A Feasibility Study of Cyclophosphamide, Trastuzumab, and...

Research Article

A Feasibility Study of Cyclophosphamide, Trastuzumab, andan Allogeneic GM-CSF–Secreting Breast Tumor Vaccine forHER2þ Metastatic Breast Cancer

Gang Chen1,2, Richa Gupta1,2, Silvia Petrik1,2, Marina Laiko1,2, James M. Leatherman1,2, Justin M. Asquith1,2,Maithili M. Daphtary1,2, Elizabeth Garrett-Mayer9, Nancy E. Davidson10, Kellie Hirt1,2, Maureen Berg1,2,Jennifer N. Uram1,2, Tianna Dauses1,2, John Fetting1,2, Elizabeth M. Duus1,2,4, Saadet Atay-Rosenthal1,2,Xiaobu Ye1,2,5, Antonio C. Wolff1,2, Vered Stearns1,2, Elizabeth M. Jaffee1,2,3,4,7,8, and Leisha A. Emens1,2,6

AbstractGranulocyte-macrophage colony-stimulating factor (GM-CSF)–secreting tumor vaccines are bioactive, but

limited by disease burden and immune tolerance. Cyclophosphamide augments vaccine activity in tolerant neumice and in patientswithmetastatic breast cancer. HER2-specificmonoclonal antibodies (mAb) enhance vaccineactivity in neu mice. We hypothesized that cyclophosphamide-modulated vaccination with HER2-specific mAbsafely induces relevant HER2-specific immunity in neumice and patients with HER2þ metastatic breast cancer.Adding both cyclophosphamide and theHER2-specificmAb7.16.4 to vaccinationmaximizedHER2-specificCD8þ

T-cell immunity and tumor-free survival in neu transgenic mice. We, therefore, conducted a single-arm feasibilitystudy of cyclophosphamide, an allogeneic HER2þ GM-CSF–secreting breast tumor vaccine, and weeklytrastuzumab in 20 patients with HER2þ metastatic breast cancer. Primary clinical trial objectives were safetyand clinical benefit, in which clinical benefit represents complete response þ partial response þ stable disease.Secondary study objectives were to assess HER2-specific T-cell responses by delayed type hypersensitivity (DTH)and intracellular cytokine staining. Patients received threemonthly vaccinations, with a boost 6 to 8months fromtrial entry. This combination immunotherapy was safe, with clinical benefit rates at 6 months and 1 year of 55%[95% confidence interval (CI), 32%–77%; P¼ 0.013] and 40% (95%CI, 19%–64%), respectively.Median progression-free survival and overall survival durations were 7 months (95% CI, 4–16) and 42 months (95% CI, 22–70),respectively. Increased HER2-specific DTH developed in 7 of 20 patients [of whom 4 had clinical benefit (95% CI,18–90)], with a trend toward longer progression-free survival and overall survival in DTH responders. Polyfunc-tional HER2-specific CD8þ T cells progressively expanded across vaccination cycles. Further investigation ofcyclophosphamide-modulated vaccination with trastuzumab is warranted. (Clinicaltrials.gov identifier:NCT00399529) Cancer Immunol Res; 2(10); 949–61. �2014 AACR.

IntroductionThe availability of multiple drugs specific for HER2 (trastu-

zumab, lapatinib, pertuzumab, and ado-trastuzumab emtan-sine) has revolutionized the treatment of HER2þ metastaticbreast cancer (1, 2). However, not all patients benefit andsurvival gains could be further enhanced. Innovative therapiesthat complement and synergize with these drugs are urgentlyneeded to improve disease outcomes.

Immune-based therapies, including vaccines and immunecheckpoint blockade, have several unique properties (3). First,immune-based therapies recruit the host tumor-specific im-mune response rather than targeting the tumor directly. Second,the host immune system can recognize an unlimited number ofoverexpressed and/or mutated targets differentially expressedby tumor cells relative to normal tissue. Third, immune-basedtherapies confer a durable treatment response due to immuno-logic memory. Although the evaluation of immune checkpointblockade has been limited in patients with breast cancer, multi-ple vaccines have been tested. Despite a good safety profile,

1Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland.2Department of Oncology, Johns Hopkins University School of Medi-cine, Baltimore, Maryland. 3Department of Pathology, Johns HopkinsUniversity School of Medicine, Baltimore, Maryland. 4Department ofPharmacology, Johns Hopkins University School of Medicine, Balti-more, Maryland. 5Department of Neurosurgery, Johns Hopkins Univer-sity School of Medicine, Baltimore, Maryland. 6Program in Pathobiol-ogy, Johns Hopkins University School of Medicine, Baltimore, Maryland.7Program in Immunology, Johns Hopkins University School of Medicine,Baltimore, Maryland. 8Program in Cellular and Molecular Medicine,Johns Hopkins University School of Medicine, Baltimore, Maryland.9Medical University of South Carolina, Charleston, South Carolina.10University of Pittsburgh Cancer Institute and UPMC CancerCenter,Pittsburgh, Pennsylvania.

Prior Presentation: Presented in part at the 47th annual meeting of theAmerican Society of Clinical Oncology, June 3–7, 2011.

CorrespondingAuthor: LeishaA. Emens, Department of Oncology, JohnsHopkins University School of Medicine, Sidney Kimmel ComprehensiveCancer Center at Johns Hopkins, Bunting-Blaustein Cancer ResearchBuilding, 1650 Orleans Street, Room 409, Baltimore, MD 21231-1000.Phone: 410-502-7051; Fax: 410-614-8216; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-14-0058

�2014 American Association for Cancer Research.

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immune responses have been modest and inconsistently asso-ciated with clinical benefit. Most of these vaccination regimenswere limited by some combination of immune tolerance, estab-lished disease burdens, or the use of suboptimal therapeutictargets and/or vaccination regimens.

