A Novel IL6 Antibody Sensitizes Multiple Tumor Types to Chemotherapy Including Trastuzumab- Resistant Tumors Haihong Zhong1, April Davis2, Maria Ouzounova3, Rosa A

Published OnlineFirst January 7, 2016; DOI: 10.1158/0008-5472.CAN-15-0883

Cancer Tumor and Stem Cell Biology Research

A Novel IL6 Antibody Sensitizes Multiple Tumor Types to Chemotherapy Including Trastuzumab- Resistant Tumors Haihong Zhong1, April Davis2, Maria Ouzounova3, Rosa A. Carrasco1, Cui Chen1, Shannon Breen1, Yong S. Chang4, Jiaqi Huang5, Zheng Liu5, Yihong Yao5, Elaine Hurt1, Jacques Moisan1, Michael Fung5, David A. Tice1, Shawn G. Clouthier2, Zhan Xiao1, Max S. Wicha2, Hasan Korkaya3, and Robert E. Hollingsworth1

Abstract

Elevated levels of the proinflammatory cytokine IL6 are promoting tumor angiogenesis, we found that MEDI5117 associated with poor survival outcomes in many cancers. inhibited the growth of endothelial cells, which can produce Antibodies targeting IL6 and its receptor have been developed IL6 and support tumorigenesis. Notably, in tumor xenograft for chronic inflammatory disease, but they have not yet been assays in mice, we documented the ability of MEDI5117 to shown to clearly benefit cancer patients, possibly due to enhance the antitumor activities of chemotherapy or gefitinib antibody potency or the settings in which they have been in combination treatment regimens. MEDI5117 also displayed þ tested. In this study, we describe the development of a novel robust activity on its own against trastuzumab-resistant HER2 þ high-affinity anti-IL6 antibody, MEDI5117, which features an tumor cells by targeting the CD44 CD24 cancer stem cell extended half-life and potent inhibitory effects on IL6 biologic population. Collectively, our findings extend the evidence of activity. MEDI5117 inhibited IL6-mediated activation of important pleiotropic roles of IL6 in tumorigenesis and drug STAT3, suppressing the growth of several tumor types driven resistance, and offer a preclinical proof of concept for the by IL6 autocrine signaling. In the same models, MEDI5117 use of IL6 antibodies in combination regimens to heighten displayed superior preclinical activity relative to a previously therapeutic responses and overcome drug resistance. Cancer Res; developed anti-IL6 antibody. Consistent with roles for IL6 in 76(2); 480–90. 2016 AACR.

Introduction binding protein (IL6Ra chain, CD126) and a 130 kDa signal transduction glycoprotein (gp130). IL6 binds to the transmem- IL6 is produced by lymphoid and nonlymphoid cells including brane IL6Ra protein and the IL6/IL6Ra complex binds to two T and B cells, monocytes, fibroblasts, keratinocytes, as well as molecules of gp130, which then activates an intracellular signal- endothelial, mesangial, and tumor cells (1, 2). Its pleiotropic roles ing cascade. This mechanism is known as classical signaling. include the activation of T cells, induction of the acute phase IL6Ra also exists as a soluble receptor (sIL6Ra), which can inflammatory response, differentiation, and survival of plasma B combine with IL6 to trigger membrane-bound gp130 dimeriza- cells, synovial fibroblasts, and osteoclasts, and stimulation of tion and thus mediate the so-called trans-signaling (3, 4). Because growth and differentiation of hematopoietic precursor cells (3). of the ubiquitous expression of gp130 on different cell types, the IL6 acts on target cells by binding to the IL6 receptor (IL6R), which trans-signaling mechanism is attributed to the broad array of IL6 consists of two membrane-bound proteins, an 80 kDa ligand- functions in the body. Activated gp130 stimulates JAK tyrosine kinases, which phosphorylate and recruit the transcription factor STAT3 to regulate gene expression (5). 1 2 Oncology Research, MedImmune, Gaithersburg, Maryland. Transla- Many cancers exploit IL6 as a growth factor as well as a modifier tional Science, MedImmune, Gaithersburg, Maryland. 3Aileron Thera- peutics, Inc., Cambridge, Massachusetts. 4University of Michigan of the tumor microenvironment. Solid tumors such as lung, Comprehensive Cancer Center, Ann Arbor, Michigan. 5Department of ovarian, breast, and colon carcinomas produce IL6, IL6Ra and Biochemistry and Molecular Biology, Georgia Regents University, gp130, allowing them to constitutively stimulate their own growth Augusta, Georgia. in autocrine manner (6–8). Other cancers, such as multiple Note: Supplementary data for this article are available at Cancer Research myeloma and neuroblastoma, do not produce IL6, but do express Online (http://cancerres.aacrjournals.org/). IL6Ra and gp130; these tumors respond to IL6 produced in the H. Zhong and A. Davis contributed equally as first authors. tumor microenvironment in a paracrine manner (9). IL6 plays Corresponding Author: Robert E. Hollingsworth, One MedImmune Way, multiple roles in tumor progression and drug resistance (10–12). It Gaithersburg, MD 20878. Phone: 301-398-5354; Fax: 301-398-8384; E-mail: can act directly on tumor cells, or function by interacting with [email protected] and Hasan Korkaya, Georgia Regents Uni- normal cells in the tumor microenvironment, including endothe- versity Cancer Center, 1410 Laney Walker Blvd., Augusta, GA 30912; E-mail: lial, immune, and inflammatory cells. Targeting IL6 and its path- [email protected] way therefore is expected to inhibit tumors through multiple doi: 10.1158/0008-5472.CAN-15-0883 mechanisms. Several anti-IL6 antibodies are in clinical develop- 2016 American Association for Cancer Research. ment for cancer therapy. Among these, siltuximab (CNTO328) is

