ORIGINAL ARTICLE

Antibody targeting of anaplastic lymphoma kinase inducescytotoxicity of human neuroblastomaEL Carpenter1, EA Haglund1, EM Mace3, D Deng1, D Martinez1, AC Wood1, AK Chow1, DA Weiser1, LT Belcastro1, C Winter1, SC Bresler4,S Asgharzadeh5, RC Seeger5, H Zhao6, R Guo6, JG Christensen7, JS Orange2, BR Pawel1, MA Lemmon3 and YP Mosse1,2

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in neuroblastoma, a devastatingpediatric cancer of the sympathetic nervous system. Germline and somatically acquired ALK aberrations induce increasedautophosphorylation, constitutive ALK activation and increased downstream signaling. Thus, ALK is a tractable therapeutictarget in neuroblastoma, likely to be susceptible to both small-molecule tyrosine kinase inhibitors and therapeuticantibodies---as has been shown for other receptor tyrosine kinases in malignancies such as breast and lung cancer. Small-molecule inhibitors of ALK are currently being studied in the clinic, but common ALK mutations in neuroblastoma appearto show de novo insensitivity, arguing that complementary therapeutic approaches must be developed. We thereforehypothesized that antibody targeting of ALK may be a relevant strategy for the majority of neuroblastoma patients likelyto have ALK-positive tumors. We show here that an antagonistic ALK antibody inhibits cell growth and induces in vitroantibody-dependent cellular cytotoxicity of human neuroblastoma-derived cell lines. Cytotoxicity was induced in cell linesharboring either wild type or mutated forms of ALK. Treatment of neuroblastoma cells with the dual Met/ALK inhibitorcrizotinib sensitized cells to antibody-induced growth inhibition by promoting cell surface accumulation of ALK and thusincreasing the accessibility of antigen for antibody binding. These data support the concept of ALK-targeted immunotherapyas a highly promising therapeutic strategy for neuroblastomas with mutated or wild-type ALK.

Oncogene (2012) 31, 4859 -- 4867; doi:10.1038/onc.2011.647; published online 23 January 2012

Keywords: neuroblastoma; anaplastic lymphoma kinase; immunotherapy; receptor tyrosine kinase

INTRODUCTIONAnaplastic lymphoma kinase (ALK) was originally identified in anoncogenic fusion protein1 expressed in anaplastic large-celllymphoma. Other oncogenic ALK fusions have been identified inhuman cancers, including non-small-cell lung cancer,2,3 squamouscell carcinoma4 and inflammatory myofibroblastic tumors.5 The full-length ALK RTK has also been linked to neuroblastoma,6 a pediatriccancer of the sympathetic nervous system accounting for 10% ofchildhood cancer mortality.7,8 The ALK gene is amplified in 2--3% ofneuroblastoma cases.9 In addition, activating mutations within thetyrosine kinase domain of ALK were recently identified as the majorcause of familial neuroblastoma,10 also arising somatically in up to10% of sporadic cases. Amplification or mutation of ALK can lead toconstitutive autophosphorylation and activation,11 -- 13 and may beassociated with a more aggressive clinical course.10,14,15 Thesefindings argue that therapeutic manipulation of intact ALK is apromising strategy for neuroblastoma treatment.

Approaches for therapeutically targeting RTKs include mono-clonal antibodies and small-molecule tyrosine kinase inhibitors(TKIs), both of which have led to dramatic increases in survival andtime to progression in multiple cancers.16,17 The trastuzumabantibody was approved for treatment of human epidermal growthfactor receptor 2 (HER2)-overexpressing breast cancer over 10years ago, and is thought to exert its effects through blockade of

aberrant signaling by amplified HER2 and antibody-dependentcellular cytotoxicity (ADCC).18 Similarly, the epidermal growthfactor receptor (EGFR) antibody cetuximab inhibits binding ofactivating ligands and induces ADCC.19 Clinical activity of TKIs thatinhibit HER2 and EGFR has been amply demonstrated; moreover,these TKIs have been found to potentiate and enhance the activityof HER2- and EGFR-targeted antibodies in breast and lung cancer,respectively.20 -- 22

Analogous approaches should be effective for targeting intactALK. Recent studies have shown that crizotinib, a dual Met/ALKTKI, induces remarkable tumor regression in non-small-cell lungcancer patients harboring ALK translocations.23 Crizotinib is alsocurrently in early-phase clinical trial testing in patients withneuroblastoma. However, preclinical studies have shown that celllines harboring the F1174L mutation, the second most commonALK mutation seen in neuroblastoma tumors, are significantlymore resistant to crizotinib than those harboring the mostcommon mutation, R1275Q.24 -- 26 Moreover, acquired resistanceto TKIs is largely inevitable,27 and resistance mutations inoncogenic ALK fusions have already emerged in early studieswith crizotinib.28,29 These findings underline an important needfor developing additional therapeutic options in neuroblastoma,an often-lethal childhood cancer.7,30 One such option is immuno-therapy, for which proof of concept was recently demonstrated in

