epigenetic potentiation of ny-eso-1 vaccine therapy in human ovarian cancer ·...

Research Article

Epigenetic Potentiation of NY-ESO-1 Vaccine Therapy inHuman Ovarian Cancer

Kunle Odunsi1,2,7, Junko Matsuzaki1,7, Smitha R. James3, Paulette Mhawech-Fauceglia4, Takemasa Tsuji1,7,Austin Miller5, Wa Zhang3,11, Stacey N. Akers1, Elizabeth A. Griffiths6, Anthony Miliotto1, Amy Beck7,Carl A. Batt8, Gerd Ritter9, Shashikant Lele1, Sacha Gnjatic10, and Adam R. Karpf3,11

AbstractThe cancer–testis/cancer germline antigen, NY-ESO-1, is a vaccine target in epithelial ovarian cancer (EOC),

but its limited expression is a barrier to vaccine efficacy. As NY-ESO-1 is regulated by DNA methylation, wehypothesized that DNA methyltransferase inhibitors may augment NY-ESO-1 vaccine therapy. In agreement,global DNA hypomethylation in EOC was associated with the presence of circulating antibodies to NY-ESO-1.Preclinical studies using EOC cell lines showed that decitabine treatment enhanced both NY-ESO-1 expressionandNY-ESO-1–specificCTL-mediated responses. On the basis of these observations, we performed aphase I dose-escalation trial of decitabine, as an addition to NY-ESO-1 vaccine and doxorubicin liposome chemotherapy, in12 patients with relapsed EOC. The regimen was safe, with limited and clinically manageable toxicities. Bothglobal and promoter-specific DNA hypomethylation occurred in blood and circulating DNAs, the latter of whichmay reflect tumor cell responses. Increased NY-ESO-1 serum antibodies and T-cell responses were observed inthe majority of patients, and antibody spreading to additional tumor antigens was also observed. Finally, diseasestabilization or partial clinical response occurred in six of ten evaluable patients. On the basis of theseencouraging results, evaluation of similar combinatorial chemo-immunotherapy regimens in EOC and othertumor types is warranted. Cancer Immunol Res; 2(1); 37–49. �2014 AACR.

IntroductionThe ability of the immune system to recognize and reject

tumors is dependent on several factors that include theexpression of immunogenic target antigens, the generation ofhigh frequencies of tumor antigen-specific T cells with potenteffector function, and the capacity to overcome mechanismsby which tumors escape immune attack. Although several invitro and in vivo studies demonstrated that the amount andduration of antigen stimulation can influence antigen-specificT-cell responses (1), the majority of cancer immunotherapystudies have focused on the generation of tumor antigen-specific T cells without concomitant manipulation of antigen

expression. This is a critical consideration because (i) tumorantigens are generally not expressed at 100% frequency incancers, (ii) even when expressed, the antigen density may below, and (iii) expression is often heterogeneous with areas oftumor that are distinctly positive or negative for the tumorantigen. Moreover, the generation of tumor-specific immunitymay result in tumor "immunosculpting" that could influencethe ability to evade immune eradication (2). In this regard, anumber of murine and human studies indicate that the adap-tive immune response can edit tumor antigen expression (3).Similar observations have been made in human immunother-apy clinical trials, in which a proportion of patients showedevidence of immunoediting with either loss of antigen expres-sion or MHC class I expression (4, 5).

In cases where epigenetic mechanisms regulate tumorantigen expression or result in immunosculpting, treatmentwith epigenetic modulatory drugs may reverse the pheno-type. For example, edited tumor cells in a murine melanomamodel, which have lost several antigens, could be induced toreexpress antigens when treated with the DNA methyltrans-ferase inhibitors (DNMTi) 5-azacytidine (5-aza), increasingtheir susceptibility to further vaccination (6). The DNMTi 5-aza and 5-aza-20-deoxycytidine (decitabine) have recentlyentered common clinical practice for the treatment of mye-loid malignancies. Clinical approval of these agents for thetreatment of myelodysplastic syndrome followed the recog-nition that low-dose treatments resulted in progressive DNAhypomethylation and clinical responses, combined with sig-nificantly reduced systemic toxicity (7). In addition, recent

Authors' Affiliations: Departments of 1Gynecologic Oncology, 2Immu-nology, 3Pharmacology and Therapeutics, 4Pathology, 5Biostatistics,6Medicine, and 7Center for Immunotherapy, Roswell Park Cancer Institute,Buffalo; 8Department of Food Science, Cornell University, Ithaca; 9LudwigInstitute for Cancer Research, NY Branch at Memorial Sloan-Kettering;10Tisch Cancer Institute, Mount Sinai School of Medicine, New York, NewYork; and 11Eppley Institute, University of Nebraska Medical Center,Omaha, Nebraska

Note: Supplementary data for this article are available at Cancer Immu-nology Research Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Authors: Kunle Odunsi, Roswell Park Cancer Institute,Elm and Carlton Streets, Buffalo, NY 14263. Phone: 716-845-8376; Fax:716-845-1595; E-mail: [email protected]; and Adam R. Karpf,University of Nebraska Medical Center, 985950 Nebraska Medical Center,Omaha, NE 68198-5950. Phone: 402-559-6115; Fax: 402-559-4651;E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-13-0126

�2014 American Association for Cancer Research.

CancerImmunology

Research

www.aacrjournals.org 37

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work showed that low-dose 5-aza, combined with the histonedeacetylase inhibitor (HDACi) entinostat, provided clinicalbenefit for lung cancer (8). To date, clinical trials in epithelialovarian cancer (EOC) have focused on using DNMTi as ameans to restore responsiveness to platinum-based chemo-therapy (9, 10).

