Dual Inhibition of Interleukin-23 and Interleukin-17 Offersjpet.aspetjournals.org/content/jpet/354/2/152.full.pdf · Stacey Skala, Noah Ditto, Zheng Lin, XiaoXia Yang, Pete Z. Cotter,

1521-0103/354/2/152–165$25.00 http://dx.doi.org/10.1124/jpet.115.224246THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 354:152–165, August 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

Dual Inhibition of Interleukin-23 and Interleukin-17 OffersSuperior Efficacy in Mouse Models of Autoimmunity s

Paul R. Mangan, Linhui Julie Su, Victoria Jenny, Andrea L. Tatum, Caryn Picarillo,Stacey Skala, Noah Ditto, Zheng Lin, XiaoXia Yang, Pete Z. Cotter, David J. Shuster,Yunling Song, Virna Borowski, Rochelle L. Thomas, Elizabeth M. Heimrich, Brigitte Devaux,Ruchira Das Gupta, Irvith Carvajal, Kim W. McIntyre, Jenny Xie, Qihong Zhao,Mary Struthers, and Luisa M. Salter-CidDiscovery Biology, Immunoscience (P.R.M., S.S., X.Y., D.J.S., Y.S., V.B., R.L.T., E.M.H., K.W.M., J.X., Q.Z., M.S., L.M.S.-C.)Selection Technologies (L.J.S., V.J.), Immunogenicity Prediction (C.P.), Pharmacology (P.Z.C., I.C.), Discovery Assays (R.D.G.),Bristol-Myers Squibb Research and Development, Waltham, Massachusetts; Protein Science and Structure, Bristol-MyersSquibb Research and Development, Princeton, New Jersey (Z.L.); Hybridoma Research, Bristol-Myers Squibb Research andDevelopment, Redwood City, California (A.L.T., B.D.); and Wasatch Microfluidics, Salt Lake City, Utah (N.D.)

Received March 6, 2015; accepted May 22, 2015

ABSTRACTTherapies targeting either interleukin (IL)-23 or IL-17 have shownpromise in treating T helper 17 (Th17)–driven autoimmunediseases. Although IL-23 is a critical driver of IL-17, recognitionof nonredundant and independent functions of IL-23 and IL-17has prompted the notion that dual inhibition of both IL-23 andIL-17 could offer even greater efficacy for treating autoimmunediseases relative to targeting either cytokine alone. To test thishypothesis, we generated selective inhibitors of IL-23 and IL-17and tested the effect of either treatment alone compared withtheir combination in vitro and in vivo. In vitro, using a novelculture system of murine Th17 cells and NIH/3T3 fibroblasts, weshowed that inhibition of both IL-23 and IL-17 completely

suppressed IL-23–dependent IL-22 production from Th17 cellsand cooperatively blocked IL-17–dependent IL-6 secretion fromthe NIH/3T3 cells to levels below either inhibitor alone. In vivo, inthe imiquimod induced skin inflammation model, and in themyelin oligodendrocyte glycoprotein peptide–induced experi-mental autoimmune encephalomyelitis model, we demonstratedthat dual inhibition of IL-17 and IL-23 was more efficacious inreducing disease than targeting either cytokine alone. Together,these data support the hypothesis that neutralization of bothIL-23 and IL-17 may provide enhanced benefit against Th17mediated autoimmunity and provide a basis for a therapeuticstrategy aimed at dual targeting IL-23 and IL-17.

IntroductionThe IL-23/IL-17–mediated axis of inflammation is pivotal to

the development of T cell–driven autoimmune diseases,including rheumatoid arthritis, inflammatory bowel disease,ankylosing spondylitis, multiple sclerosis (MS), psoriasis, andpsoriatic arthritis (Chen et al., 2006; Uyttenhove and VanSnick, 2006; Gaffen et al., 2014). IL-23/IL-17–mediatedpathology is associated with a subset of CD41 effector T cellscalled T helper 17 (Th17) (Harrington et al., 2006). Thepathogenic function of Th17 cells is driven in part by IL-23,a heterodimeric cytokine consisting of a p19 subunit coupledto the p40 subunit that is also shared by IL-12. IL-23

promotes Th17 pathogenesis by driving expression of thehallmark Th17 cytokine, IL-17, as well as other cytokines,such as IL-22 and granulocyte monocyte colony-stimulatingfactor (GM-CSF), and by suppressing IL-10 (Murphy et al.,2003; Langrish et al., 2005; McKenzie et al., 2006; McGeachyet al., 2007; McGeachy, 2011). IL-17 (or IL-17A) is thefounding member of a family of six structurally relatedcytokines named IL-17A through F (Aggarwal and Gurney,2002; Weaver et al., 2007). The most closely related IL-17members, IL-17A and IL-17F, can be secreted as IL-17A/Aand IL-17F/F homodimers as well as an IL-17A/F heterodimer(Chang and Dong, 2007). All three are produced by Th17 cellsbut, arguably, IL-17A/A and IL-17A/F are the more proin-flammatory members (Ishigame et al., 2009). In addition toTh17 cells, IL-17 can be produced by CD81 T cells, gd T cells,and invariant natural killer T (iNKT) cells and by lymphoidtissue inducer cells (Cua and Tato, 2010). IL-17 triggers

This study was supported by Bristol-Myers Squibb.dx.doi.org/10.1124/jpet.115.224246.s This article has supplemental material available at jpet.aspetjournals.org.

ABBREVIATIONS: ANOVA, analysis of variance; BMS, Bristol-Myers Squibb; CNS, central nervous system; EAE, experimental autoimmuneencephalomyelitis; ELISA, enzyme-linked immunosorbent assay; GM-CSF, granulocyte monocyte colony-stimulating factor; HAT, hypoxanthine-aminopterin-thymidine; IFN-g, interferon-g; IL, interleukin; IMQ, imiquimod; iNKT, invariant natural killer T; MOG, myelin oligodendrocyte glycoprotein;MS, multiple sclerosis; PBS, phosphate-buffered saline; PBST, 1� PBS solution with Tween; PD, pharmacodynamic; rm, recombinant murine; TGF,transforming growth factor.

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fibroblasts, epithelial cells, endothelial cells, and macrophagesto secrete cytokines, chemokines, and growth factors, includingIL-6, IL-8, CXCL1, granulocyte colony-stimulating factor, andGM-CSF, which recruit and expand neutrophils as well asmonocytes and enhance the local inflammatorymilieu (Aggarwaland Gurney, 2002). Acknowledgment of the critical involve-ment of IL-23 and IL-17 in autoimmune disorders hasmotivated the discovery of therapeutics for targeting IL-23and IL-17 pathways.Many pharmaceutical companies have now developed

antibodies against IL-23 or IL-17 that have been largelysuccessful in clinical trials, but they have also presented someunexpected results as well. Antibodies against the commonp40 subunit shared by IL-12 and IL-23 and IL-23–specificantibodies were remarkably efficacious in psoriasis patientsand showed promising results in Crohn’s disease. However, inMS patients, the trials with anti-p40 antibodies failed (Segalet al., 2008; Longbrake and Racke, 2009; Sandborn et al.,2012; Kimball et al., 2013; Papp et al., 2013a; Gaffen et al.,2014). Agents that block IL-17 were impressively efficaciousin psoriasis, psoriatic arthritis, and ankylosing spondylitis,and elicited disease improvement in rheumatoid arthritispatients (Leonardi et al., 2012; Papp et al., 2012a,b, 2013b;Kellner and Kellner, 2013; Patel et al., 2013; Rich et al., 2013;Sigurgeirsson et al., 2014). However, trials with IL-17inhibitors in Crohn’s disease were prematurely terminatedowing to a lack of efficacy and in some cases an exacerbation ofthe disease (Hueber et al., 2012). These mixed outcomeshighlight the complexity of the IL-23–IL-17 pathways andunderscore the fact that IL-23 and IL-17 are not entirelyredundant targets.Although IL-23 drives IL-17 production from Th17 cells,

IL-23 and IL-17 can function independently of each other.IL-23 induces additional proinflammatory cytokines such asIL-22 andGM-CSF, which have been shown to bemore criticalthan IL-17 to the pathology of certain animal disease models(Liang et al., 2006; Zheng et al., 2007; van der Fits et al., 2009;Codarri et al., 2011; El-Behi et al., 2011). IL-17 can be inducedindependently of IL-23 by transforming growth factor (TGF)-band IL-6 in mice (Bettelli et al., 2006; Mangan et al., 2006;Veldhoen et al., 2006) and by T-cell receptor activation inhuman cells (unpublished observation). Other cell types canalso be stimulated to produce IL-17 without IL-23, such asiNKT cells by a-galactosylceramide, and mast cells withcomplement component 5a, lipopolysaccharide, tumor necro-sis factor-a or IgG complexes (Yoshiga et al., 2008; Hueberet al., 2010).Neutralization of IL-17 alone might block all IL-17 regard-

less of its source, but other IL-23–dependent cytokines such asIL-22 and GM-CSF would be unaltered and could contributeto disease. Blockade of IL-23 alone would be expected toinhibit all IL-23–dependent cytokines including much ofthe Th17-produced IL-17. However, not all pathogenic IL-17would be inhibited since it can be produced without IL-23 incertain situations. Therefore, dual inhibition of IL-23 andIL-17 could neutralize the IL-23–dependent cytokines as wellas the effects of IL-23–independent IL-17 leading to a com-prehensive blockade of the IL-23/IL-17 pathway and enhancedefficacy in treating disease.Targeting a more complete neutralization of the IL-23/IL-17

pathway has therefore been of interest. Although agentstargeting both IL-17 and IL-23 have been reported, a complete

understanding of the potential for improved efficacy with dualinhibition of IL-23 and IL-17 is lacking (Mabry et al., 2010). Toinvestigate if greater efficacy could be achieved with dualblockade, we generated potent and selective inhibitors ofIL-23 and IL-17A. These tools enabled us to explore theimpact of IL-23 and IL-17 inhibition alone and in combinationin vitro and in vivo. We show in a novel in vitro culturesystem, where the impact of IL-23 and IL-17 blockade could betested within the same setting, that dual inhibition causeda stronger suppression of downstream cytokines than blockadeof either cytokine alone. Furthermore, combination treatmentwith both IL-23 and IL-17 inhibitors in vivo offered superiorefficacy compared with treatment with either inhibitor alone inthe imiquimod (IMQ)-induced skin inflammation and themyelin oligodendrocyte glycoprotein (MOG) peptide–inducedexperimental autoimmune encephalomyelitis (EAE) mousemodels.