Cytokine-modified tumor cell vaccines that secrete granu-locyte-macrophage colony-stimulating factor (GM-CSF) caninduce robust T cell–dependent immunity that clears estab-lished tumors in preclinical models (4). Multiple clinicalstudies have demonstrated the single-agent safety and bioac-tivity of these agents in patients with cancer, but clinicalbenefit remains unproven (5, 6). This lack of success is probablybecause vaccination alone is not potent enough to induceimmune responses sufficiently robust to overcome immunetolerance and suppression and lyse bulky disease.More recent-ly, clinical trials combining GM-CSF–secreting vaccines withthe immune checkpoint agent ipilimumab demonstratedenhanced T-cell activation associated with survival benefit inprostate and pancreas cancer (7, 8). These trials provideclinical proof of principle for combination immunotherapies.

Standard cancer drugs may also augment immune-basedtherapies, either at approveddosesor at an immune-modulatingdose and schedule (9). We showed that low-dose cyclophos-phamide enhances a HER2þ GM-CSF–secreting vaccine in bothimmune tolerant neu transgenic mice (10), and in patients withmetastatic breast cancer (11). Low-dose cyclophosphamiderelieves the suppressive influence of CD4þCD25þ regulatoryT cells (Treg) in neu mice, allowing the recruitment of potent,tumor-specific CD8þ T cells (12, 13). The monoclonal antibody(mAb) trastuzumab also has immunomodulatory activity (14).We showed that HER2-specific mAbs can augment HER2-specific CD8þ T-cell responses and tumor-free survival in vacci-nated, tumor-bearing neumice (15); furthermore, the trastuzu-mab-like mAb 7.16.4 enhances immune priming, augmentingprimary and memory CD8þ T-cell responses in vaccinated neumice (16). In patients with breast cancer, trastuzumab-basedchemotherapy induces T-cell responses within both the periph-eral blood and tumor microenvironment (17, 18).

We, therefore, evaluated the immunologic and antitumoractivity of a GM-CSF–secreting, HER2þ whole-cell breastcancer vaccine given in sequence with immune-modulatingdoses of cyclophosphamide and weekly HER2-specific mAb intolerant neu mice, and in patients with metastatic breastcancer. The clinical study was designed to assess the safety,clinical benefit, and immunologic activity of this combinationimmunotherapy regimen in patients with measurable or eva-luable HER2þ metastatic breast cancer.

Materials and MethodsPreclinical study design

Mice. Neu-N (neu) mice were derived from the colony ofGuy and colleagues (19), bred to homozygosity as verified bySouthern blot analysis (20), and maintained at Johns HopkinsUniversity. Experiments were performed with 8- to 10-week-old mice using AAALAC-compliant protocols approved by theAnimal Care and Use Committee of the Johns Hopkins Uni-versity School of Medicine.

Cell lines andmedia. The GM-CSF–secreting vaccine celllines GM (mock) and neuGM (neu-specific), the NT2.5 neu-expressing tumor cell line, and the NT2.5B7.1 antigen-present-ing cell line were derived and grown as previously described(4, 10). GM-CSF levels were verified by ELISA, and neu and B7.1levels were verified by flow cytometry biannually. All cell linesused were regularly tested and validated to be Mycoplasmafree; no other authentication assay was performed.

Antibody purification. Hybridoma cells secreting theanti-rat neu mAb 7.16.4 (21) were grown in athymic nudemice, and ascites were collected (Harlan Bioproducts forScience). 7.16.4 was purified over a T-Gel column (PierceBiotechnology) using the Biologic LP purification system(Bio-Rad), dialyzed into PBS, and >90% purity determined bySDS-PAGE analysis using Criterion 4%–15% Tris–HCl Pre-castGels and the Criterion Cell (Bio-Rad). Nonspecific isotype-matched antibody served as the control. Antibodies wereconfirmed endotoxin free by the Limulus amebocyte lysatetest (BioWhittaker).

Murine immunotherapy experiments. Neu mice werechallenged with 5 � 104 NT2.5 tumor cells, and vaccinated3 days later. Vaccine cells were irradiated before s.c. injection inboth hind limbs and the left forelimb. Cyclophosphamide(Bristol-Myers Squibb) at 100 mg/kg and 7.16.4 at 100 mg wereinjected i.p. 1 day before vaccination; 7.16.4 was then giveni.p. weekly. The tumor and vaccine cell doses, and the cyclo-phosphamide and mAb dose and schedule, have been opti-mized previously (10, 20, 22). Mice were monitored for tumoronset and growth twice weekly, and tumors were measured intwo perpendicular dimensions with calipers.

ELISPOTS. CD8þ splenic lymphocytes were purified bynegative selection (Dynal Biotech, Invitrogen). CD8þ T cells(105) were incubated in duplicatewith 104 target cells (NT2.5B7.1cells stimulated with IFNg for 2 days) at 37�C overnight (22) onprecoated IFNg-specific ELISPOT plates and developed accord-ing to themanufacturer's protocols (R&DSystems). IFNg-secret-ing CD8þ T cells were enumerated using the Immunospotcounter (Cellular Technology, LTD). The number of spots incontrol wells was averaged and subtracted from the number ofspots in each well containing both CD8þ T cells and targets.

Statistical analysis. A Student t test was applied to deter-mine the statistical significance of differences between treat-ment groups, with P < 0.05 being significant. Analyses wereperformed using GraphPad Prism, version 3.0a for Macintosh(GraphPad Software). All experiments were repeated at leasttwice, with 5 to 10 mice per group.

Clinical study designThe clinical study was an open-label, single-arm feasibility

study to evaluate the safety and clinical benefit associated withthe administration of a fixed dose of allogeneic GM-CSF–secret-ing breast tumor vaccine cells given in sequence with low-dosecyclophosphamide andweekly trastuzumab. A Simon two-stagedesign (23) was used to evaluate 20 vaccinated patients.

The clinical study was conducted in accordance with theprinciples of Good Clinical Practice and the ethical principlesstated in the Declaration of Helsinki. It was approved by theJohns Hopkins University School of Medicine Institutional

Chen et al.

Cancer Immunol Res; 2(10) October 2014 Cancer Immunology Research950




Review Board, the NIH Recombinant DNA Advisory Com-mittee, and the FDA Center for Biologics Evaluation andResearch.