The molecular mechanisms underlying the clinical benefitsof DNMTi are incompletely defined, although it is postulatedthat DNA hypomethylation and gene reactivation are involved.A number of preclinical studies have taken an unbiasedapproach to assess the pharmacologic activity of DNMTi,focusing on gene expression. A consistent observation is thata major response to DNMTi is the activation of cancer–testis orcancer germline antigen genes (11, 12). Cancer–testis antigengenes are normally silenced by DNA methylation in somaticcells but are activated in cancer by promoter DNA hypomethy-lation (13). However, most tumors do not express these genes,and inones that do, expression is frequently heterogeneous (14).Data suggest that DNA methylation is the basis for this het-erogeneity, with tumor regions expressing these genes showingpromoter-specific and global DNAhypomethylation (15). Thesedata raise the hypothesis that DNMTi could augment cancer–testis antigen expression in tumors, which may render themmore susceptible to cancer–testis antigen vaccine efficacy(14, 16). This notion is supported by the observation thatalthough the cancer–testis antigen NY-ESO-1 is expressed inup to 40% of EOC, spontaneous immune responses are found inonly a fraction of patients (17, 18), potentially because the levelsof antigen expression are below the threshold for immunerecognition in most patients. DNMTi can induce cancer–testisantigen promoter hypomethylation and gene expression inpatients with cancer, and can augment cancer–testis antigenimmunity in cell line and animal model systems (19–22). Inagreement, a recent study showed that decitabine treatmentincreased T-cell reactivity to MAGE antigens in patients withHodgkin's lymphoma (23). A final important, and often over-looked, point is that DNMTi induces MHC class I, which couldhave a dramatic impact on antitumor immunity (11, 16, 20).

In the current study, we examined the clinical combinationof an epigenetic therapy (decitabine) with a tumor antigen-targeted cancer vaccine [NY-ESO-1 protein and granulocytemacrophage colony—stimulating factor (GM-CSF) emulsifiedin Montanide]. We tested this combination in patients withEOC at the time of relapse, as an addition to the second-linechemotherapy doxorubicin. Our data show that this novelcombination therapy is safe, promotes integrated immunolog-ic responses, and resulted in stable disease or partial responsein six of ten evaluable patients. These findings encouragetesting of similar chemo-immunotherapeutic approaches forthe treatment of EOC and other human malignancies.

Materials and MethodsPreclinical studies

Decitabine and doxorubicin liposome were obtained fromSigma. A2780 and OVCAR3 EOC cell lines were cultured asdescribed (24), and drug treated as described in the Resultssection and in figure legends. T-cell responses to drug-treated

cells were measured as described in Fig. 1D. NY-ESO-1 mRNAexpression was determined by real-time PCR (RT-PCR), andlong interspersed repeated sequence (LINE-1) and NY-ESO-1methylation were determined using sodium bisulfite pyrose-quencing, as described (15). All clinical samples were obtainedunder Institutional Review Board (IRB)-approved protocols atRoswell Park Cancer Institute (RPCI).

Inclusion criteriaThe study protocol (NCT00887796) was approved by the

IRB at RPCI (Protocol #I127008), and all patients gave inf-ormed consent. Eligible patients were women with relapsedEOC (including fallopian tube and primary peritoneal can-cer), who normally receive doxorubicin as salvage therapyfor recurrent disease. Inclusion criteria were Karnofskyperformance status of at least 70%, normal complete bloodcount and kidney–liver function, and no concomitant anti-tumor therapy or immunosuppressive drugs. Exclusion cri-teria were pregnancy, seropositivity for HIV or hepatitis Bsurface antigen, brain metastasis, clinically significant auto-immune disease, or New York Heart Association class III–IVheart disease.

Study protocolThis was an open-label study with escalating doses of

decitabine, a fixed standard dose of doxorubicin liposome(40 mg/m2), and a fixed dose of vaccine (NY-ESO-1 protein þMontanide þ GM-CSF). The treatment schedule consisted ofdecitabine on day 1, doxorubicin on day 8, and vaccine on day15. In the first cohort (3 patients), decitabine was adminis-tered at a dose of 45 mg/m2 � d1; in the second cohort (6patients), decitabine was used at 90 mg/m2 � d1; and in thethird cohort (3 patients), decitabine was used at 10 mg/m2 �d5 (days 1–5), for each 28-day cycle. The choice of decitabinedose was based on previous studies of decitabine in EOC(9, 25). For vaccination, 250 mg of NY-ESO-1 protein, admixedwith 250 mg of GM-CSF, was emulsified in Montanide andinjected s.c. Treatments were repeated every 28 days, for atotal of four cycles. After completion of the regimen, patientswith stable disease or those responding on therapy werecontinued on doxorubicin until progression. Patients wereevaluated for treatment toxicity, immune responses, and DNAmethylation changes.

Investigational agentsNY-ESO-1 protein for immunization was manufactured at

the Ludwig Institute for Cancer Research (LICR) as described(26). Incomplete Freund's adjuvant (Montanide ISA-51), con-tained mineral oil (Drakeol) and anhydro mannitol octade-canoate and was manufactured by Seppic Inc. Sargramostimwas obtained from Bayer. Decitabine was obtained from EisaiPharmaceuticals under a research agreement. Pegylated lipo-somal doxorubicin for injection was supplied as a sterile,translucent red liposomal dispersion.

DNA methylation pharmacodynamicsSamples from 2 patients in the 10 mg/m2 � d5 decitabine

cohort were obtained pretreatment and on days 8 and 15 of

Odunsi et al.

