Safety and Preliminary Evidence of Biologic Efﬁcacy of a ... · The observation that direct administration of recombi-nant DNA can generate potent immune responses in rodents established

Cancer Therapy: Clinical

Safety and Preliminary Evidence of Biologic Efficacy of aMammaglobin-A DNA Vaccine in Patients with StableMetastatic Breast Cancer

Venkataswarup Tiriveedhi1,2, Natalia Tucker1, John Herndon1, Lijin Li1, Mark Sturmoski1, Matthew Ellis3,4,Cynthia Ma3,4, Michael Naughton3,4, A. Craig Lockhart3,4, Feng Gao4,5, Timothy Fleming1,4,Peter Goedegebuure1,4, Thalachallour Mohanakumar1,4,6, and William E. Gillanders1,4

AbstractPurpose: Mammaglobin-A (MAM-A) is overexpressed in 40% to 80% of primary breast cancers.

We initiated a phase I clinical trial of a MAM-A DNA vaccine to evaluate its safety and biologic

efficacy.

Experimental Design: Patients with breast cancer with stable metastatic disease were eligible for

enrollment. Safety was monitored with clinical and laboratory assessments. The CD8 T-cell response was

measured by ELISPOT, flow cytometry, and cytotoxicity assays. Progression-free survival (PFS) was

described using the Kaplan–Meier product limit estimator.

Results: Fourteen subjects have been treated with the MAM-A DNA vaccine and no significant adverse

events have been observed. Eight of 14 subjects were HLA-A2þ, and the CD8 T-cell response to vaccination

was studied in detail. Flow cytometry demonstrated a significant increase in the frequency of MAM-A–

specific CD8 T cells after vaccination (0.9% � 0.5% vs. 3.8% � 1.2%; P < 0.001), and ELISPOT analysis

demonstrated an increase in the number of MAM-A–specific IFNg-secreting T cells (41 � 32 vs. 215 � 67

spm; P < 0.001). Although this study was not powered to evaluate progression-free survival (PFS),

preliminary evidence suggests that subjects treated with the MAM-A DNA vaccine had improved PFS

compared with subjects who met all eligibility criteria, were enrolled in the trial, but were not vaccinated

because of HLA phenotype.

Conclusion: The MAM-A DNA vaccine is safe, capable of eliciting MAM-A–specific CD8 T-cell

responses, and preliminary evidence suggests improved PFS. Additional studies are required to define

the potential of the MAM-A DNA vaccine for breast cancer prevention and/or therapy. Clin Cancer Res;

20(23); 5964–75. �2014 AACR.

IntroductionProgress in basic and translational immunology has

confirmed the importance of the immune system in cancerprevention and has renewed interest in vaccine therapy for

cancer (1). DNA vaccines are safe, well tolerated, and cantypically be given in an ambulatory facility (2). Althoughbreast cancer is commonly thought to be less immunogenicthan melanoma or renal cell cancer, there is increasingevidence of a crosstalk between the immune system andbreast cancer, and this crosstalk strongly suggests that suc-cessful development of a breast cancer vaccine could have aclinical impact. Evidence of this crosstalk includes theclinical significance of immune infiltrates in breast cancer(3), clear evidence of preexisting immune responses toseveral breast cancer antigens including HER2/neu (4–6),MUC1 (7), andMAM-A (8–10), the increased prevalence ofregulatory T cells in breast cancer patients (11), and upre-gulation of inhibitory molecules of the CD28 receptorfamily on breast cancer–specific T cells (12). Most patientswithbreast cancer are diagnosedwith local–regional diseaseand typically have no evidence of disease after standardtreatment modalities, providing a window-of-opportunityto generate effective antitumor immune responses to pre-vent recurrent disease (13). Peoples and colleagues recentlyconfirmed the potential of breast cancer vaccine therapy in

1Department of Surgery, Washington University School of Medicine, St.Louis, Missouri. 2Department of Biological Sciences, Tennessee StateUniversity, Nashville, Tennessee. 3Department of Medicine, WashingtonUniversity School of Medicine, St. Louis, Missouri. 4The Alvin J. SitemanCancer Center at Barnes-Jewish Hospital and Washington UniversitySchool ofMedicine,St. Louis,Missouri. 5Division ofBiostatistics,Washing-ton University School of Medicine, St. Louis, Missouri. 6Department ofPathologyand Immunology,WashingtonUniversitySchool ofMedicine, St.Louis, Missouri.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: William E. Gillanders, Department of Surgery,Washington University School of Medicine, 660 South Euclid Avenue, POBox 8109, Saint Louis, MO 63110. Phone: 314-747-0072; Fax: 1-314-454-5509; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-14-0059

�2014 American Association for Cancer Research.

ClinicalCancer

Research

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this clinical context, demonstrating that administration of aHER2/neu peptide vaccine was associated with a survivaladvantage in a prospective study of patients with node-positive breast cancer with no evidence of disease (14).Taken together, the dynamic interaction between breastcancer and the immune system and preliminary evidenceof the efficacy of first-generation breast cancer vaccinesprovide strong rationale for the clinical evaluation of breastcancer vaccine strategies.MGBA was first identified using a differential screening

approach directed at the isolation of novel human breastcancer–associated genes (15).MGBA encodesMAM-A, a 10-kDa glycoprotein that is related to a family of epithelialsecretory proteins. Of note, MAM-A has several uniqueproperties that make it an exceptional target for breastcancer vaccine therapy. First, MAM-A is expressed almostexclusively in breast cancer (16–19). Second, MAM-A isoverexpressed in40% to80%ofprimary breast cancers (20–24).OverexpressionofMAM-A in a significant percentage ofbreast cancer suggests that many patients with breast cancerare likely to be candidates for vaccine therapy and is par-ticularly relevant for the development of vaccine strategiesfor the prevention of breast cancer. Third, MAM-A over-expression is evident in noninvasive, invasive, and meta-static breast cancer (21). This consistency of expression inbreast cancers confirms that MAM-A is an attractive targetfor vaccine therapy. Finally, we have demonstrated thatMAM-A is capable of eliciting an immune response inpatients with breast cancer (8, 9, 25, 26), and that a DNAvaccine targeting MAM-A is capable of successfully gener-ating breast cancer immunity in preclinical models (10).The observation that direct administration of recombi-