Materials and MethodsMice. BALB/c mice were purchased from either Charles River

Laboratories (Wilmington, MA) or Harlan Laboratories (Indianapolis,IN) and C57BL/6 mice were purchased from Charles River, HarlanLaboratories, or The Jackson Laboratories (Bar Harbor, ME). Thestrain, sex, age and vendor of the mice used for each in vivo protocol isindicated within the description of each procedure below. Allprocedures involving animals were reviewed and approved by theInstitutional Animal Care and Use Committee.

Generation of IL-23p19 Mouse Adnectin. The ATI-1221 anti-mouse IL-23p19 adnectin was generated using established methods(Mitchell et al., 2014). Briefly, murine IL-23 [eBioscience, San Diego,CA; chemically biotinylated at Bristol-Myers Squibb (BMS; Princeton,NJ)] was presented to large synthetic libraries of adnectins usingmurine anti-p40 mAbs (clone C17.8; eBioscience). In this way, theIL-23–specific p19 subunit was displayed for adnectin binding. Togenerate the pegylated version of the adnectin, ATI-1249, purifiedATI-1221, was conjugated with a maleimide derivative of a 40-kDa2-branched polyethylene glycol moiety (mPEG2-MAL) via standardmaleimide chemistry at the penultimate C-terminal cysteine residueaccording to methods described previously (Mitchell et al., 2014).

Generation of Mouse IL-17RA-Fc Decoy Receptor. MouseIL17RA-Fc (mIL-17RA-Fc) was overexpressed in transiently trans-fected HEK293-6E cells. Conditioned mediumwas loaded to protein Acolumn. Captured protein was eluted by 50 mM sodium acetatebuffer, pH 3.5. The eluate was neutralized by 1M Tris immediately.Protein was further purified by Superdex 200 size-exclusion column toremove aggregates, and exchange buffer to phosphate buffered saline(PBS), pH 7.2.

Generation of Mouse IL-17 Monoclonal Antibody. BALB/cmice (Charles River Laboratories) were immunized intraperitoneallyand subcutaneously four times with recombinant murine (rm) IL-17A(R&D Systems/Bio-Techne, Minneapolis, MN) (2 mg/mouse perinjection) in Ribi adjuvant (Sigma-Aldrich, St. Louis, MO). Serumsamples were evaluated for reactivity to the immunogen by enzyme-linked immunosorbent assay (ELISA) using rmIL-17 (R&D Systems).The mice received two final boosts of 2 mg/mouse per injection of rmIL17on days 3 and 2 before fusion. B cells from spleens harvested frommouse #230891were then fused (ratio of 1:1) withmouse�63.Ag8.653myeloma cells (CRL-1581; American Type Culture Collection,Manassas, VA) by electrofusion (Hybrimmune, ECM 2001; HarvardApparatus, Holliston, MA). The resulting hybridomas were platedout in flat-bottom 96-well plates (200 ml/well), and incubated for 7 daysin hypoxanthine-aminopterin-thymidine (HAT) selection medium[Dulbecco’s modified Eagle’s medium supplemented with 10% fetalbovine serum, 2 mM glutamine, 5% BM-Condimed H1 HybridomaCloning Supplement (Roche, Atlanta, GA), and 1� HAT] and an

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additional 7 days in 1� sodium hypoxanthine and thymidineselection medium (same medium supplemented with 1� sodiumhypoxanthine and thymidine instead of HAT). Hybridoma super-natants were then screened by ELISA for antigen specificity (seebelow). Antigen-specific clones were subcloned by limiting dilution toachieve stability andmonoclonality. Clones of interest were expandedand antibodies were purified from culture supernatant over protein Acolumn. Hybridoma clones were preserved by freezing in 10% DMSOfreezing media (Sigma-Aldrich).

ELISA Screening for Antigen-Specific Detection of Anti–mIL-17A mAbs. Recombinant mouse IL-17 (R&D Systems) was coated ona 96-well plate at 2 mg/ml overnight at 4°C (50 ml/well). The plate wasblockedwith 1� PBS solutionwith Tween (PBST) containing 1% bovineserum albumin (200 ml/well) for 1 hour at room temperature, thenwashed three times with 1� PBST. Undiluted hybridoma supernatantswere added to each well (50 ml/well). Plates were washed three timeswith 1� PBST before addition of anti-mouse IgG-Fc–specific horserad-ish peroxidase–conjugated antibody (Jackson ImmunoResearch, WestGrove, PA) diluted 1:2000 in 1� PBST with 1% bovine serum albumin(50 ml/well). Plates were incubated with the secondary antibody for1 hour at room temperature. The plates were washed six times prior toaddition of 100 ml/well of ABTS substrate (Moss Inc., Pasadena, MD),and absorbance was read at 450 nmand referenced at 620 nm. Rat anti-mouse IL-17 antibodies (clone 50104 and clone 50101; R&D Systems)were used as positive control in the ELISA assay along with irrelevantrat IgG (Sigma-Aldrich) and mouse IgG (Sigma-Aldrich) as isotypecontrols.

Mouse IL-23–Dependent Phospho-STAT3 Induction inKit225 Cells. Kit225 cells were seeded into 96-well plates at 4 �105 cells/well and were rendered quiescent in the absence of fetalbovine serum and IL-2 for 3 hours at 37°C. For inhibition doseresponses for ATI-1221 and ATI-1249, 10 pM rmIL-23 (generated atBMS), or 10 pM rmIL-23 preincubated with a range of concentrationsof ATI-1221 and ATI-1249 antagonist for 1 hour, was applied to thecells and the cells returned to the incubator for 15 minutes at 37°C toallow the phosphorylation of STAT3 (abbreviated as pSTAT3). Eachtest condition was assayed in duplicate in 96-well plates. Stimulationwas stopped by placing the cells on ice and adding ice-cold PBS.Finally, the cells were pelleted and lysed following standard protocolsand pSTAT3 production detected by ELISA (Cell Signaling Technol-ogy, Danvers,MA). Results expressed as percent inhibition of pSTAT3levels and IC50s were determined using GraphPad Prism software(GraphPad Software, San Diego, CA).

Mouse IL-23–Dependent Cytokine Induction in PrimaryTh17 Cells. To differentiate mouse Th17 cells for analysis, CD41

T cells were enriched from the pooled spleen and lymph nodes ofC57/B6 mice (Harlan Laboratories) by magnetic bead selection(Miltenyi Biotec Inc., San Diego, CA). CD41 cells were coculturedwith CD4-depleted splenocytes that were irradiated with 3000 rads.T cells were activated with anti-CD3 (clone 145-2C11, generated atBMS) in presence of 5 ng/ml recombinant human TGF-b1 (R&DSystems) and 20 ng/ml recombinant mouse IL-6 (R&D Systems) alongwith 2 mg/ml of anti–IL-4 (clone 11B11; eBioscience) and 2 mg/mlanti–interferon-g (anti–IFN-g) (clone XMG1.2; eBioscience) neutral-izing antibodies. After 6 days in culture, the polarized Th17 cells wereharvested, reseeded in a 96-well Falcon plate and stimulated with20 ng/ml rmIL-23 (R&D Systems) and 5 ng/ml rmIL-2 (eBioscience)along with 2 mg/ml of anti–IL-4 (clone 11B11; eBioscience) and 2 mg/mlanti–IFN-g (clone XMG1.2; eBioscience) neutralizing antibodies for4 days. The conditioned media from these cultures were harvestedand assayed for both IL-17A and IL-22 concentrations by ELISA(R&D Systems). The IL-23–dependent response was measured bycalculating the difference between the level of cytokine induced by thecombination of rmIL-2 and rmIL-23 and the baseline level induced byrmIL-2 alone. To test the activity of ATI-1221 and ATI-1249 forinhibiting IL-23–dependent responses, a dose range of each inhibitorwas added during restimulation of the Th17 cells with rmIL-2 andrmIL-23. Results are expressed as percent inhibition of IL-17A or

IL-22 levels and IC50s were determined using XLfit software (IDBusiness Solutions Ltd., London, UK).

