A Novel IL6 Antibody Sensitizes Multiple Tumor Types to ...Tumor and Stem Cell Biology A Novel IL6...

Tumor and Stem Cell Biology

A Novel IL6 Antibody Sensitizes Multiple TumorTypes to Chemotherapy Including Trastuzumab-Resistant TumorsHaihong 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 areassociated with poor survival outcomes in many cancers.Antibodies targeting IL6 and its receptor have been developedfor chronic inflammatory disease, but they have not yet beenshown to clearly benefit cancer patients, possibly due toantibody potency or the settings in which they have beentested. In this study, we describe the development of a novelhigh-affinity anti-IL6 antibody, MEDI5117, which features anextended half-life and potent inhibitory effects on IL6 biologicactivity. MEDI5117 inhibited IL6-mediated activation ofSTAT3, suppressing the growth of several tumor types drivenby IL6 autocrine signaling. In the same models, MEDI5117displayed superior preclinical activity relative to a previouslydeveloped anti-IL6 antibody. Consistent with roles for IL6 in

promoting tumor angiogenesis, we found that MEDI5117inhibited the growth of endothelial cells, which can produceIL6 and support tumorigenesis. Notably, in tumor xenograftassays in mice, we documented the ability of MEDI5117 toenhance the antitumor activities of chemotherapy or gefitinibin combination treatment regimens. MEDI5117 also displayedrobust activity on its own against trastuzumab-resistant HER2þ

tumor cells by targeting the CD44þCD24� cancer stem cellpopulation. Collectively, our findings extend the evidence ofimportant pleiotropic roles of IL6 in tumorigenesis and drugresistance, and offer a preclinical proof of concept for theuse of IL6 antibodies in combination regimens to heightentherapeutic responses and overcome drug resistance. Cancer Res;76(2); 480–90. �2016 AACR.

IntroductionIL6 is produced by lymphoid and nonlymphoid cells including

T and B cells, monocytes, fibroblasts, keratinocytes, as well asendothelial,mesangial, and tumor cells (1, 2). Its pleiotropic rolesinclude the activation of T cells, induction of the acute phaseinflammatory response, differentiation, and survival of plasma Bcells, synovial fibroblasts, and osteoclasts, and stimulation ofgrowth and differentiation of hematopoietic precursor cells (3).IL6 acts on target cells by binding to the IL6 receptor (IL6R),whichconsists of two membrane-bound proteins, an 80 kDa ligand-

binding protein (IL6Ra chain, CD126) and a 130 kDa signaltransduction glycoprotein (gp130). IL6 binds to the transmem-brane IL6Ra protein and the IL6/IL6Ra complex binds to twomolecules of gp130, which then activates an intracellular signal-ing cascade. This mechanism is known as classical signaling.IL6Ra also exists as a soluble receptor (sIL6Ra), which cancombine with IL6 to trigger membrane-bound gp130 dimeriza-tion and thusmediate the so-called trans-signaling (3, 4). Becauseof the ubiquitous expression of gp130 on different cell types, thetrans-signaling mechanism is attributed to the broad array of IL6functions in the body. Activated gp130 stimulates JAK tyrosinekinases, which phosphorylate and recruit the transcription factorSTAT3 to regulate gene expression (5).

Many cancers exploit IL6 as a growth factor as well as a modifierof the tumor microenvironment. Solid tumors such as lung,ovarian, breast, and colon carcinomas produce IL6, IL6Ra andgp130, allowing them to constitutively stimulate their own growthin autocrine manner (6–8). Other cancers, such as multiplemyeloma and neuroblastoma, do not produce IL6, but do expressIL6Ra and gp130; these tumors respond to IL6 produced in thetumor microenvironment in a paracrine manner (9). IL6 playsmultiple roles in tumor progression and drug resistance (10–12). Itcan act directly on tumor cells, or function by interacting withnormal cells in the tumor microenvironment, including endothe-lial, immune, and inflammatory cells. Targeting IL6 and its path-way therefore is expected to inhibit tumors through multiplemechanisms. Several anti-IL6 antibodies are in clinical develop-ment for cancer therapy. Among these, siltuximab (CNTO328) is

1Oncology Research, MedImmune, Gaithersburg, Maryland. 2Transla-tional Science, MedImmune, Gaithersburg, Maryland. 3Aileron Thera-peutics, Inc., Cambridge, Massachusetts. 4University of MichiganComprehensive Cancer Center, Ann Arbor, Michigan. 5Department ofBiochemistry and Molecular Biology, Georgia Regents University,Augusta, Georgia.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

H. Zhong and A. Davis contributed equally as first authors.

Corresponding Author: Robert E. Hollingsworth, One MedImmune Way,Gaithersburg, MD 20878. Phone: 301-398-5354; Fax: 301-398-8384; E-mail:[email protected] and Hasan Korkaya, Georgia Regents Uni-versity Cancer Center, 1410 Laney Walker Blvd., Augusta, GA 30912; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-15-0883

�2016 American Association for Cancer Research.

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the most advanced and is being tested in the clinic for multipletypes of cancer. Published data suggest that anti-IL6 antibodytherapies have acceptable safety and tolerability (13–15).