nant DNA can generate potent immune responses inrodents established the field of DNA vaccines in the early1990s (27). Since that time, DNA vaccines have remainedan area of intense research interest, and vaccines targetinginfectious disease and cancer have progressed into clinicaltrials. DNA vaccination offers several potential advantages.First, the presence of the full-length cDNAprovidesmultiplepotential epitopes, thus avoiding the need for patient selec-

tion based on MHC restriction. Second, bacterial plasmidDNA contains immunostimulatory unmethylated CpGmotifs that may act as potent immune adjuvants (28,29). Finally, DNA vaccines are relatively easy to preparewith high purity and high stability relative to proteins andother biologic agents, facilitating clinical translation of thisplatform, particularly in early-phase clinical trials. In clin-ical trials of infectious disease and cancer, DNA vaccinestrategies have been shown to be safe and effective indeveloping immune responses to malaria (30), HIV (31),and prostate cancer (32). Several reviews have recently beenpublished summarizing progress in the field (2, 33, 34).

To explore the potential of a novel MAM-A DNA vaccine,we initiated an open-label phase I clinical trial in patientswithmetastatic breast cancer. Patientswith stablemetastaticdisease were eligible for enrollment. Subjects were treatedwith three intramuscular injections of 4mg plasmid at one-month intervals using an FDA-approved carbon dioxide–powered jet delivery device. Safety was closely monitoredwith eight or more clinical and laboratory assessments inthe first 24 weeks of the trial. The immune response to thevaccine was measured by ELISPOT analysis, multiparam-eter flow cytometry, and cytotoxicity assays. Evidence ofbiologic efficacy was assessed by measuring progression-free survival (PFS).

Materials and MethodsStudy rationale and objectives

We recently initiated a single-institution, open-label,phase I clinical trial of a novel MAM-A DNA vaccine inpatients with breast cancer with stable metastatic disease(ClinicalTrials.gov identifier: NCT00807781). The primaryobjective of the trial was to evaluate the safety of theMAM-ADNA vaccine. Secondary and exploratory objectives includ-edmeasuring theCD8 T-cell response to the vaccine and theimpact on PFS. The MAM-A DNA vaccine is composed of aclosed circular DNA plasmid based on the pING parentalvector and is designed to express the human MAM-A breastcancer–associated antigen under a strong viral promoter(Fig. 1).

Study participantsThe phase I clinical trial was approved by the Siteman

Cancer Center Protocol Review and Monitoring Commit-tee and the Washington University School of MedicineHuman Studies Committee. A written informed consentwas obtained from all subjects before enrollment/partic-ipation in the study. Men or women 18 years or older withmetastatic breast cancer were eligible for enrollment. Eli-gible subjects had metastatic breast cancer that had beenstable for at least 30 days after the last dose of chemother-apy, or that had been stable for at least 30 days onendocrine therapy, documented adequate organ and mar-row function, ECOG status of less than or equal to 2, andnegative urine or serum b-HCG pregnancy test for womenof reproductive age. Subjects were considered ineligible ifthey had received an investigational drug within 30 days of

Translational RelevanceMAM-A is an important breast cancer–associated anti-

genwith exquisite tissue specificity.We initiated aphase Iclinical trial to evaluate the safety and efficacy of aMAM-A DNA vaccine. Patients with breast cancer with stablemetastatic disease were eligible. We demonstrate that (i)the MAM-A DNA vaccine is safe, with no significantadverse events observed, (ii) there is a significant increasein the frequency and number of MAM-A–specific CD8 Tcells after vaccination, and (iii) preliminary evidencesuggests improved progression-free survival. Additionalstudies are required to define the clinical potential of aMAM-A DNA vaccine for breast cancer treatment orprevention.

Phase I Clinical Trial of Mammaglobin-A DNA Vaccine

www.aacrjournals.org Clin Cancer Res; 20(23) December 1, 2014 5965



enrollment, had known brain metastases, known allergy,or history of serious adverse reaction to vaccines, autoim-mune disease requiring management with immunosup-pression, or history of failing greater than two chemother-apy regimens for metastatic disease. After enrollment, theprimary breast cancers of the subjects were evaluated byimmunohistochemistry (IHC) for MAM-A expression(only subjects with MAM-Aþ cancers were eligible forvaccination), and their HLA phenotype was determined

(initially, only subjects with HLA-A2 and/or HLA-A3 wereeligible for vaccination to facilitate immunemonitoring asimmunodominant epitopes have been identified for thesealleles; refs. 8–10). Screen failures were not vaccinated, butwere followed for progression of disease. Subjects who hadscreen failures based on HLA phenotype, withdrawal ofconsent, and inability to perform MAM-A immunohisto-chemical staining were considered to be appropriate forinclusion in a comparator group.

Figure 1. The MAM-A DNA vaccineis a plasmid DNA vaccine designedto drive MAM-A expression with astrong viral promoter. The pINGparental vector contains thefollowing elements: (i) a eukaryoticpromoter and enhancer from theTowne strain of CMV; (ii) apolylinker region to facilitatecloning; (iii) donor and acceptorsplice sites and a poly adenylationsignal sequence derived from thebovine growth hormone gene; (iv)the ColE1 origin of replication and(v) a gene conferring kanamycinresistance.

