The Prostate 69:938 ^ 948 (2009)

VaccinationwithRecombinantAdenoviruses andDendritic Cells Expressing Prostate-SpecificAntigensIs Effective in ElicitingCTL and SuppressesTumorGrowth inthe Experimental ProstateCancer

Sol Kim,1 Jee-Boong Lee,1 Geon Kook Lee,2 and Jun Chang1*1Divisionof Life and Pharmaceutical Sciences,EwhaWomansUniversity, Seoul, SouthKorea

2Departmentof Pathology,National Cancer Center,Goyang, SouthKorea

BACKGROUND. Prostate cancer is currently the most commonly diagnosed cancer in menand the second leading cause of cancer-related death in men in the US. Immunologicalapproaches may provide an alternative option for prevention and treatment of prostate cancer.METHODS. To develop vaccine against prostate cancer using mouse model, we constructedthree recombinant adenoviruses expressing prostate-specific membrane antigen (rAd/PSMA),prostate stem cell antigen (rAd/PSCA) and six-transmembrane epithelial antigen of theprostate (rAd/STEAP), that were specifically up-regulated in the transgenic murine prostatecancer.RESULTS. Male C57BL/6 mice were immunized by intravenous injection of theserecombinant adenoviruses and subsequently by subcutaneous injection of dendritic cellspulsed with TRAMP-C1 tumor lysate. After subcutaneous challenge with TRAMP-C1 cells,tumor growth was significantly delayed in the immunized mice compared to the control group.Surprisingly, significant numbers of STEAP-specific CD8 T cells were detected in the peripheralblood and the spleen of immune mice using MHC I tetramers, and injection of rAd/STEAPalone followed by pulsed DC was sufficient to inhibit tumor growth. Therapeutic vaccinationalso significantly delayed the growth of pre-established tumors.CONCLUSION. Our results suggest that STEAP is a good immunologic target antigen againstprostate cancer and our vaccination regimen successfully elicits anti-tumor CTL responses andsuppresses tumor growth. More studies will expedite the development of this vaccine towardclinical application. Prostate 69: 938–948, 2009. # 2009 Wiley-Liss, Inc.

KEY WORDS: vaccine; prostate cancer; STEAP; CTL

INTRODUCTION

Prostate cancer (CaP) is the most common malecancer and the second leading cause of cancer-relateddeaths in men [1]. Despite the curative treatments forthe patients with localized disease by surgical resectionand radiotherapy treatment, many patients will even-tually develop metastases. In particular, there is nocurative treatment for patients who develop recurrentdisease after conventional surgery or radiation therapyor those who have metastatic disease at the time ofdiagnosis. Although hormone ablation therapy offerspalliation, the majority of diseases eventually progress

to hormone-refractory prostate cancer and unrespon-sive to further hormonal therapy. Therefore, novelapproaches are required for the prevention and/oreffective management of patients with advanced CaP.

Grant sponsor: Ewha Womans University Research Grant of 2005.

*Correspondence to: Jun Chang, College of Pharmacy, EwhaWomans University, 11-1 Dae-Hyun Dong, Seo-Dae-Mun Gu, Seoul120-750, South Korea. E-mail: tcell@ewha.ac.krReceived 12 August 2008; Accepted 23 January 2009DOI 10.1002/pros.20942Published online 6 March 2009 in Wiley InterScience(www.interscience.wiley.com).

1 2009 Wiley-Liss, Inc.

Immunotherapy is a promising method in thetreatment of CaP, aiming to eradicate cancer cells byinducing systemic immunity to specific antigensexpressed by CaP cells as well as normal prostate [2].It is now well-established that there is a strongcorrelation between the development of cytotoxic Tlymphocytes (CTL) and tumor rejection. Immunother-apeutic strategies capable of inducing specific T-cellresponses rely on well-defined tumor-associated targetantigens and an effective approach to deliver theantigens. Over the past years, several prostate-specificgene products have been reported, including prostate-specific antigen (PSA), prostate stem cell antigen(PSCA) [3], prostate-specific membrane antigen(PSMA) [4], prostatic acid phosphatase (PAP) [5], andrecently identified six-transmembrane epithelial anti-gen of the prostate (STEAP) [6]. These prostate-specificproteins may be served as a source of antigenicpeptides, allowing CTL to identify and eliminate CaP.