In this study, we further elucidate the critical role of IL6 incancer biology inmultiple solid tumor types andprovide evidencethat IL6 is an anticancer target in mono- and especially combi-nation therapy settings. MEDI5117 is a human monoclonalantibody (mAb) that potently binds and neutralizes human IL6.It was engineered to have increased persistence in circulationcompared with unmodified antibodies through incorporation ofYTEmutations in the Fc region (16, 17). MEDI5117 inhibited IL6signaling and suppressed the growth of lung, breast, and ovariantumor cells in vitro and in vivo with higher potency than siltux-imab. Inhibition of tumor growth was significantly increasedwhenMEDI5117was used in combinationwith gefitinib, taxanes,or other chemotherapeutics. Furthermore,MEDI5117 suppressedthe growth of IL6-dependent trastuzumab-resistant breasttumors, and this was associated with a reduction of cancer stemcells (CSC). These results extend our understanding of the role ofIL6 in tumor biology anddemonstrate the potential ofMEDI5117for the treatment of multiple cancers.

Materials and MethodsCells and reagents

Cancer cell lines were obtained from the ATCC. Cell lineauthentication was conducted by STR-based DNA fingerprintingand multiplex PCR. IMPACT tests were also performed on all celllines.

CellTiterGlo reagents were obtained fromPromega. An enzymelinked immunosorbent assay (ELISA) was developed in ourlaboratories to detect free IL6 in tumor lysates. Human IL6 andsIL6R ELISA kits were purchased from R&D Systems. Recombi-nant human IL6 and sIL6R proteins were obtained from R&DSystems. Antibodies to detect STAT3 and phospho-STAT3-Tyr705were obtained from Cell Signaling Technology and gp130 anti-body was obtained from Santa Cruz Biotechnology. MCF-7/IL6–overexpressing cell line was kindly provided by Dr. MercedesRincone (University of Vermont, Burlington, VT; ref. 18). Siltux-imab (CNTO328) was generated inhouse based on publishedsequence information.

siRNA KnockdownA reverse transfectionprotocolwas used to analyze the effects of

IL6 and IL6R knockdown. Briefly, siRNA pools targeting IL6 andIL6R (Dharmacon) were prepared at a final concentration of 50nmol/L inoptiMEM(Invitrogen).DU145 cellswere then added tothe siRNAs at a density of 5,000 cells per well in 96-well plates inmedium containing 10% heat-inactivated FBS (Life Technolo-gies). After 48 hours, transfected cells were washed with ice-coldPBS and then lysed by adding Laemmli reducing buffer (BostonBioProducts). Cell lysates were then subjected to Western blotanalysis for phosphorylated STAT3 (pSTAT3). Conditionedmedi-um was analyzed in duplicate with human IL6 and soluble IL6RELISA kits (R&D Systems). For proliferation assays, CellTiterGlo reagentwas usedfivedays after transfection and luminescencewas measured using an EnVision 2104 Multilabel Reader(PerkinElmer).

Cell proliferation analysisFor DU145 cells, cell proliferation was assessed by three-

dimensional tumor spheroid assay. Cells were plated at a density

of 8,000 cells per well. Plates were spun down and incubatedovernight to allow cells to assemle into compact spheroids. Two-day-old spheroids were treated with human recombinant IL6 orsIL6R proteins (R&D Systems) with MEDI5117 at varying con-centrations. After 7-day incubation, proliferation was quantifiedusing the CellTiter-Glo assay using an EnVision 2104 MultilabelReader (PerkinElmer).

For human umbilical vein endothelial cells (HUVEC), cellswere cultured as monolayers. Recombinant proteins and anti-body were added on next day. After 72 hour incubation, prolif-eration was quantified using the CellTiter-Glo assay.

mRNA profilingTotal RNA was extracted from snap-frozen xenograft tumor

samples using the ZR RNA MicroPrep Kit (Zymo Research).Xenograft tumors were treated with 30 mg/kg of MEDI5117 orIgG isotype control IgG1 for a total of two doses. Generatingbiotin-labeled amplified cRNA was accomplished using the Mes-sageAmpTM Premier RNA Amplification Kit (Ambion) and usedfor gene expression with Affymetrix Human Genome U133 Plus2.0 GeneChip microarrays. Canonical pathway enrichment anal-yses were conducted using Ingenuity Pathway Analysis. All micro-array data are deposited in GEO repository under accessionnumber is GSE62941: (http://www.ncbi.nlm.nih.gov/geo/que-ry/acc.cgi?token¼ujajyuyovbqzdyf&acc¼GSE62941).

Mouse xenograft studiesAll animal procedures were conducted in accordance with all

appropriate regulatory standards under protocols approved bythe Medimmune Institutional Animal Care and Use Committee.

For the MCF-7 xenograft study, 0.36 mg of an estrogen pelletwas implanted subcutaneously into the left flank of each femaleathymic nudemouse. The effect of the estrogen pellet lasted for 60days in vivo. Two million MCF7 or MCF7/IL6 tumor cells weresuspended in 100 mL of BME (6mg/mL) before injection. Allmicereceived an orthotopic inoculation of tumor cells into the mam-mary fat pad.

For in vivo efficacy studies, 5 million NCI-H1650, KPL-4,DU145, or MDAH2774 cells in 50% Matrigel were inoculatedsubcutaneously into each female athymic nude mouse. Whentumors reached approximately 150 to 200 mm3, mice wererandomly assigned into groups (10 mice per group). MEDI5117or siltuximab was administered intraperitoneally (i.p.) twice perweek at indicated doses. Tumor volumes were measured twiceweekly with calipers. Tumor growth inhibition was calculated onthe last day of study relative to the initial and final mean tumorvolume of the control group.

For in vivo mechanism of action and pharmacodynamicstudies, a single dose of MEDI5117 was administered when tu-mors reached approximately 400 mm3. Tumor and serum sam-ples were collected 4 hours after dosing.