Tiriveedhi et al.

Clin Cancer Res; 20(23) December 1, 2014 Clinical Cancer Research5966



Vaccination scheduleStudy subjects were vaccinated with intramuscular injec-

tions of 4mgplasmidDNAvaccine at day1 (week1), day29� 7 (week 4), and day 57� 7 (week 8) with at least 21 daysbetween vaccinations. All vaccinationswere given intramus-cularly using an FDA-approved carbon dioxide–powered jetdelivery device (Needle Free Biojector 2000).

Safety monitoring and study proceduresSafety was closely monitored after vaccination with eight

or more clinical and laboratory assessments in the first 24weeksof the trial. After each vaccination, study subjectswerecarefully observed for 60 minutes. Vital signs were moni-tored and adverse events were recorded. The injection sitewas inspected for evidence of a local reaction. Subjects weregiven a "Diary Card" where they recorded temperature andsymptoms daily for 5 days. Laboratory tests performed onthe day of vaccination included complete blood count,comprehensive metabolic panel, and serum or urine preg-nancy test (in women of childbearing potential). Patientswere followed actively for 52 weeks after vaccination, andthen until disease progression or death, whichever occurredfirst. Toxicity was graded according to the National CancerInstitute Common Terminology Criteria for Adverse Eventsversion 3.0. Subjects had standard-of-care clinical assess-ments and imaging studies (CT scans) during follow-up toassess tumor stability.Peripheral blood specimens were obtained for immune

monitoring and other correlative studies before vaccinationand at serial time points after vaccination. Peripheral bloodmononuclear cells (PBMC) were isolated from heparinizedblood by Ficoll–Hypaque density gradient centrifugation(Pharmacia) and stored at �135�C until evaluation. TheCD8 T cells were isolated from PBMC by positive selectionusing a MACS Bead Isolation Kit (Miltenyi Biotec Inc.;ref. 35).

Breast cancer cell lines and cell cultureThe breast cancer cell lines, UACC-812 (MAM-Aþ/HLA-

A2þ), MCF-7 (MAM-A�/HLA-A2þ), MDA-MB-415 (MAM-Aþ/HLA-A2�), and MDA-MB-134 (MAM-A�/HLA-A2�),were obtained from the ATCC. The cell lines were orderedin 2004 by Dr. Mohanakumar’s laboratory. No specificauthentication of the cell lines was performed in our lab-oratory. All experiments were performed on the cell linesbelow 30th passage. Breast cancer cell lines were cultured inRPMI1640 culture medium at 37�C in 5% CO2 incubatoruntil they were 80% confluent. The presence of MAM-A inthe cell lines was confirmed by reverse transcriptase-PCR(Supplementary Fig. SS1) and Western blot analysis (datanot shown). siRNA oligonucleotides and monoclonal anti-bodies used in the cell culture inhibition studies wereobtained from Santa Cruz Biotechnology.

ELISPOT assayPBMCs were cultured overnight in complete RPMI1640

and viability was determined by Trypan blue exclusion.PBMC with viability of at least 90% were used for ELISPOT

analysis (10, 36). CD8 T cells were enriched by MACS beadnegative selection using immunomagnetic separation cock-tails (StemCell Technologies). PurifiedCD8T cells (2� 105

cells, >90% purity) were cultured in triplicate with MAM-A2.1 (LIYDSSLCDL, 20 mg/mL) in 96-well ELISPOT plates(Multiscreen IP plate) precoated with IFNg monoclonalantibody (mAb; 4 mg/mL) in the presence of autologous-irradiated T-cell–depleted PBMCs (3 � 104) for 48 to 72hours in humidified 5% CO2 at 37�C. Subsequently, theplates were washed and developed to detect the number ofspots in individual wells using an ImmunoSpot analyzer(Cellular Technology). CD8 T cells plus autologous irradi-ated T-cell–depleted PBMCs cultured in medium withoutMAM-A2.1 peptide were used as a negative control, whereasCD8 T cells plus autologous irradiated T-cell–depletedPBMCs cultured with phytohemagglutinin (5 mg/mL) wereused as a positive control. The number of spots in controlwells was subtracted from the number of spots in experi-mental wells and reported in the final results as spots permillion cells (spm � SEM).

Flow cytometryWe previously demonstrated that LIYDSSLCDL, desig-

nated MAM-A2.1, is an immunodominant HLA-A2–restricted epitope (10, 36).MAM-A2.1 tetramerswere devel-oped by Beckman Coulter Immunomics to monitor theMAM-A–specific CD8 T-cell response after MAM-A DNAvaccination. An HLA-A2 tetramer incorporating an unrelat-ed peptide from influenza (Flu), GILGFVFTL, was alsoprepared and used as a control. Tetramers were used tostain target cells at a concentration of 10 mL per 200 mL finalvolumeofCD8T cells (1�106CD8T cells/mL). Antibodiesused for flow cytometry included CD8-FITC (BD Bios-ciences), MAM-A2.1/Tetramer-PE, and Flu-peptide/Tetra-mer-PE. Samples were analyzed using a FACSCalibur/LSRIIflow cytometer (Becton Dickinson), and cell sorting wasperformed using a Vantage cell sorter (Becton Dickinson).Data were analyzed using BD FACSDiva software. Gateswere set according to isotype controls.

Cytotoxicity assayThe ability of MAM-A–specific CD8 T cells to lyse breast

cancer cell lines was determined using an LDH cytotox-icity assay (Promega). Breast cancer cells (5 � 103 cells)in 100 mL of complete medium were plated in triplicatecultures in round bottom 96-well plates in the presence ofvarying numbers of CD8 T cells (E:T ratios of 6.25:1 to50:1) and incubated at 37�C in a humidified 5% CO2

incubator for 4 hours. Controls were breast cancer targetcells alone or CD8 T cells isolated from normal subjects.Maximum release was determined by adding Triton X-100(1%) to the target cells. A colorimetric measurement ofthe released LDH was developed as per manufacturer’sinstructions and measured by spectrophotometer at450 nm. The percent-specific lysis was calculated usingthe formula [(experimental LDH release � spontaneousLDH release)/(maximum LDH release � spontaneousLDH release)] � 100.