In the current study, we have investigated the utilityof recombinant adenovirus vaccines and dendritic cell-based vaccine and evaluated the efficacy of vaccinationthrough prime-boost strategy in an experimentalmodel of prostate cancer. Our results demonstrate thatour vaccine regimen successfully suppresses tumorgrowth in both preventive and therapeutic models andSTEAP-specific CD8 T-cell response is important forthe control of tumor growth.

MATERIALSANDMETHODS

Mice andCell Lines

Five- to 6-week-old, pathogen-free, male and femaleC57BL/6 mice were purchased from Charles RiverLaboratories. Mice were kept under specific pathogen-free condition of our institute’s animal facility andall animal studies were performed according to ourInstitutional Animal Care and Use Committee.TRAMP-C1 prostate tumor cells were cultured in highglucose Dulbecco’s modified Eagle medium (DMEM)supplemented with 5% fetal bovine serum (FBS;HyClone), 5% Nu-serum (BD Biosciences), 10 nmol/Ldihydrotestosterone and 5 mg/ml insulin (Sigma).293T, EL4 and B16F10 cells were maintained in DMEMsupplemented with 10% FBS.

Construction of RecombinantAdenovirus

The cDNA for murine PSCA, PSMA, and STEAPwere prepared from mRNA samples of TRAMP-C1 cellline by RT-PCR amplification. The following primerpairs were used: PSCA (sense, CGG CTC GAG GCCACC ATG AAG ACA GTC TTC TTT CTC CTG; anti-sense, CGC TCT AGA CTA CAG ACG GCT GGA GCCCCA C), PSMA (sense, TAA CTC GAG GCC ACC ATG

TGG AAC GCA CTG CAG GAC A; anti-sense, CGGTCT AGA TTA AGC TAC TTC CAT CAG AGT CTCTGC TGC AGC TTG CAC), and STEAP (sense, TTACTC GAG GCC ACC ATG GAG ATC AGT GAC GATGTT AC; anti-sense, CGC TCT AGA CTA CAA CCTGGA GGC CAT CTC AG). To construct plasmidsencoding prostate antigens, Kpn I/Xho I fragmentcontaining each cDNA fragment was inserted into theKpn I/Xho I-digested parental plasmid pShuttle-CMV.Replication-defective adenoviruses (serotype 5) weregenerated by insertion of foreign sequences by homol-ogous recombination and subsequent purification ofrecombinant progeny as described previously [7].Briefly, the shuttle vector plasmid was electroporatedinto electrocompetent BJ5183 cells carrying thepAdEasy-1 adenoviral genomic DNA. Isolatedrecombinant adenoviral DNA was transfected into293T cells and viral plaques were isolated andexpanded, then the recombinant viruses were identi-fied by Western blotting analysis. The prostate antigenswere identified in the cell lyate of infected 293T cellsusing mouse mAb to HA tag (Roche). The adenovirusexpressing IL-12 mutant, rAd/IL-12m, has beendescribed elsewhere [8]. The viruses were amplifiedand purified by double cesium gradient ultracentrifu-gation as recommended by the protocol. The viral titerwas determined by either TCID50 or plaque assay.Recombinant adenovirus without inserted gene wasused as a control.