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the most advanced and is being tested in the clinic for multiple of 8,000 cells per well. Plates were spun down and incubated types of cancer. Published data suggest that anti-IL6 antibody overnight to allow cells to assemle into compact spheroids. Two- therapies have acceptable safety and tolerability (13–15). day-old spheroids were treated with human recombinant IL6 or In this study, we further elucidate the critical role of IL6 in sIL6R proteins (R&D Systems) with MEDI5117 at varying con- cancer biology in multiple solid tumor types and provide evidence centrations. After 7-day incubation, proliferation was quantified that IL6 is an anticancer target in mono- and especially combi- using the CellTiter-Glo assay using an EnVision 2104 Multilabel nation therapy settings. MEDI5117 is a human monoclonal Reader (PerkinElmer). antibody (mAb) that potently binds and neutralizes human IL6. For human umbilical vein endothelial cells (HUVEC), cells It was engineered to have increased persistence in circulation were cultured as monolayers. Recombinant proteins and anti- compared with unmodified antibodies through incorporation of body were added on next day. After 72 hour incubation, prolif- YTE mutations in the Fc region (16, 17). MEDI5117 inhibited IL6 eration was quantified using the CellTiter-Glo assay. signaling and suppressed the growth of lung, breast, and ovarian in vitro in vivo tumor cells and with higher potency than siltux- mRNA profiling fi imab. Inhibition of tumor growth was signi cantly increased Total RNA was extracted from snap-frozen xenograft tumor fi when MEDI5117 was used in combination with ge tinib, taxanes, samples using the ZR RNA MicroPrep Kit (Zymo Research). or other chemotherapeutics. Furthermore, MEDI5117 suppressed Xenograft tumors were treated with 30 mg/kg of MEDI5117 or the growth of IL6-dependent trastuzumab-resistant breast IgG isotype control IgG1 for a total of two doses. Generating tumors, and this was associated with a reduction of cancer stem biotin-labeled amplified cRNA was accomplished using the Mes- cells (CSC). These results extend our understanding of the role of sageAmpTM Premier RNA Amplification Kit (Ambion) and used IL6 in tumor biology and demonstrate the potential of MEDI5117 for gene expression with Affymetrix Human Genome U133 Plus for the treatment of multiple cancers. 2.0 GeneChip microarrays. Canonical pathway enrichment analyses were conducted using Ingenuity Pathway Analysis. All micro- Materials and Methods array data are deposited in GEO repository under accession Cells and reagents number is GSE62941: (http://www.ncbi.nlm.nih.gov/geo/que- Cancer cell lines were obtained from the ATCC. Cell line ry/acc.cgi?token¼ujajyuyovbqzdyf&acc¼GSE62941). authentication was conducted by STR-based DNA fingerprinting and multiplex PCR. IMPACT tests were also performed on all cell Mouse xenograft studies lines. All animal procedures were conducted in accordance with all CellTiterGlo reagents were obtained from Promega. An enzyme appropriate regulatory standards under protocols approved by linked immunosorbent assay (ELISA) was developed in our the Medimmune Institutional Animal Care and Use Committee. laboratories to detect free IL6 in tumor lysates. Human IL6 and For the MCF-7 xenograft study, 0.36 mg of an estrogen pellet sIL6R ELISA kits were purchased from R&D Systems. Recombi- was implanted subcutaneously into the left flank of each female nant human IL6 and sIL6R proteins were obtained from R&D athymic nude mouse. The effect of the estrogen pellet lasted for 60 Systems. Antibodies to detect STAT3 and phospho-STAT3-Tyr705 days in vivo. Two million MCF7 or MCF7/IL6 tumor cells were were obtained from Cell Signaling Technology and gp130 anti- suspended in 100 mL of BME (6 mg/mL) before injection. All mice body was obtained from Santa Cruz Biotechnology. MCF-7/IL6– received an orthotopic inoculation of tumor cells into the mam- overexpressing cell line was kindly provided by Dr. Mercedes mary fat pad. Rincone (University of Vermont, Burlington, VT; ref. 18). Siltux- For in vivo efficacy studies, 5 million NCI-H1650, KPL-4, imab (CNTO328) was generated inhouse based on published DU145, or MDAH2774 cells in 50% Matrigel were inoculated sequence information. subcutaneously into each female athymic nude mouse. When tumors reached approximately 150 to 200 mm3, mice were siRNA Knockdown randomly assigned into groups (10 mice per group). MEDI5117 A reverse transfection protocol was used to analyze the effects of or siltuximab was administered intraperitoneally (i.p.) twice per IL6 and IL6R knockdown. Briefly, siRNA pools targeting IL6 and week at indicated doses. Tumor volumes were measured twice IL6R (Dharmacon) were prepared at a final concentration of 50 weekly with calipers. Tumor growth inhibition was calculated on nmol/L in optiMEM (Invitrogen). DU145 cells were then added to the last day of study relative to the initial and final mean tumor the siRNAs at a density of 5,000 cells per well in 96-well plates in volume of the control group. medium containing 10% heat-inactivated FBS (Life Technolo- For in vivo mechanism of action and pharmacodynamic gies). After 48 hours, transfected cells were washed with ice-cold studies, a single dose of MEDI5117 was administered when tu- PBS and then lysed by adding Laemmli reducing buffer (Boston mors reached approximately 400 mm3. Tumor and serum sam- BioProducts). Cell lysates were then subjected to Western blot ples were collected 4 hours after dosing. analysis for phosphorylated STAT3 (pSTAT3). Conditioned medium was analyzed in duplicate with human IL6 and soluble IL6R ELISA kits (R&D Systems). For proliferation assays, CellTiter CSC analyses Glo reagent was used five days after transfection and luminescence For tumorsphere assays, single cells were plated on ultra-low 5 was measured using an EnVision 2104 Multilabel Reader attachment plates at a density of 1 10 /mL and grown for 7 days (PerkinElmer). in a mammocult medium (Stem Cell Technologies). After the treatment of primary spheres with drugs, they were dissociated Cell proliferation analysis into single-cell suspension and plated at a density of 5103– For DU145 cells, cell proliferation was assessed by three- 1104/mL. Secondary spheres were counted after 5 to 7 days in dimensional tumor spheroid assay. Cells were plated at a density culture and evaluated the efficacy of the drug treatment.