Received 17 May 2011; revised 16 December 2011; accepted 17 December 2011; published online 23 January 2012

1Division of Oncology and Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 2Department of Pediatrics, Perelman School of Medicine,University of Pennsylvania, Philadelphia, PA, USA; 3Children’s Hospital of Philadelphia Research Institute, Philadelphia, PA, USA; 4Department of Biochemistry and Biophysics, University ofPennsylvania Perelman School of Medicine, Philadelphia, PA, USA; 5Division of Hematology and Oncology, Children’s Hospital of Los Angeles, Saban Research Institute, University ofSouthern California, Los Angeles, CA, USA; 6Biostatistics and Data Management Core, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA and 7Departmentof Cancer Biology, Pfizer Global Research and Development, La Jolla Laboratories, La Jolla, CA, USA. Correspondence: Dr YP Mosse, Division of Oncology, Center for Childhood CancerResearch, Children’s Hospital of Philadelphia, Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Colket Translational Research Building, 3501 Civic CenterBoulevard, Office 3056, Philadelphia, PA 19104-4318, USA. E-mail: mosse@chop.edu

Oncogene (2012) 31, 4859 -- 4867& 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12

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a phase 3 trial of high-risk neuroblastoma patients using GD2antibodies.31 We therefore sought to identify antibody-basedstrategies for therapeutic targeting of ALK. We show here that ALKantibodies inhibit the growth of neuroblastoma cells, anddemonstrate the utility of combining ALK antibodies with TKIsas a potentially important therapeutic strategy. Our findingsprovide a strong rationale for the immediate development ofclinical grade ALK antibodies.

RESULTSALK is widely expressed in neuroblastoma tumors and cell linesSuccessful immunotherapy requires the targeted antigen to beexpressed selectively (or at much greater levels---for ubiquitouslyexpressed receptors) in tumors, but not in normal tissue. Thetargeted antigen must be expressed on the majority of tumors forimmunotherapy to be relevant to a large proportion of patients,and expression levels should correlate with disease severity. IntactALK is normally found only in the developing embryonic andneonatal brain,32 a finding confirmed by the lack of consistent ALKstaining of a normal tissue microarray (TMA; Supplementary Table 1),which suggests that ALK is a valuable target for immunotherapy.To assess ALK expression in primary patient tumors, we analyzedour own collection33 as well as data from the TARGET initiative(Therapeutically Applicable Research to Generate Effective Treat-ments: http://target.cancer.gov/). ALK mRNA expression is seen intumors from patients with clinically aggressive disease, especiallyin those with high-risk metastatic disease and/or MYCN amplifica-tion (Figure 1a; P¼ 5.06E-05 for high-risk MYCN-amplifiedneuroblastoma (HRA) versus low risk (LR), P¼ 0.0022 for HRAversus high-risk MYCN non-amplified neuroblastoma (HRN),P¼ 0.0211 for HRN versus LR). We next analyzed a TMA ofdiagnostic neuroblastomas and ganglioneuroblastomas. Westained samples from 126 patients for native ALK expressionand graded staining intensity on a scale of 0 -- 3 (representativesamples shown in Figure 1b) and percent positivity. Among thesamples analyzed, 109 (86.5%) were ALK positive, with 75 samples(59.5%) having either moderate or strong staining. As shown inFigure 1c, ALK expression was significantly stronger in patients withINSS stage 4 (P¼ 0.0108; top panel) or amplified MYCN (P¼ 0.0065;‘A’ in bottom panel). These data argue that ALK is expressed in themajority of neuroblastoma tumors, and that expression levels arehigher in those patients with the worst prognoses.

We next used confocal microscopy to assess cell surface ALKexpression on primary tumors. ALK was readily detected on 13 of16 tumors analyzed, and co-localization of ALK with the cellsurface protein cadherin was confirmed by the generation ofPearson’s correlation coefficient values (Supplementary Table 2and Figure 1d). Although microscopy using the nuclear stain 4’,6-diamidino-2-phenylindole (DAPI) and anti-cadherin antibodygenerated Pearson’s correlation coefficient values o0.5, reflect-ing a known lack of co-localization, ALK and cadherin Pearson’scorrelation coefficient values ranged from 0.6 to 40.8 for 11 ofthe 13 ALK-positive tumors. Abundant cell surface ALK expressionwas also confirmed by flow cytometry (Figure 1e) for cell linesexpressing wild-type ALK (NB1, EBc1 and IMR5), R1275Q-mutatedALK (1643) or F1174L-mutated ALK (SY5Y, Kelly), but not thecontrol SKNAS cell line shown. Plasma membrane ALK expressioncorresponded closely with relative ALK mRNA expression(Figure 1e), and was also observed in immunofluorescencemicroscopy studies (Supplementary Figure 1). These data suggestwidespread expression and cell surface localization of full-lengthALK in neuroblastoma primary tumors and cell lines.