Preparation of Tumor Lysate-PulsedDendritic Cells

Dendritic cells were generated from bone marrowcells of naıve C57BL/6 mice by culturing in completeRPMI medium containing 10% FBS supplemented with10 ng/ml recombinant GM-CSF, 10 ng/ml IL-4 (R&D),50 mmol/L 2-mercaptoethanol. On day 3, fresh cyto-kines were added, and the total culture volume wasincreased by 50% with the addition of fresh medium.After 5 days of culture, non-adherent cells wereharvested by gentle pipetting, and DCs were enrichedby density centrifugation over PerColl medium. Theresulting DC population was analyzed by flow cytom-etry and each preparation was �80% positive for theexpression of MHC II, CD11c, CD80, and CD86 (datanot shown). After purification, DCs were resuspendedto 1.0� 106 cells/ml in complete medium containingTRAMP-C1 lysate (DC to tumor cell ratio was 1 to 3)and further incubated for 18 hr. The tumor cell lysatewas prepared by three cycles of rapid freezing in liquidnitrogen and thawing at 378C. The lysate was thencentrifuged to remove particulate cellular debris. Afterpulsing, the DCs were collected, washed several timesin HBSS, and resuspended in HBSS to 5.0� 106 cells/mlfor injection into mice.

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Immunization and TumorChallenge

Male C57BL/6 mice were i.v. injected with 1� 108

PFU of recombinant adenoviruses containing rAd/PSMA, rAd/PSCA, rAd/STEAP, and rAd/IL-12m in aratio of 3:3:3:1, or control adenovirus. On 2 weeks afterpriming with recombinant adenovirus, the mice wereboosted by s.c. injection of 5� 105 tumor lysate-pulseddendritic cells. Control animals were injected with thesame number of unloaded DCs. Mice were thenchallenged with 2� 106 TRAMP-C1 cells s.c. into thevertebral flank 14 days following last vaccination.Tumor volumes were monitored using the formula:m1

2�m2� 0.5236, where m1 and m2 representedthe minimum, maximum diameters of the tumors. Fortherapeutic immunization, 2� 106 TRAMP-C1 cellswere first injected s.c. into the vertebral flank, and afterthe mice developed palpable tumor (�3mm diameter),1� 108 PFU of recombinant adenoviruses were i.v.injected. Two weeks after adenovirus injection, tumorlysate-pulsed DCs were s.c. administered and thetumor sizes were recorded two or three times a week.

Peptides and Preparation ofMHCITetramers

The peptides were synthesized commercially andpurified by reverse phase HPLC (Thermo). MHC Imonomer complexes were generated using a proce-dure described by D. Busch and E. Pamer (YaleUniversity, New Haven, CT). Briefly, H-2Kb or H-2Db

heavy chain proteins and human b2-microglobulinwere expressed in E. coli and purified from inclusionbodies. Soluble H-2b monomers were generated in thepresence of high concentrations of relevant peptides.Complexes were then biotinylated by BirA ligase(Avidity, Denver, CO) and purified by Superdex-75gel filtration and Mono-Q anion exchange chromatog-raphy. The biotinylated monomer complexes weretetramerized with PE-labeled streptavidin (MolecularProbes, Eugene, OR). Tetramers were stored at 5 mg/ml at 48C in PBS (pH 8.0) containing 0.02% sodiumazide, 1 mg/ml pepstatin, 1 mg/ml leupeptin, and0.5 mM EDTA and optimal concentration for stainingwas determined by titration.

FlowCytometryAnalysis

Cells were resuspended in FACS buffer (1% FBS,0.03% sodium azide in PBS) at 107 cells/ml. 5� 105 cellswere stained with antibodies against CD4 (cloneRM4-5), CD8 (53-6.7 or CT8a), or CD44 (IM7), andPE-conjugated tetramers for 40 min at 48C. Tetramerswere used at optimal dilutions determined by titration.To enumerate the number of IFN-g-producing cells,intracellular cytokine staining (ICS) assay was per-formed. Briefly, 106 lymphocytes were cultured in

culture tube in a volume of 1 ml with 5� 105 EL4 cellspulsed with various peptides for 5 hr at 378C. BrefeldinA (10 mg/ml; Sigma, St. Louis, MO) was added for theduration of the culture period to facilitate intracellularcytokine accumulation. Cells were first stained forsurface markers, washed, fixed and permeabilizedwith FACS buffer containing 0.5% saponin (Sigma–Aldrich, Seoul, Korea). Then, cells were stained withanti-IFN-g (XMG1.2) or its control isotype antibody (ratIgG1). Data were collected on a FACSCaliburTM (BDBiosciences, San Jose, CA). Gates were set on lympho-cytes by forward and side scatter profiles, and thedata were analyzed using CellQuestTM Pro (BDBiosciences), FlowJoTM software (Windows version5.7.2, TreeStar, San Carlos, CA), and WinMDI version2.9 software (The Scripps Research Institute, La Jolla,CA).