Western blot analysisTotal proteins were extracted from cells with lysis buffer

[50mmol/L HEPES (pH 7.6), 150 mmol/L NaCl, 1% TritonX-100, 30 mmol/L Na4P2O7, 10% glycerol, 1 mmol/L ben-zamidine, 1 mmol/L dithiothreitol, 10 mg of leupeptin/mL,1 mmol/L phenylmethylsulfonyl fluoride, 50 mmol/L NaF,1 mmol/L sodium orthovanadate, 10 mmol/L sodium pyro-phosphate decahydrate, and 10mmol/L b-glycerophosphate(SigmaAldrich). After cell lysis, the supernatantwas collectedafter centrifugation at 15,000� g for 15minutes at 4�C (37).Protein concentrationwas determined with a Bradford AssayKit from Bio-Rad. Total proteins were separated on a 4%–12% SDS-polyacrylamide gradient gel and transferred onto anitrocellulose membrane. The membranes were blockedovernight at 4�C in Tris-buffered saline with 0.05% Tween20 (5% nonfat milk in 10 mmol/L Tris-HCl, 100 mmol/LNaCl, 0.1% Tween 20, pH 7.6). The membranes were incu-bated first with primary antibodies diluted at 1:1,000 inblocking buffer at room temperature for 2 hours and thenwith a horseradish peroxide–conjugated secondary IgGmAbdiluted at 1:5,000 for 1 hour. All primary and secondaryantibodies were obtained from Santa Cruz Biotechnology.The membrane was developed using a chemiluminescencekit (Millipore) and analyzed using Bio-Rad Universal HoodII. Densitometric analysis was done using the software pro-vided by Bio-Rad.

Gene expression analysisRelative expression of HLA-A2,MAM-A, NKG2D,DAP-10,

perforin, and actin were analyzed using 6-carboxyfluores-cein–labeled quantitative real-time reverse transcription PCRprimers (Applied Biosystems) as per the manufacturer’srecommendations (25). Briefly, totalRNAwas extracted from1 � 106 cells using TRIzol reagent (Sigma Aldrich). RNAsamples were quantified by absorbance at 260 nm. The RNAwas reverse-transcribed and RT-PCRwas performed in a finalreaction volume of 20 mL using iCycler 480 Probes Master(RocheDiagnostics). Each sample was analyzed in triplicate.Cycling conditions consisted of an initial denaturationstep at 95�C for 15 minutes, followed by 40 cycles at 95�Cfor 30 seconds, followed by 61�C for 1 minute.

Statistical analysisAs a phase I study, the data analysis was primarily descrip-

tive in nature. The differences in CD8 T-cell frequencies overtime (pre- vs. postvaccination) were compared using two-way ANOVA for repeated measurement data or Friedmanrank-sum test as appropriate, and results were expressed inmean � SEM. Correlation analysis was performed usingSpearman rank test. PFS of the vaccinated patients wasdescribed using Kaplan–Meier product limit estimator andcompared with a subset of screen failures who were consid-ered to be appropriate for comparative analysis (HLA phe-notype) by log-rank test. PFS was calculated on the basis ofthe date of study registration. The distributions of demo-graphic and clinical characteristics between two groups werealso compared by two-sample t test or Fisher exact test asappropriate. All analyses were two-sided and significance

was set at a P value of 0.05. Statistical analyses were per-formed using statistical packages SAS 9.2 (SAS Institute).

ResultsStudy subjects and vaccine safety

A total of 53 subjects were consented and enrolled inphase I clinical trial between December 2009 and May2013. Following enrollment, the HLA phenotypes of thesubjects were determined and breast cancer specimenswere evaluated for MAM-A protein expression. Fourteensubjects met all eligibility criteria and were vaccinated.Thirty-nine subjects were screen failures. Ten of the 14vaccinated subjects were followed for at least 52 weeksand detailed immune monitoring was completed in 8 ofthe 14 vaccinated subjects. The remaining 6 vaccinatedsubjects were not HLA-A2þ, and detailed immune mon-itoring is ongoing in these patients, pending validation ofepitopes and reagents for the specific HLA allelesexpressed by these subjects. Data from all 14 vaccinatedsubjects are included in the safety analyses and in theanalyses of PFS.

Of the 39 screen failures, 9 were excluded based on HLAphenotype, 2 were excluded because of withdrawal of con-sent, and one was excluded because of inability to performimmunohistochemical staining for MAM-A expression.These 12 subjects met all other inclusion/exclusion criteriaand were considered suitable for comparative analysis. The27 screen failures that were not considered suitable forcomparative analysis were excluded because their breastcancers were MAM-A–negative (8/27), insurance denial(6/27), disease progression before completion of screeningstudies (4/27), lost to follow up (4/27), or ineligibilitysecondary to failing more than two prestudy chemotherapyregimens, dialysis, locally advanced disease, noncomplianceor concurrent trastuzumab therapy (1 each). Of the vacci-nated subjects, the median age was 51 years. Ninety-threepercent were female, 86% were white, and 100% were ERþ.All subjects had received endocrine therapy and themajorityhad bone-only metastatic disease (57%; Table 1). Similardemographicswerenoted in the comparative analysis screen-failure group. None of the baseline demographic or clinicalcharacteristics were significantly different between the vacci-nated and comparative analysis screen failure groups. Ofnote, screen failure and vaccinated patients received similartherapies after enrollment (Supplementary Fig. S6).