An ALK antibody induces growth inhibition and cytotoxicityWe next asked whether an antagonistic ALK monoclonal anti-body34 can inhibit growth of a neuroblastoma cell line driven by

expression of activated ALK. We first treated F1174L-expressingSY5Y cells with a 3-log dose range of a mixture of two antagonisticALK antibodies (mAb30 and mAb49) and measured growth.Antibody treatment resulted in significant dose-dependentgrowth inhibition as compared with cells treated with controlimmunoglobulin---whether we used the mAb30/mAb49 mixture(Figure 2a) or the individual antibodies (not shown). We nexttreated a panel of well-characterized neuroblastoma cell linesharboring wild-type (IMR5), mutated (1643 and SY5Y), amplified(NB1) or low/undetectable ALK (SKNAS) with a fixed concentrationof mAb30/mAb49 ALK antibody mixture, and saw a directcorrelation between ALK expression level and antibody-inducedcytotoxicity (Figure 2b), with the ALK amplified line NB1 showingthe greatest sensitivity to antibody treatment, and SKNAS showingno growth inhibition. As an additional negative control, we treatedretinal-pigmented epithelial-1 cells---a non-neuroblastoma, ALK-negative, neural crest-derived cell line---with ALK antibody ormurine immunoglobulin, and saw no growth inhibition (Supple-mentary Figure S2).

Immune cell-mediated ADCC has been shown to be importantfor the mechanism of action of the GD2 antibody in neuroblas-toma, and this effect is substantially enhanced in the presence ofinterleukin-2 (IL-2).35 To explore whether an ALK antibody couldalso induce an immune-mediated anti-tumor response in neuro-blastoma, we conducted in vitro ADCC assays using normal donorperipheral blood lymphocytes as effectors and neuroblastoma celllines as targets. Treatment with ALK antibody greatly enhancedcytotoxicity in NB1 cells induced by lymphocytes preincubatedwith IL-2 (Figure 2c, left panel). SY5Y cells also showed antibody-enhanced cytotoxicity in this assay (Figure 2c, middle panel),although less than seen for NB1 cells, possibly because of thelower cell surface ALK levels seen by flow cytometry (Figure 1e).No ADCC was detected when SKNAS cells that lack cell surfaceALK expression were used as targets (Figure 2c, right panel). Thus,antibody targeting of ALK can both inhibit cell growth and inducecytotoxicity of neuroblastoma cell lines harboring either wild-typeor mutated forms of activated ALK.

Crizotinib treatment induces cell surface accumulation of ALKTreatment with a combination of TKIs and therapeutic antibodiestargeting the same tumor antigen, such as EGFR20 or HER221 hasbeen shown to enhance the tumor growth inhibition andcytotoxicity elicited by either agent alone. One possible mechan-ism for this is cell surface accumulation of the targeted RTKresulting from RTK stabilization upon TKI binding.36,37 Certainactivating RTK mutations, including several in c-Kit, may destabi-lize the RTK and cause intracellular accumulation that can bereversed by TKI treatment---leading to increased cell surfaceexpression.38,39

To explore the effects of TKIs on surface ALK levels inneuroblastoma, we treated SY5Y cells with crizotinib and thenused surface biotin labeling of membrane proteins to quantifychanges in cell surface ALK. As shown in Figure 3a, crizotinibtreatment increased the levels of ALK protein detected at the cellsurface by 2.15 times over that found on vehicle-treated cells.Flow cytometry studies also showed a substantial crizotinib-induced increase in cell surface ALK levels for SY5Y cells(Figure 3a), with a 51.4% increase in mean fluorescence intensityfor cells treated with crizotinib (mean fluorescenceintensity¼ 636) compared with vehicle (mean fluorescenceintensity¼ 420). This phenomenon was dose dependent(Figure 3c), and increased over time (Figure 3d). To control forthe possibility that crizotinib binding induces conformationalchanges in ALK that stabilize or enhance exposure of the epitopeto which our staining antibody (mAb14) binds (increasingfluorescence signal), we repeated this experiment using an anti-ALK antibody (mAb46) that binds to an epitope distant from that

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of mAb14.34 Crizotinib-induced enhancement of cell surface ALKlevels was also observed in this experiment (Supplementary FigureS3), arguing that crizotinib functions similarly to other TKIs inpromoting cell surface accumulation of its target RTK.