Patient selectionTwenty-two patients with HER2þ metastatic breast can-

cer were enrolled at the Johns Hopkins Sidney KimmelComprehensive Cancer Center (Baltimore, MD) betweenNovember 14, 2006, and July 13, 2009; 20 patients weretreated on study. Eligible patients had an Eastern Cooper-ative Oncology Group (ECOG) performance status of 0 to 1,and a histologic diagnosis of HER2þ breast cancer by IHC 3þstaining or gene amplification �2.0 by FISH. Prior chemo-therapy must have been completed �28 days before vacci-nation; concurrent endocrine and/or bisphosphonate ther-apy was allowed. Other requirements included a cardiacejection fraction �45%, adequate end-organ function, andnegative HIV and pregnancy tests. Stable treated centralnervous system disease was allowed. Key exclusion criteriaincluded past/current autoimmune disease, non–protocol-specific treatment or parenteral steroids within 28 days ofvaccination, and past/current second malignancy (exceptsuperficial nonmelanoma skin cancer, bladder cancer,tamoxifen-related endometrial cancer cured by hysterecto-my, and cervical carcinoma in situ).

Study plan and interventionEligibility determination. Written informed consent was

obtained from each research participant. Baseline studiesincluded computed tomography of the chest, abdomen, andpelvis; bone scans; complete blood count with differential;chemistry profile; absolute eosinophil count; and echocardio-gram or multiple gated acquisition scan.Treatment plan. Patients received three monthly vacci-

nations, with a boost 6 to 8 months from trial entry. Duringactive vaccination cycles, patients received cyclophosphamide,300 mg/m2 on day �1 and 5 � 108 vaccine cells on day 0, on abackbone of weekly trastuzumab given at the standard dose(2 mg/kg, with a 4 mg/kg loading dose as necessary); trastu-zumab was given on day �1 the week of vaccination. Duringthe interim period between cycles 3 and 4, trastuzumab couldbe given weekly (2 mg/kg) or every 3 weeks (6 mg/kg).Cyclophosphamide-modulated vaccination was given every4 to 6 weeks for three cycles, with a fourth cycle 6 to 8 monthsafter the first cycle. Patients with disease progression weretaken off study.Vaccinations. Vaccine development and manufacturing

have been described in another publication (24). Briefly, theparent cell lines T47D (HER2low) and SKBR3 (HER2high) weregenetically modified by plasmid DNA transfection to se-crete GM-CSF. Clinical lots were prepared from two subclonedcell lines secreting bioactive levels of GM-CSF, 2T47D-V, and3SKBR3-7. On day 0, serum-free, cryopreserved, irradiatedvaccine cells were thawed and mixed to create a HER2þ

vaccine that secreted GM-CSF levels of about 300 ng/106 cellsper 24 hours. Vaccine cells were injected intradermally, evenlydistributed over three lymph node areas. Anesthetic lidocainecream was applied to the injection sites before vaccination.

EndpointsToxicity assessment. Toxicities were graded using the

NCI's Clinical Trials Common Terminology Criteria forAdverse Events Version 3.0 (CTCAE v.3.0). Toxicity monitoringincluded clinical assessment and complete blood counts ondays 3 and 7; chemistry profilesweremeasured before and aftereach cycle and on day 7.

Clinical benefit rate and survival outcomes. The primaryendpoint of clinical benefit was defined as complete responseþ partial response þ stable disease (CR þ PR þ SD) at 6months; clinical benefit at 1 year was also determined. Explor-atory endpoints of progression-free survival and overall sur-vival were defined as time to first disease progression or deathfrom the eligibility date.

Analysis of serum cytokine levels. Serumwas collected atbaseline and after each vaccination, and on days 0, 1, 2, 3, 4, 7,and 14 of each cycle. Serumwas separated fromwhole blood bycentrifugation, and frozen in 1-mL aliquots at �80�C. SerumGM-CSF levels were measured as described previously (11).Serum TGFb was measured by Luminex pre- and postvacci-nation according to the manufacturer's protocols.

Assessment of delayed-type hypersensitivity using HER2peptides. One hundred micrograms each of two MHCclass II HER2 epitopes (p369 and p776; ref. 25), with mutatedk-ras as a negative control, was injected intradermally on theback as previously described (11). Erythema and indurationwere assessed 2 to 3 days after injection.

Delayed-type hypersensitivity (DTH) was considered posi-tive if induration increased by �5 mm from baseline.

Collection, preparation, and cryopreservation of periph-eral blood mononuclear cells. Whole blood (200 mL) wascollected in heparin tubes pre- and postvaccination; 20 mLwhole blood was collected on days 0, 1, 2, 3, 4, 7, and 14.Peripheral blood mononuclear cells (PBMC) were isolated byFicoll-Hypaque gradient centrifugation; interface cells wereharvested and washed twice. PBMCs were resuspended inAIM-V medium (Invitrogen) with 10% dimethylsulfoxide, fro-zen at �80�C for 1 to 2 days, and stored in liquid N2 untilanalysis (8).

Assessment of Treg and myeloid-derived suppressorcells. Treg and myeloid-derived suppressor cell (MDSC)analyses were performed with modifications to publishedmethods (26–28). Briefly, 106 PBMCs were stained for 30minutes at 4�C with: Pacific blue-CD4 (BD Pharmingen),FITC-CD25 (BD Pharmingen), PE-Cy7-CD45RA (eBioscience),PerCP-Cy5.5-CCR7 (BD Pharmingen), APC-CTLA-4 (BD Phar-mingen), FITC-Lineage cocktail (BioLegend), APC-HLA-DR(BD Pharmingen), PE-Cy7-CD33 (eBioscience), and/or PE-CD11b (eBioscience). For Treg analysis, cells were fixed for40 minutes at 4�C and permeabilized with TranscriptionFactor Buffer Set (BD Pharmingen), followed by staining withPE-FoxP3 (BD Pharmingen) for 30 minutes at 4�C. Data wereacquired with a Gallios cytometer (Beckman Coulter) andanalyzed using the FlowJo V7.6.5 software (TreeStar, Inc.).