Cancer Immunol Res; 2(1) January 2014 Cancer Immunology Research38



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0.1 μmol/L 0.5 μmol/L 1 μmol/L (−)

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Figure 1. Preclinical studies supporting the use of decitabine tomodulateNY-ESO-1 vaccine efficacy. A, LINE-1 is hypomethylated inNY-ESO-1–seropositiveEOC patients. LINE-1 methylation was determined by pyrosequencing in EOC samples from patients positive or negative for serum NY-ESO-1 antibodiesat the time of diagnosis. NY-ESO-1 seropositivity was defined as a reciprocal titer of more than 100 by ELISA. B, effect of decitabine (DAC) anddoxorubicin (Doxil) treatments on NY-ESO-1 promoter methylation in EOC cells. A2780 and OVCAR3 cell lines were treated with PBS (control), 0.1 mmol/Ldecitabine for 48 hours, 0.1 mg/mL doxorubicin for 24 hours, or both drugs, in either sequence. gDNA was harvested 72 hours after the initial drug treatment,and used to determine NY-ESO-1 promoter methylation by pyrosequencing. C, effect of decitabine and doxorubicin treatment on NY-ESO-1 mRNAexpression in EOC cells. EOC cells were treated as described in B, and RNA was harvested and used to measure NY-ESO-1 expression by RT-PCR.GAPDH was amplified as a positive control. D, NY-ESO-1–specific CD8þ T-cell response following decitabine and/or doxorubicin treatment. OVCAR3(HLA-A2þNY-ESO-1�) cells were treated with the indicated concentrations of decitabine, doxorubicin, or decitabine followed by doxorubicin, as described inB. A NY-ESO-1–specific HLA-A2–restricted CD8þ T-cell clone was stimulated for 6 hours with OVCAR3 cells following harvest and wash out of reagents.IFN-g production andCD107 expression onHLA-A2/NY-ESO-1157-165 tetramerþCD8þ T cells was determined by flow cytometry. As a positive control, CD8þ

T-cell responses were tested against NY-ESO-1157-165 peptide (0.4 mg/mL)-pulsed OVCAR3 cells. Values in quadrants indicate percentages of cells. Avalue of 1% or more was considered positive. E, NY-ESO-1 expression following drug treatment and removal. A2780 cells were treated with the indicatedconcentrations of decitabine or decitabine þ doxorubicin, and cells were extracted at the indicated time points following drug removaland used to determine NY-ESO-1 expression by RT-PCR.

Decitabine and NY-ESO-1 Vaccine for Ovarian Cancer

www.aacrjournals.org Cancer Immunol Res; 2(1) January 2014 39



each treatment cycle for DNA methylation analyses. Wholeblood and serum were collected from patients and processedwithin 24 hours. Serum was spun down to remove debris, andthe supernatant was recovered. Plasma, peripheral bloodmononucleated cells (PBMC), and granulocytes were obtainedby fractionating whole blood using continuous density gradi-ent centrifugation andLymphocyte SeparationMedium (LSM).Blood samples were diluted in Hank's buffered salt solution(HBSS), layered on LSM, and centrifuged to obtain the topplasma layer, the middle PBMC layer, and the bottom gran-ulocyte/red blood cell (RBC) layer. After recovery of the plasma,PBMCs were washed with HBSS, and the cell pellet wasresuspended in RPMI medium. The granulocyte/RBC layerwas incubated with RBC lysis buffer, and the remaininggranulocyte pellet was washed with HBSS and resuspendedin RPMI medium. Genomic DNA was extracted from eachcomponent using the QIAamp DNA Blood Mini Kit (Qiagen).DNA methylation of the LINE-1 and NY-ESO-1 was determinedby bisulfite pyrosequencing as described (15).

Humoral responses to NY-ESO-1 and other tumorantigens

NY-ESO-1–specific antibodiesweremeasured by ELISA usingserum collected on days 29, 57, 85, and 113, and 6 months afterthe last vaccination, as described (17, 27). In addition, serumantibody titers for 22 unrelated recombinant proteins weredetermined by ELISA as described (27). Recombinant proteinsincluded NY-ESO-1/CTAG1B, LAGE-1/CTAG2, MAGEA1,MAGEA3,MAGEA4,MAGEA10, CT7/MAGEC1, CT10/MAGEC2,CT45/RP13-36C9.1, CT46/HORMAD1, CT47/RP6-166C19.1, Ki-67/MKI67, KRAS, SCP1/SYCP1, SOX2, SPANXA1, SSX1, SSX2,SSX4, p53/TP53, XAGE1B, and DHFR (27, 28). Specificity wasdetermined by comparing seroreactivity among the variousantigens tested, including the ubiquitously expressed nontumorantigen, dihydrofolate reductase (DHFR). A reciprocal titer wasestimated from optical density readings of serially dilutedplasma samples as the maximal dilution significantly reactingto antigen (28). Reciprocal titers of more than 100 were con-sidered significant. Negative control sera from healthy indivi-duals, and positive control sera for each antigen from patientswith cancer, were included in all assays.