Crizotinib sensitizes cells to ALK antibody-mediated growth inhibitionTo test the hypothesis that crizotinib-induced accumulation of cellsurface ALK sensitizes cells to ALK antibody treatment, we nextcompared the ability of the ALK antibody to inhibit growth ofSY5Y cells alone or together with crizotinib. Treatment withcrizotinib alone (at a sub-IC50 dose of 333 nM) or ALK antibodyalone led to measurable (but limited) growth inhibition(Figure 4a). However, combined TKI and antibody treatment had

a significantly larger inhibitory effect as compared with TKI(Po0.0001) or antibody alone (Po0.001), leading to almostcomplete growth inhibition of SY5Y cells. Increases in total ALKlevels were also seen by western blotting (Figure 4b) in cellstreated with crizotinib (alone or in combination with ALKantibody), consistent with the results shown in Figure 3. On theother hand, phosphorylated ALK levels were substantiallydiminished by crizotinib treatment, alone or together withantibody (Figure 4b), suggesting that the TKI stabilizes ALK whilesimultaneously blocking its activation. We also consideredwhether crizotinib-induced upregulation of cell surface ALK mightpromote antibody-mediated ADCC. To address this, we repeatedthe in vitro ADCC assays using SY5Y cells preincubated withcrizotinib or vehicle as targets. As shown in Figure 4c, crizotinib

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Figure 1. ALK expression in neuroblastoma. (a) ALK expression in 229 neuroblastoma patient tumors analyzed by Affymetrix Human Exon Arrayand normalized using quantile normalization (high-risk MYCN amplified (HRA), n¼ 64; high-risk MYCN non-amplified (HRN), n¼ 141; low-risk(LR), n¼ 24). (b) Representative images for immunohistochemical staining of ALK in neuroblastoma patient tumors. ALK staining was positiveoverall in 109 of 126 (86.5%) samples analyzed. In all, 17 samples showed no positive staining (upper left panel); 35 showed weak staining(upper right panel); 55 showed moderate staining (lower left panel); and 19 showed strong staining (lower right panel). (c) Box plots showing10th percentile, 90th percentile and mean ALK score by immunohistochemistry for INSS stage (top panel) and MYCN status (bottom panel; A,MYCN amplified; N, MYCN non-amplified). **Po0.01; *Po0.05. Dots indicate data-points outside of percentiles. (d) Representative confocalmicrographs of formalin-fixed paraffin-embedded primary neuroblastoma patient tumor slides showing staining for ALK and the cell surfaceantigen cadherin. All samples showed strong plasma membrane cadherin staining, and of 17 samples stained, 1 exhibited strong ALK staining(ALK score¼ 3; top row), 10 showed intermediate ALK staining, 2 had weak staining and 3 had no ALK staining (ALK score¼ 0, bottom row). Barrepresents 10mm. (e) Comparison of flow cytometry results with ALK mRNA expression levels for several neuroblastoma cell lines. Gray barsshow flow cytometry results for ALK cell surface staining. White bars represent an ALK mRNA expression ‘index’ (relative expression) measuredas ALK levels relative to HPRT1. Inset shows mean fluorescence intensity of NB1 cells for ALK staining (black line) and isotype control (gray line).

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preincubation significantly increased ADCC at effector to targetratios of 50:1 (crizotinib-treated mean¼ 55.7±9.3%; vehicle-treatedmean¼ 31.1±14.7%; P¼ 0.0331) and 25:1 (crizotinib-treated

mean¼ 33.7±2.5%; vehicle-treated mean¼ 17.3±7.9%; P¼ 0.0262),providing further evidence for the ability of crizotinib to sensitizeneuroblastoma cells to ALK antibody treatment.

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Figure 2. ALK antibody-induced growth inhibition and ADCC of neuroblastoma cells. To measure growth inhibition upon antibody exposure,cell lines were plated in 96-well plates and treated with anti-ALK (mAb30 plus mAb49) or a negative control murine IgG1. (a) Growth inhibitionof SY5Y cells treated with indicated amounts of anti-ALK as compared with control Ig. (b) The indicated cell lines were treated with 10 mg/mlALK antibody, and growth inhibition was measured after 144 h. (c) ADCC was measured using an in vitro assay as described in Materials andmethods, in which IL-2-activated peripheral blood lymphocytes were co-incubated with neuroblastoma cells in the presence (black line) orabsence (gray line) of 1 mg/ml ALK antibody. Shown are percent (%) cytotoxicities at the indicated effector:target ratios when NB1 cells (leftpanel), SY5Y cells (middle panel) or cell surface ALK-negative SKNAS cells (right panel) were used as targets.

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Figure 3. Effect of crizotinib on cell surface ALK expression. (a) SY5Y cells were incubated for 24 h with vehicle or 1000 nM crizotinib, thenbiotin labeled, precipitated with NeutrAvidin beads and immunoblotted for ALK (upper left panel) or cadherin (lower left panel). The results ofdensitometric analysis of ALK protein, normalized to cadherin, are shown in the bar chart (right panel), and indicate an increase in total ALKfrom 1.39 to 2.91 in arbitrary units. (b) Representative flow cytometry results showing mean fluorescence intensity (MFI) for SY5Y cellsincubated for 72 h with either vehicle (medium gray line; MFI¼ 420) or 1000 nM crizotinib (black line; MFI¼ 636), resulting in a 51.4% increasein MFI for crizotinib- versus vehicle-treated cells. The single-peaked light gray line represents the isotype control. (c) Concentrationdependence of the percent change in cell surface ALK, as measured by MFI, for cells incubated for 72 h with varying concentrations ofcrizotinib as compared with vehicle. (d) Time course of percentage change in cell surface ALK levels (over that seen for vehicle treatment)when cells were incubated with 1000 nM crizotinib.

