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Production of a human neutralizing monoclonal antibody and its crystal

structure in complex with ectodomain 3 of the interleukin-13 receptor α1

Article  in  Biochemical Journal · February 2013

DOI: 10.1042/BJ20121819 · Source: PubMed

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Biochem. J. (2013) 451, 165–175 (Printed in Great Britain) doi:10.1042/BJ20121819 165

Production of a human neutralizing monoclonal antibody and its crystalstructure in complex with ectodomain 3 of the interleukin-13receptor α1Nicholas T. REDPATH*†1, Yibin XU*†1, Nicholas J. WILSON‡1, Louis J. FABRI‡1, Manuel BACA‡1,2, Arna E. ANDREWS‡,Hal BRALEY‡, Ping LU§, Cheryl IRELAND§, Robin E. ERNST§, Andrea WOODS§, Gail FORREST§, Zhiqiang AN§3,Dennis M. ZALLER§, William R. STROHL§4, Cindy S. LUO*, Peter E. CZABOTAR*†, Thomas P. J. GARRETT*,Douglas J. HILTON5*†, Andrew D. NASH‡6, Jian-Guo ZHANG*†5,6 and Nicos A. NICOLA*†5,6

*The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia, †Department of Medical Biology, University of Melbourne, Parkville, VIC 3010,Australia, ‡CSL Limited, Parkville, VIC 3052, Australia, and §Merck Research Laboratories, West Point, PA 19486, U.S.A.

Gene deletion studies in mice have revealed critical roles for IL(interleukin)-4 and -13 in asthma development, with the lattercontrolling lung airways resistance and mucus secretion. Wehave now developed human neutralizing monoclonal antibodiesagainst human IL-13Rα1 (IL-13 receptor α1) subunit that preventactivation of the receptor complex by both IL-4 and IL-13. Wedescribe the crystal structures of the Fab fragment of antibody10G5H6 alone and in complex with D3 (ectodomain 3) ofIL-13Rα1. Although the structure showed significant domainswapping within a D3 dimer, we showed that Arg230, Phe233, Tyr250,

Gln252 and Leu293 in each D3 monomer and Ser32, Asn102 and Trp103

in 10G5H6 Fab are the key interacting residues at the interface ofthe 10G5H6 Fab–D3 complex. One of the most striking contactsis the insertion of the ligand-contacting residue Leu293 of D3 into adeep pocket on the surface of 10G5H6 Fab, and this appears to bea central determinant of the high binding affinity and neutralizingactivity of the antibody.

Key words: asthma, interleukin-13, interleukin-4, monoclonalantibody, receptor, TH2 immune response.

INTRODUCTION

Asthma is a heterogeneous and chronic inflammatory diseaseof the airways that is characterized by airway inflammation,reversible airway obstruction, mucus hypersecretion, airwayhyper-responsiveness and airway wall remodelling [1]. In asignificant proportion of patients, the disease manifests as anallergic hypersensitivity dominated by an immune responsepolarized to the TH2 phenotype [2]. This activation results inthe secretion of TH2 cytokines [IL (interleukin)-4, IL-13 andIL-5] that either directly or indirectly trigger many of thepathologies associated with this disease including IgE production,eosinophilia, mucus secretion by lung epithelial cells and airwaysmooth muscle hyper-responsiveness. IL-4 is thought to beimportant in skewing the T-cell response to the TH2 type and inpromoting antibody class switching to the IgE type. Whereas IL-13 has similar effects to IL-4, it also has direct roles in stimulatingmucus secretion by lung epithelial cells. IL-5 is the major cytokineaffecting eosinophil production and activation [3].

The similarity of the biological effects of IL-4 and IL-13 ispartly explained by the use of common cell-surface receptor

complexes: they each can bind to and activate a receptor complexmade up of IL-13Rα1 (IL-13 receptor α1) and IL-4Rα (IL-4receptor α). On the other hand, only IL-4 can bind to an alternativesignalling receptor complex made up of IL-4Rα and the commonγ chain receptor (γ c) [4,5].

Although IL-5-gene-deleted mice have reduced eosinophilnumbers, they nonetheless display airway hyper-reactivity,independent of IL-5, in an ovalbumin-induced model of asthma[6]. In human trials, anti-IL-5 antibodies did not completelyeliminate eosinophils from the airways and did not inhibitantigen-induced late airway responses [7,8]. Similarly, despiteearly promise, clinical trials targeting IL-4 inhibition have beendisappointing [3].

Although clinical trials with anti-IL-13 reagents are still atan early stage [9], several lines of evidence suggest that IL-13and IL-13Rα1 are critically involved in asthmatic responses.Genetic studies in mice have shown that IL-13 plays criticalroles in the regulation of IgE synthesis, mucus hypersecretion,sub-epithelial fibrosis and eosinophil infiltration into the lung,and it has been associated with the regulation of chemokinereceptors including CCR5 (CC chemokine receptor 5) [10]. In

Abbreviations used: CCL, CC chemokine ligand; CDR, complementarity-determining region 3; Ches, 2-(N-cyclohexylamino)ethanesulfonic acid; ECD,extracellular domain; FBS, fetal bovine serum; HCDR, heavy-chain CDR; HEK, human embryonic kidney; HRP, horseradish peroxidase; IL, interleukin; IL-4Rα, IL-4 receptor α; IL-13Rα1, IL-13 receptor α1; h, human; LCDR, light-chain CDR; NHDF, neonatal human dermal fibroblast; Ni-NTA, Ni2 + -nitrilotriacetate;PBMC, peripheral blood mononuclear cell; PBST, PBS containing Tween 20; PEG, poly(ethylene glycol); RMSD, root mean square deviation; RU, relativeunits; STAT6, signal transducer and activator of transcription 6; TARC, thymus- and activation-related chemokine; VH, variable heavy; VL, variable light.

1 These authors contributed equally to this work.2 Present address: Medimmune, LLC, Gaithersberg, MD 20878, U.S.A.3 Present address: UT Health Science Center, Houston, TX 77030, U.S.A.4 Present address: Janssen Pharmaceuticals at Johnson & Johnson, Radnor, PA 19087, U.S.A.5 Nicos Nicola, Douglas Hilton and Jian-Guo Zhang hold patents on the IL-13 receptor.6 Correspondence may be addressed to any of these authors (email nash@csl.com.au, zhang@wehi.edu.au or nicola@wehi.edu.au).Co-ordinates for 10G5H6 Fab and 10G5H6 Fab–ectodomain 3 complex have been deposited in the PDB under codes 4HWE and 4HWB respectively.

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166 N. T. Redpath and others

addition, genetic polymorphisms in IL-13, its receptor subunitsor its signalling molecules have been associated with the severityof human asthmatic disease [11–13].

We originally cloned the mouse IL-13Rα1 subunit and showedthat the high-affinity IL-13 receptor consisted of this subunit aswell as the IL-4Rα subunit. We also showed that this receptorcomplex was an alternative receptor for IL-4 [4]. The humanequivalent was subsequently cloned and shown to have identicalfunctional properties [14,15]. Structural studies have revealed themolecular details of IL-13 and IL-4 recognition by the identicalreceptor complex of IL-13Rα1 and IL-4Rα [5]. Although thesestructures look very similar, the molecular assembly order isdifferent and can lead to differential biological responses in thesame cell in response to the two cytokines. Clearly there would bepotential benefit in a therapeutic approach that targets both IL-13and IL-4 that are strongly associated with asthma.

In the present paper, we describe the production of fully humanneutralizing monoclonal antibodies against h (human) IL-13Rα1,show that they inhibit the binding and biological activities of IL-13 and IL-4 on human cells and define the binding epitope andstructural details of the binding interaction.

EXPERIMENTAL

Animal experimentation

All animal experiments were approved by the animal ethicscommittee of the Walter and Eliza Hall Institute in accordancewith the NHMRC (National Health and Medical ResearchCouncil) Australian code of practice for the care and use ofanimals for scientific purposes.