Analysis of NY-ESO-1–specific T cellsSample processing, freezing, and analysis were performed in

the ImmuneAnalysis Facility of the Center for Immunotherapyat RPCI. Assay performance and data reporting conformed toMinimal Information about T Cell Assays (MIATA) guidelines.A pool of synthetic overlapping 20- to 25-mer NY-ESO-1 pep-tides covering the entire NY-ESO-1 protein sequence was usedfor in vitro stimulation. Purified CD8þ or CD4þ T cells were pr-sensitized with peptide-pulsed irradiated autologous PBMCdepleted of CD4þ and CD8þ T cells, as described (29). Targetcells for analysis were activated T-cell antigen-presenting cells(T-APCs) or autologous EBV-B cells. The frequency of NY-ESO-1–specific CD8þ or CD4þ T cells was assessed by ELISPOT orintracellular cytokine staining, as described (30). For ELISPOTassays, responseswere considered positivewhen spot numbersin the presence of target cells significantly exceeded the cutoff

value (>50 spots/50,000 cells), and were at least 3 times morethan that spot count of unpulsed target cells. The averagenumber of spots against no-peptide-pulsed cells was 45. In asubset of patients with relevant HLA alleles, the frequencies ofCD8þ T cells were analyzed using human leukocyte antigen(HLA) multimers HLA-A2/NY-ESO-1157–165, HLA-B35/NY-ESO-194–102, and HLA-Cw3/NYESO-192–100. HLA multimerswere manufactured at the Peptide Synthesis Facility of theLICR (Lausanne, Switzerland). Responses were consideredsignificant if tetramer-positive cells were 0.1% or more ofCD8þ T cells, and at least 3 times more than the percentageobtained with control tetramer.

Molecular typing of HLA class I and IIHLA typing was performed at the HLA typing laboratory

of RPCI as described (31), using primers obtained fromGenovision.

Statistical analysisPatient demographic and disease characteristics were sum-

marized using frequencies and descriptive statistics. The plat-inum-free interval was defined as the number of monthsbetween the last platinum-based chemotherapy treatment andenrollment on study, and patients with an interval less than 6months were considered resistant (32). Associations betweenmethylation levels of NY-ESO-1 and LINE-1 were quantifiedusing the Pearson test. Differences in LINE-1 methylation inNY-ESO-1 antibody-positive and -negative patients wereassessed using the Welch two-tailed t test. Statistical analyseswere performed using Graphpad Prism 5.0.

ResultsDNA hypomethylation in EOC is associated withspontaneous immune responses to NY-ESO-1

Previously we found that (i) NY-ESO-1 is expressed in lessthan 50% of patients and is often focal or heterogeneous in thelesions in which it is expressed (17); (ii) NY-ESO-1 inter- andintratumor heterogeneity is associated with promoter-specificand global DNA methylation (15); (iii) decitabine treatmentinduces NY-ESO-1 hypomethylation and expression in EOCcells (15); and (iv) loss of NY-ESO-1 expression in EOC occursduring NY-ESO-1 vaccine therapy (33). These data raised thepossibility that decitabine may enhance the efficacy of NY-ESO-1 vaccines in EOC. To evaluate this hypothesis, we deter-mined whether DNAmethylation status in EOC is predictive ofspontaneous immune response to NY-ESO-1, by measuringtumor LINE-1 methylation and circulating NY-ESO-1 antibo-dies in matched EOC patient samples. The LINE-1 repetitiveelement comprises approximately 17% of the genome and isregulated by DNAmethylation.We previously validated LINE-1methylation as a biomarker for global DNAmethylation statusin EOC, and showed that LINE-1 methylation is significantlycorrelated withNY-ESO-1 promotermethylation in this disease(34). NY-ESO-1–seropositive EOC had significantly reducedtumor LINE-1 methylation compared with seronegativepatients (Fig. 1A), consistent with the hypothesis that DNAhypomethylation promotes NY-ESO-1–specific immuneresponses in EOC.

Odunsi et al.




Combination decitabine þ doxorubicin chemotherapypromotes NY-ESO-1 promoter hypomethylation, geneexpression, and immune recognition in EOC cell linesWe sought to develop a clinical regimen combining epige-

netic therapy with NY-ESO-1 vaccine. Because combinationtherapy of this type has not been tested before and safety isuncertain, we developed an initial test of a decitabine þ NY-ESO-1 vaccine in the setting of recurrent EOC, which isuniformly fatal, as an add-on to a standard second-line che-motherapy, doxorubicin liposome.We initially used two NY-ESO-1-negative human EOC cell

lines, A2780 and OVCAR3, to determine the optimal sequencingof decitabine and doxorubicin treatment. Treatment with dec-itabine followed by doxorubicin, but not the reverse sequence,resulted in NY-ESO-1 promoter hypomethylation and NY-ESO-1gene induction (Fig. 1B and C). In addition, this sequenceresulted in the recognition of EOC cells by NY-ESO-1–specificCTLclones, consistentwith functional presentationofNY-ESO-1epitopes on EOC cells posttherapy (Fig. 1D). There was alsoevidence that doxorubicin potentiated CTL responses to equiv-alent doses of decitabine (Fig. 1D). Also supporting the combi-nation treatment strategy is that decitabine potentiates apo-ptotic responses to doxorubicin in solid tumor cell lines (35).Although CTL clones produced more than 70% IFN-g responseagainst peptide-pulsedmelanoma cell lines (Supplementary Fig.S1), the response against peptide-pulsed OVCAR3 was compar-atively less (Fig. 1D), suggesting that ovarian cancer cell lines areinherently less immunogenic than melanoma. To determinehow long-lived NY-ESO-1 induction is following drug treatment,we measured NY-ESO-1 using RT-PCR in A2780 cells up to 1month after drug treatment (Fig. 1E). We observed continuedNY-ESO-1 expression throughout this time, albeit with diminish-ing expression at later time points (Fig. 1E).