In the current study, positive MAM-A expression was aninclusion criteria. MAM-A expression was quantified usingthe Allred scoring system (38), which is a composite scorebased on the proportion of cells staining positive, and thestaining intensity. MAM-A IHC was performed on 36 sub-jects, as some subjects were identified as screen failuresbefore MAM-A IHC could be performed. The average Allredscore for the 36 subjects was 3.9 (n ¼ 36, includes screenfailures). Twenty-four of 36 subjects (66%) had positiveMAM-A expression (defined as Allred score 3–8). The aver-age proportion score for the 36 subjects was 2.4 (n ¼ 36,includes screen failures).

Tiriveedhi et al.




Vaccine safety was closely monitored following vaccina-tion with eight or more clinical and laboratory assessmentsin the first 24 weeks of the trial. The most common grade 1toxicity was malaise/flu-like symptoms (4/14). Other grade1 toxicities potentially attributable to vaccination includedvaccine site tenderness (1/14), rash (1/14), and precipita-tion of a shingles episode, (designated as "infection"; 1/14).A shingles episode treated with Valtrex (also designated as"infection")was the only grade 2 toxicity (1/14). Therewereno grade 3 or 4 toxicities reported (Table 2).

Mammaglobin-A DNA vaccination elicits MAM-A2.1–specific CD8 T cellsWe previously demonstrated that LIYDSSLCDL, designat-

ed MAM-A2.1, is an immunodominant HLA-A2–restrictedepitope derived fromMAM-A (10, 36). In preclinical studies,

we observed that MAM-A2.1–specific CD8 T cells expandedfollowing MAM-A DNA vaccination of HLA-A2 transgenicmice (36). In the current study, we performed tetramer andELISPOT analyses to measure the MAM-A2.1–specific CD8T-cell response to vaccination in HLA-A2þ subjects. Thefrequency of CD8þ/MAM-A2.1þ T cells significantlyincreased following vaccination (0.9% � 0.5% vs. 3.8% �1.2%; P < 0.001; Fig. 2A, top), with longitudinal analysisdemonstrating significant responses in 6 of 8 subjects. Twosubjects (023 and 030) did not show a significant increase inthe frequency of CD8þ/MAM-A2.1þ T cells. Of note, subject023had a significant frequency of CD8þ/MAM-A2.1þ T cellsbefore vaccination that increased marginally after vaccina-tion (2.8% prevaccination vs. 3.4% at 12 months), whereassubject 030 had a low frequency of CD8þ/MAM-A2.1þ Tcells before vaccination that marginally increased after

Table 2. Adverse events

Toxicity Grade 1 Grade 2 Grade 3 Grade 4

Vaccine site tenderness 1 (7.1) 0 0 0Malaise/flu-like symptoms 4 (28.6) 0 0 0Rash 1 (7.1) 0 0 0Infection 1 (7.1) 1 (7.1) 0 0

Table 1. Patient demographics and baseline characteristics

Baseline characteristics Vaccinated (n ¼ 14) Screen failure (n ¼ 12) P

Age, y: mean � SD (median and range) 50.5 � 11.1 (48.6, 33–70) 54.5 � 12.1 (55.0, 33–80) 0.38Gender, n (%) 0.99Male 1 (7.1) 0Female 13 (92.9) 12 (100)

Race, n (%) 0.99White 12 (85.7) 10 (83.3)Black 1 (7.1) 1 (8.3)Unknown 1 (7.1) 1 (8.3)

Biomarker profile, n (%)ERþ 14 (100) 10 (83.3) 0.20Her-2þ 3 (21.4) 1 (8.3) 0.59Triple negative 0 (0) 2 (16.7) 0.20

Prior therapy, n (%)Chemotherapy 9 (64.3) 9 (75) 0.68Endocrine therapy 14 (100) 10 (83.3) 0.20Radiation 7(50) 5 (41.7) 0.71Surgery 12 (85.7) 12 (100) 0.48

Site of disease, n (%)Bone-only 8 (57.1) 4 (33.3) 0.26Viscera-only 0 (0) 2 (16.7) 0.20Lymph nodes-only 1 (7.1) 0 (0) 0.99Multiple sites 4 (28.6) 7 (58.3) 0.23

Screen fails, n (%)HLA typing 9 (75)Withdrew consent 2 (16.7)Tissue unavailable 1 (8.3)





vaccination (0.7% vs. 1.6% at 12 months). ELISPOT anal-yses demonstrated that the number of MAM-A2.1–specificCD8 T cells capable of IFNg secretion increased significantlyafter vaccination in 6of 8 subjects (41� 32 vs. 215� 67 spmCD8 T cells; P < 0.001; Fig. 2B, bottom). Consistent with thetetramer analyses, no significant change in the frequency ofIFNgþ CD8 T cells was noted in subjects 023 (241 vs. 277spm) or 030 (18 vs. 41 spm). Taken together, these datasuggest thatMAM-ADNA vaccination is capable of inducingpotent MAM-A–specific CD8 T-cell responses in patientswith breast cancer.

MAM-A–specific CD8 T cells induced after vaccinationare cytotoxic

To assess whether vaccine-induced MAM-A–specific CD8T cells are capable of lysing MAM-Aþ breast cancers, weperformed in vitro cytotoxicity assays against breast cancercell lines. Purified CD8 T cells from responders were testedagainst a panel of breast cancer cell lines including UACC-812 (MAM-Aþ/HLA-A2þ), MCF-7 (MAM-A�/HLA-A2þ),MDA-415 (MAM-Aþ/HLA-A2�), and MDA-134 (MAM-A�/HLA-A2�). MAM-A and HLA-A2 expression was con-firmed in breast cancer cell lines by RT-PCR (SupplementaryFig. S1).CD8Tcells purified fromPBMCsof responders at12months after vaccination were able to lyse MAM-Aþ/HLA-A2þUACC-812 breast cancer cells (44.3� 13.9%at 50:1 E/Tratio), but not control breast cancer cell lines (MCF-7, 2.2�0.8%, MDA-415, 1.8� 0.6% or MDA-134, 1.5� 0.4%; Fig.3A). These studies confirm theMAM-A–specific cytotoxicity,and MHC class I restriction to HLA-A2, of the MAM-A DNAvaccine-induced CD8 T cells.