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ALK antibody improves sensitivity to a broad range of crizotinibdosesWe next explored whether ALK antibody treatment could improvesensitivity of SY5Y cells to growth inhibition by crizotinib across arange of doses. We treated SY5Y cells with a 4-log range ofcrizotinib in the presence or absence of ALK antibody. Figure 5ashows that the addition of ALK antibody significantly enhancedgrowth inhibition at all crizotinib doses, except for the highestdose of 10 000 nM, a supralethal dose at which the majority ofTKI effect is likely to be off-target. These data also reveal thataddition of the ALK antibody shifts the crizotinib dose-responsecurve. Although 333 nM crizotinib is required to achieve 25%growth inhibition when added alone (Figure 5a), the sameeffect (mean¼ 33.7±2.8%) can be achieved with just 10 nM

crizotinib when antibody is included. As suggested by this finding,antibody treatment reduced the IC50 for crizotinib treatment ofSY5Y cells from 3018 nM (crizotinib alone) to 1745 nM for dualtreatment (Figure 5b). This is a particularly important result forSY5Y cells and other neuroblastoma cell lines that harbor anF1174L ALK mutation, as we have shown in other studies that thismutation causes resistance to crizotinib inhibition at typicalinhibitor levels.24 The data in Figure 5a reveal that simultaneousapplication of antibody treatment can restore crizotinib sensitivityin these cells.

Dual ALK targeting enhances programmed cell deathTo determine the effect of ALK targeting on cell cycle progression,we next analyzed the impact of TKI and/or antibody exposure onSY5Y cellular DNA content by flow cytometry. As shown inFigure 6a, and quantified in Figure 6b, treatment with antibodyalone led to a small, but significant, increase in the G0/G1 fraction(antibody-treated mean¼ 69.2±0.5%; vehicle-treated mean¼

65.7±0.3%) and a small but significant decrease in the sub G0fraction (antibody-treated mean¼ 11.4±0.3%; vehicle-treatedmean¼ 13.7±0.2%), suggesting that the main mechanism ofantibody action may be G1 arrest. On the other hand, dualantibody and TKI ALK targeting led to large and significantincreases in the sub-G0 fraction (mean¼ 38.0±0.4%), suggestinginduction of programmed cell death as the dominant mechanismof dual ALK targeting. Treatment with crizotinib alone alsoincreased the sub-G0 fraction, but to a smaller extent than seenwith dual treatment, consistent with the findings above that anti-ALK potentiates crizotinib effects.

DISCUSSIONCure rates among children with high-risk neuroblastoma haveshown only modest improvement, despite dramatic escalations inthe intensity of therapy provided, and survivors of modernhigh-risk neuroblastoma therapy are at risk for major morbid-ities.40,41 New approaches are required for treating these patients,and advances in our understanding of its molecular basis haveidentified tractable therapeutic targets that may respond to novelagents. One promising approach is to target the RTK ALK, whichcan be inhibited by TKIs and may also be a valuable target forimmunotherapy. A recent phase 3 clinical trial using an antibodyagainst the disialoganglioside GD2 has shown the promise oftargeted immunotherapy in neuroblastoma.42 We show here thatALK is also expressed in the vast majority of neuroblastoma cases,suggesting that it may represent another tractable immunother-apy target. Moreover, similar to GD2, ALK expression is largelylimited to tumor tissue,32 making it an ideal immunotherapytarget, while minimizing the risk of cytotoxicity in nonmalignanttissue and simplifying patient selection.

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Figure 4. Dual antibody/TKI targeting of ALK. SY5Y cells were treated with either 333 nM crizotinib or 10 mg/ml anti-ALK antibody(mAb30þmAb49) or both. As negative control, cells were treated with equal volumes of dimethyl sulfoxide and 10 mg/ml murine IgG1. (a) Cellgrowth was monitored by Real-Time Cell Electronic Sensing. (b) Immunoblot analysis of native ALK protein levels (upper panel) and phospho-ALK (middle panel). b-Actin levels are shown as a loading control (lower panel). (c) Effect of crizotinib pretreatment on anti-ALK antibody-mediated ADCC was measured. SY5Y cells were preincubated in the presence of crizotinib or vehicle for 48 h, harvested and then used astarget cells in the in vitro ADCC assay. **Po0.01 and *Po0.05.