Phase I trial of decitabineþNY-ESO-1 vaccine in patientsreceiving doxorubicin for recurrent EOCTwelve patients with relapsed EOC gave written informed

consent and were entered into a phase I clinical trial(NCT00887796). The trial design involved fixed doses of doxo-rubicin and NY-ESO-1 vaccine, with dose escalation of deci-tabine (seeMaterials andMethods). Unlike previous NY-ESO-1vaccine trials, tumor NY-ESO-1 expression was not used asan inclusion criterion, and 4 of 11 (36%) patients expressedNY-ESO-1 at baseline (Table 1). Patient characteristics aresummarized in Supplementary Table S1. The median ageat study entry was 59 years, and the median time fromdiagnosis to study entry was 21 months. Nine of 12 (75%)patients received at least two previous lines of chemother-apy. Eleven of 12 (92%) patients had poorly differentiatedtumors, 12 of 12 (100%) were Federation Internationale desGynaecologistes et Obstetristes (FIGO) stage III, and 11 of 12(92%) displayed serous histology. On the basis of Gyneco-logic Oncology Group clinical response criteria (32), 10 of 11(91%) patients with available information were consideredplatinum resistant.The regimenwaswell tolerated. Themost frequent grade 3/4

adverse event was neutropenia, and only 1 patient experiencedfebrile neutropenia (Supplementary Table S2). Vaccine injec-

tion site reactions were common, but only two were grade 3events. All adverse events were clinically manageable. Weobserved three serious adverse events: septicemia, intestinalobstruction, anddehydrationwere observed at the 45mg/m2�d1, 90 mg/m2 � d1, and 10 mg/m2 � d5 decitabine doses,respectively. All three of these adverse events were consideredunrelated to the study drugs. Dose-limiting toxicity wasobserved in 1 patient (grade 4 neutropenia) at the 90 mg/m2

dose, requiring a 25% dose reduction of decitabine.

Effects on DNA methylationWe obtained samples from 2 patients in the 10 mg/m2 �

d5 decitabine cohort to confirm the effect of the regimen onDNA methylation. We collected gDNA from surrogate normaltissues (PBMCs and granulocytes), as well as from circulatingDNA present in serum and plasma, which likely is comprised oftumor cell DNA (36). Indeed, LINE-1methylation in serum hasbeen used as a pharmacodynamic marker for DNMTi in othersolid tumor trials (37). We used quantitative bisulfite pyrose-quencing to measure LINE-1 and NY-ESO-1 promoter methyl-ation. DNA from PBMCs and granulocytes showed significanthypomethylation of both LINE-1 and NY-ESO-1 promotersposttherapy (Fig. 2A–D). Hypomethylation was pronouncedin granulocytes, likely due to the rapid cycling rate and shorterhalf-life of these cells as compared with PBMCs (Fig. 2A and C).Significant hypomethylation of LINE1 and NY-ESO-1 alsooccurred in plasma and serum DNA, suggesting tumor cellhypomethylation (Fig. 2A–D). In general, greater hypomethy-lation occurred at day 8 of each cycle as compared with day 15,consistent with remethylation following decitabine withdraw-al, as observed previously (19). The effect on LINE-1 and NY-ESO-1 methylation was highly correlated (Fig. 2E and F).

Humoral responsesSpontaneous and treatment-induced humoral immune

responses in patients were investigated first by measuringserum immunoglobulin G against recombinant full-lengthNY-ESO-1 protein using ELISA (Fig. 3A; Table 1). Four patients(# 01, 02, 03, and 06) were baseline seropositive for anti-NY-ESO-1 antibody and all remained seropositive during thecourse of therapy. Among the 6 baseline seronegative patientsfor which informationwas available (Pts. # 05, 07, 08, 09, 10, 11),five (83%) converted to seropositive, and this occurred rapidlyafter two to three cycles (Fig. 3A; Table 1). A comparison ofmaximal NY-ESO-1 antibody titers in each cohort indicated nosignificant difference between cohorts 2 and 3 (Fig. 3B). Cohort1 had higher NY-ESO-1 antibody titers than cohorts 2 and 3(Fig. 3B), likely reflecting their seropositivity at baseline (Fig.3A; Table 1).

To determine whether the treatment resulted in antigenspreading, we tested humoral immune responses against apanel of 22 recombinant proteins, including cancer–testisantigens (LAGE1, MAGEA, MAGEC, SSX, XAGE, CT45, CT46,CT47, SPANX), mutational antigens (TP53), and embryonic/stem cell antigens (SOX2), for recognition by ELISA, asdescribed previously (27). Overall, de novo serum reactivity toantigens not included in the vaccine was observed in 3 of 3patients analyzed before and after therapy, with titers ranging





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Odunsi et al.




from 130 to 450 (Fig. 3C). Antibodies against recombinantDHFR, measured as a control, were not detected. These dataindicate that the combination regimen leads to a broadenedprofile of antitumor immune responses in vivo.

T-cell responsesPreexisting NY-ESO-1–specific CD8þ T cells were detectable

by IFN-g ELISPOT assays in 2 of 10 (20%) patients with EOCanalyzed (Fig. 4A; Table 1). These 2 patients were in cohort 1,and both showed further increases in NY-ESO-1–specific CD8þ

T cells following treatment (Fig. 4A). There was de novoinduction of NY-ESO-1–specific CD8þ T-cell responses in 3additional patients (2 in cohort 2 and 1 in cohort 3; Fig.4A; Table 1). Thus, 5 of 11 (45%) evaluable patients hadantigen-specific CD8þ T-cell responses following therapy (Fig.4A; Table 1). Preexisting NY-ESO-1–specific CD4þ T-cellresponses were more widespread, and were detected in sevenof ten (70%) evaluable patients (Fig. 4A; Table 1). Theseresponses persisted during therapy and, in addition, there wasde novo induction of CD4þ T cells in 2 patients (Pts. #09 and 10;

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Figure 2. In vivo DNA methylation pharmacodynamics. Two patients in the 10 mg/m2 � d5 decitabine cohort were used to assess DNA methylationpharmacodynamics during therapy. Whole blood was collected pretreatment and at day 8 and 15 of each of the first four cycles of therapy. PBMCs,granulocytes, serum, and plasma samples were prepared and used for gDNA isolation. LINE-1 and NY-ESO-1 promoter methylation were determined bypyrosequencing. A and B, LINE-1methylation in samples from patients #10 and #11. C and D,NY-ESO-1methylation in samples from patients #10 and #11.E and F, association between LINE-1 methylation and NY-ESO-1 methylation in samples from patients #10 and #11. C1–C4 refers to the therapy cyclenumber. Error bars indicate � 1 SD. Pearson correlation statistics are shown in E and F.