CSC analysesFor tumorsphere assays, single cells were plated on ultra-low

attachment plates at a density of 1�105/mL and grown for 7 daysin a mammocult medium (Stem Cell Technologies). After thetreatment of primary spheres with drugs, they were dissociatedinto single-cell suspension and plated at a density of 5�103–1�104/mL. Secondary spheres were counted after 5 to 7 days inculture and evaluated the efficacy of the drug treatment.

IL6 Blockade in Combination and Drug-Resistant Settings

www.aacrjournals.org Cancer Res; 76(2) January 15, 2016 481




For CD44þCD24� CSC flow cytometry analyses, single-cellsuspensions of residual tumors cells isolated from drug-treatedmicewere incubatedwithfluorophore-conjugatedCD44orCD24antibodies alone or in combination on ice for 30minutes, washedwith Hank's Balanced Salt Solution (HBSS), and resuspended inDAPI containing HBSS buffer for flow cytometry analyses. Gatecompensation was performed with single color–stained cells andthe CD44þCD24� phenotype analyses were performed using thedouble stained cells.

Tumor reimplantation studies were conducted using cellsrecovered from the residual tumors of treated animals. Tumorswere excised, chopped, and processed with collagenase for 1 to 2hours at 37�C. Cells were then washed with PBS, trypsinized, andpassed through a 40 mm filter. The single cells were labeled withH-2Kd antibody to distinguish human cells frommouse cells andDAPI and then sorted with flow cytometry. Live human tumorcells were reimplanted orthotopically into mouse mammary fatpads to determine tumor initiation capacity. Tumors were mea-sured weekly using luciferase bioluminescense and by caliper.CSC frequencies were calculated by limiting dilution analysesusing the Walter and Eliza Hall Institute formula (http://bioinf.wehi.edu.au/software/elda/; ref. 19).

ResultsIL6 is a potent growth factor and its downstream signaling isactivated in multiple cancer types

IL6 is known to be a potent growth factor in some cancer celllines, and we observed this in the models we studied as well. Forexample, siRNA knockdown of either IL6 or IL6 receptor stronglyinhibited the proliferation of DU145 prostate cancer cells, as wellas their levels of activated STAT3, the downstream effector of theIL6 signaling pathway (Supplementary Fig. S1A). Similarly, over-expression of IL6 increased the growth rate of MCF-7 cancer cellsin vitro and in vivo (Supplementary Fig. S1B).

Although activation of IL6 signaling can occur through para-crine mechanisms, we found evidence of autocrine IL6 signalingin several cancer cell lines (Fig. 1 and Supplementary Fig. S2).MDAH2774 (ovarian cancer), NCI-H1650 and NCI-H1975(non–small cell lung cancer, NSCLC), DU145 (prostate cancer),and Detroit 562 (head and neck cancer) cells produced IL6 and

sIL6R. Membrane-bound IL6R and gp130 and intracellularpSTAT3 were detected in these cell lines as well. These compo-nents allow these cell lines to stimulate their own growth throughan IL6-dependent autocrine loop. In contrast, MDA-MB-468 andMCF-7 (breast cancer) cells do not secrete IL6 and MDA-MB-231(breast cancer) andU87MG (glioblastoma) cells secrete relativelyhigh levels of IL6, but lack IL6R on the surface.

An anti-IL6 antibody, MEDI5117, inhibits cancer cell andendothelial cell proliferation

MEDI5117 is a human, high affinity, IgG1 monoclonal anti-body (mAb) that neutralizes human IL6. The parental antibody(CAT-6001) was obtained by screening phage display humanantibody libraries for binding to recombinant human IL6 and forinhibition of the IL6/IL6R interaction (14). We then createdMEDI5117 by incorporating three amino acid substitutions,M252Y, S254T, and T256E (YTE), into the Fc region of CAT-6001 to increase the antibody's half life in vivo. Pharmacokineticstudies in cynomolgus monkeys showed that the half-life ofMEDI5117 was extended by 3-fold (28.4 vs. 8.4 days) andclearance was reduced by 4-fold (3.02 vs. 12.1 mL/kg/day) whencompared with CAT-6001 (16, 17). The half-life of MEDI5117 inhumans was 84 days, significantly longer than other anti-IL6antibodies, such as siltuximab whose half-life was 17.8 days.MEDI5117 binds IL6 with high affinity. In the KinExa assay,MEDI5117 had a Kd < 1 pmol/L, while siltuximab had a Kd ¼6.25 pmol/L. By ELISA, MEDI5117 had an EC50¼ 6.5 pmol/L forhuman IL6 binding as measured, more potent than that ofsiltuximab (EC50 ¼ 14.1 pmol/L). MEDI5117 cross reacts withcynomolgus monkey IL6, but not with IL6 from other species,including dogs, rats, and mice.

The effect of MEDI5117 on the proliferation of cancer cells wasinvestigated in DU145 prostate cancer cells using a spheroidformation assay. Stimulating DU145 cells with recombinanthuman IL6 resulted in increased cell proliferation as measuredby the number of spheroids grown in serum-free medium forseven days. The addition of sIL6Ra alone had little effect onDU145 cell proliferation. Addition of IL6 in combination withsIL6Ra stimulated cell growth to the same extent as IL6 treatmentalone. This finding indicates that the proliferative effect of IL6 on

Figure 1.The IL6 pathway is hyperactivated inseveral cancer cell lines. Conditionedmedium from various cancer cell lineswas collected and analyzed induplicate. Human IL6 and soluble IL6Rwere measured by ELISA, membraneIL6R expression was analyzed by flowcytometry, and protein levels ofgp130, pSTAT3, STAT3, and GAPDHwere assessed by Western blotting.