Immunization of MedarexTM mice with hIL-13Rα1 and selection ofhybridomas

Mice transgenic for human immunoglobulin genes (Medarex)[16] were immunized intraperitoneally with 50 μg of the hIL-13Rα1 ECD (extracellular domain) [17] in complete Freund’sadjuvant and then boosted repeatedly with hIL-13Rα1 ECDformulated in either incomplete Freund’s adjuvant or PBS. Beforefusion, mice were injected intravenously with 10 μg of hIL-13Rα1 ECD in PBS and, 4 days later, spleens were harvestedand fusions performed as previously described [18,19].

Hybridoma supernatants were screened for hIL-13Rα1 bindingactivity using a standard ELISA format with plate-bound ECD,and for IL-13 antagonist activity in the TARC (thymus- andactivation-related chemokine) release assay described below.Selected hybridomas were cloned by limit dilution.

Affinity maturation of 10G5 by phage display

Affinity maturation of monoclonal antibody 10G5 was achievedby randomly mutating amino acids within the variable H(heavy-chain) and L (light-chain) CDR (complementarity-determining region) 3 followed by expression and selection usingphage display (Supplementary Table S1 at http://www.biochemj.org/bj/451/bj4510165add.htm). Libraries were constructed asdescribed previously [20] using ‘stop template’ versions ofpFab10G5 for the Kunkel mutagenesis method [21] withmutagenic oligonucleotides designed to simultaneously repair thestop codons and introduce mutations at the designed sites.The mutagenesis reactions were then electroporated intoEscherichia coli SS320 then phage production was initiated withaddition of M13-KO7 helper phage (Invitrogen) before incubation

at 30 ◦C for 18 h. Sequencing analysis of approximately 100clones from each of the small-scale libraries showed 100%diversity of mutant clones at the designed amino acid locations.

10G5 HCDR3 and LCDR3 libraries were panned against IL-13Rα1. Briefly, four rounds of selection were performed byincubating phage with decreasing concentrations of immobilizedbiotinylated hIL-13Rα1 (10, 1, 0.1 and 0.01 μg) in 2% (w/v)dried skimmed milk in PBST (PBS containing 0.1% Tween 20)for 20 min at room temperature (22 ◦C). Downstream panningsteps were carried out on the basis of standard protocols [22].Individual clones were picked after the second and third roundof selection and the Fab cassette and the light chain were PCR-amplified and sequenced essentially as described in [23]. PhageELISA was used as the primary assay to determine the abilityof the phage-bound recombinant Fab fragments to recognizebiotinylated hIL-13Rα1 immobilized on streptavidin plates.

Site-directed mutagenesis and phage display of hIL-13Rα1 andhIL-13Rα1 fragments

The ECD of hIL-13Rα1 (fibronectin-like domains D1, D2 andD3) (GenBank® accession number NP_001551) [14] as well assequences encoding D2–D3 or individual domains (D1, aminoacids 26–105; D2, amino acids 106–202; D3, amino acids 203–319) were directionally cloned into a phagemid vector (phg3-1) and used subsequently as the templates for all expressionconstructs.

IL-13Rα1 constructs were transfected into E. coli and phageproduction was initiated by addition of M13-KO7 helperphage (Invitrogen) before incubation at 30 ◦C for 18 h. Phage washarvested by precipitation with 4% PEG [poly(ethylene glycol)]600/0.5 M NaCl and resuspended in 100 μl of PBS. IL-13Rα1-expressing phage supernatants were tested for antibody bindingby ELISA plates coated with 10G5 (or control antibodies) withdetection by 100 μl HRP (horseradish peroxidase)-conjugatedanti-M13 antibody diluted 1:2500 in 1% (w/v) dried skimmedmilk in PBS (GE Healthcare). After washing the plates threetimes with PBST, the signal was developed for 2–3 min using100 μl/well of TMB/E (3,3′,5,5′-tetramethybenzidine) substrate(Chemicon International), the reaction was stopped upon additionof 2 M phosphoric acid (50 μl/well), and absorbance wasmeasured at 450 nm.

Determination of antibody-binding affinity by surface plasmonresonance

Antibody-binding affinity was performed using a BiacoreTM

4000 biosensor (GE Healthcare). In one set of experiments,goat anti-(human IgG) (Invitrogen H10500) was immobilizedon a CM5 sensorchip following the manufacturer’s instructionsto approximately 7000 RU (relative units). Antibodies 10G5or 10G5-6 were captured on spots 1 and 5 of each flowcell for 2 min at 0.3μg/ml to approximately 200 RU. SolublehIL-13Rα1 was injected over each flow cell for 5 min anddissociation was monitored for a further 30 min. The analysiswas performed with receptor prepared in a 2-fold dilution seriesbetween 40 and 1.25 nM, with each concentration analysedtwice. The analysis was performed at a flow rate of 30 μl/minin 10 mM Hepes, 150 mM NaCl, 3 mM EDTA and 0.005%Tween 20 (pH 7.4) at 37 ◦C. Responses from spots 2 and 4 ofeach flow cell (in which 10G5 or 10G5-6 were not captured,but otherwise treated identically), were subtracted from thoseof spots 1 and 5 respectively. Responses from blank injectionswere then subtracted from those of all other samples to produce

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Structure of the IL-13Rα1 ectodomain 3–Fab complex 167

double-referenced data suitable for kinetic analysis. Double-referenced sensorgrams were fitted using non-linear regressionto a model describing 1:1 kinetics, including a term for masstransport limitation. The Rmax value was fitted locally withassociation rate (ka), dissociation rate (kd) and equilibriumdissociation constant (Kd) fitted globally. In another set ofexperiments, to analyse the effect of mutation of Leu293 in hIL-13Rα1, 10G5H6 Fab was coupled directly to a CM5 sensorchipto a level of approximately 1000 RU and background subtractionwas achieved using a flow cell on which an unrelated Fab fragmenthad been immobilized.

Determination of antibody-binding affinity by KinExA

All KinExA experiments were carried out at room temperature.The antigen hIL-13Rα1 was serially 2-fold diluted into a 1× assaybuffer (1 mg/ml BSA in PBS, pH 7.4) containing a fixed amountof IgG or Fab. Samples were allowed to reach equilibrium bypre-incubation for 12–24 h and free antibody was measured using1 μg/ml Cy5 (indodicarbocyanine)-labelled mouse anti-(humanFab) or goat anti-(human Fcγ ) for IgG. Samples were run induplicate for each experiment. At least two curves were run usingdifferent antibody concentrations, one close to the predicted Kd

and one at least 3-fold higher. The equilibrium titration data wereanalysed using a 1:1 binding model provided by KinExA software.N-curve analysis for final Kd measurement was performed usingKinExA software.

Protein production

hIL-13Rα1 D3–His6 was expressed in plasmid pET26b, whichcontains a pelB periplasmic localization signal sequence in framewith the expressed protein, in E. coli BL21(DE3)pLysS cells.Cells were grown to a D600 of 0.8–1, induced with 0.1 mM IPTG(isopropyl β-D-thiogalactopyranoside) then grown overnight at19 ◦C. Since the majority of D3–His6 was secreted into themedium, the concentrated cell supernatant was purified onan Ni-NTA (Ni2 + -nitrilotriacetate) column (Qiagen) followedby Q-Sepharose ion-exchange and Superdex 75 gel-filtrationchromatography. In order to remove the C-terminal His6 tag, theprotein was treated with 30 units of carboxypeptidase A/mg ofD3 (Sigma) for 6 h at 37 ◦C before separation on a Superdex 75column.

hIL-13Rα1 D1–D3 was expressed as a secreted N-terminalFLAG–His6-tagged protein in Sf21 insect cells using establishedprotocols [24] and purified from cell medium as above.

For expression of anti-hIL-13Rα1 Fab fragments, the Fablight and heavy chain fragments were co-expressed in the sameplasmid (pET26b or pBR322) with a pelB periplasmic localiza-tion signal sequence in frame with the light chain and an OmpAsignal sequence in-frame with the heavy chain fragment. Therewas also a His6 tag at the C-terminus of the heavy chain fragment.The Fab was purified on Ni-NTA, followed by an hIL-13Rα1 D3affinity column.