Fig. 4A and Table 1). In a subset of applicable patients forwhomprevalidatedHLA tetramerswere available, the frequen-cies of CD8þT cells were further analyzed usingHLA tetramersfor HLA-A2/NY-ESO-1157-165, HLA-B35/NY-ESO-194-102, HLA-B35/NY-ESO-194-104, and HLA-Cw3/NY-ESO-192-100. As shownin Fig. 4B, EOC patients #03 (baseline seropositive) and #06(baseline seronegative) showed expansion of NY-ESO-1–spe-cific CD8þ T cells by day 57, although the magnitude ofthe NY-ESO-1–specific cells had waned 6 months followingtherapy.

We determined the epitope clusters of therapy-inducedCD8þ T and CD4þ T cells by testing overlapping 20-merpeptides against NY-ESO-1 in ELISPOT assays. Figure 5Aillustrates the responses from patients #03 and #06 before andafter therapy, and at the time of follow-up (i.e., 6 months afterthe last treatment). In all patients, therapy-induced CD8þ T-cell epitopes were clustered in the p81–110 and 119–143regions of the NY-ESO-1 protein (Fig. 5B). Notably, CD4 epi-topes showed evidence of epitope broadening following ther-apy and spanned the 51–180 region (Fig. 5).

To assess whether therapy-induced CD8þ T cells were capa-ble of recognizing naturally processed NY-ESO-1 antigen, weused NY-ESO-1–expressing cancer cell lines as targets. Poly-clonal populations of NY-ESO-1–specific CD8þ T cells wereenriched by sorting tetramer-reactive cells or IFN-g–producingcells using IFN-g capture reagents. Although treatment-inducedpolyclonal CD8þ T cells recognized partially HLA-matched NY-ESO-1þ melanoma cell lines (MZ-MEL-9, SK-MEL-95, SK-MEL-139, SK-MEL-52), the SK-MEL-29 (B35�ESO�) line was notrecognized (Supplementary Fig. S2). To characterize CD4þ Tcells, NY-ESO-1–specific CD4þ T cells were enriched on thebasis of CD154 expression after restimulation with NY-ESO-1peptide, and tested for Th1/Th2 polarization and protein rec-ognition, as described (38). Th1 and Th2 differentiation wasdetermined on the basis of IFN-g and interleukin (IL)-4 pro-duction upon stimulation with NY-ESO-1–pooled peptides. Asshown in Supplementary Fig. S2, Th1 differentiation increasedfollowing treatment, and therapy-induced NY-ESO-1–specificCD4þ T cells recognized the full-length NY-ESO-1 protein.

Clinical responsesA total of 10 patients were evaluable for clinical response

using immune-related response criteria (modified ResponseEvaluation Criteria in Solid Tumors; ref. 39; Table 2). Notably,5 patients had stable disease (50%) and 1 patient had a partialresponse/disease remission (10%). The median duration ofstable disease was 6.3 months (range, 3.9–7.8 months), andthe partial response duration was 5.8 months. The clinicalresponse in the three different decitabine cohorts appeareddistinct (Table 2). In the first cohort (45 mg/m2), 2 of 2evaluable patients showed stable disease or partial response/remission. Similarly, in the third cohort (10 mg/m2� d5), 2 of2 evaluable patients showed stable disease. However, in thesecond cohort and the highest dose level (90 mg/m2), only 2of 6 patients (33%) showed stable disease. Finally, it wasnotable that only one of five patients showing stable diseaseor partial response expressedNY-ESO-1pretreatment (Tables 1and 2).

DiscussionHere, we report a novel therapeutic regimen for patients

with EOC that is applicable to other humanmalignancies. Theapproach utilizes an epigenetic therapy, decitabine, known toinduce cancer–testis antigen gene expression, in combinationwith a vaccine targeted against the activated antigen, in thiscase NY-ESO-1. We used this novel combination as an add-onto a standard EOC chemotherapy agent, doxorubicin, althoughthis is not a requisite part of the strategy. In addition to

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Figure 3. Humoral responses to NY-ESO-1 and other tumor antigens. A,NY-ESO-1 antibody titers in serum were measured by ELISA. Patientswith EOC receiveddecitabine at 45mg/m2�d1 (cohort 1), 90mg/m2�d1(cohort 2), or 10mg/m2� d5 (cohort 3), followed by doxorubicin on day 8,and vaccination on day 15, for each 28 day cycle. Patients #01, #02, #03,and #06 were baseline seropositive for NY-ESO-1 antibody. B,comparison of maximal NY-ESO-1 antibody titers in each patient cohortat days 57 or 85. Bars indicate median � SD. C, serum reactivityagainst a panel of 22 unrelated recombinant proteins was tested forrecognition by ELISA. A positive response was considered morethan100 reciprocal titer. FU, follow-up.

Odunsi et al.