Zhong et al.

Cancer Res; 76(2) January 15, 2016 Cancer Research482




DU145 spheroids occurs through classical signaling involvingthe membrane-bound receptor. Treatment with MEDI5117completely blocked IL6-induced spheroid growth (Fig. 2A, left).Proliferationwas not blocked by treatmentwith a control isotype-matched IgG1 mAb.

HUVEC cells do not expressmembrane bound IL6Ra (20), andtherefore IL6 stimulation of HUVECs relies on trans-signaling(21). Adding recombinant human IL6 or sIL6R alone had onlymodest effects on HUVEC proliferation. However, adding IL6with sIL6R together resulted in significantly increased HUVEC

proliferation (Fig. 2A, right). Treatment with MEDI5117 blockedthe HUVEC proliferation induced by IL6 and sIL6R. Thus,MEDI5117 can inhibit proliferation of cancer cells by blockingclassical IL6 signaling and proliferation of endothelial cells byblocking IL6 trans-signaling in vitro.

The inhibitory activities of MEDI5117 and siltuximab werecompared in both DU145 cells and TF-1 (erythroleukemic) cells.Treatment with MEDI5117 or siltuximab blocked IL6-induced cellproliferation in a dose-dependent manner (Fig. 2B); however,MEDI5117wasmore potent than siltuximab based on IC50 values.

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Figure 2.MEDI5117 inhibited cell proliferation andblocked STAT3 activation in cancer cell lines and endothelial cells. A, DU145 spheroidswere grown in serum-freemediumandstimulated with recombinant human IL6 and soluble IL6R, or IL6 þ sIL6R at the indicated concentrations in the presence of 20 mg/mL control IgG antibodyor MEDI5117 (left). Treatments were performed in triplicate and mean spheroid numbers and standard deviations were calculated. A, DU145 cells and HUVECswere incubated with recombinant human IL6 and sIL6R along with 20 mg/mL control antibody or MEDI5117 (right). Proliferation was measured using the CellTiterGlo 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 thepresence ofMEDI5117 or siltuximab (CNTO328). C andD,MEDI5117, siltuximab, and a control IgGwere added directly to the cells for a final concentration of 30 mg/mLin the presence of 10 ng/mL of IL6, sIL6R, or IL6/sIL6R. After 24 hours, cells were lysed and subjected to Western blot analysis.






IL6 signaling involves activation of the transcription factor,STAT3. To analyze effects on STAT3, several different cancer celllines and HUVECs were treated with MEDI5117, siltuximab, or acontrol IgG1 mAb (Fig. 2C, left). Basal levels of pSTAT3 (it'sactivated form) were suppressed by MEDI5117 in each of the celllines; treatment with siltuximab resulted in less efficient suppres-sion. Addition of recombinant human IL6 activated STAT3 inboth the MDAH2774 and DU145 cancer cell lines, and pretreat-ment with MEDI5117 blocked this activation (Fig. 2C, right). InDU145 cells, the level of pSTAT3 in MEDI5117-treated cells waseven lower than the basal level of pSTAT3 in untreated cells.MEDI5117 treatment reduced basal pSTAT3 levels, and reversedIL6 or IL6/sIL6R induced STAT3 activation in HUVECs (Fig. 2D).

These results corroborate that MEDI5117 can inhibit both clas-sical and trans-signaling stimulated by IL6.

Neutralization of IL6 by MEDI5117 inhibits tumor growth inxenograft models

MEDI5117 was evaluated in NSCLC (NCI-H1650), ovarian(MDAH-2774), and prostate (DU145) xenograft models in vivo(Fig. 3A and Supplementary Fig. S3). These tumor models pro-duce IL6 and bear cell surface IL6R. MEDI5117 at 30mg/kg alonecaused tumor growth inhibition (DTGI) of 51% (P ¼ 0.004) inNCI-H1650 and 59% DTGI (P ¼ 0.007) in MDAH2774. Dosesbetween 3 and 30 mg/kg showed comparable DTGI with the 30mg/kg dose. A low dose of 1 mg/kg was insufficient to inhibit

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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 administeredvia 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 extractsand 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 hoursafter the last dose of MEDI5117 by staining with an anti-pSTAT3 antibody. Quantification of pSTAT3-expressing cells in tumor and stroma components was doneon four randomly chosen fields. � , P < 0.05. D, Ki67 staining was performed to measure the proliferation of xenograft tumors cells. The percentage of Ki67-positivenuclei 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 werecounted in three vascularized areaswithin the tumor (right). Meanvessel density and lumen area for CD31was determined as themean of the three counts. � ,P<0.05.

Zhong et al.





tumor growth.MEDI5117 treatment was well tolerated inmice asno body weight loss was observed compared with control groups.The antitumor activity ofMEDI5117was also found tobe superiorto that of siltuximab inDU145 andMDAH2774 xenograftmodels(Supplementary Fig. S3).

Free human IL6 levels were measured in NCI-H1650 xenografttumors in both control IgG1 and MEDI5117-treated mice.MEDI5117 treatment suppressed human IL6 levels in both serumand tumor lysates in vivo. In addition, MEDI5117 treatmentresulted in more than 50% reduction of pSTAT3 levels in tumorcell lysates as detected by ELISA (Fig. 3B). Immunohistochemistryconfirmed this reduction, and also showed pSTAT3 reduction intumor cells and stromal cells within and surrounding the tumor(Fig. 3C). Cell proliferation was also reduced, as assessed byimmunohistochemistry with anti-Ki67 antibodies (Fig. 3D).