Crystallization and structural determination

The final crystals of 10G5H6 Fab were obtained through a fewrounds of microseeding and crystallized in 1.2 M (NH4)2SO4,0.5% PEG8000 and 0.1 M Hepes (pH 7.2). For the 10G5H6 Fab–hIL-13Rα1 D3 complex, the protein was concentrated to an A280 of17 and the crystals grown from 1.6 to 2.0 M Li2SO4, 0.1 M Ches[2-(N-cyclohexylamino)ethanesulfonic acid] buffer (pH 9.5) bythe hanging-drop vapour-diffusion method.

Data from 10G5H6 Fab crystals were collected on a homesource at 100 K and processed with HKL2000 [25]. The structurewas solved by molecular replacement using PHASER [26], usingone Fab of PDB code 1T04 as a search model. The model waschecked for stereochemical correctness using RAMPAGE of theCCP4 suite [27]. The Ramachandran plot indicated that 97.1% ofresidues fall in the favoured regions and 2.9 % fall in the allowedregions.

Data from 10G5H6 Fab–hIL-13Rα1 D3 crystals were collectedat 100K on the MX2 beamline at the Australian Synchrotron andprocessed with the XDS package [28]. Structures were solvedby molecular replacement using uncomplexed 10G5H6 Fab as asearch model with PHASER [26], whereas D3 was gradually builtinto the model starting from two strands. Structure refinement wascarried out with PHENIX [29] and AutoBuster (BUSTER version2.10.0; Global Phasing) and manual rebuilding was undertakenusing COOT [30]. The Ramachandran plot indicated that 94.7 %of residues fall in the favoured regions, 4.9 % fall in the allowedregions and 0.4% (Ala241 and Ala304) fall in outlier regions.

Cell-based bioassays

STAT6 luciferase reporter assay

A HEK (human embryonic kidney)-293 cell line stablytransfected with a luciferase reporter gene under the controlof a STAT6 (signal transducer and activator of transcription6) promoter was stably transfected with hIL-13Rα1 and hIL-4Rα (293-STAT6-Luc-IL-13R). These cells were plated at 5×104

cells/well in 96-well flat-bottomed plates in RPMI 1640 medium,10% FBS (fetal bovine serum) and GlutaMAXTM (Invitrogen).Recombinant hIL-13 or hIL-4 (R&D Systems) was added andcells were incubated at 37 ◦C for 18 h under 5% CO2. Luciferaseactivity was determined by Britelite Plus Kit (PerkinElmer) andthese cells responded to both hIL-13 and hIL-4 equivalently withan EC50 of ∼1 ng/ml.

Eotaxin [CCL (CC chemokine ligand) 11] release

NHDF (neonatal human dermal fibroblast) cells (Lonza CC-2509)were maintained in FGMTM-2 medium (Lonza CC-3132) at 37 ◦Cunder 5% CO2. Cells were plated in flat-bottomed 96-well platesat 105 cells per well and PMA (20 ng/ml) was added for 1 h.Recombinant hIL-13 or hIL-4 was added and cells incubated at37 ◦C for 18 h under 5% CO2 before assaying eotaxin in thesupernatant by ELISA (R&D Systems).

TARC (CCL17) release

PBMCs (peripheral blood mononuclear cells) were isolated fromnormal donors with informed consent (Australian Red Cross)and plated at 5×105 cells/well in 96-well flat-bottomed plates inRPMI 1640 medium, 10% FBS and GlutaMAXTM. RecombinanthIL-13 or hIL-4 was added and cells were incubated at 37 ◦C for48 h under 5% CO2 before assaying TARC in the supernatant byELISA (R&D Systems).

CD23 up-regulation

PBMCs were isolated and plated as above except that cellswere incubated for 18 h. Cells were then incubated with FITC-conjugated anti-CD14 and PE (phycoerythrin)-conjugated anti-CD23 antibodies (BD Biosciences) at 4 ◦C for 30 min.

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168 N. T. Redpath and others

Figure 1 10G5 antibody inhibits IL-13 binding and activity

(a) Inhibition of IL-13 binding to hIL-13Rα1. Purified hIL-13Rα1 (8 μg/ml) was incubated without (A) or with (B) 10G5 monoclonal antibody (50 μg/ml) before passing over a hIL-13 biosensorchip. (b) Inhibition of IL-13- and IL-4-induced eotaxin (CCL11) production from NHDFs. NHDFs were cultured with PMA and incubated with increasing concentrations of 10G5 before stimulationwith IL-13 (left) at various concentrations (�, 4 ng/ml; �, 20 ng/ml; �, 200 ng/ml) or IL-4 (right) at various concentrations (�, 0.2 ng/ml; �, 1 ng/ml; �, 10 ng/ml) for 18 h. Supernatants werecollected and assayed for eotaxin production. These experiments were repeated four times with representative graphs shown.

RESULTS

Generation of human neutralizing antibodies against hIL-13Rα

By immunizing MedarexTM HuMab mice with soluble hIL-13Rα1, we generated a panel of hybridoma clones thatexpress fully human antibodies against recombinant hIL-13Rα1. Characterization of the antibodies identified a candidate,designated 10G5, with strong to moderate neutralizing activityof both IL-13 and IL-4 signalling in normal human fibroblastsexpressing IL-13Rα1 and IL-4Rα (Figure 1) and with high affinityfor hIL-13Rα1 as determined by surface plasmon resonance (Kd

3.65 +− 0.04 nM, n = 4).The 10G5 antibody was subjected to affinity maturation (as

outlined in Supplementary Table S1). Initially three phage displaylibraries covering the variable HCDR3 (FPNWGSFDY) andanother three libraries covering the variable LCDR3 (QQYET)were generated. These libraries all contained >108 functionaldiversity and, between them, covered all combinations of aminoacids at every position randomized in each set. In addition to therandomization of amino acid residues in HCDR3 and LCDR3, adeletion of one amino acid residue for HCDR3 and addition of oneor two amino acid residues for LCDR3 were also made. In the firstround of optimization, the most significant leads were a F100Mmutation in HCDR3 (MPNWGSFDY; designated 10G5H6), anda separate, less potent mutant possessing a pair of changes in theC-terminal residues of HCDR3 (FPNWGSLDH). Notably, only

the first, seventh and ninth positions of HCDR3 appeared to beconducive to randomization without loss of activity. Shorteningof the HCDR3 by one amino acid residue abolished the bindingactivity of 10G5 to IL-13Rα1.

No significant potency improvement was obtained from theoriginal LCDR3 libraries (keeping the HCDR3 as wild-type),with the exception that it became obvious that insertion ofadditional residues in the very short LCDR3 of 10G5 abolishedbinding activity. A second-round LCDR3 library was generatedusing the 10G5H6 (MPNWGSFDY) mutant as the VH (variableheavy) chain to search for synergy with the modified LCDR3.A set of LCDR3 mutants was obtained containing smallneutral residues in the last two positions of LCDR3, includingQQYAT, QQYAS, QQYGS and QQYST (Supplementary TableS1), the best of which was QQYAS, as determined in anin vitro dissociation-based binding assay (results not shown).A second-round library of HCDR3 was constructed in whichthree amino acid positions were randomized (XPNWGSXDX;randomized positions indicated by X), as dictated by the first-round results. The best results from this library are shown inSupplementary Table S1. A series of specific constructs was thenmade to test various combinations; the best three of which allcontained the same sequence in HCDR3 (MPNWGSLDH) andLCDR3 (QQYAS) and possessed Kd values for hIL-13Rα1 insolution of 20–30 pM. Two of these also contained inadvertentframework mutations. 10G5-6 was the lead VH/VL (variable

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Structure of the IL-13Rα1 ectodomain 3–Fab complex 169

Figure 2 Inhibition of IL-13- and IL-4-induced chemokine production and activation of human cells and cell lines