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þ For CD44 CD24 CSC flow cytometry analyses, single-cell sIL6R. Membrane-bound IL6R and gp130 and intracellular suspensions of residual tumors cells isolated from drug-treated pSTAT3 were detected in these cell lines as well. These compo- mice were incubated with fluorophore-conjugated CD44 or CD24 nents allow these cell lines to stimulate their own growth through antibodies alone or in combination on ice for 30 minutes, washed an IL6-dependent autocrine loop. In contrast, MDA-MB-468 and with Hank's Balanced Salt Solution (HBSS), and resuspended in MCF-7 (breast cancer) cells do not secrete IL6 and MDA-MB-231 DAPI containing HBSS buffer for flow cytometry analyses. Gate (breast cancer) and U87MG (glioblastoma) cells secrete relatively compensation was performed with single color–stained cells and high levels of IL6, but lack IL6R on the surface. þ the CD44 CD24 phenotype analyses were performed using the double stained cells. An anti-IL6 antibody, MEDI5117, inhibits cancer cell and Tumor reimplantation studies were conducted using cells endothelial cell proliferation recovered from the residual tumors of treated animals. Tumors MEDI5117 is a human, high affinity, IgG1 monoclonal anti- were excised, chopped, and processed with collagenase for 1 to 2 body (mAb) that neutralizes human IL6. The parental antibody hours at 37C. Cells were then washed with PBS, trypsinized, and (CAT-6001) was obtained by screening phage display human passed through a 40 mm filter. The single cells were labeled with antibody libraries for binding to recombinant human IL6 and for H-2Kd antibody to distinguish human cells from mouse cells and inhibition of the IL6/IL6R interaction (14). We then created DAPI and then sorted with flow cytometry. Live human tumor MEDI5117 by incorporating three amino acid substitutions, cells were reimplanted orthotopically into mouse mammary fat M252Y, S254T, and T256E (YTE), into the Fc region of CAT- pads to determine tumor initiation capacity. Tumors were mea- 6001 to increase the antibody's half life in vivo. Pharmacokinetic sured weekly using luciferase bioluminescense and by caliper. studies in cynomolgus monkeys showed that the half-life of CSC frequencies were calculated by limiting dilution analyses MEDI5117 was extended by 3-fold (28.4 vs. 8.4 days) and using the Walter and Eliza Hall Institute formula (http://bioinf. clearance was reduced by 4-fold (3.02 vs. 12.1 mL/kg/day) when wehi.edu.au/software/elda/; ref. 19). compared with CAT-6001 (16, 17). The half-life of MEDI5117 in humans was 84 days, significantly longer than other anti-IL6 Results antibodies, such as siltuximab whose half-life was 17.8 days. IL6 is a potent growth factor and its downstream signaling is MEDI5117 binds IL6 with high affinity. In the KinExa assay, activated in multiple cancer types MEDI5117 had a Kd < 1 pmol/L, while siltuximab had a Kd ¼ IL6 is known to be a potent growth factor in some cancer cell 6.25 pmol/L. By ELISA, MEDI5117 had an EC50 ¼ 6.5 pmol/L for lines, and we observed this in the models we studied as well. For human IL6 binding as measured, more potent than that of example, siRNA knockdown of either IL6 or IL6 receptor strongly siltuximab (EC50 ¼ 14.1 pmol/L). MEDI5117 cross reacts with inhibited the proliferation of DU145 prostate cancer cells, as well cynomolgus monkey IL6, but not with IL6 from other species, as their levels of activated STAT3, the downstream effector of the including dogs, rats, and mice. IL6 signaling pathway (Supplementary Fig. S1A). Similarly, over- The effect of MEDI5117 on the proliferation of cancer cells was expression of IL6 increased the growth rate of MCF-7 cancer cells investigated in DU145 prostate cancer cells using a spheroid in vitro and in vivo (Supplementary Fig. S1B). formation assay. Stimulating DU145 cells with recombinant Although activation of IL6 signaling can occur through para- human IL6 resulted in increased cell proliferation as measured crine mechanisms, we found evidence of autocrine IL6 signaling by the number of spheroids grown in serum-free medium for in several cancer cell lines (Fig. 1 and Supplementary Fig. S2). seven days. The addition of sIL6Ra alone had little effect on MDAH2774 (ovarian cancer), NCI-H1650 and NCI-H1975 DU145 cell proliferation. Addition of IL6 in combination with (non–small cell lung cancer, NSCLC), DU145 (prostate cancer), sIL6Ra stimulated cell growth to the same extent as IL6 treatment and Detroit 562 (head and neck cancer) cells produced IL6 and alone. This finding indicates that the proliferative effect of IL6 on

Figure 1. The IL6 pathway is hyperactivated in several cancer cell lines. Conditioned medium from various cancer cell lines was collected and analyzed in duplicate. Human IL6 and soluble IL6R were measured by ELISA, membrane IL6R expression was analyzed by ﬂow cytometry, and protein levels of gp130, pSTAT3, STAT3, and GAPDH were assessed by Western blotting.

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A IgG1 control - 20 μg/mL IgG1 control - 20 μg/mL 200 DU145 HUVEC MEDI5117 - 20 μg/mL 100 MEDI5117 - 20 μg/mL

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0 0 –1 0 1 0123 –25 –25 Antibody concentration (nmol/L) Antibody concentration (pmol/L)

C DU145 MDA-MB-468 MDA-H2774 NCI-H1650 NCI-H1975 MDAH2774 DU145 IL6 – ++ – ++ MEDI5117 ––+ ––+ CAT-6001 MEDI5117 Control IgG CNTO328 MEDI5117 CNTO328 CNTO328 Control IgG CNTO328 MEDI5117 CNTO328 MEDI5117 Control IgG Control IgG MEDI5117 Control IgG pSTAT3 pSTAT3 STAT3 STAT3 Actin

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Figure 2. MEDI5117 inhibited cell proliferation and blocked STAT3 activation in cancer cell lines and endothelial cells. A, DU145 spheroids were grown in serum-free medium and stimulated with recombinant human IL6 and soluble IL6R, or IL6 þ sIL6R at the indicated concentrations in the presence of 20 mg/mL control IgG antibody or MEDI5117 (left). Treatments were performed in triplicate and mean spheroid numbers and standard deviations were calculated. A, DU145 cells and HUVECs were incubated with recombinant human IL6 and sIL6R along with 20 mg/mL control antibody or MEDI5117 (right). Proliferation was measured using the CellTiter Glo assay and growth induction over the untreated control was calculated. All treatments were performed in triplicate and mean, and SDs were calculated. B, the inhibitory activities of MEDI5117 and siltuximab were compared in vitro. DU145 and TF-1 cells were stimulated with recombinant human IL6 in the presence of MEDI5117 or siltuximab (CNTO328). C and D, MEDI5117, siltuximab, and a control IgG were added directly to the cells for a ﬁnal concentration of 30 mg/mL in the presence of 10 ng/mL of IL6, sIL6R, or IL6/sIL6R. After 24 hours, cells were lysed and subjected to Western blot analysis.