IL-17–Dependent CXCL1 Induction in NIH/3T3 Cells. NIH/3T3 cells (CRL-1658; American Type Culture Collection) were seededat concentration of 3 � 104 cells per well onto a 96-well flat-bottomFalcon plate. Cells were stimulated with 30 ng/ml recombinant mouseIL-17A/A (rmIL-17A/A) (generated at BMS) or 300 ng/ml recombinantmouse IL-17A/F (rmIL-17A/F) (R&D Systems) for 24 hours. Condi-tioned supernatant was harvested and tested for mouse CXCL1concentrations by ELISA (R&D Systems). The IL-17–dependentresponse was evaluated by calculating the difference between thelevel of cytokine induced by rmIL-17A/A or rmIL-17A/F and thebackground level in cultures with media alone. To test the activity ofmIL-17RA-Fc or the anti–IL-17 antibody for inhibiting IL-17–dependentresponses, a dose range of each inhibitor was added during stimulationof the NIH/3T3 cells with rmIL-17A/A or rmIL-17A/F. Results areexpressed as percent inhibition of CXCL1 levels and IC50s were deter-mined using XLfit software.

Coculture of Th17 Cells with NIH/3T3 Cells. Th17 cells weregenerated as described above and seeded at concentration of 2 � 105

cells per well onto a flat bottom plate along with 3 � 104 cells per wellNIH/3T3 cells. Cultures were stimulated with 100 ng/ml rmIL-23(R&D Systems) and 5 ng/ml rmIL-2 (eBioscience) along with 2 mg/mlof anti–IL-4 (clone 11B11; eBioscience) and 2 mg/ml anti–IFN-g (cloneXMG1.2; eBioscience) neutralizing antibodies for 4 days. The condi-tioned media from these cultures was harvested and assayed forIL-17A, IL-22, and IL-6 concentrations by ELISA (R&D Systems). Totest the impact of ATI-1221 and mIL-17RA-Fc for inhibiting cytokineresponses, each inhibitor at 20 nM concentration was added eitheralone or in combination during stimulation with rmIL-2 and rmIL-23.One-way analysis of variance (ANOVA) and Bonferroni’s post-testusing GraphPad Prism software were performed on cytokine results totest for statistical significance of differences observed.

IL-23–Dependent In Vivo Pharmacodynamic Model. FemaleC57BL/6 mice (Charles River Laboratories) at 8–9 weeks of age wereacclimated for 3 days and were randomly assigned in treatmentgroups based on body weight. In this model, animals exposed to mIL-2(eBioscience) and mIL-23 (generated at BMS) increased their serumlevels of mIL-17 as a response from polyclonally activated T cells.A priming dose with mIL-2 was administered to all groups (exceptthe control group) 24 hours before mIL-23 administration. Whencoadministered, mIL-2 and mIL-23 were diluted in PBS for a final100 ml intraperitoneal administration per mouse. Two hours beforethe start of the challenge doses of mIL-2 plus mIL-23, a single dose ofATI-1249 formulated in PBS was administered subcutaneously at thedoses indicated. Challenge doses of mIL-2 plus mIL-23 were given at0, 7, and 23 hours. Optimal amounts of mIL-2, when coinjected withmIL-23, were determined experimentally and were used to maintainmIL-17 induction throughout the evaluation period. Thus, at timepoints 0 hours and 23 hours, 5 mg mIL-2 was used, whereas 10 mg wasused at the 7-hour time point. A constant amount of 0.2 mg/mouse ofmIL-23 was coadministered at all the indicated time points. After30 hours the priming dose was experimentally determined to be thehighest peak for mIL-17 induction in the absence of anti–IL-23blockers. Thus, all treatment groups were anesthetized at 30 hoursusing 2.5% isoflurane inhalation and blood was terminally collectedvia cardiac puncture. Serum was separated by centrifugation, andserum samples were stored at –80°C analysis. SerummIL-17 cytokinelevels were evaluated by ELISA (R&D Systems), following themanufacturer’s instructions. Serum mIL-22 cytokine levels wereevaluated using EMDMillipore (Billerica, MA) LuminexMilliplex kit,following the manufacturer’s instructions. One way ANOVA andDunnett’s multiple comparison tests using GraphPad Prism softwarewere performed on cytokine results to test for statistical significanceof observed differences between treatments.

IL-17–Dependent In Vivo Pharmacodynamic Model. FemaleBALB/c mice (Harlan Laboratories) at 8–10 weeks of age were dosedsubcutaneously with the anti–IL-17 antibody at various doses. Eighteen

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hours later, mice were challenged with rmIL-17A/A (150 mg/kg)(generated at BMS) or rmIL-17A/F (150 mg/kg) (R&D Systems)subcutaneously in 0.2 ml of PBS. Two hours later, serum samples werecollected and the levels of CXCL1 were measured by ELISA (R&DSystems). One-way ANOVA and Dunnett’s multiple comparison testsusing GraphPad Prism software were performed on cytokine results totest for statistical significance of the differences observed betweentreatment.

Mouse Imiquimod-Induced Skin Inflammation Model. Fe-male BALB/c mice (Harlan Laboratories) at 6–9 weeks of age wererandomly assigned to treatment groups (n 5 8). Three days beforeinitiation of the study, mice were shaved along their backs (fromshoulder to hip) with electric clippers, and a depilation lotion (Nair;Church&Dwight Co., Ewing, NJ) was applied for complete removal ofhair. Anesthetizedmice (2.5% isoflurane inhalation) received a dose of62.5 mg/mouse of imiquimod (IMQ Cream 5%; Graceway Pharma-ceuticals, Bristol, TN) applied with a wooden applicator to the shavedskin. In the prophylactic study design beginning 18 hours before IMQtreatment, mice received either PBS, anti–IL-17 antibody (20mg/kg s.c.),ATI-1249 (20 mg/kg s.c.), or a combination of the two therapeutics.Skin thickness was measured daily using a Mitutoyo dial caliper(model no. 2412F; Mitutoyo, Kawasaki, Japan). The dorsal skin wasvisually assessed for erythema and scaling daily using the followingcriteria: 0, none; 1, slight; 2, moderate; 3, marked; 4, severe. Skinthickness was calculated as the percent change in thickness frombaseline measurement taken before the start of IMQ application foreach animal. The results are expressed as mean 6 S.E.M. for eachgroup. Data calculations were performed in Microsoft Excel (Redmond,WA), while statistical analysis (one-way ANOVA with Bonferroni’spost-test) and graphics were performed using GraphPad Prismsoftware. After seven applications of IMQ using 2.5% isofluraneinhalation, all treatment groups were anesthetized and blood wascollected via cardiac puncture. Serum was separated by centrifuga-tion and samples were stored at –80°C until analysis. Serum cytokinelevels were evaluated using Luminex multiplex kits (EMD Millipore)following the manufacturer’s instructions. One-way ANOVA andBonferroni’s post-test using GraphPad Prism software was performedon cytokine results to test for statistical significance of differencesobserved between treatment groups.

Histology for Mouse IMQ-Induced Skin InflammationModel. Dorsal skin from five animals per group was collected atnecropsy, fixed in 10% neutral buffered formalin, routinely processedto paraffin, and sectioned at 5 mm for H&E evaluation of lesionseverity. To score the severity of inflammation of the dorsal skin, anobjective scoring system was developed based on the followingparameters: extent of the lesion, the severity of hyperkeratosis,number and size of pustules, height of epidermal hyperplasia(acanthosis, measured in interfollicular epidermis), and the amountof inflammatory infiltrate in the dermis and soft tissue. Acanthosisand inflammatory infiltrate were scored independently on a scalefrom 0 to 4: 0, none; 1, minimal; 2, mild; 3, moderate; 4, marked.

Mouse Experimental Autoimmune EncephalomyelitisModel. C57BL/6 female mice at 6–8 weeks of age (The JacksonLaboratories) were immunized subcutaneously with a total of 200 mlover two areas with 3 mg/ml MOG35–55 emulsified 1:1 with 3 mg/mlMycobacterium tuberculosis (Difco Laboratories/Becton Dickinson,Franklin Lakes, NJ) in Incomplete Freund’s Adjuvant (Sigma-Aldrich) (300 mg peptide and 300 mg M. tuberculosis per mouse).Two hours later, mice were injected intraperitoneally with 300 ngpertussis toxin (in 100 ml volume). One day before immunization, micewere treated subcutaneously with PBS (every other day), the anti–IL-23adnectin, ATI-1249 (25 mg/kg every other day), the anti–IL-17 antibody(25 mg/kg 1�/week), or combination of ATI-1249 and the anti–IL-17antibody with the same regimen used in the monotherapy groups.Clinical scoring and body weight were taken three times per week.Clinical scoring system: 0.5, partial tail weakness; 1, limp tail orwaddling gait with tail tonicity; 1.5, waddling gait with partial tailweakness; 2, waddling gait with limp tail (ataxia); 2.5, ataxia with

partial limb paralysis; 3, full paralysis of one limb; 3.5, full paralysis ofone limbwith partial paralysis of a second limb; 4, full paralysis of twolimbs; 4.5, moribund; 5, death. Mean clinical score was calculated byaveraging the scores of all mice in each group. One-way ANOVA withBonferroni’s post-test were performed using GraphPad Prismsoftware on clinical score data to test for statistical significance ofthe differences observed between treatment groups.