HEK-293 IL-4R/IL-13R luciferase cells (a) and A549 human lung epithelial cells (c) were incubated with increasing concentrations of 10G5 (�) or 10G5-6 (�) then stimulated with IL-13 for18 h. HEK-293 IL-4R/IL-13R luciferase cells (b) and A549 human lung epithelial cells (d) were incubated with increasing concentrations of 10G5-6 then stimulated with IL-4 (�) or IL-13 (�)for 18 h. Cells were assayed for luciferase activity (a and b) or supernatants were collected and assayed for CCL17 production (c and d). For (e) and (f), PBMCs were prepared from normal humanblood and incubated with increasing concentrations of 10G5-6 then stimulated with IL-13 for 18 h. Supernatants were collected and assayed for CCL17 production by ELISA (e) or cells were stainedwith anti-CD14 and anti-CD23 antibodies and the percentage of CD14+ , CD23 + cells were determined by flow cytometry (f). All experiments were repeated at least four times with representativegraphs shown. Results in (a)–(e) are means +− S.E.M. from duplicate wells. mAb, monoclonal antibody.

light) combination chosen for development due to the leastnumber of framework changes. The amino acid sequences ofHCDR3 and LCDR3 for parental 10G5, 10G5H6 and 10G5-6along with their corresponding IL-13Rα1-binding affinities andcell-based potencies are shown in Table 1. Notably, a single aminoacid change from 10G5 to 10G5H6 (F100M in HCDR3) resultedin increased binding affinity of nearly 10-fold and an increase inpotency of ∼3-fold. The additional changes from 10G5H6 to10G5-6 were two amino acid changes in the light chain (E93Aand T94S) and two additional changes in the heavy chain (F106Land Y108H) that further increased binding affinity 4-fold andpotency ∼1.5-fold.

Inhibitory activities of antibodies against hIL-13Rα1

Both 10G5 and 10G5-6 were shown to completely blockbinding of IL-13 to recombinant hIL-13Rα1 in a surfaceplasmon resonance assay and to efficiently inhibit IL-13- andIL-4-stimulated production and release of the T-lymphocytechemokine TARC (CCL17) from A549 human lung epithelialcells as well as expression of a STAT6–luciferase reporter inHEK-293 cells engineered to express hIL-4Rα and IL-13Rα1(Figure 2). 10G5-6 also completely inhibited IL-13-induced up-regulation of CD23 and CCL17 release from human PBMCs(Figure 2). Both antibodies more efficiently inhibited IL-13

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170 N. T. Redpath and others

Table 1 Binding affinity and inhibitory capacity of 10G5 and affinity-maturedneutralizing antibodies

Equilibrium dissociation constants (K d) (mean +− S.D. from three replicates) for bindinghIL-13Rα1 were determined using kinetic exclusion on a KinExATM instrument and 50 %inhibitory doses (IC50) were determined by titration in the NHDF assay stimulated with IL-13 at20 ng/ml.

HCDR3 LCDR3 K d (KinExA) IC50 (NHDF assay)Antibody sequence sequence (pM) (ng/ml)

10G5 FPNWGSFDY QQYET 861 +− 17 310 +− 4110G5H6 MPNWGSFDY QQYET 99 +− 2 110 +− 2810G5-6 MPNWGSLDH QQYAS 27 +− 1 70 +− 10

action compared with IL-4 action (∼30-fold difference in EC50)(Figure 2).

The IL-13Rα1 ECD consists of three fibronectin type III-likemodules, so we prepared constructs encoding the full-lengthECD and truncated fragments that contained one or two ofthese modules. The protein constructs were displayed on M13phage as gene III fusions, which allowed for binding assaysby phage ELISA. Only phage display constructs containingthe third (membrane-proximal) fibronectin type III module(D3) showed significant binding to 10G5 (Figure 3a), showingthat the 10G5-binding epitope was localized to this regionof IL-13Rα1. Since 10G5 bound to human, but not mouse,IL-13Rα1, we generated a series of human–mouse chimaeric

molecules (again by phage display) to more precisely define thebinding epitope in the D3 region of hIL-13Rα1. The series ofchimaeras (HM1–HM11 shown in Supplementary Figure S1 athttp://www.biochemj.org/bj/451/bj4510165add.htm) allowed usto identify two linear segments of the IL-13Rα1 sequence thatcontained residues critical for antibody binding (Figure 3b).Finer mapping of the binding epitope was then carried out byindividual alanine mutations of each human/mouse difference inthese regions. This series of IL-13Rα1 ECD mutants, displayedon phage, identified Phe233, Phe249, Tyr250 and Gln252 as having thestrongest effects on antibody binding (Figure 3c).

Structures of 10G5H6 and 10G5H6 Fab–D3 complex

To refine further the epitope of 10G5 and to understand thestructural basis for the improved affinity attributed to the M100Fsubstitution in HCDR3, we crystallized the Fab fragment of10G5H6 antibody and the complex of the 10G5H6 Fab antibodywith the D3 subdomain of hIL-13Rα1. We solved the structure of10G5H6 Fab at 2.43 Å (1 Å = 0.1 nm) resolution and the structureof the complex at 2.61 Å.

The crystal structure of 10G5H6 Fab was refined to 2.43 Åwith R/Rfree factors of 17.58%/22.61% (Table 2). The final modelcontained one 10G5 Fab molecule in the asymmetric unit with sixdisordered residues (133–138) from the heavy chain. The structureof 10G5H6 showed that HCDRs and LCDRs form a deep pocketable to bind to D3 of IL-13Rα1.

Figure 3 Epitope mapping of 10G5 by phage ELISA

Phage-displayed hIL-13R constructs were tested for binding to various monoclonal antibodies. 10G5 or anti-IL-13R control antibodies [8B4, a D2-specific monoclonal (control 1), and 2B6, aD1/2-specific monoclonal (control 2)] were coated on to plates and phage-displayed ECDs of the hIL-13R (a), phage-displayed human–mouse (HM) mutant receptors (b) or phage-displayedhIL-13R-containing point mutations (c) were incubated with the coated antibodies. Bound receptor was detected with a HRP-conjugated anti-phage antibody. (a) Results are A 450 values. (b andc) Results for 10G5 binding to mutant receptors is expressed as log binding index as determined from the following equation: log[100×(EC50 10G5 receptor mutant/EC50 control 1 receptormutant)/(�EC50 10G5 receptor mutants/�EC50 control 1 receptor mutants)]. The experiments were repeated twice. Representative histograms are shown.

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Structure of the IL-13Rα1 ectodomain 3–Fab complex 171

Table 2 Data collection and refinement statistics of 10G5H6 and 10G5H6Fab–D3

Values in parentheses represent highest resolution shell.

10G5H6 10G5H6–Fab D3

Data collectionSpace group I422 P4122Cell dimensions

a, b, c (A) 121.36, 121.36, 152.1 123.48, 123.48,181.43α, β , γ (◦) 90, 90, 90 90, 90, 90

Resolution (A) 50–2.43 (2.52–2.43) 50–2.61 (2.76–2.61)Rsym or Rmerge 12.8 (45.8) –I/σ I 28.1 (7.0) 5.71 (0.26)Completeness (%) 99.6 (96.9) 98.5 (98.7)Redundancy 14.3 (13.6) 4.8 (4.3)CC(1/2)* – 98.5 (11.2)

RefinementResolution (A) 42.9–2.43 47.2–2.61Number of reflections 21 607/1107 42 736/2148Rwork/R free 17.53/22.32 19.67/22.42Number of atoms

Protein 3209 4071Ligand/ion 80 130Water 243 77

B-factorsProtein 40.13 69.44Ligand/ion 66.55 114.79Water 43.21 48.35

RMSDsBond lengths (A) 0.010 0.010

*See [33]. This dataset was initially cut off at 3.0 A (I/σ I = 1.2) resolution and subsequentlyreprocessed to 2.61 A (I/σ I = 0.26) with XDS-XSCALE using the CC(1/2) statistic to determinethe useful resolution limit.