It is important to note that MEDI5117 does not bind tosystemic mouse IL6, which is known to regulate tumor microen-vironment and contribute to xenograft tumor growth. This mayexplain the partial tumor growth inhibition we observed in vivo,and also suggests that our results underestimate the antitumoreffects that MEDI5117 would exert in cancer patients. To confirmthat MEDI5117 could inhibit systemic IL6 in vivo, a model wasgenerated in which human IL6 was administered by intraperito-neal injection intomale C57/B/6/J mice. Systemic administrationof IL6 is known to trigger an elevation of acute phase proteins suchas haptoglobulin. MEDI5117 dose dependently inhibited thehaptoglobulin levels induced by human IL6, with significantinhibition 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 thanthat of MEDI5117 (Supplementary Fig. S4). Therefore,MEDI5117wasmore potent than siltuximab in inhibiting humanIL6 in vivo.

MEDI5117 was also tested in the MDA-MB-468 model, whichdoes not secrete IL6, and the U87MGmodel, which produces IL6but does not express surface IL6R. No TGI was observed in thesemodels (data not shown), suggesting an active IL6 autocrine loopis the key determinant of sensitivity to anti-IL6 cancer activity inxenograft models.

MEDI5117 inhibits expression of IL6 target genes andangiogenic genes

To further understand the mechanism of action of MEDI5117,whole genome mRNA profiling was performed on untreated orMEDI5117-treated tumor xenograft samples. Table 1 lists thegenes whose expression was most downregulated withMEDI5117 treatment in the three responsive models (DU145,NCI-H1650, and KPL-4). All of these genes are known to betranscriptional regulation targets of the IL6/JAK/STAT3, AKT, andMAPK pathways. Among these, SOCS3 (suppression of cytokinesignaling 3) mRNA was found to be consistently downregulatedby MEDI5117 in all three models. The expression of SOCS3 isinduced by cytokines such as IL6, IL10, and IFNg . In contrast, no

modulationof IL6–related pathway gene expressionwas observedin the nonresponsive models (NCI-H1975, U87MG; data notshown).

We explored effects on gene expression further using a humanangiogenesis gene array. MEDI5117 treatment inhibited theexpression of several genes involved in angiogenesis such as KDR,PECAM-1, and VEGF as shown in Table 2. The downregulation ofKDR and PECAM-1 expression was confirmed by qRT-PCRanalysis (Fig. 3E). We also found reductions in CD31 expression,blood microvessel density, and the size of vessel lumens inNCI-H1650 tumors after MEDI5117 treatment (Fig. 3F). Thisdemonstrates that IL6 is involved in angiogenesis in some tumortypes, and extends our previous findings studying ovarian cancercells (22). Thus, the antitumor effects of MEDI5117 involveinhibition of tumor cell growth and suppression of angiogenesis.

Neutralization of IL6 with MEDI5117 enhances antitumoractivities of chemotherapeutic agents and gefitinib

We tested the antitumor efficacy of MEDI5117 in combinationwith several standard-of-care therapies in various mouse cancermodels. All of the combination treatments we tested were welltolerated. The effect of combining MEDI5117 with taxol, a stan-dard-of-care for breast, ovarian, NSCLC, and prostate cancers(23), was evaluated in the NCI-H1650 xenograft models. Treat-ment with either MEDI5117 or taxol alone inhibited tumorgrowth by 50% and 92% at the end of treatment, respectively.Combination treatment with MEDI5117 plus taxol resulted in

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 4.49E�02 SOCS3, HP, C3, C4B, CP, SERPINA1, FGB, FGANCI-H1650 4.51Eþ00 1.24E�01 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 5.06E�02 SOCS3, SOD2, C4B, SERPINA3, OSMR, FGB, LBP, FGA, A2M

Table 2. MEDI5117 treatment inhibited angiogenic gene expression

Gene% Inhibition withMEDI5117 treatment

CD44 41%CDH5 31%COL15a1 48%FGF2 35%IFNb 43%KDR 44%NRP2 25%TNF 32%VEGF 22%COL4a2 35%FBLN5 39%FLT3 29%HEY1 34%IL12a 43%ITGaVb3 38%PDGFRb 42%PECAM1 56%PROX1 47%SERPINC1 56%TEK 52%TGFa 24%TIMP2 32%

NOTE: DU145 cells were treated with IgG1 control mAb or MEDI5117. mRNA wasisolated and was then subjected to a human angiogenesis array.






tumor regression (DTGI¼ 102%), with 2 of 10mice experiencingcomplete tumor regression. Tumor regrowth was monitoredfollowing the discontinuation of the treatments. Animals in thetaxol only treatment group showed rapid regrowth of theirtumors. However, the regrowth of the tumors in mice treatedwith the combination of MEDI5117 and taxol was significantlydelayed. The two mice that had complete tumor regressionremained tumor free at the end of the regrowth phase (Fig. 4A,left). Free human IL6 and pSTAT3 levels were measured in NCI-H1650 xenograft mice treated with MEDI5117, taxol, and acombination of both (Fig. 4A, middle and right). Taxol treatmentresulted in upregulation of human IL6 and pSTAT3 comparedwith baseline levels, indicating activation of the IL6R inflamma-tory pathway. In contrast, treatment with MEDI5117 suppressedbasal IL6 and pSTAT3 and counteracted taxol-stimulated pathwayactivation.