NY-ESO-1, other epigenetically regulated cancer–testis anti-gens for which vaccines are available could be targeted usingthis approach. Because cancer–testis antigens are regulated byDNA methylation, histone acetylation, and other epigeneticmarks, agents other thandecitabine (e.g., HDACi) could also betested in combination with cancer–testis antigen vaccines(14). In addition to EOC, other cancers, for example,melanomaand lung, are candidates for this approach, as cancer–testisantigens are epigenetically regulated in most human tumors,and cancer–testis antigen vaccines are in an advanced stage ofclinical development in these malignancies (14).A number of preclinical observations supported this

approach. NY-ESO-1 expression is regulated by DNA methyl-ation in EOC cells and tumor tissues, and decitabine treatmentinducesNY-ESO-1 expression in EOCcell lines (15). In addition,heterogeneous intratumor expression of NY-ESO-1, which

frequently occurs in EOC and other tumor types, is associatedwith DNA methylation of the NY-ESO-1 promoter and also theglobal methylation status of the tumor cells (15). Notably, wefound that NY-ESO-1 serum antibodies in patients with EOC atbaseline are associated with LINE-1 hypomethylation inmatched tumors. This observation provides support to thenotion that global DNA methylation status plays a key role inregulating NY-ESO-1 expression and immune recognition invivo.

Because of an unknown safety profile, we tested the regimenin patients with recurrent EOC, as an add-on to second-linedoxorubicin chemotherapy. To determine the proper sequenc-ing of decitabine and doxorubicin, we conducted treatmentstudies on EOC cell lines, and found that decitabine treatmentmust precede doxorubicin to hypomethylate and activateNY-ESO-1. This finding is consistent with a requirement for

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Figure 4. T-cell responsesto NY-ESO-1. CD8þ andCD4þ T cells isolated fromPBMCs were independentlystimulated with CD8�CD4� cellspulsed with NY-ESO-1–pooledpeptides and cultured for14 to 15 days. A, IFN-gproduction from CD8þ orCD4þ T cells was determined byELISPOT assay againstNY-ESO-1–pooled peptide-pulsedphytohemagglutinin (PHA)-blastedCD4þ T cells. The number ofIFN-g spots indicated reflectssubtraction of the number of spotsagainst peptide-unpulsed target.B, NY-ESO-1–specific CD8þ T-cellfrequency was assessed by flowcytometry following staining withthe indicated tetramers. FU,follow-up.





continued DNA synthesis to incorporate decitabine and toremodel DNA methylation, as doxorubicin treatment likelyblocks decitabine incorporation and cell cycling. Importantly,EOC cells treated with the decitabine þ doxorubicin combi-nation promoted NY-ESO-1 antigen-restricted CTL recogni-tion of EOC cell lines, and the effect of drug treatment on NY-ESO-1 expression was longlasting.

Two patients enrolled in the trial, at the 10 mg/m2 � d5decitabine dose level, were used to evaluate pharmacodynamiceffects on DNA methylation. In both patients, there was amarked reduction of methylation of the LINE-1 and the NY-ESO-1 promoters in blood cells, and hypomethylation of thetwo regions were remarkably correlated. Because of logisticalconstraints, we were unable to obtain tumor tissues postther-apy to study DNA methylation or NY-ESO-1 expression. How-ever, we observed robust hypomethylation in circulating DNAobtained from serum and plasma posttherapy. Because circu-

lating DNA in patients with cancer is derived from tumor cells(36), our data suggest that decitabine treatment causes DNAhypomethylation in clinically relevant target cells. These find-ings are also consistent with the prior demonstration thatdecitabine treatment leads to hypomethylation of genesdetected in plasma DNA, which is hypermethylated in plati-num-resistant EOC (9).

NY-ESO-1–specific antibodies were generated in two thirds(67%) of thebaseline seronegative patients. Remarkably,denovoinduction of humoral immune responses against the unrelatedantigens MAGEA4, CT45, CT47, CT55, and SOX2 was alsoobserved, indicative of antigen spreading. Similar evidence ofantigen spreading was not observed in our previous NY-ESO-1vaccine trials in patients with EOC (18, 40). In a previous report,sera from patients vaccinated with the recombinant NY-ESO-1protein sometimes showed nonspecific reactivity against otherrecombinant proteins (41), and the possibility was raised that

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Figure 5. Epitopes of CD4þ

and CD8þ T cells pre- andposttherapy. NY-ESO-1–specificCD8þ and CD4þ T-cell responsesagainst 20-mer peptides weretested by ELISPOT assaysfollowing in vitro stimulation. A,responses of CD8þ and CD4þ Tcells pretherapy, posttherapy, andat the time of follow-up (FU; 6months after the last treatment),from patients #03 and #06. Forpatient #03, the posttreatment timepoint shown for both CD8þ andCD4þ is day 113. For patient #06,the posttreatment time pointsshown are d57 and d85 for CD8þ

and CD4þ, respectively. B,summary of NY-ESO-1 epitopesrecognized by CD8þ or CD4þ Tcells. A positive response wasdefined as a spot count more than50 out of 50,000 cells, and at leastmore than 3 times spot count ofunpulsed targets.

Odunsi et al.




there could be reactivity against bacterial components or His6-tag in the vaccine preparation. We ruled this out in the currentstudy, as no antibody responses were observed against theubiquitous control protein (DHFR). There are several potentialmechanisms by which antigen spreading may have been facil-itated with the current regimen. The first is upregulation ofadditional tumor antigens and/or MHC class I by decitabine,leading to improved recognition by the immune system (16).The second is chemotherapy-induced immunogenic cell death,which can lead to cross-presentation of tumor antigens (42).Finally, the destruction of tumor cells by NY-ESO-1–specificimmunitymay release additional tumor antigens, leading to thegeneration of multiple heteroclitic serologic responses (43).Humoral immune responses posttherapy were accompa-

nied by antigen-specific CD8þ T-cell responses in 50% of thepatients. In contrast, with the exception of patients vaccinatedwith recombinant NY-ESO-1 protein and CpG in Montanide(44), less than 50% of the patients vaccinated with NY-ESO-1protein in previous trials developed antigen-specific CD8þ T-cell responses (44–46). Consistent with previous studies (44),we found that the specificity of the CD8þ T-cell responses wasclustered in the central region of the NY-ESO-1 protein. More-over, therapy-induced CD8þ T-cell lines from patients recog-nized naturally processed full-length antigen and tumor tar-gets. Although antigen-specific CD4þ T-cell responses werepresent at baseline in the majority of patients, the regimen ledto broadening of epitopes, which persisted during the follow-up period. In addition, the combination therapy led to skewingtoward Th1 differentiation of NY-ESO-1–specific CD4þ T cells,as comparedwith baseline preexisting antigen-specific CD4þTcells. Together, our results reveal integrated induction of

antibody, CD8þ, and CD4þ T cells, along with antigen spread-ing, using this novel regimen.