For the 10G5H6 Fab–D3 complex, the initial model containingonly 10G5H6 Fab was refined using AutoBuster. A gooddifference Fo − Fc electron density map was built manually withtwo polyalanine strands. Through iterative rounds of refinementand model building, the whole D3 domain was placed into thefinal model. The structure of the 10G5H6 Fab–D3 complexwas determined at 2.61 Å resolution with R/Rfree factors of19.67%/22.42% (Table 2). The asymmetric unit contained onecopy of the D3 domain bound to one Fab fragment of 10G5H6(Figure 4a) (solvent content approximately 75 %). The structurehas a number of interesting features, especially in the D3 domain(Figures 4a and 4b). The most striking are the swapping of theN-terminal strands A and B and C-terminal strand G between twoD3 monomers (Figure 4b and see below). There are also five Chesbuffer molecules found in the complex; two of the Ches moleculesoccupied the hydrophobic pocket in D3 that was created by theswapping of B–C and F–G loops.

The crystal structures of 10G5H6 Fab in both complexed anduncomplexed states aligned well with an RMSD (root mean squaredeviation) of 0.68 Å for 423 Cαs. Upon D3 binding, only minorconformational changes (<1 Å) were seen in most of the CDRs,with the exception of HCDR3. The most important residues(Asn102 and Trp103) from HCDR3 are involved in six hydrogenbonds and extensive van der Waals contacts in the interface (seebelow) and undergo an approximate 2 Å shift upon D3 binding.The side chain of Arg60 from the heavy chain undergoes a shift tostabilize the 310 helix at the C-terminus of D3. Another importantresidue, Tyr92, in LCDR3 undergoes a dramatic side-chain shiftto contact Leu293 of D3 (Figure 4d).

Interface of the 10G5H6 Fab–D3 complex

The antibody–antigen interface buries a total surface area of2199 Å2 (1137 Å2 of D3 accessible surface area and 1062 Å2

of 10G5H6). Analysis of the complex structure indicated that 22residues of D3 participate in the antibody–antigen interaction.These residues belong to four discontinuous fragments: Pro202

and Asp203 at the N-terminus of D3 form two hydrogen bondswith Asp56 and Ser57, both from the heavy chain (Figures 5aand 5b); residues 226–235 from the B–C loop and strand C;residues 248–253 from strand C′; and residues 289–296 from theC-terminus of strand F followed by the F–G loop which containsa 310 helix (residues 292–294) (Figure 5a). The total interactionbetween 10G5H6 Fab and D3 consists of 16 hydrogen bonds(<3.5 Å), three salt bridges and 197 van der Waals contacts (<4Å). All 16 hydrogen bonds involve HCDRs 1–3 and LCDRs1–3 of 10G5H6. As for D3, strands C, C′ and the F-strand-loop participate in forming four, five and five hydrogen bondsrespectively.

Arg230 from the B–C loop forms three salt bridges with Asp56

HCDR2 and a hydrogen bond with Trp34 of HCDR1 (Figure 5b).Phe233 from strand C of D3 is a key residue and its phenyl groupmakes extensive van der Waals contact with the side chain ofTrp103 (HCDR3) of 10G5H6 Fab heavy chain (Figure 5b). Anotherimportant residue from strand C is Leu232, which forms a hydrogenbond with Asn102 (H3) of 10G5H6 Fab (Figure 5b). The phenylgroup of Tyr250 of strand C′ of D3 stacks with the side chain ofTrp103 (H3) as well. Gln252 of strand C′ forms four hydrogen bondswith Ser32 (H1) and Asn102 (H3), both from the heavy chain of10G5H6 Fab (Figure 5c). In addition, the main-chain oxygen atomof Val248 of D3 hydrogen-bonds with the OG atoms of Ser54 (L2).One of the most striking contacts is the insertion of Leu293 of D3into the deep pocket on the surface of 10G5H6 Fab (Figure 5d).Leu293, in the middle of a 310 helix, was positioned approximately4 Å from five residues that line the pocket of 10G5H6 (Trp34,Trp48, Val51 and Met100 in the heavy chain fragment andTyr92 in the light chain) (Figure 5d and Supplementary FigureS2 at http://www.biochemj.org/bj/451/bj4510165add.htm). Thebackbone oxygen atom of Leu293 of D3 hydrogen-bonds with NEof Arg60 in the heavy chain fragment, which also forms anotherhydrogen bond with the main-chain oxygen atom of Tyr295. Asn291,another important residue in D3, forms two hydrogen bonds withTyr92 (LCDR3) and Asn102 (HCDR3). Additionally, Thr290 of D3makes one hydrogen bond with Tyr33 (LCDR1) (Figure 5d).

Comparison of D3 with that in intact receptor

The D3 domain of the 10G5H6 Fab–D3 complex displays someunusual features. The global alignment between D3 within the10G5H6 Fab–D3 complex and D3 within the IL-13 receptorcomplex (PDB code 3BPO) was broken because of the extensiveN- and C-terminal strand swapping in the 10G5H6 Fab–D3complex. The B–C loop is stretched out and residues 226–229form a helix. This extended B–C loop separates strands A and Bfrom strands C, C′, E and F so that the strands A and B belongto one monomer and strands C, C′, E and F belong to anothermonomer (Figure 4b). In addition, the extended F–G loop allowsswapping of the G strand. The extensive domain swapping favoursa more stable dimeric D3.

Although the overall alignment between D3 of the 10G5H6Fab–D3 complex and D3 of the IL-13 receptor complex wasbroken, strands C, C′ and F align well with an RMSD of 0.87Åfor 23 Cαs.

The key epitope residues Arg230, Phe233, Tyr250, Gln252 and Leu293

are located close to or on these three strands (Figure 6a). Phe233

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172 N. T. Redpath and others

Figure 4 Structure of the 10G5H6 Fab–D3 complex

(a) Ribbon diagram of 10G5H6 Fab–D3 complex (D3, green; light chain, yellow; heavy chain, cyan). (b) Structure of the D3 domain-swapped dimer in the complex. (c) Ribbon diagram ofdomain-swapped dimeric D3 with two 10G5 Fabs. (d) Comparison of the CDRs of unbound 10G5H6 (light chain, marine; heavy chain, pink) and bound 10G5H6 (light chain, yellow; heavy chain,cyan) with large conformational change residues indicated.

fitted well (RMSD of 1.6Å for Cα) between the two D3 complexesand the Cα of Tyr250 and Gln252 shifted 2.3 and 3.5 Å respectively.The Cα of Arg230 shifted 4.5 Å, but the biggest shift (5.6 Å forCα) seen was for Leu293 (Figure 6b).

The B–C loop in the D3 domain of the full-length receptoris stabilized by interactions with both IL-13 (Arg230 of theD3 domain forms hydrogen bonds with Phe107 and Arg108

of IL-13 and the disulfide bond Cys231–Cys294 of D3 is invan der Waals contact with Phe107 of IL-13) and the D2domain (Asn226 of D3 hydrogen-bonds with the main-chainoxygen atom of Asn112 in D2, whereas Phe227 in D3 interactswith Leu113 in D2) [5]. It is possible that in the absenceof these stabilizing interactions, the B–C loop may be quitemobile.

Since Leu293 in D3 is an important contact residue for bindingIL-13 and IL-4 [5] and appears to make a significant shift inour structure relative to that in the full-length ECD [5], wetested the binding of 10G5H6 Fab to wild-type compared withL293A mutant full-length hIL-13Rα1 ECD by surface plasmonresonance. The mutant receptor bound 10G5H6 with nearly 100-fold lower affinity than did wild-type (Kd 52 nM comparedwith 0.57 nM) (Figure 7), most of the change in affinity beingdue to a greater than 30-fold increase in the kd of the L293Amutant compared with wild-type. Similar results were seenwith L293A D3 domain compared with wild-type D3 domain(Supplementary Figure S3 at http://www.biochemj.org/bj/451/bj4510165add.htm).