DU145 spheroids occurs through classical signaling involving proliferation (Fig. 2A, right). Treatment with MEDI5117 blocked the membrane-bound receptor. Treatment with MEDI5117 the HUVEC proliferation induced by IL6 and sIL6R. Thus, completely blocked IL6-induced spheroid growth (Fig. 2A, left). MEDI5117 can inhibit proliferation of cancer cells by blocking Proliferation was not blocked by treatment with a control isotype- classical IL6 signaling and proliferation of endothelial cells by matched IgG1 mAb. blocking IL6 trans-signaling in vitro. HUVEC cells do not express membrane bound IL6Ra (20), and The inhibitory activities of MEDI5117 and siltuximab were therefore IL6 stimulation of HUVECs relies on trans-signaling compared in both DU145 cells and TF-1 (erythroleukemic) cells. (21). Adding recombinant human IL6 or sIL6R alone had only Treatment with MEDI5117 or siltuximab blocked IL6-induced cell modest effects on HUVEC proliferation. However, adding IL6 proliferation in a dose-dependent manner (Fig. 2B); however, with sIL6R together resulted in signiﬁcantly increased HUVEC MEDI5117 was more potent than siltuximab based on IC50 values.

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IL6 signaling involves activation of the transcription factor, These results corroborate that MEDI5117 can inhibit both clas- STAT3. To analyze effects on STAT3, several different cancer cell sical and trans-signaling stimulated by IL6. lines and HUVECs were treated with MEDI5117, siltuximab, or a control IgG1 mAb (Fig. 2C, left). Basal levels of pSTAT3 (it's Neutralization of IL6 by MEDI5117 inhibits tumor growth in activated form) were suppressed by MEDI5117 in each of the cell xenograft models lines; treatment with siltuximab resulted in less efﬁcient suppres- MEDI5117 was evaluated in NSCLC (NCI-H1650), ovarian sion. Addition of recombinant human IL6 activated STAT3 in (MDAH-2774), and prostate (DU145) xenograft models in vivo both the MDAH2774 and DU145 cancer cell lines, and pretreat- (Fig. 3A and Supplementary Fig. S3). These tumor models pro- ment with MEDI5117 blocked this activation (Fig. 2C, right). In duce IL6 and bear cell surface IL6R. MEDI5117 at 30 mg/kg alone DU145 cells, the level of pSTAT3 in MEDI5117-treated cells was caused tumor growth inhibition (DTGI) of 51% (P ¼ 0.004) in even lower than the basal level of pSTAT3 in untreated cells. NCI-H1650 and 59% DTGI (P ¼ 0.007) in MDAH2774. Doses MEDI5117 treatment reduced basal pSTAT3 levels, and reversed between 3 and 30 mg/kg showed comparable DTGI with the 30 IL6 or IL6/sIL6R induced STAT3 activation in HUVECs (Fig. 2D). mg/kg dose. A low dose of 1 mg/kg was insufﬁcient to inhibit

1,200 A IgG1 control B Free human IL6 levels Free human IL6 levels pSTAT3 levels

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20 25 30 35 40 45 50 55 MEDI5117 MEDI5117 MEDI5117 Days after implantation Control IgG1 Control IgG1 Control IgG1

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Figure 3. Inhibition of growth of NCI-H1650 xenograft tumors in vivo by MEDI5117. A, NCI-H1650 cells were implanted into nude mice and MEDI5117 was administered via intraperitoneal injection at a dose of 30 mg/kg, twice per week. Ten animals were used for each group. B, free IL6 and pSTAT3 levels in tumor extracts and serum dosed with MEDI5117 were assessed by ELISA four hours after the last dose of MEDI5117. C, pSTAT3 levels in tumor lysates were analyzed four hours after the last dose of MEDI5117 by staining with an anti-pSTAT3 antibody. Quantiﬁcation of pSTAT3-expressing cells in tumor and stroma components was done on four randomly chosen ﬁelds. , P < 0.05. D, Ki67 staining was performed to measure the proliferation of xenograft tumors cells. The percentage of Ki67-positive nuclei from three randomly chosen areas was evaluated. , P < 0.05, IgG1 control mAb dosed at 30 mg/k, and MEDI5117 was dosed at 30 mg/kg. E, angiogenesis-related genes in treated and untreated tumors were analyzed by qRT-PCR. F, CD31 was detected by immunohistochemistry (left). Vessels were counted in three vascularized areas within the tumor (right). Mean vessel density and lumen area for CD31 was determined as the mean of the three counts. , P < 0.05.

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Table 1. MEDI5117 treatment conferred speciﬁc inhibition of IL6-related signaling pathways -log(P value) Ratio Genes that are downregulated with MEDI5117 treatment DU145 8.37Eþ00 4.49E02 SOCS3, HP, C3, C4B, CP, SERPINA1, FGB, FGA NCI-H1650 4.51Eþ00 1.24E01 IL6ST, MAP2K6, SOCS3, TCF4, ITIH3, MAP2K7, SERPING1, FN1, TNFRSF1A, RRAS, SERPINA3, PIK3R3, IKBKB, HMOX1, SHC1, JUN, CRABP2, AKT3, SERPINA1, OSMR, SERPINE1, RBP5 KPL-4 7.79Eþ00 5.06E02 SOCS3, SOD2, C4B, SERPINA3, OSMR, FGB, LBP, FGA, A2M