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Similar to the GD2 antibody, and several clinically successfulantibodies such as trastuzumab and cetuximab, we find that anALK antibody can mediate ADCC of ALK-positive neuroblastomacells. In our in vitro ADCC system, neuroblastoma cells were killedwhen treated with 1 mg/ml anti-ALK antibody, a lower dose thanthe 10 mg/ml clinically achieved in trastuzumab-treated breastcancer patients,43 and well below the trough plasma antibodyconcentrations achieved in two phase I studies of lung cancer

patients treated with cetuximab.44,45 Our experiments were notdesigned to determine the optimal ADCC dose, but nonethelessargue that antibody targeting of ALK is likely to be an importanttherapeutic avenue based on its ADCC effects alone. Additionalstudies will be required to determine the minimum ALK antibodydose at which ADCC can be induced, the range of effectiveconcentrations and dependence of ADCC sensitivity on ALKexpression level and genotype.

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Figure 5. Effects of an antagonist ALK antibody on crizotinib dose -- response curve. SY5Y cells were treated with crizotinib at the indicateddoses, either alone or in combination with 10 mg/ml (total) ALK antibody mAb30 and mAb49. Cell growth was measured at day 7 using Real-Time Cell Electronic Sensing. (a) Comparison of growth inhibition for monotherapy with dual ALK targeting: white bars represent crizotinibalone and gray bars represent crizotinib treatment in the presence of 10 mg/ml anti-ALK. **Po0.01 and *Po0.05. (b) IC50 was calculated over arange of 10 doses of crizotinib alone (circles) or crizotinib plus 10 mg/ml anti-ALK (squares), yielding IC50 values of 3018 nM and 1745 nM,

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Figure 6. Cell cycle analysis of inhibitor-treated cells. SY5Y cells were treated with 1000 nM crizotinib, 10 mg/ml antibody, both, or vehicle/IgG1,and were then harvested, fixed, stained with propidium iodide and analyzed by flow cytometry. (a) Representative histograms showingproportion of cells in sub-G0/apoptosis, G0/G1 and G2/mitosis. (b) Quantification of flow cytometry results. **Po0.01 and *Po0.05.

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Unlike GD2 antibodies, ALK antibodies bind to and inhibit anoncogenic receptor---permitting an additional immune cell-independent component of its inhibitory mechanism. ALKoverexpression or mutation leads to its hyperactivation, autopho-sphorylation and elevated downstream signaling,13 as also seenfor HER2 and EGFR in breast and lung cancer.46 The oncogenicrole of full-length ALK in neuroblastoma was originally describedwhen activating mutations were discovered in the germline ofpatients with familial neuroblastoma, and were subsequentlyfound to be somatically acquired.9,10,47,48 More recently, it wasshown that high levels of ALK expression are associated withdecreased survival.49 Thus, ALK signaling---enhanced by mutationor overexpression---has an important role in initiation and/ormaintenance of neuroblastoma. ALK inhibition with crizotinib orother TKIs is likely to be an important tool in treating ALK-dependent neuroblastoma, but it is already clear that certainALK-activating mutations reduce sensitivity to this drug.24

Antagonistic ALK antibodies may offer a parallel approach toALK inhibition, just as has been reported for antibodies thattarget HER2 or EGFR. We show here that an antagonist ALKantibody inhibits growth of neuroblastoma cells over a range ofdoses, all of which are below (or equal to) those reported to beclinically achievable in patients with trastuzumab or cetuximab.The ALK antibody inhibits growth of neuroblastoma cell linesthat harbor either amplified/overexpressed ALK (NB1) or ALKwith the most common constitutively activating mutations(R1275Q in 1643 cells and F1174L in SY5Y cells). Notably, SY5Ycells are relatively resistant to TKI targeting of ALK, but respondto antibody inhibition. ALK antibody therapy may therefore berelevant for patient tumors exhibiting a broad range of ALKexpression levels and aberrations.

Our results strongly argue for dual antibody and TKI targeting ofALK---the utility of which has been demonstrated for several otheroncogenic RTKs.21,22,50 -- 52 For non-small-cell lung cancer expres-sing erlotinib-resistant EGFRT790M, such dual targeting was theonly therapeutic approach among several studied that inducedtumor regression.53 The parallels between T790M-mutated EGFRand (crizotinib resistant) F1174L-mutated ALK are robust,24

suggesting that dual antibody/TKI therapy may be particularlyimportant in this case. Binding of crizotinib promotes cell surfaceaccumulation of ALK, as has been observed for several othertherapeutically targeted TKIs---a likely key mechanism for theefficacy of dual TKI/antibody targeting.21 Combined antibody/TKItreatment in F1174L-expressing cells led to almost completegrowth inhibition, induced a significantly higher level of apoptosisthan either treatment alone and reduced the IC50 for crizotinibtreatment by half. This implies that dual ALK targeting may be arelevant therapeutic strategy for decreasing the dose-dependenttoxicities associated with TKI therapy and may even providepotential for delaying or preventing TKI resistance.