DISCUSSION

In the present paper, we have described the generationand neutralizing activities of humanized antibodies thatrecognize the D3 domain of hIL-13Rα1. The 10G5 antibodyand the affinity-matured derivatives (10G5H6 and 10G5-6)inhibited the biological actions of both IL-13 and IL-4,including chemokine production by human cell lines, activationand chemokine production by human PBMCs and eotaxin releasefrom normal human dermal fibroblasts at doses below 10 μg/ml.The differential inhibitory doses of antibody required to inhibitIL-13 compared with IL-4 could be a result of the different orderof assembly of the ternary complexes with antibody inhibiting thefirst step of IL-13 receptor assembly, but only the second step ofIL-4 receptor assembly [5].

Crystal structures were determined for the Fab fragment of10G5H6 alone and in complex with the D3 domain of hIL-13Rα1. Surprisingly, the latter structure did not consist of theexpected D3 monomer in complex with Fab 10G5H6, but rathera dimer in complex with two Fab fragments (Figure 4c). Relativeto the published structure of D3 in hIL-13Rα1, our D3 fragmentdisplayed N- and C-terminal domain swapping that was intrinsicto dimer formation. Since the swapped A, B and G strandsshowed extensive crystal lattice packing contacts, it is likelythat this domain swapping favoured crystal formation in ourexperiments. It was observed during the purification of hIL-13Rα1 D3 that it existed as both monomeric and dimeric forms

c© The Authors Journal compilation c© 2013 Biochemical Society

Structure of the IL-13Rα1 ectodomain 3–Fab complex 173

Figure 5 The interface of 10G5H6 Fab–D3

(a) The four fragments containing the epitope of D3 (green) lie on the 10G5 surface. Important residues on D3 are shown as sticks except for Leu293 which is shown as balls. The key residues on10G5H6 are also labelled. (b–d) The hydrogen-bonding interaction network between 10G5H6 and D3. Oxygen, nitrogen and sulfur atoms are coloured red, blue and yellow/orange respectively. Ineach case, D3 elements are in green, 10G5H6 heavy chain is in cyan and 10G5H6 light chain is in yellow.

Figure 6 Comparison of the D3 domain structures seen in IL-13/IL-13Rα1 (PDB code 3BPO) or 10G5H6 Fab–D3 complexes

(a) Overall superimposition of D3 dimer from our complex (green and blue) with D3 from the holoreceptor complex 3BPO (magenta). (b) The relative orientations of Leu293 and other residues fromour structure (green) and 3BPO (magenta) are superimposed.

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174 N. T. Redpath and others

Figure 7 Effect of Leu293 mutation in D3 on binding affinity to 10G5H6

Binding of full-length ECDs of wild-type or L293A hIL13Rα1 to immobilized 10G5H6 Fab measured using surface plasmon resonance. The concentrations (nM) of receptor used are indicated.Representative sensorgrams are shown and the mean +− S.D. K d values are shown (n = 3).

that could be separated by gel filtration. It seems likely, giventhe results of the structural determination, that the dimeric D3observed during purification resulted from domain swapping.Although monomeric D3 was isolated for use in 10G5H6 complexformation, the possibility cannot be excluded that there wasdimeric D3 in the preparation or that dimer formation occurredsubsequent to isolation of the monomer, either before or duringcrystallization.

Three-dimensional domain swaps of this type were firstobserved for diphtheria toxin and ribonuclease A oligomerizationand have now been observed for a large number of differentproteins [31,32].

The major deviations (other than the domain swap) betweenthe D3s in the two structures involved a 3.7 Å movement ofCys231 and a 4.5 Å movement of Arg230 with a consequent 5.6Å movement of the important IL-13-binding residue Leu293.Comparison of 10G5H6 Fab alone or in complex with D3indicated some similarly dramatic movements especially of lightchain Tyr92 which forms part of the binding pocket for Leu293 ofD3. Leu293 (Leu319 in the nomenclature of LaPorte et al. [5]) isa key interacting residue of IL-13Rα1 forming Site IIa contactswith both IL-4 and IL-13 [5]. Since 10G5H6 Fab bound with highaffinity to both D3 and the full-length ectodomain of hIL-13Rα1and, in both cases, the affinity of this interaction was decreasedapproximately 100-fold by the L293A mutation, it is likely thatthe B–C loop structure in the receptor, in which Leu293 is located,displays significant mobility. Indeed the B–C loop conformationis among the most variable in fibronectin III domain structuresof other receptors and in hIL-13Rα1, it makes direct interactions

with bound ligands. It is possible therefore that the B–C loop inthe unliganded receptor has a conformation quite different fromthat seen in the liganded state.

Superimposition of our complex structure on the D3 domainof the holoreceptor complex [5] indicates that the Fabfragment occupies much of the space normally occupied bythe D2 domain of the IL-4Rα chain (Supplementary FigureS4 at http://www.biochemj.org/bj/451/bj4510165add.htm). It istherefore likely that 10G5 inhibits IL-13 and IL-4 binding andbiological activity both by binding ligand-contact residues in D3(Leu293), thus inhibiting ligand binding, as well as occupying thesite required for IL-13Rα1 D3 to interact with ligand and withIL-4Rα D2.

The first affinity-matured version of 10G5 (10G5H6)contained a single amino acid change of Phe100 to methionine.Computational replacement of Met100 in the 10G5H6 complexstructure with phenylalanine resulted in steric overlapwith the important contact residue Leu293 of hIL-13Rα1,suggesting a plausible explanation for the increased affinity of10G5H6 (Supplementary Figure S5 at http://www.biochemj.org/bj/451/bj4510165add.htm).

Most other changes in the HCDR3 region resulted in loss ofbinding because they included the contact residues Asn102 or Trp103

or residues adjacent to them. However, mutations F106L andY108H in HCDR3 increased affinity, presumably by subtly alter-ing the distance or orientation of these contact residues. Similarlymutated residues in the LCDR3 region that increased bindingaffinity (E93A and T94S) are not ligand-contact residues, but aredirectly adjacent to the contact residues Gln91 and Tyr92 (Figure 5).

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Structure of the IL-13Rα1 ectodomain 3–Fab complex 175

In summary, we have reported the generation of humanneutralizing antibodies against hIL-13Rα1 that inhibit IL-13 andIL-4 actions and thus may find clinical use in treating asthma.The structure of the antigen–antibody complex revealed thekey antibody-contact residues in hIL-13Rα1 required to inhibitIL-13 binding, especially Leu293. Furthermore, mutation analysisshowed how the binding affinity of the antibody could beimproved.

AUTHOR CONTRIBUTION

Arna Andrews, Louis Fabri, Hal Braley, Andrea Woods, Cindy Luo, Thomas Garrett, PingLu, Cheryl Ireland, Robin Ernst and Gail Forrest designed and performed experiments.Peter Czabotar interpreted experiments. Nicholas Redpath, Yibin Xu, Nicholas Wilson,Manuel Baca, Douglas Hilton, Andrew Nash, Jian-Guo Zhang, Zhiqiang An, Dennis Zaller,William Strohl and Nicos Nicola designed experiments, interpreted and summarized data.Nicos Nicola wrote the paper with input from all authors.

ACKNOWLEDGEMENTS

We thank P.M. Colman and M.C. Lawrence for structural insights and continual support.We also thank the IL-13R antibody team at Merck Research Laboratories for their collectivecontribution to the project. Data were collected on the MX1 and MX2 beamlines at theAustralian Synchrotron, Melbourne, VIC, Australia. Crystallization trials were performedat the Bio21-C3 (Collaborative Crystallisation Centre). N.J.W., A.E.A., L.J.F., H.B., M.B.and A.N.D. are or were employees of CSL Ltd. P.L., C.I., R.E.E., A.W., G.F., Z.A., D.M.Z.and W.R.S. are or were employees of Merck, Sharp and Dohme.