tumor growth. MEDI5117 treatment was well tolerated in mice as modulation of IL6–related pathway gene expression was observed no body weight loss was observed compared with control groups. in the nonresponsive models (NCI-H1975, U87MG; data not The antitumor activity of MEDI5117 was also found to be superior shown). to that of siltuximab in DU145 and MDAH2774 xenograft models We explored effects on gene expression further using a human (Supplementary Fig. S3). angiogenesis gene array. MEDI5117 treatment inhibited the Free human IL6 levels were measured in NCI-H1650 xenograft expression of several genes involved in angiogenesis such as KDR, tumors in both control IgG1 and MEDI5117-treated mice. PECAM-1, and VEGF as shown in Table 2. The downregulation of MEDI5117 treatment suppressed human IL6 levels in both serum KDR and PECAM-1 expression was confirmed by qRT-PCR and tumor lysates in vivo. In addition, MEDI5117 treatment analysis (Fig. 3E). We also found reductions in CD31 expression, resulted in more than 50% reduction of pSTAT3 levels in tumor blood microvessel density, and the size of vessel lumens in cell lysates as detected by ELISA (Fig. 3B). Immunohistochemistry NCI-H1650 tumors after MEDI5117 treatment (Fig. 3F). This confirmed this reduction, and also showed pSTAT3 reduction in demonstrates that IL6 is involved in angiogenesis in some tumor tumor cells and stromal cells within and surrounding the tumor types, and extends our previous findings studying ovarian cancer (Fig. 3C). Cell proliferation was also reduced, as assessed by cells (22). Thus, the antitumor effects of MEDI5117 involve immunohistochemistry with anti-Ki67 antibodies (Fig. 3D). inhibition of tumor cell growth and suppression of angiogenesis. It is important to note that MEDI5117 does not bind to systemic mouse IL6, which is known to regulate tumor microen- Neutralization of IL6 with MEDI5117 enhances antitumor vironment and contribute to xenograft tumor growth. This may activities of chemotherapeutic agents and gefitinib in vivo explain the partial tumor growth inhibition we observed , We tested the antitumor efficacy of MEDI5117 in combination and also suggests that our results underestimate the antitumor with several standard-of-care therapies in various mouse cancer fi effects that MEDI5117 would exert in cancer patients. To con rm models. All of the combination treatments we tested were well in vivo that MEDI5117 could inhibit systemic IL6 , a model was tolerated. The effect of combining MEDI5117 with taxol, a stan- generated in which human IL6 was administered by intraperito- dard-of-care for breast, ovarian, NSCLC, and prostate cancers neal injection into male C57/B/6/J mice. Systemic administration (23), was evaluated in the NCI-H1650 xenograft models. Treat- of IL6 is known to trigger an elevation of acute phase proteins such ment with either MEDI5117 or taxol alone inhibited tumor as haptoglobulin. MEDI5117 dose dependently inhibited the growth by 50% and 92% at the end of treatment, respectively. fi haptoglobulin levels induced by human IL6, with signi cant Combination treatment with MEDI5117 plus taxol resulted in inhibition being noted at doses of 78 mg/kg and higher. Siltux- imab had a similar effect at a dose of 266 mg/kg, much higher than Table 2. MEDI5117 treatment inhibited angiogenic gene expression that of MEDI5117 (Supplementary Fig. S4). Therefore, % Inhibition with MEDI5117 was more potent than siltuximab in inhibiting human in vivo Gene MEDI5117 treatment IL6 . CD44 41% MEDI5117 was also tested in the MDA-MB-468 model, which CDH5 31% does not secrete IL6, and the U87MG model, which produces IL6 COL15a1 48% but does not express surface IL6R. No TGI was observed in these FGF2 35% models (data not shown), suggesting an active IL6 autocrine loop IFNb 43% KDR is the key determinant of sensitivity to anti-IL6 cancer activity in 44% NRP2 25% xenograft models. TNF 32% VEGF 22% MEDI5117 inhibits expression of IL6 target genes and COL4a2 35% FBLN5 angiogenic genes 39% FLT3 29% To further understand the mechanism of action of MEDI5117, HEY1 34% fi whole genome mRNA pro ling was performed on untreated or IL12a 43% MEDI5117-treated tumor xenograft samples. Table 1 lists the ITGaVb3 38% genes whose expression was most downregulated with PDGFRb 42% MEDI5117 treatment in the three responsive models (DU145, PECAM1 56% PROX1 NCI-H1650, and KPL-4). All of these genes are known to be 47% SERPINC1 56% transcriptional regulation targets of the IL6/JAK/STAT3, AKT, and TEK 52% MAPK pathways. Among these, SOCS3 (suppression of cytokine TGFa 24% signaling 3) mRNA was found to be consistently downregulated TIMP2 32% by MEDI5117 in all three models. The expression of SOCS3 is NOTE: DU145 cells were treated with IgG1 control mAb or MEDI5117. mRNA was induced by cytokines such as IL6, IL10, and IFNg. In contrast, no isolated and was then subjected to a human angiogenesis array.

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tumor regression (DTGI ¼ 102%), with 2 of 10 mice experiencing alimta þ carboplatin, doxorubcin, and topotecan (Supplemen- complete tumor regression. Tumor regrowth was monitored tary Fig. S5). Combination treatments led to enhanced inhibition following the discontinuation of the treatments. Animals in the or delay in tumor growth compared with either agent alone. In taxol only treatment group showed rapid regrowth of their some cases (Supplementary Figs. S4B and S4D), the combination tumors. However, the regrowth of the tumors in mice treated efficacy was less pronounced compared with that seen in other with the combination of MEDI5117 and taxol was significantly experiments, and in these cases we observed lower induction of delayed. The two mice that had complete tumor regression IL6 secretion and signaling by the chemotherapy agent. This remained tumor free at the end of the regrowth phase (Fig. 4A, reinforces the hypothesis that patients could be selected for IL6 left). Free human IL6 and pSTAT3 levels were measured in NCI- combination therapy based on assessment of therapy-induced IL6 H1650 xenograft mice treated with MEDI5117, taxol, and a upregulation. combination of both (Fig. 4A, middle and right). Taxol treatment The effects of combining MEDI5117 and gefitinib were exam- resulted in upregulation of human IL6 and pSTAT3 compared ined in the NCI-H1650 xenograft model (Fig. 4C). Both with baseline levels, indicating activation of the IL6R inflamma- MEDI5117 alone and gefitinib alone inhibited tumor growth, tory pathway. In contrast, treatment with MEDI5117 suppressed although gefitinib was more effective during the treatment phase basal IL6 and pSTAT3 and counteracted taxol-stimulated pathway (53% DTGI for gefitinib vs. 39% DTGI for MEDI5117). The activation. combination of MEDI5117 and gefitinib enhanced antitumor The combination of MEDI5117 with taxotere was evaluated in activity (70% DTGI). In addition, the combination treatment led the KPL-4 breast cancer orthotopic model (Fig. 4B) and DU145 to the strongest posttreatment antitumor activity. prostate xenograft model (Supplementary Fig. S5A). MEDI5117 þ alone had a modest effect on KPL-4 tumor growth (DTGI ¼ 29%). MEDI5117 inhibits a trastuzumab-resistant HER2 breast Taxotere was highly efficacious causing regression during the tumor model dosing phase; however, tumors regrew once the treatment was We previously reported that activation of an IL6 inflammatory discontinued. Combining MEDI5117 with taxotere resulted in loop mediates trastuzumab resistance in HER2-overexpressing complete tumor regression, which lasted for more than two breast cancer cells (24, 25). BT474 cells, which contain amplified months (Fig. 4B). HER2, are sensitive to trastuzumab, but PTEN knockdown and MEDI5117 was also tested in combination with other non- long-term trastuzumab treatment–generated cells (called BT474- taxane chemotherapeutics including gemcitabine þ cisplatin, PTEN-LTT cells) that resisted killing by trastuzumab. Compared