Histology for Mouse EAE Model. At the end of study, intactspinal columns were removed and fixed in 10% neutral bufferedformalin. Three transverse sections of the lumbar segment of spinalcord were excised and routinely processed to paraffin. Paraffin-embedded tissues were sectioned at 3 mm for H&E, and 6 mm for themyelin-specific stain Luxol fast blue. Slideswere analyzed in a blindedfashion for severity of inflammation and demyelination. A histologicscore of 0 to 5 was assigned separately for each lesion characteristicfor all three transverse sections of spinal cord evaluated per animal. Ascore of 0 indicates no lesions/infiltrate; 1, minimal; 2, mild; 3,moderate; 4, marked; and 5, severe. Average scores of inflammationand demyelination were calculated and reported.

ResultsThe Anti-Murine IL-23–Specific Adnectins ATI-1221

and ATI-1249 Potently Inhibit IL-23–DependentResponses In Vitro. To selectively and specifically neutral-ize murine IL-23, a murine IL-23p19–specific adnectin wasgenerated using mRNA display (Roberts and Szostak, 1997).An adnectin is a recombinant protein derived from the tenthtype III domain of fibronectin that has been modified to bindspecifically to a selected target. The functional potency ofthe adnectins was evaluated for inhibition of murineIL-23–dependent pSTAT3 signaling in the human cell lineKit225. As a result of the 70% homology between mouse andhuman IL-23, stimulation of Kit225 cells with murine IL-23induced a robust 8- to 9-fold induction of pSTAT3 abovebackground (Oppmann et al., 2000). Using this assay weobtained a series of mIL-23 binding adnectins. To improve theaffinity of these molecules against mIL-23, an optimization-selection was carried out using increased selective pressure,including lowering target concentration. From this optimizationprocess, ATI-1221 was identified as a potent IL-23–specificinhibitor with subnanomolar binding affinity to IL-23 (Supple-mental Table 1). In the Kit225 assay, ATI-1221 dose de-pendently inhibited the IL-23–induced activation of pSTAT3with an IC50 of 1.5 nM (Fig. 1, A and B).To test ATI-1221 for activity against IL-23–dependent

responses in primary cells, an assay was established forinducing IL-23–driven cytokine secretion from differentiatedmouse Th17 cells. T cells were polarized to a Th17 phenotypeby incubation with TGF-b1 and IL-6 for 6 days. The Th17 cellswere harvested and recultured with IL-2 and IL-23 to induceIL-17A and IL-22. The addition of IL-2 was required tomaintain cell viability and enable robust cytokine productionbut did not strongly induce IL-17A or IL-22 by itself. Additionof IL-23 to IL-2 typically caused a 2- to 3-fold induction ofIL-17A and at least a 15-fold induction of IL-22. Addition ofa range of concentrations of ATI-1221 during stimulationof Th17 cells with IL-2 and IL-23 caused potent inhibitionof both IL-23–dependent IL-17A and IL-22, with comparableIC50 values of 1.6 and 1.9 nM, respectively (Fig. 1, A, C, and D).To increase the pharmacokinetic properties of ATI-1221,

the adnectin was reformatted as a PEGylated molecule andrenamed ATI-1249. To ensure that PEGylation did not affect


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potency, it was tested for functional activity in vitro in theIL-23–induced pSTAT3 assay in Kit-225 cells and also theprimary Th17 cell assay. Similar to the unformatted adnectin,ATI-1249 bound IL-23 with subnanomolar affinity andinhibited pSTAT3 in a dose-dependent manner with an IC50

of 2.6 nM (Supplemental Table 1; Fig. 1, A and B). Inthe primary Th17 cell assay, ATI-1249 also inhibitedIL-23–dependent cytokine secretion from Th17 cells with anIC50 of 1.9 nM for inhibition of IL-17A and 2.3 nM forinhibition of IL-22 (Fig. 1, A, C, and D). Therefore, PEGylationof ATI-1249 did not adversely affect the potency of theanti–IL-23 adnectin. The specificity of ATI-1221 and ATI-1249 for IL-23 over IL-12 was confirmed by demonstratingthat ATI-1221 and ATI-1249 had no effect on IL-12–dependent mouse Th1 differentiation and secretion of IFN-gcompared with an anti-p40 antibody (Supplemental Fig. 1).Together these data established ATI-1221 and ATI-1249 aspotent and selective inhibitors of IL-23–dependent responsesin vitro.The mIL-17RA-Fc and Anti-Mouse IL-17 Antibody

Potently Inhibit IL-17–Dependent CXCL1 In Vitro.IL-17 acts on various cell types, including epithelial cells,endothelial cells, and fibroblasts to induce numerous cyto-kines and chemokines, such as IL-6, granulocyte colony-stimulating factor, GM-CSF, IL-1b, TGF-b, tumor necrosisfactor-a, IL-8, and CXCL1 (Aggarwal and Gurney, 2002;Weaver et al., 2007). To test the impact of IL-17 neutralizationon cellular responses in vitro we generated a soluble decoyreceptor mouse IL-17RA-Fc as well as a monoclonal antibodyagainst mouse IL-17. To measure the activity and selectivityof mIL-17RA-Fc and the anti-mouse IL-17 antibody forinhibition of IL-17–dependent responses, these agents weretested for inhibition of murine IL-17A/A and IL-17A/F–dependent induction of CXCL1 from NIH/3T3 fibroblast cells.Stimulation of NIH/3T3 cells for 24 hours withmouse IL-17A/Aor IL-17A/F elicited a greater than 15- or 200-fold induction ofCXCL1, respectively. Treatment with mIL-17RA-Fc caused

a dose-dependent inhibition of IL-17A/A–induced activitywith an IC50 of 1.8 nM (Fig. 2, A and B), and IL-17A/F–inducedactivity with an IC50 of 2.6 nM (Fig. 2, A and C). The anti–IL-17antibody was slightly more potent against IL-17A and IL-17A/Fwith an IC50 of 0.9 nM and 1.2 nM, respectively (Fig. 2). Thesedata confirmed that both the mIL-17RA-Fc and the anti–IL-17antibody were potent and selective inhibitors of mouse IL-17A/Aand IL-17A/F in vitro.Dual Inhibition of IL-23 and IL-17 In Vitro Offers

More Complete Suppression of Cytokine Secretion. Toextend our findings with IL-23 and IL-17 inhibitors in distinctIL-23– and IL-17–dependent assays, we sought to test theimpact of dual inhibition of IL-23– and IL-17–dependentresponses within the same assay system. To test for potentialcooperative effects of inhibiting both IL-23 and IL-17 in vitro,a method was developed in which polarized mouse Th17 cellswere cocultured with NIH/3T3 cells. In this system, Th17 cellsserved as the IL-23–responsive population and the source ofIL-17, and the NIH/3T3 cells were the IL-17–responsive cells.Given a role for IL-6 more physiologically relevant thanCXCL1 in the development of Th17-mediated pathology, IL-6was used as the primary readout of IL-17–dependent activityin the NIH/3T3 cells (Yao et al., 1995; Hwang et al., 2004; Yenet al., 2006; Chiricozzi and Krueger, 2013). Addition of IL-23induced IL-17A production from the Th17 cells, which in turnhad a downstream effect of driving cytokines from NIH/3T3cells, including IL-6 (Fig. 3, A and C). Notably, in parallel toIL-17 production, IL-23 could also drive IL-22 production fromTh17 cells, providing an additional readout of IL-23 function(Fig. 3B). Using this method, the impact on cytokine pro-duction of blocking IL-23 and IL-17 alone or in combinationwas tested. A dose of the anti–IL-23 adnectin ATI-1221 andthe mIL-17RA-Fc fusion was chosen on the basis of thepotency in the initial functional in vitro assessment such thatsaturating levels of each agent were used corresponding toapproximately 10-fold over the IC50 in the primary cell–basedassays described above.

Fig. 1. Mouse anti–IL-23 adnectins ATI-1221and ATI-1249 potently inhibit IL-23–dependentresponses in vitro. (A) Average IC50 valuescalculated from at least four experiments foranti–IL-23 adnectins in the IL-23–dependentpSTAT3 induction assay in Kit225 cells andthe IL-23–dependent cytokine secretion assayin polarized Th17 cells. (B) IC50 curves shownwere generated from one study and arerepresentative of at least four replicate stud-ies testing the inhibition of IL-23–inducedpSTAT3 by ATI1221 and ATI-1249 in theIL-23–dependent pSTAT3 induction assayin Kit225 cells. Data points are the percentinhibition calculated from the average valueof duplicate wells. Error bars representS.E.M. (C and D) IC50 curves shown weregenerated for one study and are representa-tive of least four replicate studies testingthe inhibition of IL-23–dependent IL-17A(C) and IL-22 (D) by ATI1221 or ATI-1249 inthe IL-23–dependent cytokine secretion assayin polarized Th17 cells. For (C) and (D) datapoints are the percent inhibition calculatedfrom the average value of triplicate wells.

