stateof the receptor, as manymutations located in the transmembrane(TM) domains (primarily TM5 and TM6, data shown inSupplemental Table 3); cysteine residues in the extracellular
Fig. 3. Inhibition of FLAG-tagged nanobody binding to CHO-CXCR2 cellsby monovalent nanobodies (A) using FLAG-tagged 163E3 and (B) usingFLAG-tagged 127D1. Data presented are representative of three experi-ments performed once.
Fig. 4. Effect of generating biparatopic nanobody construct on potencyin CXCL1-stimulated [35S]GTPgS binding assay using CHO-CXCR2 cellmembranes. Data are presented as the mean 6 S.E.M. of three separateexperiments.
Fig. 5. Schild experiments performed using CXCL1-stimulated [35S]GTPgSbinding assay with CHO-CXCR2 cell membranes in the presence of a rangeof concentrations of (A) 127D1-35GS-163E3 (B) 127D1 and 163E3 mono-valent nanobodies added together. (C) Schild plots derived from data shownin (A) and (B). Data are presented as the mean 6 S.E.M. of three separateexperiments.
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loops (Supplemental Table 2) appear to affect the binding ofthis nanobody, as shown in Fig. 7. The single-point mutationsW112A (ECL1) and D274A (ECL3), that form part of theepitope for the class 2 nanobodies, were both successfullyexpressed in CHO-Flp-In cells. Both single residue substitu-tions, although they did not completely inhibit the binding of163E3, produced.10-fold loss in affinity compared with CXCR2wild-type (data not shown) with no effect on the affinity of127D1, confirming the shotgun mutagenesis data. Interest-ingly, CXCL1 stimulated [35S]GTPgS binding was completelyabolished in cells expressing these single-point mutations,
suggesting that these residues play a critical role in CXCR2receptor activation.As the class 2 nanobodies appear to use a more complex
epitope, compared with those of class 1, a more significantepitope disruptionmay be required to completely inhibit 163E3binding. This was demonstrated by generation of a CXCR2-CCR9 chimeric receptor, whereby ECL2 of the CXCR2 receptorwas replaced with the equivalent region from the CCR9 re-ceptor. The CCR9 receptor was chosen as it showed the leastsequence homology to the CXCR2 receptor in a comparison ofchemokine receptor family members. This ECL2 replacementfully inhibited the binding of all class 2monovalent nanobodies.As shown in Fig. 8, Fig. 9C, and Table 3, determination ofnanobody binding by fluorescence image analysis demonstratedthat ECL2 replacement completely inhibited binding of 163E3,while retaining binding of 127D1.Biparatopic Nanobody Binding to Cells Coexpress-
ing Mutated CXCR2 Receptors. To gain an understandingof the binding of the biparatopic nanobody at a molecularlevel, we determined the apparent affinity of the biparatopicnanobody for both the CXCR2-W15A mutant and the CXCR2-CCR9-ECL2 chimera. We predicted that due to the total lossof binding of one of its constituent building blocks, the affinityof the biparatopic nanobody would match that of the buildingblock that was still capable of binding to themutated receptor.In the case of the CXCR2-CCR9-ECL2 chimera, this is exactly
Fig. 6. Inhibition of basal [35S]GTPgS binding to CHO-CXCR2 cellmembranes using monovalent and biparatopic nanobodies. Data arepresented as the mean 6 S.E.M. of three separate experiments.
Fig. 7. Epitope mapping of CXCR2 nanobodies: snake plot representation of human CXCR2 with highlighted amino acids involved in the binding ofCXCL1, CXCL8, and nanobodies 127D1 and 163E3 [blue: Katancik et al., 2000; yellow: 127D1; and red: 163E3]. In addition, residues are highlightedthat affect the epitope of 163E3 but are believed not to directly contact the nanobody (green). Residues shown in yellow, red, and green were identifiedafter shotgunmutagenesis (Integral Molecular) and whenmutated to alanine resulted in diminished binding (#30% compared with wild-type CXCR2) ofthe respective nanobody.