IFNg and TNFa contribute to the cytolytic activity ofMAM-A–specific CD8 T cells

IFNg and TNFa have been shown to contribute to thecytolytic activity of antigen-specific CD8 T cells (39). Todetermine the impact ofMAM-ADNA vaccination on TNFaexpression in MAM-A–specific CD8 T cells, purified CD8 Tcells from vaccinated subjects were cocultured with UACC-812 breast cancer cells, and TNFa expression was deter-mined by RT-PCR and immunoblot analysis. Within thesensitivity of our detection system, we were not able toperform ELISA-based approach to TNFa detection. TNFaexpression increased 7.8-fold after DNA vaccination (rela-tive expression compared with actin 2.2 � 0.6 vs. 17.1 �3.7; Fig. 3B). Similar results were observed when TNFaexpression was assessed by immunoblot (SupplementaryFig. S2). Both IFNg and TNFa contribute to the cytolyticactivity of MAM-A–specific CD8 T cells as the ability to lyseUACC-812 cells was significantly reduced following incu-bationwithmAb specific for IFNg (16.6%�7.1%vs. 43%�15% for isotype control), TNFa (14.3% � 8.5%), or both(2.4% � 1.1%; Fig. 3C). Taken together, these resultsdemonstrate that MAM-A DNA vaccination is capable ofinducing MAM-A–specific CD8 T cells that are cytolytic andIFNg and TNFa contribute to this cytolytic activity.

NKG2D signaling contributes to the cytolytic activity ofMAM-A–specific CD8 T cells

NKG2D has been shown to be upregulated upon activa-tion of CD8 T cells (40). After vaccination, NKG2D mRNAexpression was upregulated in purified CD8 T cells culturedwith UACC-812 breast cancer cells (0.3 � 0.2 relative

Figure 2. MAM-A DNA vaccination results in expansion of MAM-A–specific CD8 T cells. PBMCs were obtained before vaccination, and at serial time pointsfollowing vaccination. The frequency and number of MAM-A–specific CD8 T cells was determined by tetramer analysis (A), and IFNg ELISPOT (B). PBMCsfrom 8 HLA-A2þ patients were stained with anti-CD8 mAb and HLA-A2/MAM-A2.1 tetramer (closed squares) or HLA-A2/Flu-M1 tetramer (open triangles).Patients were vaccinated at 0, 1, and 2 months. Data shown represent the percentage of CD8þ/tetramerþ cells as a percentage of total CD8þ T cells.Significant increases in tetramer frequencywere noted in 6 of 8 patients. The samePBMCsampleswere used to determine the number ofMAM-A2.1–specificCD8 T cells producing IFNg by ELISPOT assay. Purified CD8 T cells were stimulated with MAM-A2.1 peptide in the presence of autologous irradiated T-cell–depleted PBMCs, and the number of IFNg-producing cells was determined using an ELISPOT reader. Data are presented as the number of IFNg-producingcells per million CD8 T cells (spm).

Tiriveedhi et al.




Figure 3. MAM-A DNA vaccination induces MAM-A–specific CD8 T cells capable of specifically lysing MAM-Aþ breast cancer cell lines in a NKG2D/DAP10/perforin-dependent manner. A, CD8 T cells isolated from HLA-A2þ subjects 12 months after vaccination were tested for cytolytic activity on a panel ofbreast cancer cell lines at various E:T ratios. Only theHLA-A2þ/MAM-Aþ breast cancer cell line, UACC-812, was effectively lysed. B, CD8 T cells were purifiedfrom PBMCs at the indicated time points after vaccination, and TNFa production in response to stimulation with UACC-812 breast cancer cells wasdetermined by qRT-PCR. Similar results were observed for IFNg production (data not shown). C, UACC-812–specific cytolytic activity was determined in thepresence of antibodies to isotype control, IFNg , or TNFa. Error bars represent the mean � SEM from the 6 HLA-A2þ responders. D, UACC-812–specificcytolytic activity was determined in the presence of antibodies to isotype control, NKG2D, IFNg , or TNFa, or control or NKG2D siRNA. Error barsrepresent the mean� SEM from the 6 HLA-A2þ responders. E, purified CD8 T cells from subjects vaccinated with the MAM-A DNA vaccine were stimulatedwith UACC-812 breast cancer cells at a 50:1 T-cell to breast cancer cell ratio as indicated. Representative immunoblots for NKG2D (left); DAP10 (middle), andperforin (right). Actin expression represents a protein loading control. F, purified CD8 T cells were tested for UACC-812–specific cytolytic activity in thepresence of isotype control, NKG2D, DAP-10, or perforin antibody, or control, NKG2D, DAP-10, or perforin siRNA. Error bars represent themean�SEM fromthe 6 HLA-A2þ responders.





expression compared with actin before vaccination vs. 13.7� 2.4 at 12 months; P < 0.05; Fig. 3D). Similar increases inNKG2D protein expression were also observed (Fig. 3E). Ofnote, NKG2D mRNA and protein expression were down-regulated in the presence of anti-IFNg mAb (3.7 � 0.9relative expression compared with actin), anti-TNFa mAb(5.8 � 1.4), or both (1.3 � 0.5) suggesting important rolesfor IFNg and TNFa in the induction of NKG2D expression(Fig. 3D and Supplementary Fig. S3). Specific ablation orantibody neutralization of NKG2D significantly reduced thecytolytic activity of MAM-A–specific CD8 T cells (Fig. 3F).