Assessment of peripheral HER2-specific CD8þ T cells.T-cell analyses were conducted with modifications to pub-lished methods (29, 30). Monocytes were isolated using theMACS CD14 isolation kit (Miltenyi Biotec), and cultured in 6-

Cyclophosphamide- and Trastuzumab-Modulated Vaccination

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well plates (2� 106 cell/mL) in RPMI-1640medium containing5% pooled human serum (PHS), 50 ng/mL GM-CSF (MiltenyiBiotec), and 50 ng/mL IL4 (Miltenyi Biotec). Cells were cul-tured for 48 hours and subsequently incubated for 18 hourswith 10 ng/mL IL1b (Miltenyi Biotec), 10 ng/mL IL6 (MiltenyiBiotec), 10 ng/mL TNFa (Miltenyi Biotec), and 0.5 to 1 mg/mLPGE2 (Sigma-Aldrich). The quality of monocytic dendritic cells(MoDC) was assessed by staining with PE-Cy7-CD80 (BioLe-gend), FITC-CD83 (BioLegend), Pacific blue-CD86 (BioLe-gend), APC-HLA-DR (BD Pharmingen), and/or APC-IL-12 (BDPharmingen). Overlapping peptide mixtures were used tostimulate CD8þ T cells (JPT Peptide Technologies). PepMixHER2 (extracellular and intracellular domains) is a pool of 15-mer peptides that span the entire HER2 protein and overlap by11 amino acids; PepMix CEF Pool and PepMix HIV served aspositive and negative controls, respectively. Peptides wereresuspended in DMSO, stored at �80�C, thawed the day ofassay, and diluted to the required concentration.

Isolated, activated MoDCs were mixed with peptides at1 mg/mL in RPMI-1640 medium containing 5% PHS and incu-bated at 37�C for 2 hours. CD8þ T cells were purified fromPBMCs using the Dynabeads Untouched CD8 T-cell kit (Invitro-gen). HER2-specific CD8þ T cells were indistinguishable fromthe background by flow cytometry directly ex vivo, and were,therefore, stimulated in vitro by coculturing peptide-loadedMoDCs with autologous CD8þ T cells at a ratio of 1:3 in the

presence of IL7 (R&D Systems) and IL2 (R&D Systems) atconcentrations of 10 ng/mL and 10 U/mL, respectively. Ondays 8 and 15, T cells were restimulated with MoDCs. The cellswere refed with IL7 and IL2 every 3 days. The cells were harvest-ed on day 15 after 4 hours of restimulation and assayed by intra-cellular cytokine staining (ICS) using FITC-CD8 (eBioscience),PE-IFNg (BioLegend), APC-TNFa (BioLegend), and PE-Cy7-IL2(BD Pharmingen). Sample data were acquired by a Gallioscytometer (Beckman Coulter) and analyzed using the FlowJoVersion 7.6.5 software. The proportions of polyfunctional CD8þ

effector T-cell subsets were classified by cytokine secretion.

Statistical considerations and data analysisThe trial database was closed on October 16, 2013. A

Simon two-stage design was used (23). Nine patients were tobe enrolled and treated in the first stage. At the end of thefirst stage, if 2 or more patients demonstrated clinicalbenefit, 11 additional patients were to be enrolled andtreated. This design had an a level of 0.08 with a power of0.86 to detect a clinical benefit (CRþ PRþ SD) of 0.45 from anull hypothesis rate of 0.20. At the end of the second stage, if7 or more patients demonstrated clinical benefit, the nullhypothesis was rejected. On the basis of this study design,there was a 44% chance of early stopping if the true responserate was 0.20, and a 4% chance of early stopping if the trueresponse rate was 0.45.

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Figure 1. HER2-specific mAbs combined with HER2-specific vaccination augment tumor-free survival and HER2-specific CD8þ T-cell levels in neu mice.Neu mice (10 per group) were challenged with NT-2.5 tumor cells, and vaccinated 3 days later. A single dose of cyclophosphamide (CY) was given 1 daybefore vaccination in selected mice; weekly HER2-specific mAb (7.16.4) or nonspecific IgG was also started 1 day before vaccination. A, vaccination þ7.16.4. B, vaccination þ cyclophosphamide. C, vaccination þ cyclophosphamide þ 7.16.4. D, splenic T cells were isolated from neu mice at the end oftreatment (3–5 per group). CD8þ T cells were isolated and incubated with NT2.5B7.1 target cells for IFNg ELISPOT analysis. Circles, individual mice;bars, average value. neuGM, 3T3 cells that secrete GM-CSF and express rat HER2 (neu vaccine); GM, 3T3 cells that secrete GM-CSF (control vaccine).

Chen et al.





The study met criteria for full accrual, and data from 20patients were used in the analysis. Tumor responses (CR þ PRþ SD) were determined by RECIST 1.0. Clinical benefit wasestimated using the binomial distribution along with the 95%confidence interval (CI). Continuous data were summarizedusing mean and SD. Categorical data were represented usingpercentage or proportion. Survival probability was estimatedusing the Kaplan–Meier method (31). The CI of median survivaltimewas constructed by themethodof Brookmeyer andCrowley(32). Paired and unpaired Student t tests were used for pre- andpostvaccine comparisons and between-group comparisons. AllP values are reported as two-sided, and all analyses were con-ducted using SAS software (version 9.2 SAS Institute).

ResultsPreclinical modelingNeu mice exhibit preexisting immune tolerance specific for

rat HER2 (neu), and are a stringent model for developing clini-cally relevant cancer immunotherapies (10, 20, 22). We firstevaluated whether the trastuzumab-like mAb 7.16.4 could aug-ment the activity of neu-specific vaccination aloneor sequencedwith immune-modulating doses of cyclophosphamide in thesemice. Treating tumor-bearing neu mice with 7.16.4 and mockvaccination delayed tumor growth relative to neu-targeted

vaccination alone, which is ineffective in this model (Fig. 1).Adding cyclophosphamide or 7.16.4 to neu-specific vaccinationalso delayed tumor outgrowth, increasing tumor-free survivalfrom 0% (vaccine or 7.16.4 alone) to 10% (vaccineþ cyclophos-phamide) and 30% (vaccine þ 7.16.4; Fig. 1A and B). Addingboth cyclophosphamide and 7.16.4 to neu-targeted vaccinationincreased tumor-free survival to about 60% (Fig. 1C). Thisrelative antitumor efficacy was reflected in the numbers oftumor-specific IFNg-secreting CD8þ T cells by ELISPOT, wherethe highest levels were detected in the setting of cyclophos-phamide þ 7.16.4 þ vaccine (Fig. 1D).