The combination of MEDI5117 with taxotere was evaluated inthe KPL-4 breast cancer orthotopic model (Fig. 4B) and DU145prostate xenograft model (Supplementary Fig. S5A). MEDI5117alone had amodest effect on KPL-4 tumor growth (DTGI¼ 29%).Taxotere was highly efficacious causing regression during thedosing phase; however, tumors regrew once the treatment wasdiscontinued. Combining MEDI5117 with taxotere resulted incomplete tumor regression, which lasted for more than twomonths (Fig. 4B).

MEDI5117 was also tested in combination with other non-taxane chemotherapeutics including gemcitabine þ cisplatin,

alimta þ carboplatin, doxorubcin, and topotecan (Supplemen-tary Fig. S5). Combination treatments led to enhanced inhibitionor delay in tumor growth compared with either agent alone. Insome cases (Supplementary Figs. S4B and S4D), the combinationefficacy was less pronounced compared with that seen in otherexperiments, and in these cases we observed lower induction ofIL6 secretion and signaling by the chemotherapy agent. Thisreinforces the hypothesis that patients could be selected for IL6combination therapy basedon assessment of therapy-induced IL6upregulation.

The effects of combining MEDI5117 and gefitinib were exam-ined in the NCI-H1650 xenograft model (Fig. 4C). BothMEDI5117 alone and gefitinib alone inhibited tumor growth,although gefitinib was more effective during the treatment phase(53% DTGI for gefitinib vs. 39% DTGI for MEDI5117). Thecombination of MEDI5117 and gefitinib enhanced antitumoractivity (70% DTGI). In addition, the combination treatment ledto the strongest posttreatment antitumor activity.

MEDI5117 inhibits a trastuzumab-resistant HER2þ breasttumor model

We previously reported that activation of an IL6 inflammatoryloop mediates trastuzumab resistance in HER2-overexpressingbreast cancer cells (24, 25). BT474 cells, which contain amplifiedHER2, are sensitive to trastuzumab, but PTEN knockdown andlong-term trastuzumab treatment–generated cells (called BT474-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 withMEDI5117 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 daysfor 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), MEDI5117alone (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.

Zhong et al.





with the parental BT474, BT474-PTEN-LTT cells displayed anaggressive metastatic phenotype. This was due to the inductionof an epithelial-to-mesenchymal transition (EMT) and CSCexpansionmediated by autocrine IL6 secretion. As in our previousstudies, trastuzumab significantly inhibited the growth of paren-tal BT474 cells in vitrobut failed to do so for BT474-PTEN-LTT cells(Fig. 5A). In contrast,MEDI5117 suppressed the growthof BT474-PTEN-LTT cells but did not affect the growth of BT474 cells,suggesting that this inhibitory activity is specific to the IL6-producing BT474-PTEN-LTT cells.

We reasoned that MEDI5117 might exert these effects on theBT474-PTEN-LTT cells by sequestering free IL6 and indirectlyreducing the levels of related inflammatory cytokines. Elevatedlevels of IL6, IL8, TGFb, sIL6R, and surface IL6R in BT474-PTEN-LTT cells compared with the parental BT474 were demonstratedby ELISA (Supplementary Figs. S6A and S6B). These proteins weresignificantly reduced following a 48-hourMEDI5117 treatment invitro (Fig. 5B), confirming suppression of the IL6–related inflam-matory response.

Similar to results from our in vitro studies, parental BT474tumors were very sensitive to trastuzumab treatment in mousexenografts, while they failed to respond to the MEDI5117 treat-ment (data not shown). In contrast, MEDI5117 exerted a stronginhibition of BT474-PTEN-LTT tumor growth (Fig. 5C), whereasthis tumor was resistant to trastuzumab treatment in vivo.Furthermore, we observed that BT474-PTEN-LTT tumors

compared with the parental BT474 formed spontaneous metas-tases primarily in the lungs, which were inhibited by MEDI5117treatment (Fig. 5D). In addition, we utilized an NF-kB reporter-expressing BT474-PTEN-LTT cell line tomonitor the IL6-mediatedNF-kB activity in vivo (24). MEDI5117 treatment significantlyreduced NF-kB activity in growing tumors compared with thenegative control (R347) or trastuzumab-treated animals (Fig. 5E).The fact that the NF-kB activity correlated with tumor growthsuggests a critical role for IL6-mediated NF-kB signaling in tumorgrowth.

IL6 neutralizationbyMEDI5117 inhibits trastuzumab-resistantbreast cancer stem cells

Compared with BT474 cells, trastuzumab-resistant BT474-PTEN-LTT cells displayed an expanded CSC population charac-terized by a CD44þCD24� phenotype (24). To determine wheth-er MEDI5117 can target breast CSCs, we performed in vitrotumorsphere assays. BT474-PTEN-LTT cells displayed consider-ably higher tumorsphere forming capacity compared with theparental BT474 (Fig. 6A). MEDI5117 treatment had no effect onthe parental BT474 tumorspheres but significantly reducedtumorsphere formation by BT474-PTEN-LTT cells, indicatingthe latter depends on IL6.We also characterized the proportion ofCD44þ/CD24� CSCs in BT474-PTEN-LTT xenografts with orwithout MEDI5117 treatment by flow cytometry. Althoughparental BT474 lacks CD44þCD24� cells, these CSCs represented