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In this coculture system, stimulation with IL-2 alone led toan increase in IL-17A and IL-22 consistent with the abilityof IL-2 to act as a T-cell survival and proliferation factorpromoting increased levels of T-cell cytokines in the culture.Importantly, the low levels of IL-6 produced by the coculturesystem were also increased with the addition of IL-2,consistent with the increases in T-cell cytokines in thissystem. Of note, blockade of IL-23 did not affect the modestlyincreased IL-17A– or IL-22–cytokine production driven byIL-2 alone, suggesting that production of these cytokines wasnot dependent on IL-23 (Fig. 3, A and B). To further activatethe IL-23–IL-17 pathway, IL-23 was added to the coculturesystem and dramatically increased IL-17A as well as IL-22production, consistent with the important role of IL-23 indirectly driving these cytokines from T cells (Fig. 3A) (Lianget al., 2006). Blockade of IL-23 with the anti–IL-23 adnectinATI-1221 alone caused a greater than 95% inhibition ofIL-17A (Fig. 3A) and 92% inhibition of IL-22 (Fig. 3B).Production of IL-22 driven either by IL-2 or IL-2/IL-23 was notaffected by the inhibition of IL-17 and was blocked similarlyby either the IL-23 adnectin or the combination of the IL-23

adnectin with mIL-17RA-Fc (Fig. 3B). These data suggestminimal or no contribution of the IL-17 neutralization in thecombination treatment, which might have been expectedconsidering the exclusive dependence of IL-22 on IL-23 (Lianget al., 2006).In addition, IL-6 was detected in these cultures at baseline

(media alone) and was further increased by the addition ofIL-2 or IL-2/IL-23 (Fig. 3C). IL-23 blockade had no effect onIL-6 in the baseline or IL-2-stimulated cultures. The IL-6levels in cultures with IL-2 alone was modestly but signifi-cantly augmented approximately 25% by the addition of IL-23(Fig. 3C). While the IL-6 levels seem to be very dependent onIL-17 levels detected in the culture, the dramatically in-creased IL-17 produced in the presence of IL-23 caused onlya modest enhancement of IL-6 production from the NIH/3T3cells. Consistent with this notion, IL-23 neutralization hadonly a modest effect for blocking IL-6 driven by IL-2/IL-23 butdid reduce the IL-6 levels to the level induced by IL-2 alone.However, the amount of IL-6 in the culture was still markedlyelevated in the presence of IL-23 blockade. Notably, IL-17production was not completely eliminated in the presence

Fig. 2. Mouse anti–IL-17 inhibitors,mIL-17RA-Fc and anti–IL-17 Ab, po-tently inhibit IL-17–dependet responsesin vitro. (A) Average IC50 values calcu-lated from at least three experiments foranti–IL-17 inhibitors against IL-17A/Aand IL-17A/F–dependent induction ofCXCL1 from NIH/3T3 fibroblast cells.(B) IC50 curves shown were generatedfrom one study and are representative ofat least three replicate studies testing theinhibition of IL-17A/A–induced or (C)IL-17A/F–induced CXCL1 by mIL-17RA-Fcand anti–IL-17 in NIH/3T3 cells stimulatedfor 24 hours. Each data point repre-sents the percent inhibition calculatedfrom the average CXCL1 concentration induplicate wells.

Fig. 3. Dual inhibition of IL-23 and IL-17 in a coculture of Th17 and NIH/3T3 cells is more effective for inhibiting cytokine production than either alone.ELISA measurements of IL-17A (A), IL-22 (B), and IL-6 (C) concentrations in conditioned supernatants collected from cocultures of polarized Th17 cellswith NIH/3T3 cells and stimulated with IL-2 alone or IL-2+IL-23 for 4 days. The impact of IL-23 and IL-17 inhibition on cytokine levels was tested byadding media alone (open bars), 20 nM ATI-1221 alone (gray bars), 20 nM mIL-17RA-Fc alone (hatched bars), or both inhibitors combined (solid bars).Graphs show data from one study and are representative of at least three comparable studies. Data are calculated as the average cytokine concentrationfrom triplicate wells. Error bars represent S.E.M. *P, 0.01 by one-way ANOVAwith Bonferroni’s post-test for multiple comparisons for IL-2 versus IL-2+IL-23 stimulation, (#) for media versus any treatment, and (^) for single inhibitor versus combination treatment. Note: All cytokine levels elevated byIL-2 or IL-2+IL-23 regardless of treatment were statistically significantly different compared with the media alone condition.


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of IL-23 neutralization (Fig. 3A). The level of IL-17 thatpersisted was actually comparable to the amount observedwith IL-2 alone, suggesting that IL-17 was produced in-dependently of IL-23. Clearly, this amount of IL-17 wassufficient to drive robust IL-6 secretion even in the presence ofIL-23 neutralization. However, the IL-6 production under allconditions, including baseline, was inhibited by IL-17 block-ade, consistent with the hypothesis that IL-6 production inthe NIH/3T3 cells was driven by IL-17 expressed either atbaseline or further induced by IL-2 and IL-2/IL-23, with an85% inhibition of IL-6 at baseline, 67% inhibition of IL-6 afterIL-2 treatment alone, and 55% inhibition in the IL-2 /IL-23costimulation observed (Fig. 3C). Of note, neutralization ofIL-17, however, did not block the levels of IL-6 in the presenceof IL-2/IL-23 to the level observed with IL-2 alone. These datasuggest that the IL-17 inhibition alone was insufficient toreduce the IL-6 levels that were augmented with IL-23.Together, these results demonstrate that IL-23 blockadealonewas ineffective for complete inhibition of IL-17–dependentIL-6 production, and IL-17 blockade alone was also inade-quate for total inhibition of IL-23–dependent responses, includ-ing production of IL-22.When both ATI-1221 and mIL-17RA-Fc were added

together in cultures stimulated with IL-23 and IL-2, the dualinhibition further reduced IL-6 levels more than 69% (Fig.3C). This reduction of IL-6 was 37% lower than the levelobserved in the presence of IL-17 blockade alone and wascomparable to the level induced by IL-2 alone (Fig. 3C). Theaugmented suppression of IL-6 levels suggest that the IL-23inhibition abolished the IL-23–dependent IL-17 as observedwith ATI-1221 alone, and the mIL-17-Fc further blocked anyresidual IL-17 secreted independently of IL-23. This compre-hensive neutralization of IL-17 with dual inhibition led to the

more profound reduction of IL-6. Despite the fact that theIL-23 adnectin was virtually ineffective for reducing IL-6levels on its own, a reduction was observed when it wascombined with IL-17 neutralization, indicating that betterblockade of the IL-17–IL-23 axis could be achieved by dualinhibition. Collectively, these data from the coculture systemdemonstrate that dual targeting of both IL-23 and IL-17 notonly completely inhibited IL-23–dependent IL-22 but also hada more dramatic effect by blocking IL-6 levels as well.The PEGylated Anti-Murine IL-23–Specific Adnectin

ATI-1249 Inhibits IL-23–Dependent Activity In Vivo.With the aim of ultimately testing the impact of IL-23 andIL-17 dual inhibition in vivo, we validated our inhibitors inIL-23– and IL-17–dependent pharmacodynamic (PD) models.First, we evaluated the activity of the PEGylated anti–IL-23adnectin ATI-1249 for inhibiting IL-23–dependent responsesin vivo. The PD model used to test the functional activity ofATI-1249 against IL-23–dependent responses in vivo involvedadministering mIL-2 and mIL-23 to mice according to aspecific regimen (Fig. 4A). IL-2 was injected alone 24 hoursbefore three consecutive treatments with IL-2 and IL-23 at0, 7, and 23 hours. Three challenges of IL-2/IL-23 wererequired to detect robust cytokine levels in the serum. At 30hours, the mice were bled and serum was processed formeasuring IL-17 and IL-22 concentrations by ELISA. Chal-lenge with IL-2 alone or mouse IL-23 alone had minimaleffects on cytokine secretion, but the combination of IL-23with IL-2 resulted in robust production of IL-17A and IL-22(Fig. 4, B and C). To test the activity of ATI-1249 for inhibitionof these IL-23–dependent cytokines, mice were treated witha range of ATI-1249 concentrations 2 hours before the firstchallenge with IL-2/IL-23 (Fig. 4A). In these mice, a doseresponse was observed, with maximal reduction of both IL-17

Fig. 4. Mouse anti–IL-23 adnectin ATI-1249 inhibits IL-23–dependent cytokines in vivo. (A) Experimental design of the IL-23–dependent PD assay, (B)ELISAmeasurements of IL-17A, and (C) IL-22 in serum collected frommice challenged by intraperitoneal injection with one dose of IL-2 alone and threesubsequent doses of IL-2+IL-23 at 24, 31, and 47 hours after the first treatment with IL-2. To test the impact of IL-23 inhibition, mice were dosed bysubcutaneous injection with a range of ATI-1249 concentrations 2 hours before the first challenge with IL-2+IL-23. A terminal bleed was performed andserumwas processed for analysis at 56 hours. The results are expressed as the mean IL-17 or IL-22 level in serummeasured in five mice per group. Errorbars represent S.E.M. #P, 0.05 by one-way ANOVA with Dunnett’s multiple comparison test for IL-2+IL-23 versus either IL-2+PBS or the IL-23+PBScontrols. *P , 0.05 by one-way ANOVA with Dunnett’s multiple comparison test for ATI-1249–treated groups versus untreated IL-2+IL-23 control.