FUNDING

The work was supported by a Merck, Sharp & Dohme Research Grant (2009-2010), theAustralian National Health and Medical Research Council [grant numbers 257500 and461219], the Australian Cancer Research Foundation, the Victoria Government OperationalInfrastructure Scheme, and the Australian Government Independent Research InstitutesInfrastructure Support Scheme.

REFERENCES

1 Holgate, S. T. (2012) Innate and adaptive immune responses in asthma. Nat. Med. 18,673–683

2 Wenzel, S. E. (2012) Asthma phenotypes: the evolution from clinical to molecularapproaches. Nat. Med. 18, 716–725

3 Finkelman, F. D., Hogan, S. P., Hershey, G. K., Rothenberg, M. E. and Wills-Karp, M.(2010) Importance of cytokines in murine allergic airway disease and human asthma. J.Immunol. 184, 1663–1674

4 Hilton, D. J., Zhang, J. G., Metcalf, D., Alexander, W. S., Nicola, N. A. and Willson, T. A.(1996) Cloning and characterization of a binding subunit of the interleukin 13 receptorthat is also a component of the interleukin 4 receptor. Proc. Natl. Acad. Sci. U.S.A. 93,497–501

5 LaPorte, S. L., Juo, Z. S., Vaclavikova, J., Colf, L. A., Qi, X., Heller, N. M., Keegan, A. D.and Garcia, K. C. (2008) Molecular and structural basis of cytokine receptor pleiotropy inthe interleukin-4/13 system. Cell 132, 259–272

6 Hogan, S. P., Koskinen, A., Matthaei, K. I., Young, I. G. and Foster, P. S. (1998)Interleukin-5-producing CD4+ T cells play a pivotal role in aeroallergen-inducedeosinophilia, bronchial hyperreactivity, and lung damage in mice. Am. J. Respir. Crit. CareMed. 157, 210–218

7 Kips, J. C., O’Connor, B. J., Langley, S. J., Woodcock, A., Kerstjens, H. A., Postma, D. S.,Danzig, M., Cuss, F. and Pauwels, R. A. (2003) Effect of SCH55700, a humanizedanti-human interleukin-5 antibody, in severe persistent asthma: a pilot study. Am. J.Respir. Crit. Care Med. 167, 1655–1659

8 Leckie, M. J., ten Brinke, A., Khan, J., Diamant, Z., O’Connor, B. J., Walls, C. M., Mathur,A. K., Cowley, H. C., Chung, K. F., Djukanovic, R. et al. (2000) Effects of an interleukin-5blocking monoclonal antibody on eosinophils, airway hyper-responsiveness, and the lateasthmatic response. Lancet 356, 2144–2148

9 Corren, J., Lemanske, R. F., Hanania, N. A., Korenblat, P. E., Parsey, M. V., Arron, J. R.,Harris, J. M., Scheerens, H., Wu, L. C., Su, Z. et al. (2011) Lebrikizumab treatment inadults with asthma. N. Engl. J. Med. 365, 1088–1098

10 Mitchell, J., Dimov, V. and Townley, R. G. (2010) IL-13 and the IL-13 receptor astherapeutic targets for asthma and allergic disease. Curr. Opin. Invest. Drugs 11, 527–534

11 Beghe, B., Hall, I. P., Parker, S. G., Moffatt, M. F., Wardlaw, A., Connolly, M. J., Fabbri, L.M., Ruse, C. and Sayers, I. (2010) Polymorphisms in IL13 pathway genes in asthma andchronic obstructive pulmonary disease. Allergy 65, 474–481

12 Black, S., Teixeira, A. S., Loh, A. X., Vinall, L., Holloway, J. W., Hardy, R. and Swallow, D.M. (2009) Contribution of functional variation in the IL13 gene to allergy, hay fever andasthma in the NSHD longitudinal 1946 birth cohort. Allergy 64, 1172–1178

13 Tamura, K., Arakawa, H., Suzuki, M., Kobayashi, Y., Mochizuki, H., Kato, M., Tokuyama,K. and Morikawa, A. (2001) Novel dinucleotide repeat polymorphism in the first exon ofthe STAT-6 gene is associated with allergic diseases. Clin. Exp. Allergy 31, 1509–1514

14 Aman, M. J., Tayebi, N., Obiri, N. I., Puri, R. K., Modi, W. S. and Leonard, W. J. (1996)cDNA cloning and characterization of the human interleukin 13 receptor α chain. J. Biol.Chem. 271, 29265–29270

15 Caput, D., Laurent, P., Kaghad, M., Lelias, J. M., Lefort, S., Vita, N. and Ferrara, P. (1996)Cloning and characterization of a specific interleukin (IL)-13 binding protein structurallyrelated to the IL-5 receptor α chain. J. Biol. Chem. 271, 16921–16926

16 Lonberg, N. (2005) Human antibodies from transgenic animals. Nat. Biotechnol. 23,1117–1125

17 Miloux, B., Laurent, P., Bonnin, O., Lupker, J., Caput, D., Vita, N. and Ferrara, P. (1997)Cloning of the human IL-13R α1 chain and reconstitution with the IL4Rα of a functionalIL-4/IL-13 receptor complex. FEBS Lett. 401, 163–166

18 Galfre, G., Howe, S. C., Milstein, C., Butcher, G. W. and Howard, J. C. (1977) Antibodiesto major histocompatibility antigens produced by hybrid cell lines. Nature 266, 550–552

19 O’Reilly, L. A., Cullen, L., Moriishi, K., O’Connor, L., Huang, D. C. and Strasser, A. (1998)Rapid hybridoma screening method for the identification of monoclonal antibodies tolow-abundance cytoplasmic proteins. BioTechniques 25, 824–830

20 Sidhu, S. S., Lowman, H. B., Cunningham, B. C. and Wells, J. A. (2000) Phage display forselection of novel binding peptides. Methods Enzymol. 328, 333–363

21 Kunkel, T. A., Roberts, J. D. and Zakour, R. A. (1987) Rapid and efficient site-specificmutagenesis without phenotypic selection. Methods Enzymol. 154, 367–382

22 Barbas, 3rd, C. F., Kang, A. S., Lerner, R. A. and Benkovic, S. J. (1991) Assembly ofcombinatorial antibody libraries on phage surfaces: the gene III site. Proc. Natl. Acad. Sci.U.S.A. 88, 7978–7982

23 Hoet, R. M., Cohen, E. H., Kent, R. B., Rookey, K., Schoonbroodt, S., Hogan, S., Rem, L.,Frans, N., Daukandt, M., Pieters, H. et al. (2005) Generation of high-affinity humanantibodies by combining donor-derived and synthetic complementarity-determining-region diversity. Nat. Biotechnol. 23, 344–348

24 Xu, Y., Kershaw, N. J., Luo, C. S., Soo, P., Pocock, M. J., Czabotar, P. E., Hilton, D. J.,Nicola, N. A., Garrett, T. P. and Zhang, J. G. (2010) Crystal structure of the entireectodomain of gp130: insights into the molecular assembly of the tall cytokine receptorcomplexes. J. Biol. Chem. 285, 21214–21218

25 Otwinowski, Z. and Minor, W. (1997) Processing of X-ray diffraction data collected inoscillation mode. Methods Enzymol. 276, 307–326

26 McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. andRead, R. J. (2007) Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674

27 Winn, M. D., Ballard, C. C., Cowton, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan,R. M., Krissinel, E. B., Leslie, A. G., McCoy, A. et al. (2011) Overview of the CCP4 suiteand current developments. Acta Crystallogr. Sect. D: Biol. Crystallogr. 67, 235–242

28 Kabsch, W. (2010) XDS. Acta Crystallogr. Sect. D: Biol. Crystallogr. 66, 125–13229 Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J.