BT474-PTEN-LTT

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Figure 5.MEDI5117 shows specific therapeutic activity against a trastuzumab-resistant breast tumor model. A, sensitivity of BT474 cells and BT474-PTEN-LTT cells totrastuzumab 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 vivogrowth inhibition of BT474-PTEN-LTT tumors treated with trastuzumab and MEDI5117. D, H&E staining of primary tumors and lungs from animals bearingBT474 cells, BT474-PTEN-LTT cells, or BT474-PTEN-LTT cells treated with MEDI5117. E, an NF-kB reporter–expressing BT474-PTEN-LTT cell line wasused 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.






about 75% of the BT474-PTEN-LTT cell population (Fig. 6B). Inline with our in vitro tumorsphere data, the number of CD44þ

CD24� CSCs was substantially lower in BT474-PTEN-LTT xeno-grafts when animals were treated withMEDI5117 compared withthose treated with the negative control antibody, R347. It isunclear whether, in addition to reducing the CD44þCD24� cellfraction, MEDI5117 also drives differentiation to the CD44þ

CD24þ phenotype.To further determine the effect of the MEDI5117 on CSCs, we

next performed reimplantation assays using cells taken fromBT474-PTEN-LTT tumors treated for eight weekswith the negativecontrol antibody, trastuzumab, or MEDI5117. Recipient micewere implanted with either 500 or 5,000 cells and monitoredfor 12 weeks. Under these conditions, both trastuzumab andcontrol-treated tumor cells were able to initiate secondary tumors,although tumors did not grow in all recipient mice (Fig. 6C). Incontrast, MEDI5117-treated primary tumors failed to grow uponsecondary reimplantation in any of the recipient mice, suggestingelimination of the CSC population. Data from this experimentwas used to estimate the frequency of CSCs after each treatment aswell (Fig. 6D). These results demonstrate that IL6 depletion byMEDI5117 diminishes the CSC population, which contributes tooverall tumor growth suppression of our trastuzumab-resistantmodel.

DiscussionIL6 is a pleiotropic cytokine that was initially characterized as a

key component of inflammatory responses. Therapeutics target-ing the IL6 pathway are being developed for inflammatory andautoimmune diseases. Tocilizumab, an anti-IL6 receptor anti-body, was approved in 2009 for the treatment of rheumatoidarthritis (26, 27). Siltuximab, a chimeric mouse-human anti-IL6antibody, was approved to treat patients with Castleman disease(FDA.gov press release for siltuximab approval, April 24, 2014).

Numerous studies have revealed a role for IL6 in various typesof cancer as well. Elevated levels of IL6 are associated with poor

prognosis in cancers of the lung, colon, kidney, bladder, andothers (28–30). Thus, therapeutics targeting IL6 and IL6 signalingcomponents are also being tested in clinical trials for cancer.Siltuximab has been shown to have modest benefit in treatingcancers (13, 31, 32). Clazakizumab (ALD518) improves cancer-related fatigue and cachexia in NSCLC patients (33, 34). Elsili-momab (BE-8), a murine anti-IL6 mAb, could not efficientlyblock IL6 levels due to its short half-life and neutralization bya human anti-mouse immune response, and has generatedmixedresults in clinical trials (35). MEDI5117 is a fully human mAbwith no potential immunogenicity issues in human and has ahigher binding affinity for human IL6 (Kd < 1 pmol/L) relative toother anti-IL6 mAbs. Compared with siltuximab, MEDI5117showed enhanced in vitro cell killing (Fig. 2B) and pathwaysuppression (Fig. 2C) and inhibited systemic IL6 in vivo moreefficiently (Supplementary Fig. S3). In mouse xenograft models,MEDI5117 treatment resulted in an improvement in tumorgrowth inhibition compared with siltuximab (SupplementaryFig. S3). However, mouse models do not permit the significanthalf-life extension afforded by our engineered YTE mutation inMEDI5117. The YTE mutation extends the half-life of MEDI5117in humans almost five-fold compared with siltuximab. Thus,MEDI5117 is expected to have significantly better clinical activityand require less frequent dosing in patients compared withsiltuximab and other non-YTE IL6-blocking antibodies.

We found that MEDI5117 inhibited tumor cell growth in vitroand in vivo. The mechanism of this inhibition involved effects onIL6-mediated growth signaling through the IL6R/JAK/STAT3pathway. In addition, repression of endothelial cell growth andangiogenesis was observed after neutralization of IL6. Gilbert andHemann reported that in a mouse model of Burkitt lymphoma,doxorubicin treatment induced production of IL6 by endothelialcells in blood vessels, which promoted the survival of residualtumor cells and eventual tumor relapse (36). ThatMEDI5117 alsoinhibits IL6 activity and growth of human endothelial (HUVEC)cells suggests that this activity would contribute to its antitumoreffects in humans. Furthermore, extending previous results (37),

Reimplanta�on assay at limi�ng dilu�onInoculated cell numbers

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A B

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Figure 6.MEDI5117 targets the breast cancerstem cell population in vitro and inmouse xenografts. A, breasttumorsphere formation of BT474 cellsBT474-PTEN-LTT cells treated withMEDI5117 or an isotype controlantibody. B, residual tumors fromisotype or MEDI5117 treated animalswere evaluated by flow cytometry forthe presence of CD44þCD24� breastCSCs. C, secondary tumor reinitiationwas assessed by removing BT474-PTEN-LTT cells removed from tumor-bearing mice that were treated foreight weeks with an isotype controlantibody, trastuzumab, or MEDI5117and reimplanted into new recipientmice. The table lists number of mice inwhich secondary tumors were formedout of the five mice in each treatmentgroup. D, the frequency of breast CSCwas calculated on the basis of thesecondary tumor initiation capacity ofresidual tumors.