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and IL-22 obtained at 5 mg/kg of ATI-1249, corresponding to98 and 88% reductions, respectively (Fig. 4, B and C). Thesedata demonstrate that ATI-1249 caused potent inhibitionof IL-23–dependent cytokine responses in vivo and suggestthat good pharmacodynamic coverage of IL-23 was obtainedduring the 32 hours of the experiment post-adnectin dosing.The Mouse Anti–IL-17 Antibody Inhibits IL-17A/A–

and IL-17A/F–Dependent Activity In Vivo. Next, wetested our anti–IL-17 inhibitors for activity in vivo. Becausethe anti–IL-17 antibody was slightly more potent againstIL-17A/A and IL-17A/F activity than the mIL-17-RA decoyreceptor in vitro, we tested the activity of anti–IL-17 antibodyfor inhibition of IL-17A/A and IL-17A/F responses in vivo. ThePD model established for investigating IL-17–dependentresponses in vivo involved subcutaneous challenge of micewith mouse IL-17A/A or IL-17A/F followed by blood collectionat 2 hours after treatment. Serum was subsequently processedand tested for CXCL1 levels by ELISA. Mice challenged witheither IL-17A/A or IL-17A/F exhibited a robust secretion ofCXCL1 into the serum (Fig. 5, A and B). Administering a rangeof anti–IL-17 antibody concentrations 18 hours before eitherIL-17A/A or IL-17A/F challenge caused significant inhibition ofCXCL1. With IL-17A/A challenge, the anti–IL-17 antibodycaused a dose-dependent inhibition of CXCL1, in which thehighest dose tested, 1.0 mg/kg, blocked 92% of the untreatedcontrol (Fig. 5A). The anti–IL-17 antibody was less potent inblocking IL-17A/F–induced CXCL1 in vivo but still signifi-cantly inhibited secretion at the highest doses tested, both the0.2 and 1.0 mg/kg doses showing approximately 50% inhibitionof CXCL1 production (Fig. 5B). These data demonstrated thatthe anti–IL-17 antibody had potent activity against bothIL-17A/A and IL-17A/F activity in vivo, with good pharmaco-dynamic coverage through the 20 hours of the experiment.Dual Inhibition of Mouse IL-23 and IL-17 Offers

Superior Efficacy in the Imiquimod-Induced SkinInflammation Model. Treatment of mouse skin with IMQleads to skin inflammation, including increases in skinthickness and scaling, that has been reported to be dependent

on IL-17 and IL-23 on the basis of the protection from thesesymptoms in mice deficient for IL-23p19 and IL-17 receptor(van der Fits et al., 2009). Therefore, we tested whetherinhibition of both IL-23 and IL-17 might offer superior efficacycompared with inhibition of either alone in the IMQ skininflammation model. In this model, IMQ was applied to theskin on backs of shaved mice for 7 consecutive days. SuccessiveIMQ treatments caused a rapid increase in skin thickness in thefirst 4 days with a greater than 70% change from baseline at day4 (Fig. 6A). Skin scaling also increased, reaching a score of 3 byday 7 (Fig. 6B). Histologic examination of the skin showed thatthere were minimal-to-moderate epidermal hyperplasia (acan-thosis) and moderate-to-marked mixed-cell infiltrates of thedermis in the back skin of IMQ-inducedmice (Fig. 6, C, G, andH).Preventative dosing of both inhibitors in this model was

performed at doses expected to give robust suppression of thetarget cytokines. On the basis of the nearly maximal activityof this antibody at 1 mg/kg in the IL-17–dependent PDmodel,complete neutralization of IL-17A would be expected at thehigh dose (20 mg/kg) used in this model. Similarly, neutral-ization of IL-23 by 20 mg/kg ATI-1249 would be expected onthe basis of the near maximal suppression of IL-23–mediatedeffects at 5 mg/kg in the IL-23–dependent PDmodel describedearlier. Treatment with the anti–IL-17 antibody 1 day beforeIMQ application and every other day thereafter had nosignificant effect on preventing skin thickening or scaling(Fig. 6, A and B). In contrast, treatment with a similarregimen of ATI-1249 caused reduced thickness starting at day5 and was 38% lower than the vehicle control at day 7 but didnot reach statistical significance compared with the vehiclecontrol. However, ATI-1249 did have a significant effect forreducing skin scaling by day 6 (Fig. 6B). Dosed simulta-neously, the combination of ATI-1249 and anti–IL-17 anti-body caused an effect similar to that of ATI-1249 alone forreducing skin thickening initially and by day 7 had a superioreffect. Notably, only the dual treatment induced a statisticallysignificant reduction in skin thickening compared with thevehicle control. Amore dramatic effect of the combination dosingwas observed on the skin scaling score where no increase inscaling was observed beyond 4 days with combination treat-ment. Moreover, although treatment with ATI-1249 alone andtogether with anti–IL-17 antibody caused significant reduc-tions in the scaling score, the combination therapy was stillcomparatively superior (Fig. 6B). The suboptimal efficacy of themonotherapies and the superiority of the combination treat-ment were further supported by histologic evaluation of skincollected from treated mice (Fig. 6, C–F). In the presence ATI-1249 or the anti–IL-17 antibody alone, there was no significanteffect on the acanthosis score. The combination treatment, onthe other hand, reduced acanthosis 55% compared with thecontrol or either of the monotherapies but did not reachstatistical significance (Fig. 6G). For the dermal infiltrateanalysis, the groupmean scores were reduced 31% byATI-1249alone and 38% by anti–IL-17 antibody alone comparedwith thevehicle control group (Fig. 6H). With the combination treat-ment, the infiltrates were significantly reduced by 69%compared with vehicle and over 50% compared with eitherATI-1249 or anti–IL-17 antibody alone.Analysis of serum cytokines in IMQ challenged mice

showed that several IL-23 and IL-17–associated cytokineswere increased, including IL-17A, IL-22, and IL-6 (Fig. 7,A–C). However, owing to the local nature of the inflammation

Fig. 5. Mouse anti–IL-17 antibody inhibits IL-17–dependent responses invivo. ELISA measurements of CXCL1 in serum from mice challengedsubcutaneously with (A) IL-17A/A or (B) IL-17A/F. To test the impact ofIL-17 inhibition, mice were dosed with anti–IL-17 antibody by sub-cutaneous injection 18 hours before challenge with IL-17A/A or IL-17A/F.Two hours after challenge, serum was collected for analysis. The resultsare expressed as mean CXCL1 level in serum measured in four mice percohort. Error bars represent S.E.M. *P , 0.05 by one-way ANOVA withDunnett’s multiple comparison test for anti–IL-17 antibody treatmentversus “no inhibitor,” which for (A) was mIgG isotype control and for (B)was PBS.


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in the skin in this model, the overall levels of the cytokinesremained low and exhibited considerable interanimal vari-ability.Nonetheless, a reduction of cytokine levelswas observed

in the serum from treated animals, which were in line withexpectations from the in vitro experiments. In mice treatedwith ATI-1249 alone, the group mean concentration for IL-17A

Fig. 6. Dual inhibition of IL-23 and IL-17 offers superior efficacy in the IMQ-induced skin inflammationmodel. Three days before IMQ challengemice wereanesthetized with isoflurane and their back hair was removed. One day before IMQ application and every other day thereafter animals were dosedsubcutaneously with PBS, 20mpk ATI-1249 alone, 20mpk anti–IL-17 antibody alone, or both combined. Imiquimod was applied every day from days 0 to 6.(A) Skin thickness and (B) scaling was measured daily. Results are expressed as the mean values for eight animals per cohort. Error bars represent S.E.M.*P , 0.05 by one-way ANOVA with Bonferroni’s post-test for multiple comparisons for any treatment versus vehicle, (#) anti–IL-17 antibody versus ATI-1249, or (+) anti–IL-17 antibody versus the combination treatment. ***P , 0.001 by one-way ANOVA with Bonferroni’s post-test for multiple comparisonsfor the combination treatment versus vehicle. At necropsy, back skin was prepared for histology and analyzed for acanthosis, and dermal infiltrate byneutrophils, eosinophils, plasma cells, lymphocytes, and melanocytes. Representative images were scored as follows: (C) PBS–acanthosis, 3; dermalinfiltrate, 4; (D) ATI-1249–acanthosis, 2; dermal infiltrate, 2; (E) anti–IL-17 Ab–acanthosis, 2; dermal infiltrate, 3; (F) combination–acanthosis: 1; dermalinfiltrate, 1. From five animals in each treatment group the average histology score was calculated for (G) acanthosis and (H) dermal infiltrate. Error barsrepresent S.E.M. *P , 0.01 between the combination treatment versus PBS by Kruskal-Wallis test with Dunn’s post-test for multiple comparisons.