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what was observed (Fig. 9C and Table 3). In the case of theCXCR2-W15Amutant, thedata showthatalthough thebiparatopicnanobody shows approximately a 10-fold loss in affinity withthis mutation compared with CXCR2 wild-type, it exhibits afivefold increased affinity compared with monovalent 163E3.One possible explanation could be that the binding of the
163E3 moiety of the biparatopic nanobody brings the 127D1moiety within close enough proximity to the N-terminus of theCXCR2-W15A receptor and as such allows some binding,althoughwithmuch reduced affinity, to the other key residueswithin its epitope (F11 and/or F14). The CXCR2-CCR9-ECL2chimeric receptor was successfully combined with the W15Apoint mutant in one cell line, and Fig. 8 shows binding curvesof both monovalent and biparatopic nanobodies to this cellline. Figure 9D and Table 3 show that for this cell line theaffinities of both monovalent nanobodies and biparatopicnanobody are comparable to the affinities observed with theCHO-CXCR2 wild-type cells. Most importantly, the statisticsconfirm that the biparatopic nanobody binds equally well toboth CXCR2wild-type and CXCR2-W15A:CXCR2-CCR9-ECL2cells.Given the reduced affinity of the biparatopic nanobody
for the individual CXCR2-W15A and CXCR2-CCR9-ECL2 celllines, themost likely explanation for reinstatement of its affinityon the combined cell line is binding across two different mutantCXCR2 receptors. Essentially, these data indicate that theclass 1 nanobody moiety 127D1 of the biparatopic construct is
binding to the wild-type N-terminal region of the CXCR2-CCR9-ECL2 receptor and that the class 2 nanobody 163E3 moiety isbinding to thewild-typeECL2 region of theCXCR2-W15Areceptor.
DiscussionIn this study we describe the generation and characteriza-
tion of highly potent, selective, and efficacious nanobodiesagainst the chemokine receptor CXCR2. Interestingly, thesenanobodies exhibit a very different mechanism of inhibitionas compared with small-molecule antagonists of CXCR2, whichhave been shown to bind to an intracellular binding site anddisplay an allosteric mechanism of action (Bradley et al., 2009;Salchow et al., 2010).A broad panel of CXCR2 nanobodies was identified, all of
which fell into two distinct classes, based on whether theybound to a 1–19 amino acid peptide derived from the first 19residues of the CXCR2 receptor. Nanobodies that were able tobind the peptide were termed class 1; these had higher potenciesthan the other class of nanobodies, but they were unable to fullyinhibit CXCL1 binding and function, particularly at high agonistconcentrations. The class 2 nanobodies did not bind the 1–19peptide and were of lower potency compared with class 1. How-ever, they were able to fully inhibit CXCL1 binding and functionand appeared to be competitive in Schild analysis experiments.Both classes of nanobody displayed inverse agonism, class 1 toa lesser extent, but this suggests they can both inhibit the active
Fig. 8. Nanobody binding to mutated CXCR2receptors expressed in CHO cells (blue fluores-cent nuclear counterstainingwithHoechst dye)andmeasured usingAlexa647-conjugated anti-His antibody, as shown by red fluorescent cellmembrane staining.
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conformation of the receptor. In addition, it was demonstratedthat nanobodies from different classes did not compete with eachother for binding to CXCR2.
Shotgunmutagenesis studies confirmed that these two classesof nanobodies bind to distinct, nonoverlapping regions on theCXCR2 receptor (Fig. 7). The epitope of the class 1 nanobodies
Fig. 9. Effect of CXCR2 receptor mutations on affinity of monovalent and biparatopic nanobodies measured using Alexa647-conjugated anti-Hisantibody on (A) CHO-CXCR2wild-type cells, (B) CHO-W15A cells, (C) CHO-CXCR2-CCR9-ECL2 cells, and (D) CHO-CXCR2-W15A:CXCR2-CCR9-ECL2cells. Data are presented as the mean 6 S.E.M. of three separate experiments.