Clinical trialPatient characteristics. On the basis of this preclinical

model, we conducted a clinical trial testing cyclophosphamide-modulated vaccination with trastuzumab in patients withHER2þ metastatic breast cancer. The intervention and datacollection schedules are shown in Fig. 2. Twenty eligiblepatients were vaccinated, with an age range of 34 to 69 years(Table 1). All had HER2þ metastatic disease, 13 had estrogenreceptor–positive and/or progesterone receptor–positive dis-ease. The mean disease-free interval to relapse from firstdiagnosis was 32 months (range, 0–130 months); 7 (35%)patients presented with metastatic breast cancer at diagnosis.

Overall trial schema

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Figure 2. Study schema. Patients received up to four vaccination cycles. Cyclophosphamide (C) was given on day �1, and vaccine on day 0. Trastuzumab(T) was givenweekly during active vaccine cycles, and timed to coincidewith cyclophosphamide administration on day�1. During the interim period betweencycles 3 and 4, trastuzumab could be given weekly or every 3 weeks as per the standard of care. GM-CSF levels and immunity were measured as indicated.Skin biopsies on days þ3 and þ7 were from the largest vaccine site.






Eighteen patients (90%) received prior trastuzumab formetastatic breast cancer, and 3 patients received priortrastuzumab as adjuvant therapy. Eleven (55%) patientswere on concurrent endocrine therapy, and the majority(70%) received concurrent bisphosphonate therapy for skel-etal metastasis. The mean and median sums of the longesttumor diameter at study entry were 40.7 mm and 24.5 mm,respectively.

All patients (100%) received at least one vaccination, 15(75%) received at least three vaccinations, and 8 (40%) receivedall four vaccinations. Two patients came off-study for reasonsunrelated to disease progression, one after three vaccine cyclesdue to relocation, and another after two vaccine cycles due to

study demands. A third off-study event was related to both avaccine-related serious adverse event and simultaneous dis-ease progression. All other off-study events before cycle 4 weredue to breast cancer progression.

Toxicity. The most common adverse events were localvaccine site reactions, including erythema, induration, pruritus,and/or discomfort (Table 2). These self-limited local reactionsoccurred in all individuals, and increased in intensity butnot duration with subsequent vaccination cycles. The mostcommon systemic adverse events were fatigue, urticaria, pru-ritus, and fever. Seven patients (35%) developed urticariadistant from the vaccine site; of these, 3 (15%) developed grade2–3 urticaria that required parenteral intervention with diphen-hydramine and/or steroids. Of these, one was a vaccine-relatedserious adverse event requiring overnight hospitalizationand steroid treatment. No evidence of clinically significantautoimmunity was detected. Despite the use of trastuzumab,no decrement in cardiac function was detected (data notshown).

SerumGM-CSFpharmacokinetics. SerumGM-CSF levelswere measured as an indicator of the vaccine's life span fol-lowing injection. Serum GM-CSF levels peaked at 48 hours,with peak levels persisting across all four cycles as previouslyobserved (11). Trastuzumab did not alter the timing of the peakor the kinetics of GM-CSF decay (data not shown).

Clinical outcomes. The clinical benefit rates of weeklytrastuzumab with cyclophosphamide-modulated vaccinationat 6 months and 1 year were 11 of 20 (55%; 95% CI, 32%–77%;P ¼ 0.013) and 8 of 20 (40%; 95% CI, 19%–64%), respectively.One partial response and no complete responses wereobserved. Overall median progression-free survival and overallsurvival were 7 months (95% CI, 4–16) and 42 months (95% CI,22–70), respectively (Fig. 3A). The 5-year survival rate was 6 of20 (30%, 95% CI, 12–54).

Immunologic analyses. We previously demonstrated thefeasibility of using DTH responses specific for HER2-derivedpeptides and HER2-specific antibody responses by ELISA asimmune response biomarkers for this cell-based vaccine (11).Both because our preclinical data demonstrate a correlation ofHER2-specific CD8þ T cells with tumor-free survival in vacci-nated neu mice, and because trastuzumab confounds theassessment of HER2-specific antibody responses, we assessedHER2-specific DTH and measured HER2-specific CD8þ T cellsby ICS as biomarkers of vaccine-induced immunity in thisstudy. HER2-specific DTH developed in 7 of 20 patients (35%;95%CI, 15%–59%); 4 of these (57%; 95%CI, 18%–90%) showed aclinical benefit. There was a trend toward longer progression-free survival and overall survival for patients who developedHER2-specific DTH following vaccination (Fig. 3B and C),although this was not statistically significant due to the smallsample size of this study. Median progression-free survival forDTH-positive versus DTH-negative patients was 9 months(95% CI, 4–46) versus 4 months (95% CI, 3–14; P ¼ 0.58).Median overall survival durations for DTH-positive versusDTH-negative patients were 70 months (95% CI, 40–70) versus29 months, respectively (95% CI, 13—not reached; P ¼ 0.11).Larger randomized studies are required to rigorously evaluatethe possible relationship between DTH and survival.

Table 1. Patient characteristics

CharacteristicsNumber ofpatients %

Total number of patients 22 enrolled20 vaccinated20 evaluable

Age, yMedian 52Range 34–69

ER-positive or PR-positive tumor 13 65HER2-positive tumor 20 100Metastatic disease at diagnosis 7 35DFI to relapse (months)a

Mean 32Median 28Range 0–130

Sites of distant metastasisBone 14 70Liver 5 25Lung 4 20Lymph node (distant) 3 15Brain 1 5

Sum of the longest tumordiameter at study entryMean 40.7 mmMedian 24.5 mm

Prior lines of chemotherapy formetastatic diseaseMedian 1Range 0–6Mean 1.25

Prior trastuzumab therapyAdjuvant 3 15Metastatic 18 90

Prior lapatinib therapy 2 10Concurrent endocrine therapy 11 55Concurrent bisphosphonate therapy 14 70

Abbreviations: DFI, disease-free interval; ER, estrogenreceptor; PR, progesterone receptor.a13 patients who did not present with metastatic disease atdiagnosis.