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and IL-22 but not IL-6 was reduced. The anti–IL-17 antibodydid not reduce IL-22 or IL-6. However, treatment with thecombination of ATI-1249 and the anti–IL-17 antibody causeddecreases in both IL-22 and IL-6 serum levels (Fig. 7, B and C).These effects on serum cytokines in this in vivo modelcorroborate the cooperative effects of the combination treat-ment observed in vitro. Collectively, data from the analysis ofthe skin thickness, scaling, histology, and serum cytokinelevels all suggest that dual inhibition of IL-23 and IL-17 in theIMQ skin inflammation model offered superior efficacycompared with neutralization of either IL-23 or IL-17 alone.Dual Inhibition of Mouse IL-23 and IL-17 Offers

Superior Efficacy in the MOG Peptide-Induced EAEModel. Similar to the IMQ-induced skin inflammationmodel, the IL-23–IL-17 axis has been shown to be central toinhibition of disease development inmodels of central nervoussystem (CNS) inflammation (Hofstetter et al., 2005; Langrishet al., 2005; Chen et al., 2006; Kroenke et al., 2008; Ishigameet al., 2009; Hu et al., 2010; El-Behi et al., 2011; Kap et al.,2011). To test whether dual inhibition of IL-23 and IL-17offers superior efficacy compared with either inhibitor alonefor treatment of CNS inflammation, we tested ATI-1249 andthe anti–IL-17 antibody dosed alone or in combination ina MOG peptide–induced EAE model. In this model, 25 mg/kgof each agent was used. On the basis of IL-23 and IL-17challenge models, the dose of inhibitors used was 5- and25-fold over the maximal efficacious dose for good pharmaco-dynamic coverage in mice, respectively.Induction of EAE in control animals resulted in rapid

weight loss starting at day 9 along with onset of clinicalsymptoms that reached maximum severity by 12–14 days(Fig. 8, A and B). Treatment with ATI-1249 or the anti–IL-17antibody alone administered just before disease onset at day 7caused a modest reduction in disease score compared tovehicle-treated animals. Either inhibitor dosed by itself alsohad a modest effect for preventing weight loss (Fig. 8, A andB). Whereas the protection by the anti–IL-17 antibody wasmaintained to day 20, protection waned in mice treated withATI-1249 alone. When both ATI-1249 and the anti–IL-17antibody were administered together at day 7, the combina-tion treatment caused a more dramatic reduction in theseverity of clinical disease and prevented weight loss moresignificantly than either treatment alone (Fig. 8, A and B).Histologic examination of the spinal cords from EAE mice

treated with ATI-1249 and the anti–IL-17 antibody alone or incombination corroborated the superiority of the dual treat-ment for preventing disease (Fig. 8, C–F). Dosed alone,

ATI-1249 or the anti–IL-17 antibody provided partial pro-tection on the basis of inflammation and demyelination scores(Fig. 8, G and H). In contrast, the combination of ATI-1249 orthe anti–IL-17 antibody yielded a more significant effect inreduction of spinal cord inflammation and demyelinationscores (Fig. 8, G and H). These data demonstrated that in theMOG peptide–induced EAEmodel the dual inhibition of IL-23and IL-17 offered a superior effect compared with eitherinhibitor alone for reduction of overall disease severity byall measures: clinical scores, body weight loss, spinal cordinflammation, and demyelination.

DiscussionThe generation of potent and specific neutralizing mole-

cules against mouse IL-23p19 and IL-17 enabled us to probethe impact of IL-23 and IL-17 inhibition alone and incombination and, for the first time, demonstrate a cooperativeeffect of dual inhibition on cytokine production in vitro andenhanced efficacy in vivo. These findings are remarkablegiven the established connection between IL-23 and IL-17that might suggest that these cytokines are redundant(Murphy et al., 2003; Langrish et al., 2005; Luger et al.,2008). Yet, IL-23 induces additional cytokines, such as IL-22,that have distinct functions (Liang et al., 2006). Consistentwith the biology of IL-23 in mice, we showed that committedTh17 cells stimulated with IL-23 secreted robust levels ofIL-17A and IL-22. Notably, the secretion of IL-22 is unique tomouse Th17 cells polarized with TGF-b1 and IL-6. In humanT cells, the addition of TGF-b has been shown to inhibit IL-22but drive production of IL-26, which is related to IL-22 and isalso induced by IL-23 (Wilson et al., 2007; Manel et al., 2008).Both IL-22 and IL-26 have been associated with humandiseases, especially in psoriasis, in which both cytokines areelevated in lesional psoriatic skin (Wilson et al., 2007). Inour studies, the production of IL-22 coincident with IL-17Ain the mouse Th17 cells afforded two readouts that could beused tomeasure IL-23 function. Incubationwith an unformattedanti–IL-23p19-selective adnectin, ATI-1221, as well as aPEGylated form, ATI-1249, potently and comparably in-hibited both IL-23–dependent cytokines, IL-17A and IL-22.In vivo, dosing ATI-1249 into IL-23–challenged mice alsoblocked production of IL-17 and IL-22. These data confirmedthe in vitro and in vivo activity of these adnectins and the rolefor IL-23 in driving not only IL-17 but also IL-22.In a similar way, IL-17 can act independently of IL-23. In

mice, IL-17 can be produced in the absence of IL-23 by Th17

Fig. 7. Dual inhibition of IL-23 andIL-17 enhances inhibition of serum cyto-kines in the IMQ-induced skin inflam-mation model. Serum was analyzed byELISA for (A) IL-17A, (B) IL-22, and (C)IL-6 concentrations. Data are presentedas the mean cytokine concentration from10 animals per cohort in the presence ofvehicle (open bars), ATI-1249 alone (graybars), anti–IL-17 alone (hatched bars), orboth inhibitors combined (solid bars). Errorbars represent S.E.M. *P , 0.05 by one-way ANOVA with Bonferroni’s post-testformultiple comparisons for any treatmentversus vehicle or (#) anti–IL-17 antibodyversus ATI-1249.


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cells during differentiation, as well as by iNKT and mast cells(Bettelli et al., 2006; Mangan et al., 2006; Veldhoen et al.,2006; Yoshiga et al., 2008; Hueber et al., 2010). To assess the

impact of IL-17 blockade, two inhibitors of IL-17 weregenerated, a mouse decoy receptor, mIL-17RA-Fc, and amonoclonal antibody against murine IL-17A. Both agents

Fig. 8. Dual inhibition of IL-23 and IL-17 offers superior efficacy in the MOG peptide–induced EAEmodel. Mice were immunized withMOG peptide emulsifiedwith incomplete Freund’s adjuvant and two injections of pertussis toxin. Mice were treated subcutaneously with PBS (every other day), ATI-1249 (25mg/kg everyother day), anti–IL-17 antibody (25 mg/kg once per week), or combination of ATI-1249 and anti–IL-17 antibody (the same regimen used in the monotherapygroups). Dosingwas initiated the day before immunization. Superior efficacy is demonstratedwith the combination of ATI-1249 and the anti–IL-17 antibody basedon (A) clinical scores and (B) body weight. Results are expressed as the mean values for 14 animals per cohort. Error bars represent S.E.M. *P, 0.05 by one-wayANOVA with Bonferroni post-test for multiple comparisons for any treatment versus vehicle, (#) anti–IL-17 antibody versus ATI-1249 (^) ATI-1249 versus thecombination treatment or (+) anti–IL-17 antibody versus the combination. Representative images are transverse sections of lumbar spinal cord frommice inducedwith MOG35–55 EAE, stained with luxol fast blue, and H&E counterstain to demonstrate reduction in inflammation (arrow), active demyelinating lesions (arrowhead), and tissue damage (*) comparing treatment with (C) PBS, (D) ATI-1249, (E) anti–IL-17 Ab, and (F) combination therapy. Scale bar, 100 mm. A cumulativescorewas calculated for (G) inflammation and (H) demyelination for each treatment group. Results are expressed as themean values for seven animals per cohort.Error bars represent S.E.M. *P, 0.01 between combination treatment compared with PBS by Kruskal-Wallis test with Dunn post-test for multiple comparisons.

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blocked IL-17A/A– and IL-17A/F–induced responses in NIH/3T3 cells in vitro. Moreover, in an IL-17–dependent PDmodelin vivo, dosing the anti–IL-17 antibody into mice beforechallenge with IL-17A/A and IL-17A/F inhibited CXCL1induction by either cytokine. Together, these data confirmedthe activity of our key tool inhibitors for neutralizing IL-17Aactivity in vitro and in vivo and enabled us to assess theimpact of any IL-23–independent functions of IL-17.Using these inhibitors of IL-23 and IL-17, the relative

effects of each agent alone and in combination was tested ina coculture system where exogenous IL-23 drove IL-17 produc-tion from Th17 cells, which in turn induced cytokine productionfrom NIH/3T3 cells. Here, we chose to measure IL-6 ratherthan CXCL1 induction from NIH/3T3 cells on the basis of thedemonstrated role for IL-6 in autoimmune disease (Yao et al.,1995; Hwang et al., 2004; Yen et al., 2006; Chiricozzi andKrueger, 2013). Using this system, we confirmed the keyfunctional role of IL-23 for parallel induction of IL-22 and IL-17and the key functional role of IL-17 in the induction of IL-6, butwe also revealed some critical limitations of neutralizing eitherIL-23 or IL-17 alone. Blocking IL-23 only partially inhibitedIL-17 produced by the cytokine and, therefore, had a modestnonsignificant effect on the IL-6 produced in the system.Conversely, inhibiting IL-17 was more efficacious for blockingIL-6 but did not inhibit IL-22 produced by IL-23. Thecombination treatment showed good neutralization of IL-22and, interestingly, greater inhibition of IL-6 than eithertreatment alone, perhaps reflecting that when the level ofIL-17 is decreased by IL-23 blockade, inhibition of the cytokineproduction by the residual IL-17 is more easily inhibited.Remarkably, even the basal IL-6 produced in the coculturesystem was extremely sensitive to IL-17 blockade, with a near-complete suppression of IL-6 by the anti–IL-17 antibody. Theprofound inhibition of proinflammatory cytokines with dualinhibition suggested that this comprehensive blockade mightoffer enhanced efficacy for treating disease.To investigate whether dual inhibition of IL-17 and IL-23

might lead to improved efficacy in vivo, we tested the anti–IL-23adnectin ATI-1249 and the anti–IL-17 antibody in the IMQ-induced skin inflammation model. Mice deficient in IL-23p19or the IL-17 receptor are protected from IMQ-induced scalingand skin thickening, demonstrating a pivotal role for theIL-23/IL-17 axis in this model (van der Fits et al., 2009).Protection in IL-23p19–deficient mice stems from dramati-cally reduced IL-17A and IL-17F. In accord with these data,we showed that treatment with the anti–IL-23 adnectin inIMQ-challenged mice reduced skin thickening and scalingand, by histologic examination, scores for acanthosis andcell infiltrates. In contrast, treatment with the anti–IL-17antibody had little or no effect on the IMQ-induced disease.The lack of efficacy was surprising given the demonstratedsuppression of disease in IL-17 receptor–deficient mice. Yet,the selectivity of our antibody for IL-17A/A and IL-17A/F butnot IL-17F/F could be responsible for this outcome (Supple-mental Table 2) and are consistent with studies demonstrat-ing an association of IL-17F with skin inflammation (vander Fits et al., 2009; Fujishima et al., 2010). Moreover,IL-17RA–deficient mice likely elicit a more comprehensiveblockade of all forms of IL-17, including IL-17F, since IL-17A/A,IL-17A/F, and IL-17F/F all require IL-17RA for signaling (Gaffen,2009). Follow-on studies using pharmacologic inhibitors ofIL-17A and IL-17F would be required to confirm this hypothesis.