Generation of Ag-Specif|c CTL andCell-MediatedCytotoxicityAssay

Splenocytes from the immune mice were in vitrostimulated by adding 10 mM peptides. Effector cellsused in CTL assays were prepared from these culturesby Ficoll-Hypaque density gradient purification. Pep-tide-pulsed EL4 target cells (5� 104/well) were addedto serial dilutions of effector cells in 96-well round-bottom plates at E:T ratios of 1:1 to 50:1. After 4 hrat 378C, cytotoxicity was quantified by measurementof the cytosolic lactate dehydrogenase (LDH) in theculture supernatant (n¼ 3) using cytotoxicity detectionkit (Roche). Specific lysis for each E:T ratio is expressedas: specific lysis¼ [(experimental release)� (spontane-pontaneous release)/(target maximum� target spon-taneous release)]. Spontaneous LDH release in theabsence of CTL was <10% of the maximal cellularrelease by detergent lysis. All experimental proceduresand assays were performed at least two times, withsimilar results.

Histochemistry

For immunohistochemistry, excised tumor tissueswere fixed with 10% buffered formalin before beingembedded in paraffin blocks. Eight-micrometer-thick sections were cut on a microtome and collectedonto positively charged slides. After rehydration, slideswere treated with 10% normal mouse serum in PBScontaining 0.1% Tween 20 for 30 min, and washed andincubated with primary anti-CD8b Ab (clone H35-17.2;Santa Cruz Biotechnology) for 1 hr at room temper-ature. Sections were then incubated for 1 hrwith secondary anti-mouse Ab and visualized withperoxidase/diaminobenzidine detection (ChemMatedetection kit; Ventana) for 2–10 min, as needed.

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Parallel sections were stained with H&E (Sigma–Aldrich). Sections were examined using a NikonMicrophot-FXA microscope, and representative imageswere captured using a Sony DKC-ST5 digital camera.

Statistical Analysis

Comparisons between groups were analyzed usinga Student’s t-test (Sigma Plot 8.0 software). Value ofP< 0.05 was accepted for significance.

RESULTS

Construction of RecombinantAdenoviral VectorsExpressing ProstateAntigens

E1, E3-deleted replication-defective adenoviralrecombinants carrying prostate antigen genes wereconstructed as described in the Materials and Methods(Fig. 1A). The backbone vector is derived fromadenovirus type 5, pAdEasy-1 [7]. The introducedgenes also encode influenza HA tag sequence, and theexpression of transgene by rAd/PSCA, rAd/PSMA,and rAd/STEAP adenovirus was confirmed by West-ern blotting analysis using anti-HA tag antibody(Fig. 1B).

ProphylacticVaccinationDelaysTumorGrowth andInduces Antigen-Specif|c CD8TCells

To evaluate the efficacy of antigen-specific vaccina-tion against prostate cancer, male C57BL/6 mice wereimmunized with recombinant adenoviruses and tumorlysate-pulsed DCs in combined strategy, as shown inFigure 2A. A separate group of mice was also treatedwith empty vehicle and unloaded DCs as a negativecontrol for comparison. After TRAMP-C1 challenge,the growth of tumor mass was significantly delayedin the immune mice compared to the control mice(P< 0.001; Fig. 2B). These results suggest that prophy-lactic vaccination with recombinant adenovirus andpulsed DCs might provide potential protection againsttumor challenge.

To examine whether the immunization elicitsprostate antigen-specific T-cell responses, spleen cellsfrom the immune mice were in vitro stimulated withthe mixture of overlapping peptides covering PSCA,PSMA, and STEAP, and IFN-g-producing cells weremeasured by intracellular cytokine staining. IFN-g-positive CD8 T cells were only detected in the immunegroup, when compared to the control mice (Fig. 3A).However, IFN-g-producing CD4 T cells were notobserved in either group when splenocytes werestimulated with tumor lysate-pulsed DCs.