Chen et al.





HER2-specific CD8þ T-cell function before and after vaccina-tion was assessed in vitro by ICS using commercially availablepeptide mixtures containing overlapping epitopes spanningthe entire HER2 protein, a representative analysis is shownin Fig. 4A. Both the average level of CD8þ T cells secreting IFNg(0.501% versus 0.785%, P < 0.0001), IL2 (0.567% versus 0.683%,P ¼ 0.0006) and TNFa (0.751% versus 1.525%, P < 0.0001),and the proportion of polyfunctional CD8þ T cells secretingmore than one cytokine (0.023% versus 0.735%, P < 0.0001)increased across the vaccination cycles (Fig. 4B and C). Inexploratory analyses, increased polyfunctional CD8þ T cellswere possibly associated with longer progression-free survival(HR, 0.44; 95% CI, 0.2–0.98; P ¼ 0.04) and OS (HR, 0.43; 95% CI,0.13–1.36; P ¼ 0.15).We also assessed peripheral markers of immune suppres-

sion by FACS (Treg and MDSC) and Luminex (TGFb).Although an association between decreased TGFb levelsand immunity in patients treated with a HER2 peptidevaccine and concurrent trastuzumab was reported previ-

ously (33), we found no difference in serum TGFb levelsbefore and after vaccination (43,125 versus 46,936 pg/mL,P¼ 0.21). Tregs decreased across all vaccine cycles, and bothoverall and CTLA-4þ Tregs decreased from baseline topostvaccine cycle 1; MDSCs also decreased from baselineto postvaccine cycle 1 (Fig. 5). Exploratory analyses sug-gested that neither baseline nor decreased levels of periph-eral Tregs were associated with survival benefit. However,decreased numbers of peripheral MDSCs were possiblyassociated with both longer progression-free survival(HR, 0.5; 95% CI, 0.29–0.85; P ¼ 0.010) and overall survival(HR, 0.52; 95% CI, 0.31–0.89; P ¼ 0.016) baseline levels ofMDSCs were not. To further explore possible associationsbetween immunologic biomarkers and survival, a subgroupanalysis of 12 patients was performed comparing 6 indivi-duals with OS � 24 months with 6 individuals with OS � 60months. Both decreased MDSCs and increased polyfunc-tional T cells following vaccination were possibly associatedwith OS, with P values of 0.012 and 0.028, respectively.

Table 2. Summary of treatment-related adverse events

Adverse eventAll grades Grade 3 or 4

Patients, n % Patients, n %

Local vaccine site reactionsErythema/induration 20 100 0 0Pruritus 20 100 0 0Pain/soreness/tenderness 17 85 0 0Local rash at/near vaccine site 7 35 1 5Blister formation 5 25 0 0Hyperpigmentation 4 20 0 0Ecchymosis (bruising) 3 15 0 0Local edema 2 10 0 0Vaccine site flare 2 10 0 0Groin tightness 1 5 0 0

Systemic toxicitiesFatigue 8 40Urticaria 7 35 2 10Pruritus (distant from vaccine site) 6 30 1 5Fever 5 25Flu-like symptoms/myalgia 4 20Lymphadenopathy 4 20Abdominal pain 3 15 1 5Rash (distant from vaccine site) 3 15 1 5Achiness/malaise 3 15Chills 3 15Dizziness 2 10Anorexia 1 5Erythema (distant from vaccine site) 1 5Headache 1 5Nausea 1 5Arm pain 1 5Cancer site pain 1 5Leg tenderness 1 5

NOTE: Data are given as any incident per patient, for a maximum of 20 counts per event.






DiscussionThe results from this study of a HER2þ GM-CSF–secreting

breast tumor vaccine with low-dose cyclophosphamide andweekly trastuzumab supports the following seven conclusions.First, in the clinically relevant neu transgenic mouse, addingboth cyclophosphamide and 7.16.4 (a trastuzumab-like mAb)to vaccination increases tumor-free survival and IFNg-secret-

ing tumor-specific CD8þ T cells relative to adding eithercyclophosphamide or 7.16.4 alone to vaccination. Second, upto four sequential cyclophosphamide-modulated vaccinationsare safe and well tolerated in patients with HER2þ metastaticbreast cancer receiving weekly trastuzumab. Third, this immu-notherapy was associated with a 6-month clinical benefit rateof 55%, and median progression-free survival and overallsurvival of 7 months and 42 months, respectively. This studymet its primary endpoint, as the target therapeutic outcomewas a 6-month clinical benefit rate of 45%. Fourth, thiscombination immunotherapy induced new or increasedHER2-specific T-cell immunity in patients with HER2þ met-astatic breast cancer as measured by HER2-specific DTH andCD8þ T cells by ICS. There was a trend toward longerprogression-free survival and overall survival in patientswho developed HER2-specific DTH relative to those whodid not. Fifth, polyfunctional HER2-specific CD8þ T cellsprogressively expanded across the vaccination cycles. Sixth,both Tregs and MDSCs decreased across the vaccinationcycles. Finally, hypothesis-generating analyses suggest thatboth decreased MDSCs and a greater proportion of poly-functional HER2-specific CD8þ T cells may be associatedwith longer progression-free survival and overall survival.A comparative trial powered for survival will be required torigorously evaluate these associations.

Trastuzumab has revolutionized the management ofHER2þ breast cancer, and remains the major backbone oftherapy for early- and late-stage disease (1, 2, 14). However,intrinsic and acquired trastuzumab resistance remains a sig-nificant limitation to its clinical utility. In patients, trastuzu-mab-based chemotherapy is associatedwith increased levels ofHER2-specific antibodies and T cells (17), and intratumorallymphoid nodules develop in the setting of neoadjuvant tras-tuzumab-based chemotherapy (18). These observations sug-gest that leveraging the ability of trastuzumab to activateadaptive immunity could increase its efficacy. One clinicalstudy combined a peptide vaccine with weekly trastuzumab,demonstrating increased levels of HER2-specific T cells, epi-tope spreading, and decreased serum levels of the immune-suppressive cytokine TGFb (33). We showed that HER2-spe-cificmAbs alone induce low levels of neu-specific T cells in neutransgenic mice. The addition of HER2-specific mAbs to neu-specific GM-CSF–secreting vaccination more effectively aug-ments neu-specific CD8þ effector T-cell function and prolongstumor-free survival in these mice (15). We further showed thatthe trastuzumab-like mAb 7.16.4 augments locoregionalimmune priming through binding the Fc receptor of dendriticcells in similarly vaccinated neumice, augmenting CD8þ T-cellproliferation, cytokine secretion, and central memory devel-opment (16). Here, we build on these prior studies to show thatthe combination of low-dose cyclophosphamide, trastuzumab,and a GM-CSF–secreting HER2þ allogeneic cell-based vaccineis safe, bioactive, and associated with clinical benefit inpatients with HER2þ metastatic breast cancer.