Figure 4. In vivo efficacy of MEDI5117 in combination with taxanes or gefitinib. A, IL6 and pSTAT3 were analyzed in NCI-H1650 tumors after 21 days of treatment with MEDI5117 alone (30 mg/kg dosed twice per week for 3 weeks), taxol alone (10 mg/kg dosed daily for five doses), or the combination of MEDI5117 with taxol; N ¼ 10 animals/group. B, antitumor efficacy was assessed in the KPL-4 model using MEDI5117 with or without taxotere dosed at 10 mg/kg every four days for five doses. C, MEDI5117 plus gefitinib combination was studied in the NCI-H1650 model, using gefitinib alone (7.5 mg/kg dosed daily for 14 days), MEDI5117 alone (30 mg/kg dosed twice per week for four weeks) or the combination of gefitinib with MEDI5117. N ¼ 10 animals/group; CR, complete regression; PO, oral administration.

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with the parental BT474, BT474-PTEN-LTT cells displayed an compared with the parental BT474 formed spontaneous metas- aggressive metastatic phenotype. This was due to the induction tases primarily in the lungs, which were inhibited by MEDI5117 of an epithelial-to-mesenchymal transition (EMT) and CSC treatment (Fig. 5D). In addition, we utilized an NF-kB reporter- expansion mediated by autocrine IL6 secretion. As in our previous expressing BT474-PTEN-LTT cell line to monitor the IL6-mediated studies, trastuzumab significantly inhibited the growth of paren- NF-kB activity in vivo (24). MEDI5117 treatment significantly tal BT474 cells in vitro but failed to do so for BT474-PTEN-LTT cells reduced NF-kB activity in growing tumors compared with the (Fig. 5A). In contrast, MEDI5117 suppressed the growth of BT474- negative control (R347) or trastuzumab-treated animals (Fig. 5E). PTEN-LTT cells but did not affect the growth of BT474 cells, The fact that the NF-kB activity correlated with tumor growth suggesting that this inhibitory activity is specific to the IL6- suggests a critical role for IL6-mediated NF-kB signaling in tumor producing BT474-PTEN-LTT cells. growth. We reasoned that MEDI5117 might exert these effects on the BT474-PTEN-LTT cells by sequestering free IL6 and indirectly IL6 neutralization by MEDI5117 inhibits trastuzumab-resistant reducing the levels of related inflammatory cytokines. Elevated breast cancer stem cells levels of IL6, IL8, TGFb, sIL6R, and surface IL6R in BT474-PTEN- Compared with BT474 cells, trastuzumab-resistant BT474- LTT cells compared with the parental BT474 were demonstrated PTEN-LTT cells displayed an expanded CSC population charac- þ by ELISA (Supplementary Figs. S6A and S6B). These proteins were terized by a CD44 CD24 phenotype (24). To determine wheth- significantly reduced following a 48-hour MEDI5117 treatment in er MEDI5117 can target breast CSCs, we performed in vitro vitro (Fig. 5B), confirming suppression of the IL6–related inflam- tumorsphere assays. BT474-PTEN-LTT cells displayed consider- matory response. ably higher tumorsphere forming capacity compared with the Similar to results from our in vitro studies, parental BT474 parental BT474 (Fig. 6A). MEDI5117 treatment had no effect on tumors were very sensitive to trastuzumab treatment in mouse the parental BT474 tumorspheres but significantly reduced xenografts, while they failed to respond to the MEDI5117 treat- tumorsphere formation by BT474-PTEN-LTT cells, indicating ment (data not shown). In contrast, MEDI5117 exerted a strong the latter depends on IL6. We also characterized the proportion of þ inhibition of BT474-PTEN-LTT tumor growth (Fig. 5C), whereas CD44 /CD24 CSCs in BT474-PTEN-LTT xenografts with or this tumor was resistant to trastuzumab treatment in vivo. without MEDI5117 treatment by flow cytometry. Although þ Furthermore, we observed that BT474-PTEN-LTT tumors parental BT474 lacks CD44 CD24 cells, these CSCs represented

AB C BT474 200 IL6 IL8 TGFb BT474-PTEN-LTT xenogras BT474-shPTEN-LTT 10,000 30,000 800 150 Trastuzumab 8,000 600 * * 20,000 100 6,000 ** 400 pg/mL 4,000 ** 10,000 50 200 2,000 % of Surviving cells

0 0 0 0 R347 Trastuzumab MEDI5117 R347 MEDI5117 R347 MEDI5117 R347 MEDI5117 Weeks

DETumor Lung F NF-κB reporter acvity

40X BT474 Mouse body weight Trastuzumab 24

22 R347 Trastuzumab 20 BT474- MEDI5117 PTEN-LTT 18 Met weight (g) Body 16

56789 Weeks Weeks BT474- PTEN-LTT +MEDI5117

Figure 5. MEDI5117 shows speciﬁc therapeutic activity against a trastuzumab-resistant breast tumor model. A, sensitivity of BT474 cells and BT474-PTEN-LTT cells to trastuzumab and MEDI5117 analyzed in vitro. B, IL6, IL8, and TGFb levels measured by ELISA in BT474-PTEN-LTT cells treated with MEDI5117 in vitro.C,in vivo growth inhibition of BT474-PTEN-LTT tumors treated with trastuzumab and MEDI5117. D, H&E staining of primary tumors and lungs from animals bearing BT474 cells, BT474-PTEN-LTT cells, or BT474-PTEN-LTT cells treated with MEDI5117. E, an NF-kB reporter–expressing BT474-PTEN-LTT cell line was used to measure the activation of the NF-kB pathway by MEDI5117 or trastuzumab in vivo. F, body weights of BT474-PTEN-LTT cells treated with MEDI5117, trastuzumab, or an isotype control antibody, R347.