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Fig. 1. Construction of defective adenoviral recombinantsexpressingprostateantigens.A:Theconstructionschemeofadeno-viralrecombinantsexpressingPSCA,STEAP,orPSMA.B:Westernblotting analysis of rAd/PSCA, rAd/PSMA, or rAd/STEAP-infected293TcellsusingmousemAbspecific toHAtag sequence.

Fig. 2. Immunization strategy and induction of anti-tumor pro-tection.A:The scheme of immunization.Groups of10maleC57BL/6micewereinjectedwithrAdfollowedbytumorlysate-loadedDCs,andwere challengedwithTRAMP-C1cells.B:Theresults depict thesizeof tumordevelopedafterchallenge.Resultsarepresentedas themean tumor size over time. Similar results were obtained in twoseparate studies.

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Identif|cation ofNovel CTLEpitopes inPSMAand STEAP

In order to identify potential H-2b-restricted CTLepitopes within PSCA, PSMA, and STEAP proteins,we processed sequences by two epitope predictionalgorithms available on the World Wide Web, referredto as ‘‘SYPFEITHI’’ (University of Tubingen; www.syfpeithi.de) [9] and ‘‘BIMAS’’ (Bioinformatics and

Molecular Analysis Section of the National Institutesof Health; bimas.dcrt.nih.gov/molbio/hla_bind) [10].According to the information by SYPFEITHI, naturallypresented epitopes might be among the top-scoring 2%of all peptides predicted. We then decided to followboth predictions for the synthesis of peptides shown inTable I, representing the top 2% of predicted peptidesby each method.

A panel of 20mer overlapping peptides of PSCA andSTEAP proteins as well as a set of peptides derivedfrom PSCA, STEAP, and PSMA proteins, predicted byalgorithms for the H-2b MHC class I alleles weresynthesized (Table I). Splenocytes from the immunemice were isolated 2 weeks after boosting immuniza-tion and peptides were tested for their capability toinduce IFN-g production in ICS assays. The syntheticpeptide set was initially screened as mixture of allpeptides, followed by assays with individual peptidesof positive pools. We initially found substantial CD8T-cell responses against a mixed pool of peptides, butno detectable CD4 T-cell response upon tumor lysate-pulsed stimulators (Fig. 3A). Next, to identify individ-ual epitopes, splenocytes from the immune mice werestimulated once with all mixed peptides for 6 days,and then with each individual peptide in ICS assays.Substantial CD8 T-cell responses were detected againsttwo sequences from the STEAP protein (mSTEAP/5-13, and mSTEAP/186-193) and two sequencesfrom the PSMA protein (PSMA/214-222, and PSMA/587-595) (Fig. 3B). Only weak responses weredetected against mSTEAP/82-90 and mSTEAP/327-335 peptides.

The epitope prediction algorithm indicated thatthree 9-mer sequences, mSTEAP/5-13 (DDVTNPEQL),PSMA/214-222 (NMVKNVQLA), and PSMA/587-595(FELANSIVL), are H-2Db-restricted, while 8-mermSTEAP/186-193 (RSYRYKLL) is H-2Kb-restricted.To confirm the correct restriction elements for thesepeptides, we tested the stabilizing capability of thepeptides with appropriate MHC class I molecules in arefolding assay and the folding efficiency was deter-mined by gel filtration and SDS–PAGE analysis.As shown in Table I, efficient stabilization of the three9-mer peptides and the 8-mer STEAP peptide to H-2Db

and H-2Kb, respectively, was observed. These peptideswere selected for analyzing prostate-specific CD8 T-cellresponses after immunization.