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was formed of three key residues within the N-terminus of thereceptor, and the epitope of the class 2 nanobodies was a moreconformationally complex epitope formed primarily by ECL1and ECL3, although ECL2 was also demonstrated to beimportant in maintaining epitope conformation. A study byKatancik et al. (2000) identified amino acid residues of theCXCR2 receptor that are critical for the binding of CXCL1and CXCL8 (also shown in Fig. 7). Four key amino acids wereidentified in the N-terminus of the receptor, in very closeproximity to the class 1 nanobody epitope, and three key aminoacid residues in ECL1, which are positioned either side of keyresidueswhich form part of the class 2 nanobody epitope. Thesedata suggest that the nanobodies inhibit key interactionswithin the binding sites for CXCL1 and CXCL8 at the CXCR2receptor. These data also suggest that the binding sites ofCXCL1 and CXCL8 span two distinct regions of the receptor,which indicates that it might be possible to inhibit only onebinding site while potentially retaining agonist binding at theother site. It is therefore possible that the class 1 nanobodiescan bind to the N-terminus of the CXCR2 receptor, while eitherCXCL1 (or CXCL8) is simultaneously bound to the ECL1region of the receptor, resulting in “partial” competition of thisclass of nanobodies with the chemokines. Partial competitiveinhibition has previously been proposed for a number ofantagonists of class B GPCRs and has been described in termsof the Charnière effect (Hoare, 2007). More recently it has beenshown that this mode of inhibition can result in incompleteinhibition curves, as we observed in this study for the class 1nanobodies, which could be misinterpreted as an allostericeffect (Vauquelin et al., 2015).The two classes of nanobody also behaved very differently
when bivalent constructs were generated; for the class 1nanobodies, this resulted in large increases in potency comparedwith the monovalent molecules, suggesting simultaneous bindingof the linkedmonomers to epitopes that are close enough togetherto allow this to occur (model 1 as described by Vauquelin andCharlton (2013)). In contrast, the class 2 nanobodies did not showany strong increase in potency as bivalent constructs, suggestingthat the two building blocks do not bind simultaneously as theepitopes are too far apart (model 2, as described by Vauquelin andCharlton, 2013). Receptor dimerization, specifically the concept ofasymmetric activation of GPCR dimers (Damian et al., 2006),could also be an explanation for the data we have observed withthe bivalent nanobodies. Asymmetric activation proposes thatthere is one active conformation and one inactive conformation ofeach monomer within the dimer after binding of the agonist. Ourdata suggest that the epitope for the class 2 nanobodies is more
complex than for the class 1 nanobodies and also that it is moresensitive to changes in conformation. Applying asymmetricactivation to the binding of a bivalent class 2 nanobody toCXCR2 would mean that the binding of one of the nanobodiesto a CXCR2 monomer within the dimer could produce anallosteric conformational change that inactivates the otherCXCR2 receptor and thus prevents the simultaneous binding ofthe second linked nanobody.The generation of the biparatopic nanobody formed by
linking together a building block from each class resultedin the most desirable pharmacologic profile, combining highpotency and efficacy in one molecule. When discussing bindingmodes of the bivalent nanobodies identified in this study, theassumption was that to see gains in potency, simultaneousbinding of the linked nanobodies to two epitopes on two separatereceptors had to occur. As the epitopes for the two classes ofnanobodies are distinct and nonoverlapping, the individualbuilding blocks (in a biparatopic format) could bind simulta-neously within one receptor, or across two separate receptors.Competition assays with the two different classes of monovalentnanobodies demonstrated that they do not compete with eachother. To investigate whether a biparatopic nanobody couldalso bind across two separate receptors, we generated a cell linethat expressed two CXCR2 receptor variants, one with a muta-tion in the N-terminal (class 1) nanobody epitope and a chimericreplacement of a key region of the class 2 nanobody epitope.Each of these mutated receptors could only bind one of thetwo building blocks comprising the biparatopic nanobody. Asanticipated, the affinity of the biparatopic nanobody for thiscell line was similar to that for wild-type CXCR2, revealingthat the biparatopic nanobody can bind across two CXCR2receptors. There is evidence that the CXCR2 receptor can formdimers (Trettel et al., 2003) as well as CXCL8 (Horcher et al.,1998) and more recently CXCL1 (Ravindran et al., 2013).Although we cannot definitively rule out that overexpression ofour mutant receptors in a recombinant system has “forced” anotherwise unnatural receptor dimerization or increased receptorcolocalization to occur, the fact that we observe significant gainsin potency with the biparatopic and bivalent class 1 formatsin the neutrophil assays suggests that the receptors are alsosufficiently colocalized/dimerized in an endogenous system.Overall, our data suggest that it may be possible for thebiparatopic nanobodies to bind within one CXCR2 receptoror across two receptors.Comparisons can bemade between our data and that produced
for nanobodies binding the CXCR4 or CXCR7 chemokine receptors(Jähnichen et al., 2010; Maussang et al., 2013). N-terminal CXCR7
TABLE 3Nanobody affinity measured using Alexa647-conjugated anti-His antibody on wild-type and mutatedCXCR2 receptor CHO cell lines
Nanobody
pKd of Nanobody Binding to CHO Cell Lines
CXCR2 WT CXCR2-W15A CXCR2-CCR9-ECL2CXCR2-W15A:CXCR2-CCR9-
ECL2
127D1 (class 1) 9.02 6 0.06a/a No binding 9.18 6 0.09a/a 9.07 6 0.10a/a
163E3 (class 2) 8.44 6 0.21b/a 8.14 6 0.18a/a No binding 8.11 6 0.07b/a
127D1-35GS-163E3(biparatopic)
9.65 6 0.14c/a 8.72 6 0.08b/b 8.99 6 0.05a/b 9.71 6 0.16c/a
WT, wild type.Values annotated with the same superscript letter are not statistically significantly different for a given receptor
subtype across different nanobodies (values before /) or for a given nanobody across different receptors (values after /)(P , 0.05; Tukey’s honest significant difference test after two-way analysis of variance).