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ABBT474-shPTEN-LTT Figure 6. 400 ** ** MEDI5117 targets the breast cancer 100 stem cell population in vitro and in * CD44+/CD24– mouse xenografts. A, breast 300 BT474 80 CD44+/CD24+ tumorsphere formation of BT474 cells BT474-shPTEN-LTT BT474-PTEN-LTT cells treated with NS ** 60 MEDI5117 or an isotype control 200 antibody. B, residual tumors from

% of Cells 40 isotype or MEDI5117 treated animals were evaluated by ﬂow cytometry for 100 þ 20 the presence of CD44 CD24 breast

Number of spheres/10K cells CSCs. C, secondary tumor reinitiation 0 0 was assessed by removing BT474- R347 MEDI5117 R347 MEDI5117 PTEN-LTT cells removed from tumor- bearing mice that were treated for CDeight weeks with an isotype control antibody, trastuzumab, or MEDI5117 Reimplantaon assay at liming diluon Cancer stem cell frequency and reimplanted into new recipient Inoculated cell numbers Treatment CSC frequency (1/N) mice. The table lists number of mice in Treatment 500 5,000 which secondary tumors were formed Trastuzumab 8,080 out of the ﬁve mice in each treatment R347 0/5 2/5 MEDI5117 Inf. group. D, the frequency of breast CSC Trastuzumab 2/5 1/5 was calculated on the basis of the MEDI5117 0/5 0/5 R347 11,063 secondary tumor initiation capacity of residual tumors.

about 75% of the BT474-PTEN-LTT cell population (Fig. 6B). In prognosis in cancers of the lung, colon, kidney, bladder, and þ line with our in vitro tumorsphere data, the number of CD44 others (28–30). Thus, therapeutics targeting IL6 and IL6 signaling CD24 CSCs was substantially lower in BT474-PTEN-LTT xeno- components are also being tested in clinical trials for cancer. grafts when animals were treated with MEDI5117 compared with Siltuximab has been shown to have modest benefit in treating those treated with the negative control antibody, R347. It is cancers (13, 31, 32). Clazakizumab (ALD518) improves cancer- þ unclear whether, in addition to reducing the CD44 CD24 cell related fatigue and cachexia in NSCLC patients (33, 34). Elsili- þ fraction, MEDI5117 also drives differentiation to the CD44 momab (BE-8), a murine anti-IL6 mAb, could not efficiently þ CD24 phenotype. block IL6 levels due to its short half-life and neutralization by To further determine the effect of the MEDI5117 on CSCs, we a human anti-mouse immune response, and has generated mixed next performed reimplantation assays using cells taken from results in clinical trials (35). MEDI5117 is a fully human mAb BT474-PTEN-LTT tumors treated for eight weeks with the negative with no potential immunogenicity issues in human and has a control antibody, trastuzumab, or MEDI5117. Recipient mice higher binding affinity for human IL6 (Kd < 1 pmol/L) relative to were implanted with either 500 or 5,000 cells and monitored other anti-IL6 mAbs. Compared with siltuximab, MEDI5117 for 12 weeks. Under these conditions, both trastuzumab and showed enhanced in vitro cell killing (Fig. 2B) and pathway control-treated tumor cells were able to initiate secondary tumors, suppression (Fig. 2C) and inhibited systemic IL6 in vivo more although tumors did not grow in all recipient mice (Fig. 6C). In efficiently (Supplementary Fig. S3). In mouse xenograft models, contrast, MEDI5117-treated primary tumors failed to grow upon MEDI5117 treatment resulted in an improvement in tumor secondary reimplantation in any of the recipient mice, suggesting growth inhibition compared with siltuximab (Supplementary elimination of the CSC population. Data from this experiment Fig. S3). However, mouse models do not permit the significant was used to estimate the frequency of CSCs after each treatment as half-life extension afforded by our engineered YTE mutation in well (Fig. 6D). These results demonstrate that IL6 depletion by MEDI5117. The YTE mutation extends the half-life of MEDI5117 MEDI5117 diminishes the CSC population, which contributes to in humans almost five-fold compared with siltuximab. Thus, overall tumor growth suppression of our trastuzumab-resistant MEDI5117 is expected to have significantly better clinical activity model. and require less frequent dosing in patients compared with siltuximab and other non-YTE IL6-blocking antibodies. in vitro Discussion We found that MEDI5117 inhibited tumor cell growth and in vivo. The mechanism of this inhibition involved effects on IL6 is a pleiotropic cytokine that was initially characterized as a IL6-mediated growth signaling through the IL6R/JAK/STAT3 key component of inflammatory responses. Therapeutics target- pathway. In addition, repression of endothelial cell growth and ing the IL6 pathway are being developed for inflammatory and angiogenesis was observed after neutralization of IL6. Gilbert and autoimmune diseases. Tocilizumab, an anti-IL6 receptor anti- Hemann reported that in a mouse model of Burkitt lymphoma, body, was approved in 2009 for the treatment of rheumatoid doxorubicin treatment induced production of IL6 by endothelial arthritis (26, 27). Siltuximab, a chimeric mouse-human anti-IL6 cells in blood vessels, which promoted the survival of residual antibody, was approved to treat patients with Castleman disease tumor cells and eventual tumor relapse (36). That MEDI5117 also (FDA.gov press release for siltuximab approval, April 24, 2014). inhibits IL6 activity and growth of human endothelial (HUVEC) Numerous studies have revealed a role for IL6 in various types cells suggests that this activity would contribute to its antitumor of cancer as well. Elevated levels of IL6 are associated with poor effects in humans. Furthermore, extending previous results (37),