Nevertheless, neutralizing both IL-23 and IL-17 causeda cooperative effect to reduce IMQ-induced pathology,especially in disease scores for scaling and histologic scoresfor cellular infiltrate. These findings suggest that, althoughblocking IL-23 alone might significantly inhibit cytokinesecretion and downstream responses, the added inhibition ofIL-17A leads to a more robust blockade of cytokine, includingany from IL-23–independent sources. Notably, the serum IL-6and IL-22 levels induced in IMQ-challenged animals in thepresence of inhibitors of both IL-23 and IL-17 were reducedcomparably or more than either inhibitor alone. These in vivodata are remarkably consistent with our in vitro data demon-strating a similar cooperative effect of these inhibitors forreducing IL-6 and IL-22 levels. Taken together, our dataconfirm the role of IL-23 and IL-17 in IMQ-induced skininflammation and demonstrate that dual blockade of bothIL-23 and IL-17 offers enhanced efficacy against disease.The involvement of IL-23 and IL-17 in skin inflammation

has now been well validated by clinical trials in psoriasis,where treatments with antibodies against either IL-23 orIL-17 demonstrated impressive efficacy in patients. In phaseII studies, antibodies against IL-23p19, IL-17, and theIL-17RA receptor subunit, all caused .75% disease clearance[Psoriasis Area and Severity Index (PASI) 75] in 70–80% ofpatients. Almost half also reached PASI 100 or 100% diseaseclearance (Papp et al., 2012a,b, 2013a,b; Rich et al., 2013;Gaffen et al., 2014). Our data showing efficacy from targetingIL-23 alone in the IMQ-induced model substantiate the role ofIL-23 in both mouse and human skin inflammation. Yet, thelack of efficacy from neutralizing IL-17 alone in the IMQmodel highlight potential differences between the animal andhuman pathology. Antibodies against IL-17A specifically arehighly efficacious for treating psoriasis, suggesting a signifi-cant role for IL-17A in human but not mouse disease (Pappet al., 2012a,b; Patel et al., 2013; Rich et al., 2013). It isprobable that, although IL-23 and IL-17 levels are known tobe elevated in the lesional skin of psoriasis patients, therelative involvement of each in the development of the diseasein mice and humans is different (Nograles et al., 2008;Johansen et al., 2009; Martin et al., 2013). Because the mousemodel does not precisely parallel human disease, the directapplicability of our findings to psoriasis remains uncertain.Nevertheless, the cell types, cytokines, and mechanismsinvolved in the IMQ model are comparable to those in humandisease. Therefore, our data suggest that blocking IL-23 andIL-17 simultaneously might offer superior anti-inflammatoryactivity against some elements of skin inflammation. Still, themagnitude of the response to all the mechanisms targetingthe IL-23/IL-17 pathway in clinical trials raises the questionof whether there is room in human psoriasis for increasedefficacy through a more comprehensive targeting of thesepathways.Beyond skin pathology, a critical role for both IL-23 and

IL-17 has also been described in models of CNS inflammation,leading us to investigate whether dual targeting might offeran enhanced efficacy in the MOG peptide–induced EAEmodel. Mice that are genetically deficient for IL-23p19 orIL-17, or receive treatment with IL-23p19 or IL-17 neutral-izing agents, have shown reduced disease in various EAEmodels (Hofstetter et al., 2005; Langrish et al., 2005; Chenet al., 2006; Kroenke et al., 2008; Ishigame et al., 2009; Huet al., 2010; El-Behi et al., 2011; Kap et al., 2011). Consistent


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with these data, treatment of MOG peptide–challenged micewith either the anti–IL-23 adnectin alone or the anti–IL-17antibody alone significantly abrogated disease compared withcontrols as measured by decreased clinical score, maintenanceof body weight, and histology readouts of reduced inflamma-tion and demyelination. Even though each of the inhibitorsalone demonstrated a reduction in the disease, the combina-tion treatment dramatically reduced all disease manifesta-tions to levels significantly lower than the suppression byeither ATI-1249 or the anti–IL-17 antibody alone. Thisoutcome further supports our assertion that a dual blockadeof IL-23 and IL-17 offers better efficacy than blocking eithercytokine alone in murine models of CNS inflammation.Though clinical trials testing neutralizing agents against

either IL-23 or IL-17 yielded uniformly positive outcomes inpsoriasis patients, the results from these inhibitors in MSpatients have been mixed. Despite the universal effect ofIL-23 or IL-17 neutralization in reducing EAE, only the IL-17antibodies have shown early positive data in clinical trialsof MS, and trials with agents against IL-12p40 failed todemonstrate any efficacy (Segal et al., 2008; Longbrake andRacke, 2009). These findings may be surprising, given thesignificant impact of both anti–IL-23p19 and anti–IL-17antibodies for preventing CNS inflammation in mice, butthey highlight again the discrepancies between the mousemodels and human disease (Chen et al., 2006; Uyttenhove andVan Snick, 2006). Yet, IL-23 and IL-17 brain expression levelshave been associated withMS, in which IL-23p19 is expressedin active MS lesions and IL-17A is overexpressed in patientbrain biopsies (Li et al., 2007; Tesmer et al., 2008). As with theIMQ model, the cell types and cytokines involved in the EAEmodel are comparable to those in human disease. Therefore,our data demonstrating a remarkably suppressive effect ofdual IL-23 and IL-17 neutralization of EAE disease maysuggest that a similar combination could lead to morepromising efficacy in MS relative to the modest or no efficacyseen clinically with the individual agents.Collectively, our findings demonstrate that comprehensive

neutralization of both IL-23– and IL-17–dependent cytokinesthrough dual blockade of IL-23 and IL-17 leads to enhancedefficacy for reducing disease in the IMQ skin inflammationand MOG peptide–induced EAE disease models. Comparisonof our datawith clinical data in psoriasis andMS for anti–IL-23and anti–IL-17 therapies, as well as the published data inmurine models of skin and CNS inflammation, suggests thatthe balance of the role of IL-17 and IL-23 may be different inanimal models compared with human disease. However,assessment of the dual targeting of IL-17 and IL-23 in thesemurine models does provide proof of principle and suggeststhat, even given the potential human-to-murine difference,the more complete blockade obtained with dual targeting willprovide better efficacy in human disease. These data providea basis for pursuing a dual targeting strategy for IL-23 andIL-17, either through separate dosing of neutralizing agentsor perhaps with a bispecific molecule. The generation ofa stable bispecific molecule with specificity for IL-23 andIL-17 has been described and while the function of each IL-23and IL-17 neutralizing arm was demonstrated in separateassays there was no validation that the combined effect ofneutralizing both IL-17 and IL-23 was more efficacious thanmonotherapies either in vitro or in vivo (Mabry et al., 2010).Our data from both in vitro and in vivo models suggest that

a more complete blockade of IL-23– and IL-17–dependentresponses can be achieved through dual inhibition and thatthis profound inhibition of Th17 responses might offerenhanced benefit for treating certain autoimmune disorders.

Authorship Contributions

Participated in research design: Mangan, Su, Ditto, Lin, Yang,Cotter, Shuster, Borowski, Carvajal, Das Gupta, Xie, Zhao.

Conducted experiments: Mangan, Jenny, Picarillo, Skala, Ditto,Yang, Cotter, Shuster, Borowski, Thomas.

Contributed new reagents or analytic tools: Su, Jenny, Tatum, Lin,Devaux, Das Gupta.

Performed data analysis: Mangan, Yang, Cotter, Shuster, Song,Borowski, McIntyre, Xie, Heimrich.

Wrote or contributed to the writing of the manuscript: Mangan, Su,Cotter, Shuster, Heimrich, Devaux, Carvajal, McIntyre, Xie,Struthers, Salter-Cid.

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Address correspondence to: Dr. Paul R. Mangan, K4.813C, Route 206 andProvince Line Road, Bristol-Myers Squibb Company, Princeton NJ 08543.E-mail: [email protected]


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Dual Inhibition of Interleukin-23 and Interleukin-17 Offersjpet.aspetjournals.org/content/jpet/354/2/152.full.pdf · Stacey Skala, Noah Ditto, Zheng Lin, XiaoXia Yang, Pete Z. Cotter,