The NKG2D adapter protein DAP-10 has been shown tobe an important downstreammediator of NKG2D-inducedcytolytic activity (41), and perforin has been identified as akey cytotoxic effector molecule released by activated CD8 Tcells (42). To determine whether DAP-10 and/or perforincontribute to NKG2D-mediated cytolytic activity after vac-cination, we assessed DAP-10 and perforin expression inMAM-A–specificCD8T cells after coculturewithUACC-812breast cancer cells. Expression of DAP-10 and perforin wereincreased in purified CD8 T cells following vaccination(DAP-10: 0.5� 0.3 relative expression compared with actinbefore vaccination vs. 9.3 � 2.7 at 12 months; P < 0.05;perforin: 1.7� 0.6 relative expression compared with actinbefore vaccination vs. 16.5� 4.1 at 12months, P< 0.05; Fig.3E, middle and right panels, and Supplementary Figs. S4and S5). Specific ablation or antibody neutralization ofNKG2D was associated with downregulation of DAP-10expression (Supplementary Fig. S4), and reduced perforinexpression (Supplementary Fig. S5), suggesting a criticalrole for NKG2D in the regulation of DAP-10 and perforinexpression inMAM-A–specificCD8T cells after vaccination.Similarly, antibodies to IFNg , TNFa, or both stronglyinhibited the expression of both DAP-10 (SupplementaryFig. S4) and perforin (Supplementary Fig. S5) in CD8 T cellscultured with UACC-812 cells.

Specific ablation of NKG2D or DAP10 expression wasassociated with a significant reduction in MAM-A–specificCD8 T-cell cytolytic activity (DAP-10: 44.3% � 13.9% vs.3.8%� 1.6%; P < 0.05; NKG2D: 44.3%� 13.9%vs. 4.7%�1.3%; P < 0.05; Fig. 3F). Cytotoxicity was mediated almostexclusively through perforin, as specific ablation or anti-body neutralization of perforin reduced cytolytic activity toalmost zero. On the basis of these results, we conclude thatMAM-A DNA vaccination is capable of inducing MAM-A–specific CD8 T cells with significant cytolytic activity. Thiscytolytic activity is dependent on NKG2D, DAP10, andperforin expression.

Preliminary evidence suggests that MAM-A DNAvaccination is associated with improved PFS

Although this study was not powered to evaluate theimpact ofMAM-A vaccination on PFS, clinical outcomewasone of the exploratory objectives. We compared progres-sion-free survival between subjects who received the vaccineand subjects who met all eligibility criteria and wereenrolled in the trial but were screen failures because of HLAphenotype. PFS was determined by blinded review of case

report forms, medical records, and diagnostic imagingresults. PFS of the vaccinated patients was described usingKaplan–Meier product limit estimator and comparedwith asubset of screen failures by log-rank test. MAM-A vaccina-tion was associated with a prolonged PFS (6-month PFS53% vs. 33%; P ¼ 0.011; Fig. 4).

DiscussionPreclinical studies by our group (10) and others (43–45)

have demonstrated that MAM-A is an exceptional target forbreast cancer vaccine therapy based on its exquisite tissuespecificity (16–19), overexpression in 40% to 80%of breastcancers (20–24), and evidence of preexisting immunity inpatients with breast cancer (8, 9, 25, 26). On the basis ofthese preclinical studies, we initiated a phase I clinical trialto evaluate the safety and biologic efficacy of a novelplasmid MAM-A DNA vaccine. The MAM-A DNA vaccineis based on the pING vector, drivingMAM-A expression by astrong viral promoter. Patients with breast cancer withstable metastatic disease were eligible for participation. Todate, 14 subjects have been successfully treated with thevaccine, including 8 HLA-A2þ subjects that were studiedwith in-depth immune monitoring analyses using PBMCcollected before, during, and after vaccination.

Figure 4. Preliminary evidence suggests that MAM-A DNA vaccination isassociatedwith improved PFS. A total of 53 patientswere consented andenrolled in the phase I clinical trial. After enrollment, 39 patients wereconsidered screen failures and 14 patients were vaccinated. Of the 39screen failures, 12 were excluded based on HLA type, withdrawal ofconsent or inability to performMAM-A IHC and were considered suitablefor comparative analysis. PFS was compared between the twogroups using Kaplan–Meier product limit estimator and log-rank test.Vaccinated patients are designated with a solid line and screen failurecontrols are designated with a dashed line. MAM-A vaccination wasassociated with a prolonged PFS (6-month PFS, 53% vs. 33%;P ¼ 0.011).

Tiriveedhi et al.




The results of the phase I clinical trial demonstrate thesafety of the MAM-A DNA vaccine and compelling prelim-inary evidence of biologic efficacy. Specifically, we havemade three important observations: (i) the MAM-A DNAvaccine is safe, with no significant adverse events observedin the trial. Themost common grade 1 toxicity wasmalaise/flu-like symptoms; (ii) there was a significant increase in thefrequency and number of MAM-A–specific CD8 T cellsfollowing vaccination, as measured by tetramer analysisand ELISPOT; and (iii) preliminary evidence suggests thatsubjects who were vaccinated had improved PFS comparedwith subjects who were enrolled in the trial but were notvaccinated because of HLA type.Induction of effective antitumor immunity requires that a