Zhong et al.





we found that MEDI5117 treatment of several tumor types in vivocould reduce tumor angiogenesis by downregulation of variousgenes including VEGF, KDR, and PECAM-1. Thus, the activity ofMEDI5117 in inhibiting tumor growth reflects the pleiotropicroles of IL6 in cancer.

IL6 overproduction occurs in response to various cancer drugtreatments and in drug-resistant tumors, and thus the full benefitsof suppressing IL6 may be achieved in these situations. We foundthat taxanes upregulated IL6 production in NSCLC and prostatecancer cells, and that the combination of taxanes withMEDI5117was significantly more efficacious than either agent alone. Thisefficacy was associated with suppression of activated STAT3, thedownstream effector of the IL6/JAK signaling axis. Similarly,MEDI5117 combined with taxotere resulted in durable tumorregression in a breast cancermodel, whereas treatment with eitheragent alone did not. Activating mutations in the EGFR canpromote IL6 production and activate JAK/STAT3 signaling inhuman lung adenocarcinomas (38). Increased IL6 secretion andconsequent autocrine survival signaling were also found to be acauseof drug resistance to the EGFR inhibitor, erlotinib, inNSCLC(12). Conversely, IL6 antibody treatment can upregulate EGFRexpression and limit ovarian tumor growth inhibition (39). Thissuggests that a combined approach blocking both the EGFR andIL6 pathways may improve response and counteract resistance.Our results showing that MEDI5117 in combination with gefiti-nib enhanced antitumor efficacy in the H1650 NSCLC modelsupports this hypothesis. We also generated gefitinib-resistantH1650 cells and found that MEDI5117 treatment restored sen-sitivity to this drug (data not shown).

Our previous work showed that activation of an IL6 feedbackloop may play a role in trastuzmab resistance in PTEN-deletedHER2þ tumors (24). We therefore tested the activity of MEDI5117in our trastuzumab-resistant breast cancer model, which produceshigh levels of IL6. Neutralization of IL6 by MEDI5117 potentlyinhibited the growth of this trastuzumab resistant tumor. In con-trast, the antibody had no activity against matched EGFR-sensitive(PTENwild-type) tumor cells that do not overproduce IL6. Growthinhibition of the trastuzumab-resistant tumor model byMEDI5117 was associated with suppression of downstream NF-kB signaling and lung metastasis. Iliopoulos and colleagues dem-onstrated that IL6 activation of NF-kB in turn drives further IL6production, creating an inflammatory positive feedback loop (40).Thus, interrupting this IL6/NF-kB activation loop may be partic-ularly important in somedrug-resistant cancers that dependon IL6.

IL6 is known to regulate the growth of some cancer stem cells,and this may contribute to treatment resistance and relapse (41).For example, IL6 stimulates the tumorigenic capacity of CSCsisolated from head and neck squamous tumors (42). Similarly,

glioma stem cells express IL6R, and targeting IL6 decreases gliomastem cell survival and tumor growth (43). This stimulation of CSCgrowth has also been found to contribute to resistance to trastu-zumab. Yang, and colleagues reported that resistance of gastriccancer cells with CSC characteristics to trastuzumab is associatedwith activation of an IL6/STAT3/Jagged-1/Notch positive feed-back loop (44). The results described in this report extend ourprevious findings (24) by showing that neutralization of IL6 byMEDI5117 can inhibit CSCs and that this is may contribute toovercoming trastuzumab resistance in breast cancer. Togetherwith the enhanced efficacy we observed when paired with othertherapeutics, these results encourage further study of MEDI5117in combination treatment scenarios and in drug-resistant cancers.

Disclosure of Potential Conflicts of InterestJ. Huang has ownership interest (including patents) and is an employee at

Medimmune/Aztrazeneca. R.E. Hollingsworth is a Senior Director at MedIm-mune. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: H. Zhong, C. Chen, Y.S. Chang, J. Huang, Y. Yao,E. Hurt, D.A. Tice, Z. Xiao, M.S. Wicha, H. Korkaya, R.E. HollingsworthDevelopment of methodology: H. Zhong, C. Chen, Y. Yao, J. Moisan,H. KorkayaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Zhong, A. Davis, M. Ouzounova, R.A. Carrasco,C. Chen, Y. Yao, J. Moisan, M. Fung, H. KorkayaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Zhong, A. Davis, M. Ouzounova, C. Chen,S. Breen, J. Huang, Z. Liu, Y. Yao, M. Fung, Z. Xiao, M.S. Wicha, H. Korkaya,R.E. HollingsworthWriting, review, and/or revision of the manuscript: H. Zhong, C. Chen,J. Huang, S.G. Clouthier, Z. Xiao, M.S. Wicha, H. Korkaya, R.E. HollingsworthAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. Zhong, S. Breen, J. Huang, S.G. Clouthier,R.E. HollingsworthStudy supervision: H. Zhong, C. Chen, Y.S. Chang, E. Hurt, D.A. Tice,S.G. Clouthier, Z. Xiao, H. Korkaya, R.E. Hollingsworth

AcknowledgmentsThe authors thank Dr. Mohammed Dar and Dr. Ronald Herbst for critical

review of the article.

Grant SupportThis work was supported by Medimmune.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 6, 2015; revised September 28, 2015; accepted October 2,2015; published OnlineFirst January 7, 2016.

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2016;76:480-490. Published OnlineFirst January 7, 2016.Cancer Res Haihong Zhong, April Davis, Maria Ouzounova, et al. Chemotherapy Including Trastuzumab-Resistant TumorsA Novel IL6 Antibody Sensitizes Multiple Tumor Types to

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