A significant challenge in optimizing combination tumorcell vaccine-based immunotherapy is the lack of clear biomar-kers for assessing immune response and drug–drug interac-tions. We previously demonstrated the feasibility of using DTH

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Figure 3. Progression-free survival (PFS) and overall survival (OS) ofvaccinated patients. A, Kaplan–Meier curves of the percentage PFS andOS in months (mPFS and mOS) from the time of eligibility determination(n ¼ 20). B, Kaplan–Meier curves of PFS stratified by DTH-positive andDTH-negative patients. C, Kaplan–Meier curves of OS stratified byDTH-positive and DTH-negative patients.

Chen et al.





Figure 4. Vaccination elicits HER2-specific CD8þ T-cell responses in patients with metastatic breast cancer as measured by ICS. A, HER2-specific CD8þ

T cells that secrete IFNg , TNFa, or IL2 are detectable by ICS in vaccinated patients. A representative patient is shown. B, the percentage of HER2-specificCD8þ T cells that secrete IFNg , TNFa, or IL2 increases across the vaccination cycles. The connected points represent the mean and SD of values acrossthe patient population, with three replicates per patient. C, the proportion of polyfunctional HER2-specific CD8þ T cells that secrete more than onecytokine increases across the vaccination cycles.






responses specific for twoMHCClass II HER2-derived peptidesand HER2-specific antibody responses by ELISA beforeand after vaccination as immune response biomarkers forthis cell-based vaccine (11). Because the use of trastuzumabhere confounds the assessment of HER2-specific antibodyresponses, we measured HER2-specific DTH and peripheralHER2-specific CD8þ T cells by flow cytometry. We found thatHER2-specific DTH developed in 7 of 20 patients (35%); 4 ofthese patients showed a clinical benefit. In addition, there wasa trend toward longer progression-free survival and overallsurvival in patients who developedHER2-specific DTH relativeto those who did not. These data are consistent with othersmall clinical trials that have shown an association betweenenhanced tumor-specific DTH and survival (34–37). Cyto-kine-secreting CD8þ T cells also increased with vaccination

in patients with metastatic breast cancer. While vaccine-induced CD8þ T cells from neu mice could be detected byELISPOT directly ex vivo, vaccine-induced CD8þ T cells frompatients with metastatic breast cancer were detected by flowcytometry only after in vitro stimulation. This differentialsensitivity in detecting vaccine-induced CD8þ T cells islikely explained by differences in the host (transgenic neumice are genetically homogenous, whereas patients withmetastatic breast cancer are genetically and biologicallyheterogeneous), the vaccine (the murine vaccine specificallytargets HER2, whereas the human vaccine delivers multipletumor antigens where responses to HER2 are used as asentinel measure of vaccine activity), the T cells (assayedfresh from mice as opposed to frozen from patients withmetastatic breast cancer), and the assays (ELISPOT is more

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Figure5. Markers of immune suppression in the peripheral blooddecrease after vaccination. A,CD4þFoxP3þTregs decrease across the vaccination cycles. B,CD4þ FoxP3þ Tregs decrease from baseline after one vaccination cycle; P ¼ 0.043. C, CD4þFoxP3þ CTLA-4þ Tregs decrease from baseline after onevaccination cycle; P ¼ 0.01. D, HLA-DRneg/linlo/CD33þ/CD11bþ MDSCs; P < 0.0001.

Chen et al.





sensitive than flow cytometry). We used ICS by flow cyto-metry to rapidly evaluate HER2-specific CD8þ T cells thatsecrete one or more cytokines in patients with metastaticbreast cancer .Polyfunctional antigen-specific T cells may be indicators of

disease control and/or survival in patients with chronic infec-tions and cancer (38–40). Polyfunctional T cells secrete mul-tiple relevant cytokines (IFNg , TNFa, and IL2) that reflect thequality of the immune response as a more informative corre-late of sustained protective immunity than T cells that secreteonly a single cytokine (IFNg or TNFa). Durable T-cell poly-functionality correlates with long-term disease control in HIV(41) and malaria patients (42), but has been less studied invaccinated patients with cancer. A telomerase peptide vac-cine given with temezolomide to patients with metastaticmelanoma induced polyfunctional T cells (43). Here, wedemonstrate the progressive expansion of polyfunctionalHER2-specific T cells across vaccination cycles, where agreater proportion of polyfunctional CD8þ T cells may beassociated with longer progression-free survival and overallsurvival. A comparative clinical study powered for survival isrequired to formally evaluate this hypothesis.Tregs and MDSCs are significant mediators of immune

tolerance and suppression in patients with cancer. We showedpreviously that low-dose cyclophosphamide depletes Tregs inneu mice, facilitating the vaccine-mediated recruitment andactivation of latent, high-avidity tumor-specific T cells andtumor rejection (10, 12, 13). Here, we demonstrate a decline inperipheral Tregs and MDSCs in vaccinated patients withmetastatic breast cancer. Several clinical trials have exploreddepleting Tregs in the setting of vaccination for cancer, withenhancement of antigen-specific immune responses to vacci-nation (11, 44–46). Notably, a randomized phase II studyevaluating a multipeptide renal cell carcinoma vaccine aloneor sequenced with low-dose cyclophosphamide demonstratedthat cyclophosphamide-modulated vaccination reduced Tregsand induced T-cell responses specific for multiple peptideantigens, increasing the OS of vaccinated patients (46). Anassociation between levels of circulating MDSCs and immuneresponse or clinical outcomes has been reported in severalimmunotherapy trials, and these results are consistent withour findings (46–49).This combination immunotherapy was safe. Given the