cancer vaccine be capable of eliciting robust type I immu-nity. In the last two decades, considerable progress has beenmade in understanding the complex regulatory and signal-ing networks that control immune responses. It is increas-ingly clear that CD8 T-cell responses are tightly regulated bythe immune system, presumably to avoid deleterious auto-immune responses. In the context of breast cancer, regula-tory networks such as the increased prevalence of T regu-latory cells (Treg; ref. 11) and upregulation of inhibitorymolecules on breast cancer–specific T cells (12) are presentthat restrain breast cancer immune responses. These regu-latory networks may also limit the T-cell response to ther-apeutic cancer vaccines. In a previous study evaluating theCD4T-cell response to vaccination in 7 of the first 9 patientsof this cohort, we demonstrated that the frequency of Tregwas significantly decreased following MAM-A DNA vacci-nation, whereas the frequency of CD4þICOShi MAM-A–specific T cellswas increased (35). TheseCD4þICOShi T cellsproduced higher levels of IFNg and lower levels of IL10 aftervaccination.We present evidence here that theMAM-ADNA vaccine is

able to induce type I immune responses despite the presenceof these regulatory networks. MAM-A DNA vaccinationresults in induction of MAM-A–specific CD8 T cells thatare capable of producing both IFNg and TNFa, and lysingMAM-Aþ breast cancer cells. MAM-A–specific CD8 T cellsalso have increased NKG2D, DAP-10, and perforin expres-sion. Of note, in CD8 T cells, NKG2D signaling has beenshown to augment proliferation and cytotoxicity uponantigen encounter, suggesting that NKG2D functions as acostimulatory molecule (46). Our studies demonstrate thatafter MAM-A DNA vaccination, there is specific upregula-tion of NKG2D and DAP-10 expression (Fig. 3). Althoughthis response is not unique to MAM-A vaccination, it doesprovide strong supporting evidence for the efficacy of theMAM-A DNA vaccine. Taken together, the results of thesetwo studies strongly suggest that theMAM-ADNAvaccine iscapable of inducing strong type I immune responses. Thehumoral response to vaccination was not measured.Current cancer vaccine clinical development paradigms

emphasize the early assessment of cancer vaccine efficacy inan appropriate clinical context (47). These paradigms havebeen endorsed in the literature and in FDA guidance docu-ments. Although treatment of patients with metastatic dis-

ease is appropriate for first-in-human studies of a novelbiologic therapeutic cancer vaccine treatment of patientswith metastatic disease does have unique limitations. First,it is very difficult to assess the biologic efficacy of cancervaccines in patients with metastatic disease, as the timeinterval from vaccine administration to subsequent diseaseprogression in patients with metastatic disease may be tooshort to develop a productive antitumor immune response.Second, patients with metastatic disease have typicallyreceived multiple prior cancer treatments, which may bedetrimental to the immune system. The fact that preliminaryevidence suggests that the MAM-A DNA vaccine was associ-ated with improved PFS in patients with metastatic breastcancer is notable, supporting ongoing clinical developmentof the MAM-A DNA vaccine. Studies to define the clinicalpotential of a MAM-A DNA vaccine are particularly impor-tant given the safety of the vaccine, evidence of biologicefficacy, exquisite tissue specificity, and near-universalexpression of MAM-A in breast cancer, and the potential ofMAM-A as a target for a breast cancer prevention vaccine.

Disclosure of Potential Conflicts of InterestT. Fleming holds a patent onmammaglobin that is ownedbyWashington

University. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: V. Tiriveedhi, M. Ellis, C. Ma, A.C. Lockhart,P. Goedegebuure, T. Mohanakumar, W.E. GillandersDevelopment of methodology: V. Tiriveedhi, J. Herndon, T. Fleming,P. Goedegebuure, T. Mohanakumar, W.E. Gillanders, Lijin LiAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): V. Tiriveedhi, N. Tucker, M. Sturmoski, M. Ellis,C. Ma, M. Naughton, A.C. Lockhart, W.E. Gillanders, Lijin LiAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): V. Tiriveedhi, N. Tucker, C. Ma,A.C. Lockhart, F. Gao, W.E. GillandersWriting, review, and/or revision of the manuscript: V. Tiriveedhi,N. Tucker, M. Ellis, C. Ma, A.C. Lockhart, F. Gao, P. Goedegebuure,W.E. GillandersAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): N. Tucker, J. Herndon, C. Ma,W.E. GillandersStudy supervision: A.C. Lockhart, W.E. GillandersOther (involved in the GMP manufacture and quality control of themammaglobin DNA vaccine): T. Fleming

AcknowledgmentsThe authors thank the Alvin J. Siteman Cancer Center at Washington

University School of Medicine and Barnes-Jewish Hospital (St. Louis, MO)for the use of the Biologic Therapy Core Facility, the Clinical Trials Core andBiostatistics Core, which provided critical support for the vaccine manufac-ture and validation, design and performance of the clinical trial, and dataanalysis, respectively. The authors are also extremely grateful to George andDiana Holway, whose generous gift provided the resources necessary to helprealize the promise of new discovery.

Grant SupportThisprojectwas fundedby researchgrantsDOD/CDMRP-BCRPW81XWH-

06-1-0677 (toW.E.Gillanders) andGateway for Cancer Research P-06-016 (toW.E. Gillanders). V. Tiriveedhi, P. Goedegeburre, and T. Mohanakumar arefundedby theFoundation forBarnes-JewishHospital.N.Tuckerwas supportedby aNCI grant T32CA009621. The SitemanCancer Center is supported inpartby NCI Cancer Center Support Grant #P30 CA91842.

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

Received January 23, 2014; revised August 18, 2014; accepted August 19,2014; published online December 1, 2014.





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2014;20:5964-5975. Clin Cancer Res Venkataswarup Tiriveedhi, Natalia Tucker, John Herndon, et al. Breast CancerMammaglobin-A DNA Vaccine in Patients with Stable Metastatic Safety and Preliminary Evidence of Biologic Efficacy of a

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