The major limitation for combining cytotoxic chemothera-py, such as doxorubicin, and immunotherapy is that cytotoxicdrugs are generally regarded as immunosuppressive due totoxicity to dividing immune cells. However, the potential fortherapeutic synergy of chemotherapy and immunotherapy hasbeen recognized (47). Recent work has shown that doxorubicinenhances the proliferation of tumor antigen-specific CD8þ

T cells in tumor-draining lymph nodes, and promotes tumorinfiltration by IL-17–secreting gd T cells and activated IFN-g–secreting CD8þ T cells (48, 49) Moreover, doxorubicin hasthe capacity to increase the permeability of tumor cells togranzyme B, thereby rendering themmore susceptible to CTL-mediated lysis, even if they do not express the antigen recog-nized by CTLs (50).

The majority of patients enrolled in this trial had multipleprevious lines of chemotherapy and were considered platinumresistant. Thus, the observed cases of stable disease and partialresponse in this population are notable. Although the currenttrial was not powered to address response rates and progres-sion-free survival, the clinical results obtained are encouraging.A limitation of our study design is our inability to delineate theindividual contributions of doxorubicin, decitabine, and vac-cination to the immunologic and clinical results. However, thisquestion can be addressed in the phase II setting.

The present study suggests that conventional chemother-apy, decitabine, and immunotherapy may promote the gen-eration of vaccine-induced immune responses. Althoughintegrated humoral and T-cell responses occurred in a sig-nificant proportion of patients with ovarian cancer in ourprevious NY-ESO-1 vaccine trials (18, 33, 40), this is our firstobservation of antigen spreading with de novo induction ofimmune responses against a broad array of unrelated tumorantigens. In addition, we observed immunologically andclinically beneficial responses regardless of the pretreatmenttumor NY-ESO-1 expression status, potentially increasing thepatient population eligible for vaccine therapy. We concludethat the current strategy may be capable of overcomingseveral potential mechanisms by which ovarian and othercancers escape immune attack.

Disclosure of Potential Conflicts of InterestE. Griffiths has received a commercial research grant from Supergen, Inc, and

has received honoraria from service on the speakers' bureau from Celgene, Inc,Alexion Pharmaceuticals, and Incyte, Inc. G. Ritter has ownership interest(including patents) as coinventor on primary affiliation assigned NY-ESO-1patents. S. Gnjatic has ownership interest (including patents) in NY-ESO-1related patents. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: K. Odunsi, G. Ritter, A.R. KarpfDevelopment of methodology: K. Odunsi, P. Mhawech-Fauceglia, S. Gnjatic,A.R. KarpfAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Odunsi, J. Matsuzaki, S.R. James, W. Zhang, S.N.Akers, A. Beck, C.A. Batt, A.R. KarpfAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):K. Odunsi, J. Matsuzaki, S.R. James, T. Tsuji, A. Miller,E. Griffiths, S. Gnjatic, A.R. KarpfWriting, review, and/or revision of themanuscript:K. Odunsi, J. Matsuzaki,S.R. James, S.N. Akers, E. Griffiths, G. Ritter, S. Lele, S. Gnjatic, A.R. Karpf

Table 2. Clinical responses

Patientnumber

Decitabinedose

Clinicalresponsea

01 45 mg/m2 � d1 N/Eb02 45 mg/m2 � d1 SD03 45 mg/m2 � d1 PR04 90 mg/m2 � d1 PD05 90 mg/m2 � d1 PD06 90 mg/m2 � d1 PD07 90 mg/m2 � d1 PD08 90 mg/m2 � d1 SD09 90 mg/m2 � d1 SD10 10 mg/m2 � d5 SD11 10 mg/m2 � d5 SD12 10 mg/m2 � d5 N/Ec

Abbreviations: N/E, not evaluable, safety assessment only;PD, progressive disease; PR, partial response/remission;SD, stable disease.aThe median duration of SD responses was 6.3 months(range, 3.9–7.8), and the PR duration was 5.8 months.bLeft study early; no posttreatment CT scan.cLeft study early; contraindicated corticosteroid use.





Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J. Matsuzaki, S.R. James, A. Miliotto, G.Ritter, S. Gnjatic, A.R. KarpfStudy supervision: A.R. Karpf

Grant SupportThisworkwas supported by theOvarianCancerResearch Fund, RPCI Alliance

Foundation, Cancer Vaccine Collaborative grant (Cancer Research Institute/

Ludwig Cancer Research), Cancer Research Institute Anna-Maria Kellen ClinicalInvestigator Award, NIH RO1CA116674, CA158318, and Eisai Pharmaceuticals.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 14, 2013; revised October 14, 2013; accepted October 24, 2013;published online January 3, 2014.

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2014;2:37-49. Cancer Immunol Res Kunle Odunsi, Junko Matsuzaki, Smitha R. James, et al. Ovarian CancerEpigenetic Potentiation of NY-ESO-1 Vaccine Therapy in Human

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