Our results point to the urgent need for development ofclinically relevant inhibitory ALK antibodies and their evaluationwith in vivo preclinical models and clinical trials. Ongoing work todevelop a clinically relevant anti-ALK antibody includes humaniza-tion to reduce immunogenicity,54 defucosylation to maximizeADCC,55 and possible conjugation to immunostimulatory cyto-kines, such as IL-2 or cytotoxic agents.56 Preclinical optimization ofanti-ALK antibody design will be crucial for maximizing success inthe clinic.

MATERIALS AND METHODSCell culture and reagentsCell lines were maintained in a 5% CO2 incubator at 37 1C in completeRPMI (Invitrogen, Carlsbad, CA, USA). Mouse monoclonal immunoglobulinIgG1 antibodies 14, 30, 46 and 49 were generated to the extracellulardomain of human ALK as previously described57 and provided by Dr Marc

Vigny. Murine IgG1 (Sigma-Aldrich, St Louis, MO, USA) and dimethylsulfoxide were used as negative controls.

Quantitative mRNA expressionRNA from primary diagnostic tumor specimens of 229 neuroblastomas wasanalyzed using Affymetrix (Santa Clara, CA, USA) Human Exon Arrays(HuEx), normalized by quantile normalization and summarized usingrobust multichip average (Affymetrix Power Tools software packageversion 1.12). ALK expression levels were obtained by averaging the coreunique probe sets for the ALK transcript (transcript ID: 2546409).Significance was determined among patients diagnosed with LR (stage 1and 2, n¼ 24), HRN (418 months of age at diagnosis, n¼ 141) and HRA(n¼ 64). The P-value was calculated using likelihood ratio w2-tests in thecontext of a general linear model. Differences between subgroups wereassessed using the Wald test. Analyses were performed using theR statistical package (R version 12.1, Auckland, New Zealand).

ImmunohistochemistryCases were selected from a review of tumors accessioned to the PathologyDepartment of the Children’s Hospital of Philadelphia (CHOP) from 1987 to2004. Specimen selection and construction of the TMA followed approvalby the CHOP Institutional Review Board. Tumors were reviewed by apediatric pathologist for adequacy, and classified using InternationalNeuroblastoma Pathology Classification criteria. The TMA comprised twoparaffin blocks and contained tumor cores from 126 patients (117neuroblastomas and 9 nodular ganglioneuroblastomas). MYCN amplifica-tion status and stage were obtained by tumor registry review. Staining wasconducted with pre-diluted anti-ALK-1 (Ventana Medical Systems,Oro Valley, AZ, USA) using pressure cooker antigen retrieval, overnightincubation and avidin -- biotin complex conjugation. An ALK staining scorewas calculated as the product of staining intensity grade (1 -- 3; weak,moderate and strong) and percentage of neuroblasts stained. The Foodand Drug Administration normal human organ TMA (US Biomax, Rockville,MD, USA) was similarly analyzed.

MicroscopyFormalin-fixed paraffin-embedded tissue slides of 16 neuroblastomapatient samples were rinsed in xylene, rehydrated with ethanol andtreated in a pressure cooker with High pH Retrieval Solution (Dako,Carpinteria, CA, USA). Slides were incubated with primary ALK-1 antibody(Ventana Medical Systems), followed by rabbit pan-cadherin antibody(Abcam, Cambridge, MA, USA). Slides were then incubated with secondaryantibodies Alexa-488 anti-Rabbit (Invitrogen) and Alexa-594 anti-Mouse(Invitrogen) and counterstained in DAPI hydrochloride (Sigma, Ronkonko-ma, NY, USA). Tissue sections were visualized using Olympus IX-81 DSUspinning disk confocal microscope (Olympus, Center Valley, PA, USA)through a 60� 1.49 NA oil immersion objective. Images were analyzed andPearson’s correlation coefficient values generated using Volocity software(Improvision, Waltham, MA, USA). To grade staining, settings weredetermined at the beginning of the experiment, so that unstained controlswere negative and single-stained controls were not emitting signaldetectable using filter sets intended for imaging other fluorophores.

ImmunofluorescenceCell lines were plated overnight on Lab Tek II Chamber Slides (NuncThermo Scientific, Roskilde, Denmark), then transferred to ice and washedwith ice-cold phosphate-buffered saline before blocking. Cells wereincubated for 60 min on a rocker table with ALK antibody mAb14, washedand incubated for 45 min with rhodamine-conjugated goat anti-mousesecondary antibody (Jackson, West Grove, PA, USA), washed and cover-slipped with DAPI-containing mounting media (Santa Cruz Biotechnology,Santa Cruz, CA) for evaluation with a fluorescence microscope.

Flow cytometryCells were kept ice cold during staining to minimize receptor endocytosis,harvested and washed, and then ALK antibody mAb14 was added for a

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10mg/ml final concentration. Donkey anti-mouse IgG PE (eBioscience,San Diego, CA, USA) was then added (2.5mg/ml). In some experiments,cells were treated with crizotinib (or dimethyl sulfoxide as a negativecontrol) and harvested at subsequent time points for flow cytometry. Inother experiments, staining was conducted with mAb46, which binds to adistinct epitope from mAb14. Cells were analyzed on an LSR II flowcytometer (BD Biosciences, San Jose, CA, USA). All results shown arerepresentative of at least three independent experiments.