J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W. et al. (2010) PHENIX: acomprehensive Python-based system for macromolecular structure solution. ActaCrystallogr. Sect. D: Biol. Crystallogr. 66, 213–221

30 Emsley, P. and Cowtan, K. (2004) Coot: model-building tools for molecular graphics. ActaCrystallogr. Sect. D: Biol. Crystallogr. 60, 2126–2132

31 Huang, Y., Cao, H. and Liu, Z. (2012) Three-dimensional domain swapping in the proteinstructure space. Proteins 80, 1610–1619

32 Liu, Y. and Eisenberg, D. (2002) 3D domain swapping: as domains continue to swap.Protein Sci. 11, 1285–1299

33 Karplus, P. A. and Diederichs, K. (2012) Linking crystallographic model and data quality.Science 336, 1030–1033

Received 4 December 2012/4 February 2013; accepted 6 February 2013Published as BJ Immediate Publication 6 February 2013, doi:10.1042/BJ20121819

c© The Authors Journal compilation c© 2013 Biochemical Society

Biochem. J. (2013) 451, 165–175 (Printed in Great Britain) doi:10.1042/BJ20121819

SUPPLEMENTARY ONLINE DATAProduction of a human neutralizing monoclonal antibody and its crystalstructure in complex with ectodomain 3 of the interleukin-13receptor α1Nicholas T. REDPATH*†1, Yibin XU*†1, Nicholas J. WILSON‡1, Louis J. FABRI‡1, Manuel BACA‡1,2, Arna E. ANDREWS‡,Hal BRALEY‡, Ping LU§, Cheryl IRELAND§, Robin E. ERNST§, Andrea WOODS§, Gail FORREST§, Zhiqiang AN§3,Dennis M. ZALLER§, William R. STROHL§4, Cindy S. LUO*, Peter E. CZABOTAR*†, Thomas P. J. GARRETT*,Douglas J. HILTON5*†, Andrew D. NASH‡6, Jian-Guo ZHANG*†5,6 and Nicos A. NICOLA*†5,6

*The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia, †Department of Medical Biology, University of Melbourne, Parkville, VIC 3010,Australia, ‡CSL Limited, Parkville, VIC 3052, Australia, and §Merck Research Laboratories, West Point, PA 19486, U.S.A.

Figure S1 Alignment of human and mouse IL-13Rα1 amino acid sequences

Conserved amino acid residues between human and mouse are depicted with a colon (:) and non-conserved residues are represented with a space (). Domains are separated by single vertical lines(|). The boxed and labelled regions (HM1–HM11) in domain 3 of hIL-13 receptor represent various mutant forms of hIL-13 receptor. The human–mouse (HM) mutants were generated where thehuman amino acid was replaced with the corresponding murine amino acid in the boxed region. A total of 11 HM IL-13 receptor constructs were generated to allow all non-conserved residues indomain 3 of hIL-13R to be replaced with the murine sequence.

1 These authors contributed equally to this work.2 Present address: Medimmune, LLC, Gaithersberg, MD 20878, U.S.A.3 Present address: UT Health Science Center, Houston, TX 77030, U.S.A.4 Present address: Janssen Pharmaceuticals at Johnson & Johnson, Radnor, PA 19087, U.S.A.5 Nicos Nicola, Douglas Hilton and Jian-Guo Zhang hold patents on the IL-13 receptor.6 Correspondence may be addressed to any of these authors (email nash@csl.com.au, zhang@wehi.edu.au or nicola@wehi.edu.au).Co-ordinates for 10G5H6 Fab and 10G5H6 Fab–ectodomain 3 complex have been deposited in the PDB under codes 4HWE and 4HWB respectively.

c© The Authors Journal compilation c© 2013 Biochemical Society

N. T. Redpath and others

Figure S2 Electron density (2Fo − Fc, 1.5σ ) surrounding Leu293 of hIL-13Rα1 at the receptor–antibody interface

Leu293 of hIL-13Rα1, Trp34, Trp48, Val51 and Met100 of the heavy chain and Tyr92 of the light chain of 10G5H6 are labelled.

c© The Authors Journal compilation c© 2013 Biochemical Society

Structure of the IL-13Rα1 ectodomain 3–Fab complex

Figure S3 Effect of Leu293 mutation in D3 on binding affinity to 10GH6

Binding of the wild-type or L293A mutant of the D3 domain of hIL-13Rα1 to immobilized 10G5H6 Fab measured using surface plasmon resonance. Concentrations of D3 used (nM) are indicated.Representative sensorgrams are shown and mean+−S.D. K d values are shown (n = 3).

c© The Authors Journal compilation c© 2013 Biochemical Society

N. T. Redpath and others

Figure S4 10G5H6 sterically obstructs IL-13 and IL-4Rα binding to IL-13Rα1

(a) Structure of the IL-13 holoreceptor complex (extracellular domain) from La Porte et al. [1]. (b) Superimposition of 10G5H6 Fab interacting with D3 of hIL-13Rα1 on the holoreceptor complexshowing overlap of the antibody with the position normally occupied by IL-4Rα.

Figure S5 Met100 in 10G5H6 contacts Leu293 from the D3 domain of hIL-13Rα1 and phenylalanine in this position would overlap Leu293

(a) Interaction of Met100 of 10G5H6 Fab with Leu293 of the D3 domain of hIL-13Rα1 (green) and surrounding residues of the heavy (cyan) and light (yellow) chains. (b) Potential overlap of theoriginal 10G5 Fab Phe100 with Leu293.

c© The Authors Journal compilation c© 2013 Biochemical Society

Structure of the IL-13Rα1 ectodomain 3–Fab complex

Table S1 Strategy used for affinity maturation of 10G5 antibody

Mutated residues are bold and italic. Positions marked X were randomized for phage display.

Antibody Library HCDR3 sequence LCDR3 sequence

10G5 FPNWGSFDY QQYETRound 1

H1.1 FPNWXXXDXH1.2 XXXXGSFDYH1.3 FPXX -XXDYL1.1 QXXXTL1.2 QQXXXTL1.3 QQXXXXT

10G5H5 FPNWGSLDH10G5H6 MPNWGSFDY10G5H10 FPNWGSLDK10G5H11 FPNWGSMDA10G5H12 FPNWGSFDY (T120I)10G5L1 QTYST10G5L2 QQYATRound 2

H2.1 XPNWGSXDX10G5H6/L2.1 MPNWGSFDY QXXXT

10G5R4-1 MPNWGSFDT10G5R4-2 MPNWGSLDH10G5R4-3 MPNWGSFDS10G5R4-4 MPNWGSLDT10G5R4-5 MPNWGSLDA10G5R4-6 MPNWGSLDN10G5R4-7 MPNWGALDS10G5R4-8 MPNWGSFDN10G5R4-9 MPNWGSLDY10G5R4-10 MPNWGSVDHSJ2-57 MPNWGSFDY QTYSTSJ2-63 MPNWGSFDY QSYSTSJ2-88 MPNWGSFDY QRYSTSJ3-68 MPNWGSFDY QQYATSJ2-81 MPNWGSFDY QQYASSJ2-74 MPNWGSFDY QQYSTSJ2-85 MPNWGSFDY QQYGSSJ3-71 MPNWGSFDY QQYSSRound 310G5-1 ARMPNWGSFDY QQYAS10G5-2 VRMPNWGSLDH QQYAS10G5-3 ARMPNWGSFDY QQYEA10G5-4 VRMPNWGSLDH (T120I) QQYAS10G5-5 VRMPNWGSLDH (T120I) QQYET10G5-6 ARMPNWGSLDH QQYAS10G5-7 ARMPNWGSLDH (T120I) QQYAS10G5-8 ARMPNWGSLDH (T120I) QQYET

REFERENCE

1 LaPorte, S. L., Juo, Z. S., Vaclavikova, J., Colf, L. A., Qi, X., Heller, N. M., Keegan, A. D.and Garcia, K. C. (2008) Molecular and structural basis of cytokine receptor pleiotropy inthe interleukin-4/13 system. Cell 132, 259–272

Received 4 December 2012/4 February 2013; accepted 6 February 2013Published as BJ Immediate Publication 6 February 2013, doi:10.1042/BJ20121819

c© The Authors Journal compilation c© 2013 Biochemical Society

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