CD8T-Cell ResponsesDetectedbyMHCITetramers

To directly measure magnitude of tumor-specificCD8 T-cell responses induced by immunization, weprepared H-2Db/STEAP/5-13, H-2Db/PSMA/214-222, H-2Db/PSMA/587-595, and H-2Kb/STEAP/186-193 MHC I tetramer complexes. Freshly isolated

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Fig. 3. Induction of specific CD8 T-cell response by vaccinationandidentificationofCTLepitopes.A:Splenocytes fromtheimmunemicewereharvested14daysafterboostimmunizationandIFN-g ICSwasperformedinthepresenceof1mg/mlofoverlappingpeptides forCD8or tumor lysate (in aresponder:tumorcellratio of1:3) forCD4T cells.Cellswere gated on lymphocytes profileby forward scatterand side scatter.B: Splenocytes were stimulated for 6 days in thepresence of1 mg/ml of overlappingpeptides and then IFN-g ICSwasperformed after stimulation with 1 mM of each indicated peptide.Cellsweregatedonforwardscatter andCD8aþ lymphocytes.

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peripheral blood lymphocytes from the immune micewere directly stained with tetramers, and the propor-tions of epitope-specific CD8 T cells were determined.In preliminary experiments, we were not able to detectany significant antigen-specific CD8 T cells in the PBLafter adenovirus immunization by tetramer staining.However, approximately 5.0% of CD8 T cells in the PBLwere specifically stained for mSTEAP/186-193 peptideafter DC boosting (at day 21), whereas no significantCD8 T-cell response specific for PSMA/587-595 and theother epitopes were observed (Fig. 4A and data notshown). We have not observed any specific stainingabove background level from the control mice (Fig. 4A),and any reactivity to irrelevant MHC-matched tet-ramers in the immune mice (data not shown), confirm-ing the specificity of the tetramer staining. Notably,mSTEAP/186-193-specific CD8 T-cell response elicitedby vaccination was even increased at day 5 after tumorchallenge and well sustained until day 44 afterchallenge (Fig. 4A,B). At all time points, mSTEAP/186-193-specific CD8 T cells exhibited CD44hi pheno-type (Fig. 4A). These results demonstrated that STEAP-specific CD8 T cells were mainly elicited by theimmunization and might be associated with activeinhibition of tumor growth as shown in Figure 2.

The previous results suggest that STEAP-specificCTL response plays a major role in controlling thegrowth of challenged tumor cells. To test whethervaccination of rAd/STEAP is sufficient for the protec-tion, the groups of mice were immunized withindividual adenovirus and the tumor growth wasmonitored as described in Figure 2. The results showedthat STEAP vaccination induced significantly delayin tumor growth compared with a group of micevaccinated with rAd/PSMA or the control adenovirus(Fig. 5). PSCA was not included in this experiment

since PSCA vaccination alone had no effect on thetumor growth (data not shown). The level of tumorgrowth inhibition by STEAP vaccination was compa-rable to that by mixed vaccination (STEAPþPSMA),indicating that STEAP-specific response is the majordeterminant controlling the tumor growth in thissetting.

Recognition of Epitope-Loaded Target Cellsand TumorCells byCTL

Since the inhibition of tumor growth requires correctrecognition of tumor antigens and cytolytic function ofCD8 T cells, the ability of peptide-specific CD8 T cellsto lyse the target cells was determined. CTLs weregenerated by in vitro stimulation of splenocytes fromthe immune mice and investigated for cytotoxic activityin LDH release assay. Restimulated CTLs efficientlylysed peptide-pulsed syngeneic target cells except forSTEAP/5-13-pulsed cells, whereas the same target cellswere not lysed by splenocytes derived from the controlmice (Fig. 6A). The amount of killing was highest formSTEAP/186-193. Another important parameter inevaluating the effectiveness of anti-tumor CTL mightbe efficient recognition of authentic tumor cellsexpressing cognate antigen-MHC complex. We there-fore tested the ability of peptide-restimulated CTLcultures to recognize endogenously processed antigenon TRAMP-C1 cells. The restimulated CTLs showedpreferential recognition of TRAMP-C1 cells comparedto B16F10 melanoma cells as a negative control inICS assay (Fig. 6B). These results demonstrated thatvaccine-induced CTL populations from the immunemice recognized endogenously processed, cognateantigen-MHC complexes. However, the recognitionefficiency of TRAMP-C1 by effector cells was relatively