Growth inhibitionWe used the Real-Time Cell Electronic Sensing system (ACEA Biosciences,San Diego, CA, USA) to measure the in vitro effect of ALK antibodies mAb30and mAb49 alone or in combination with crizotinib. Cells were plated intriplicate, and antibody and/or drug added 24 h later. Antibody treatmentwas continued for 4 days. Growth inhibition was calculated as:100� (1�(cell index treatment/cell index control)). All cell lines weremycoplasma tested and genotyped using the AmpFLSTR Identifiler kit(Applied Biosystems, Foster City, CA, USA). All experiments were conducteda minimum of three times, and results quoted as mean±s.d. Linear mixedeffect models were fitted for statistical analysis of time and treatmenteffects. To account for nonlinearity, time by treatment and time squared bytreatment interaction terms were included in the models. F tests were usedto examine the difference of the progression of cell index over timebetween each individual and the combination treatment. For IC50

calculation, cells were plated in a 5-log dose range of crizotinib alone orin combination with 10mg/ml ALK mAb30 and mAb49. GraphPad PrismVersion 5.0 (GraphPad Prism, La Jolla, CA, USA) was used to calculate theIC50 from the Real-Time Cell Electronic Sensing-generated cell index datausing the log (inhibitor) versus response---variable slope equation.

Surface biotinylation and western blotsCells were grown in 10 cm dishes until 70 -- 80% confluency, treated withcrizotinib, ALK antibody, the combination, or vehicle at the times anddoses indicated. For biotinylation, EZ-LINK Sulfo-Biotin (Pierce, Rockford, IL,USA) was used according to manufacturer instructions. Biotinylation wasperformed after 24 h treatment with 1000 nM crizotinib (or control). Cellswere washed with ice-cold phosphate-buffered saline, lysed and biotin-labeled protein precipitated with NeutrAvidin beads (Thermo Scientific,Rockford, IL, USA). Whole-cell lysates or NeutrAvidin precipitates wereharvested and immunoblotted using antibodies against ALK (CellSignaling, Danvers, MA, USA), phospho-ALK Tyr 1604 (Cell Signaling), actin(Santa Cruz Biotechnology) or cadherin (Abcam). Results shown arerepresentative of at least two independent experiments.

Propidium iodide stainingAt 24 h post plating, cells were treated with1000 nM crizotinib, 10 mg/mlALK antibody, both agents or negative controls. Antibody treatment wascontinued for four additional days. Cells were harvested, stained withpropidium iodide and analyzed on an LSR II flow cytometer.

Antibody-dependent cell-mediated cytotoxicity assayThe CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison,WI, USA) was used to assess ADCC. Normal donor peripheral bloodmononuclear cells were used as effectors and plated for 2 h to allowmonocytes to adhere. Non-adherent lymphocytes were replated incomplete RPMI containing 1000 IU/ml rhIL-2 (Chiron, Emeryville, CA,USA), collected and washed. Target NB1 or SY5Y cells were harvestedand washed. In some experiments, target SY5Y cells were preincubated48 h in crizotinib. Effector and target cells were plated in quadruplicate ateffector: target ratios of 50:1, 25:1 and 10:1, and experimental wells treatedwith 1 mg/ml ALK mAb30 and mAb49. Control wells were plated accordingto manufacturer’s specifications. After a 4-h incubation at 37 1C, cell viabilitywas assessed by an enzymatic assay quantifying lactate dehydrogenaserelease upon cell lysis. Percent (%) cytotoxicity was calculated:

Experimental� effector spontaneous� target spontaneoustarget maximum� target spontaneous

�100

Experiments were conducted comparing untreated wells with treatmentwith 1 mg/ml murine IgG1. No difference was detected between untreated(% cytotoxicity¼ 3.1%þ 0.3) and IgG1-treated wells (0.64þ 0.28%;P¼ 0.8822). Results are representative of three independent experiments,quoted as mean±standard deviation.

CONFLICT OF INTERESTThe authors declare no conflict of interest.

ACKNOWLEDGEMENTSWe thank Pfizer for their gift of crizotinib, and Dr Marc Vigny for his gift of the ALKmonoclonal antibodies 30, 49, 46 and 14. This work was supported in part by NIHGrants R01-CA140198 (YPM), 2R01 CA60104-16 (RCS), 2R01 CA60104-16S1 (RCS), theChildren’s Oncology Group, the Carly Hillman Fund (YPM), NIH Training Grant inStructural Biology T32-GM008275 (SCB), a fellowship grant from the St Baldrick’sFoundation (ACW) and the US Army Peer-Reviewed Medical Research Program(W81XWH-10-1-0212/3 to MAL/YPM).

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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