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we found that MEDI5117 treatment of several tumor types in vivo glioma stem cells express IL6R, and targeting IL6 decreases glioma could reduce tumor angiogenesis by downregulation of various stem cell survival and tumor growth (43). This stimulation of CSC genes including VEGF, KDR, and PECAM-1. Thus, the activity of growth has also been found to contribute to resistance to trastu- MEDI5117 in inhibiting tumor growth reflects the pleiotropic zumab. Yang, and colleagues reported that resistance of gastric roles of IL6 in cancer. cancer cells with CSC characteristics to trastuzumab is associated IL6 overproduction occurs in response to various cancer drug with activation of an IL6/STAT3/Jagged-1/Notch positive feed- treatments and in drug-resistant tumors, and thus the full benefits back loop (44). The results described in this report extend our of suppressing IL6 may be achieved in these situations. We found previous findings (24) by showing that neutralization of IL6 by that taxanes upregulated IL6 production in NSCLC and prostate MEDI5117 can inhibit CSCs and that this is may contribute to cancer cells, and that the combination of taxanes with MEDI5117 overcoming trastuzumab resistance in breast cancer. Together was significantly more efficacious than either agent alone. This with the enhanced efficacy we observed when paired with other efficacy was associated with suppression of activated STAT3, the therapeutics, these results encourage further study of MEDI5117 downstream effector of the IL6/JAK signaling axis. Similarly, in combination treatment scenarios and in drug-resistant cancers. MEDI5117 combined with taxotere resulted in durable tumor regression in a breast cancer model, whereas treatment with either Disclosure of Potential Conflicts of Interest agent alone did not. Activating mutations in the EGFR can J. Huang has ownership interest (including patents) and is an employee at promote IL6 production and activate JAK/STAT3 signaling in Medimmune/Aztrazeneca. R.E. Hollingsworth is a Senior Director at MedIm- human lung adenocarcinomas (38). Increased IL6 secretion and mune. No potential conflicts of interest were disclosed by the other authors. consequent autocrine survival signaling were also found to be a cause of drug resistance to the EGFR inhibitor, erlotinib, in NSCLC Authors' Contributions (12). Conversely, IL6 antibody treatment can upregulate EGFR Conception and design: H. Zhong, C. Chen, Y.S. Chang, J. Huang, Y. Yao, expression and limit ovarian tumor growth inhibition (39). This E. Hurt, D.A. Tice, Z. Xiao, M.S. Wicha, H. Korkaya, R.E. Hollingsworth suggests that a combined approach blocking both the EGFR and Development of methodology: H. Zhong, C. Chen, Y. Yao, J. Moisan, IL6 pathways may improve response and counteract resistance. H. Korkaya fi Acquisition of data (provided animals, acquired and managed patients, Our results showing that MEDI5117 in combination with ge ti- provided facilities, etc.): H. Zhong, A. Davis, M. Ouzounova, R.A. Carrasco, nib enhanced antitumor efficacy in the H1650 NSCLC model C. Chen, Y. Yao, J. Moisan, M. Fung, H. Korkaya supports this hypothesis. We also generated gefitinib-resistant Analysis and interpretation of data (e.g., statistical analysis, biostatistics, H1650 cells and found that MEDI5117 treatment restored sen- computational analysis): H. Zhong, A. Davis, M. Ouzounova, C. Chen, sitivity to this drug (data not shown). S. Breen, J. Huang, Z. Liu, Y. Yao, M. Fung, Z. Xiao, M.S. Wicha, H. Korkaya, Our previous work showed that activation of an IL6 feedback R.E. Hollingsworth Writing, review, and/or revision of the manuscript: H. Zhong, C. Chen, loop may play a role in trastuzmab resistance in PTEN-deleted þ J. Huang, S.G. Clouthier, Z. Xiao, M.S. Wicha, H. Korkaya, R.E. Hollingsworth HER2 tumors (24). We therefore tested the activity of MEDI5117 Administrative, technical, or material support (i.e., reporting or organizing in our trastuzumab-resistant breast cancer model, which produces data, constructing databases): H. Zhong, S. Breen, J. Huang, S.G. Clouthier, high levels of IL6. Neutralization of IL6 by MEDI5117 potently R.E. Hollingsworth inhibited the growth of this trastuzumab resistant tumor. In con- Study supervision: H. Zhong, C. Chen, Y.S. Chang, E. Hurt, D.A. Tice, trast, the antibody had no activity against matched EGFR-sensitive S.G. Clouthier, Z. Xiao, H. Korkaya, R.E. Hollingsworth (PTEN wild-type) tumor cells that do not overproduce IL6. Growth inhibition of the trastuzumab-resistant tumor model by Acknowledgments MEDI5117 was associated with suppression of downstream NF- The authors thank Dr. Mohammed Dar and Dr. Ronald Herbst for critical review of the article. kB signaling and lung metastasis. Iliopoulos and colleagues demonstrated that IL6 activation of NF-kB in turn drives further IL6 production, creating an inflammatory positive feedback loop (40). Grant Support Thus, interrupting this IL6/NF-kB activation loop may be partic- This work was supported by Medimmune. The costs of publication of this article were defrayed in part by the payment of ularly important in some drug-resistant cancers that depend on IL6. page charges. This article must therefore be hereby marked advertisement in IL6 is known to regulate the growth of some cancer stem cells, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. and this may contribute to treatment resistance and relapse (41). For example, IL6 stimulates the tumorigenic capacity of CSCs Received April 6, 2015; revised September 28, 2015; accepted October 2, isolated from head and neck squamous tumors (42). Similarly, 2015; published OnlineFirst January 7, 2016.

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A Novel IL6 Antibody Sensitizes Multiple Tumor Types to Chemotherapy Including Trastuzumab-Resistant Tumors

Haihong Zhong, April Davis, Maria Ouzounova, et al.

Cancer Res 2016;76:480-490. Published OnlineFirst January 7, 2016.

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