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nanobodies partially inhibited [125I]CXCL12 binding, suggestingthat within the chemokine receptor family at least, inhibitingbinding of the agonist to theN-terminus is not sufficient to fullyinhibit receptor function. In addition, when two identical non–N-terminal CXCR7 nanobodies were linked, there was nomorethan a threefold increase in affinity, whereaswith a biparatopicCXCR7 nanobody composed of building blocks that bound todistinct nonoverlapping regions of the receptor, larger increasesin potency were observed. Jähnichen et al. (2010) also observedan increase in affinity and potency with an anti-CXCR4biparatopic format, the major difference being that it wascomposed of two nanobodies that compete with each other andbind to different but overlapping epitopes in ECL2. The inverseagonism that we observe with both nanobody classes is uniquewhen compared with CXCR4, in which inverse agonism wasonly observed with the biparatopic format (Jähnichen et al.,2010). In addition, we do appear to have observedmuch greatergains in potency as a result of generating the biparatopic formatfor CXCR2, when compared with both CXCR4 and CXCR7, andthis may be due to the epitopes of the different nanobody classesand/or differences in CXCR2 receptor activation. Finally, it waspostulated for the anti-CXCR4 biparatopic nanobody that itmust be binding to two CXCR4 molecules in close proximity,given that the epitopes for the monovalent nanobodies weredistinct but overlapping (Jähnichen et al., 2010). However, webelieve that the data we have generated are the first time thatthis has been demonstrated experimentally for such a bipar-atopic nanobody specific for a G protein–coupled receptor, whoseepitopes are distinct. Finally, the potential for both intramolecularand intermolecular avid binding with the biparatopic nanobodyopens up the possibility for the inhibition of 1) monomericCXCR2, 2) homodimeric CXCR2, and 3) heterodimeric CXCR2.In other words, whatever the relevant signaling conformation(s)of the receptor is/are, the biparatopic nanobody should be able toeffectively bind and inhibit any/all of these possibilities.
Acknowledgments
The authors thank Catelijne Stortelers for her help with the DNAimmunizations, Peter Hunt for his help in generating the CXCR2-CCR9-ECL2 chimera and for the useful discussions on nanobody bindingmechanisms, Lindsay Fawcett for providing molecular biology tools forthe generation of mutated CXCR2 receptors, and Cheryl Paes, RachelFong, and Edgar Davidson from Integral Molecular for their help withgenerating Fig. 7.
Authorship Contributions
Participated in research design: Bradley, Brown, Charlton, Cromie,Dombrecht, Grot, Kent, Laeremans, Manini, Steffensen, Van Heeke,Vlerick, Willis.
Conducted experiments: Bradley, De Taeye, Grot, Laeremans,Manini, Roobrouck, Steffensen, Van den Heede, Vlerick, Willis.
Contributed new reagents or analytic tools: Bradley, De Taeye, Vanden Heede, Grot, Kent.
Performed data analysis: Bradley, Brown, Charlton, De Taeye,Dombrecht, Grot, Laeremans, Manini, Roobrouck, Steffensen, Vanden Heede, Van Heeke, Vlerick, Willis.
Wrote or contributed to the writing of the manuscript: Bradley,Brown, Charlton, Cromie, Dombrecht, Kent, Steffensen, VanHeeke.
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Address correspondence to: Steven J Charlton, School of Life Sciences,Queen’s Medical Centre, University of Nottingham, Nottingham, United Kingdom,NG7 2UH. E-mail: [email protected] or Bruno Dombrecht, AblynxNV, Technologiepark 21, 9052 Zwijnaarde, Belgium. E-mail: [email protected]
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