known cardiac toxicity of trastuzumab (14), cardiac functionwas assessed every 3 months on study. No patient developed asignificant drop in left ventricular ejection fraction. These datasuggest that the addition of cyclophosphamide-modulatedvaccination to weekly trastuzumab did not increase cardiactoxicity, although the sample size is limited. Notably, urticariawas observed in a significant number of patients in this study; 3patients required parenteral diphenhydramine and/or ster-oids. It remains unclear if urticaria may be associated withhigher levels of immunity or clinical benefit. We observedclinical benefit rates of 55% at 6 months and 40% at 1 year.The median progression-free survial in this study was 7months, the median overall survival was 42 months, and the5-year survival rate was 30%. Although the sample size is small,these clinical results compare favorably with those from other

trials of HER2-directed therapy for metastatic HER2þ breastcancer that do not include cytotoxic chemotherapy. Theseminal trials of single-agent trastuzumab for the first-lineand second/third-line treatment of patients with metastaticdisease showed clinical benefit rates of 48% and 56% at6 months (50, 51); median overall survival in these seminaltrials was 24 and 13 months. More recently, novel mAb-basedHER2-directed therapies were approved for metastatic HER2þ

breast cancer progressing on prior trastuzumab therapy (2).Concurrent pertuzumab and trastuzumab therapy is associ-ated with a clinical benefit rate of 50%, and a median progres-sion-free survival of 5.5 months (52) and ado-trastuzumabemtansine therapy is associated with a median progression-free survival and overall survival of 9.6 months and 30.9months, respectively (53).

In conclusion, this allogeneic GM-CSF–secreting breasttumor vaccine is safe and bioactive, and confers clinical benefitwhen sequenced with low-dose cyclophosphamide and stan-dard trastuzumab in patients with HER2þ metastatic breastcancer. It induces HER2-specific immunity as measured byDTH and polyfunctional CD8þ T cells, two measures of a high-quality immune response.We observed a clinical benefit rate of55% at 6 months, with progression-free survival and overallsurvival of 7 months and 42 months, respectively. Since thisstudy began, two additionalmAb-based therapies with survivalbenefit were approved for metastatic HER2þ breast cancer(pertuzumab and ado-trastuzumab emtansine). The data pre-sented here are encouraging relative to results with otherHER2-directed combination therapies, including these newagents. Further investigation of GM-CSF–secreting vaccinesin combination with low-dose cyclophosphamide and HER2-specific mAb therapies is warranted.

Disclosure of Potential Conflicts of InterestE.M. Duus is the Director of Clinical Research at Helsinn Therapeutics.

V. Stearns reports receiving a commercial research grant from MedImmune.L.A. Emens receives research funding from Genentech, Inc. L.A. Emens and E.M.Jaffee receive research funding from Roche, Inc. Under a licensing agreementbetween Aduro, Inc. and the Johns Hopkins University, the University and E.M.Jaffee are entitled to milestone payments and royalty on sales of the GM-CSF–secreting breast cancer vaccine. The terms of these arrangements are beingmanaged by the Johns Hopkins University in accordance with its conflict ofinterest policies. L.A. Emens is a member of the Cellular, Tissue and GeneTherapies Advisory Committee at the FDA. Nopotential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: E. Garrett-Mayer, L.A. EmensDevelopment of methodology: G. Chen, E. Garrett-Mayer, L.A. EmensAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Chen, R. Gupta, S. Petrik, J.M. Leatherman, N.E.Davidson, M. Berg, J. Fetting, E.M. Duus, S. Atay-Rosenthal, A.C.Wolff, V. Stearns,E.M. Jaffee, L.A. EmensAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Chen, R. Gupta, J.M. Asquith, E.M. Duus, S. Atay-Rosenthal, X. Ye, L.A. EmensWriting, review, and/or revision of the manuscript: R. Gupta, J.M. Leather-man, M.M. Daphtary, E. Garrett-Mayer, N.E. Davidson, J. Fetting, E.M. Duus,S. Atay-Rosenthal, X. Ye, A.C. Wolff, V. Stearns, E.M. Jaffee, L.A. EmensAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M. Laiko, J.M. Leatherman,J.M. Asquith, M.M. Daphtary, K. Hirt, J.N. Uram, L.A. EmensStudy supervision: J.M. Leatherman, T. Dauses, L.A. EmensOther (regulatory affairs): M.M. DaphtaryOther (review of imaging at baseline and evaluation of disease progres-sion and response to therapy): S. Atay-Rosenthal






AcknowledgmentsThe authors thank Mark Greene at the University of Pennsylvania for

providing the 7.16.4 hybridoma.

Grant SupportThisworkwas supported by theDepartment of Defense Clinical Translational

Research Award W81XWH-07-1-0485 (to L.A. Emens), Genentech, Incorporated(to L.A. Emens), and in part by American Cancer Society RSG CCE 112685 (to L.A.Emens), the Specialized Programs in Research Excellence (SPORE) in BreastCancer P50CA88843 (to N.E. Davidson, E.M. Jaffee, and L.A. Emens), and theRapid Access to Investigational Drugs (RAID) Program of the National CancerInstitute/NIH (to L.A. Emens). It was also supported by funding from the

Gateway Foundation (to L.A. Emens), the Safeway Foundation (to L.A. Emens),Avon Foundation (to E.M. Jaffee and L.A. Emens), and in part by the JohnsHopkins University Institute for Clinical and Translational Research, Climb forHope, and the Josephine A. Peiser Foundation. E.M. Jaffee is the Dana and Albert"Cubby" Professor of Oncology.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received April 1, 2014; revised July 25, 2014; accepted July 30, 2014;published OnlineFirst August 12, 2014.

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2014;2:949-961. Published OnlineFirst August 12, 2014.Cancer Immunol Res Gang Chen, Richa Gupta, Silvia Petrik, et al. Metastatic Breast Cancer

+Secreting Breast Tumor Vaccine for HER2−Allogeneic GM-CSF A Feasibility Study of Cyclophosphamide, Trastuzumab, and an

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