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TABLE I. Stabilization ofMHCbyCandidateCTLEpitopes

Epitope SequencePredicted MHC

restrictionIFN-g

production Refoldinga

PSCA/28-36 AQMNNRDCL Db — n.d.PSMA/86-94 AGTQNNFEL Db — n.d.PSMA/183-191 RTEDFFKL Kb — n.d.PSMA/214-222 MMVKNVQL Db þ þPSMA/587-595 FELANSIVL Db þ þSTEAP/5-13 DDVTNPEQL Db þ þSTEAP/82-90 SSLTFLYTL Db þ/� �STEAP/186-193 RSYRYKLL Kb þ þSTEAP/327-335 VSKINRTEM Db þ/� n.d.

aIn vitro refolding assay was performed by incubating each peptide with appropriate MHC Iheavy chain and b2 microglobulin. Correctly folded MHC I molecules were confirmed by gelfiltration chromatography with standard markers. n.d. not determined.

VaccinationAgainst Experimental ProstateCancer 943

low, when compared to peptide-pulsed EL4 target cells(Fig. 6B). This might be due to the lower level of MHCclass I expression on TRAMP-C1 cells than EL4 cells(data not shown).

Traff|cking of CD8TCells IntoTumorTissuesof the ImmuneMice

As significant amounts of STEAP-specific CD8T cells were found in the blood, we therefore examinedwhether CD8 T cells effectively trafficked into thetumor environment using the preventive experimentalsetup as described in Figure 2. Increased number ofinfiltrating mononuclear cells within tumor tissuestaken from the immune mice was observed by H&Estaining, compared to the control (Fig. 7). Immunohis-tochemistry also revealed increased numbers of infil-trating CD8bþ cells within the tumor tissues of theimmune mice relative to the control (Fig. 7). These datasuggest that the strong expansion of tumor-specificCD8 T cells and active recruitment of lymphocytesincluding CD8 T cells correlates to the inhibition oftumor growth by immunization.

ActiveTreatmentof Established TumorWithTherapeuticVaccination

To extend our findings to a more clinically relevantmodel, we next tested a potential therapeutic efficacy ofthe vaccination in tumor-bearing mice. To this end, themice were s.c. injected with TRAMP-C1 cells, and after

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Fig. 4. The kinetics of tumor antigen-specific CD8 T-cellresponses in the blood. A: Representative tetramer staining ofPBMC obtained from immune or control mice. The blood drawnfrom each indicated time pointwas stainedwith MHC I tetramersas describedinMaterials andMethods Section.Cellsweregatedonforwardscatter andCD8aþ lymphocytes.B:ThekineticsofSTEAP/186-193 tetramer-positive CD8 T cells after immunization andtumor challenge in the peripheral blood. Mean values at eachtimepointwere calculated for all immunizedmice (n¼10)andwereplottedwithmeanvalues for thecontrol (n¼ 5).

Fig. 5. STEAP immunization induces anti-tumor protection.Groupsof fivetosixmaleC57BL/6micewereinjectedwithindicatedrAdfollowedbytumorlysate-loadedDCs,andwerechallengedwithTRAMP-C1cells as describedin theMaterials andMethodsSection.The results depict the size of tumors developed after challenge.Results are presented as the mean tumor size over time. Similarresultswereobtainedin twoindependentexperiments.

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Fig. 6. Cytolytic activityandrecognitionofTRAMP-C1tumorcellsbyCTL.A:RestimulatedCTLs fromimmuneorcontrolmiceweremixedwithpeptide-pulsedEL4 targetcells in aLDHreleaseassay.B: Splenocytes fromimmunemicewere stimulatedwitheachindicatedpeptide for6 days and IFN-g production ofpeptide-specific CTLswas determinedby IFN-g ICS assay after incubationwithTRAMP-C1cells, B16F10mela-noma cells as negative control target, or peptide-pulsed EL4 cells as positive control target.Cellswere gatedon forward scatter andCD8aþ

lymphocytes.

tumor size reached about a diameter of 3 mm, micewere immunized with adenovirus and tumor lysate-pulsed DCs. The growth of tumors was then monitoredand the magnitude of the tumor-specific T-cellresponses was determined by tetramer staining andICS for IFN-g. The tumor growth rate was significantlyreduced in the immunized mice compared to thecontrol group (Fig. 8A). Weak but distinct IFN-gproducing T cells were observed from the splenocytesof the immune mice after primary immunization(Fig. 8B). However, the frequency of IFN-g-producingT cells was not increased even after boosting (Fig. 8B).

DISCUSSION

In this study, we have evaluated efficacy of prime-boost immunization strategy in preventive and ther-apeutic settings in a murine model of prostate cancer.Tumor rejection or growth delay is primarily mediatedby anti-tumor T-cell responses recognizing tumor-specific antigens (TSA) or tumor-associated antigens(TAA). In our experimental model, substantial CD8T-cell responses against STEAP are detected aftervaccination, while there was no detectable or weakCD4 T-cell response. These results support an idea thatthese STEAP-specific CD8 T cells play a major role indelaying the growth of prostate cancer cells.

STEAP is expressed predominantly in prostatecancer tissues both in human [6] and TRAMP mice[11], and thus represents potential target for immuno-therapy. STEAP is also shown to be expressed by avariety of cancers such as prostate, colon, pancreas,bladder, Ewing’s sarcoma, and ovarian, while its

expression in normal tissues is restricted to the prostate[6]. Furthermore, murine STEAP are highly homolo-gous to human STEAP at amino acid levels, suggestingthat the similar approaches are feasible for immuno-therapy of human prostate cancer. Indeed, it has beenshown that several peptides from STEAP sequencewere able to induce antigen-specific HLA-A2-restrictedCTLs [12]. With our prime-boost immunization strat-egy, we detected strong CD8 T-cell response to H-2Kb-restricted STEAP/186-193 epitope in the preventivesettings. Analysis of STEAP sequence using a combi-nation of epitope prediction algorithms has identifiedfour potential CTL epitopes including STEAP/186-193,but we failed to detect any specific CD8 T-cell responsesto three other peptides (data not shown). Two of these

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Fig. 8. Theefficacyof therapeutic vaccination againstpre-estab-lished TRAMP-C1 tumors. A: Male C57BL/6 mice (n¼10) wereinjectedwithTRAMP-C1cellsandthetumorgrowthwasmonitoredfollowing therapeutic vaccinations (prime and boost). B: Spleno-cytes from the immune mice were harvested 14 days afterboost immunization and IFN-g ICSwas performed in the presenceof 1 mg/ml of overlapping peptides for CD8 or tumor lysate (in aresponder:tumor cell ratio of1:3) for CD4 T cells.Cells were gatedonforwardscatter andCD8aþorCD4lymphocytes.

Fig. 7. Immunohistochemistry of tumor tissues isolated fromimmune mice. Solid tumor tissues were isolated and analyzed byimmunohistochemistry for thepresence ofCD8b-expressing cells.Pictures are representative for five to seven individualmice in eachgroup. [Color figure canbeviewedin the onlineissue,whichis avail-ableatwww.interscience.wiley.com.]

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three peptides scored very well in the MHC bindingprediction, while STEAP/186-193 showed moderateaffinities for MHC class I binding. It is likely thatthymocyte precursors which recognize the self-pep-tides with high affinities might have been negativelyselected, whereas precursors which recognize STEAP/186-193 peptide with moderate affinities are positivelyselected during maturation and move to the periphery.However, it seems that peripheral tolerance still hassurveillance mechanisms suppressing the activationof these cells, which might be overcome by activeimmunization of tumor-free mice. In tumor-existingenvironment, the level of tolerance seems to be higher,since weaker CD8 T-cell response to STEAP wasdetected. Our results also provide evidence thatSTEAP/186-193 peptide is naturally processed inTRAMP-C1 tumor cells and correctly presented toallow recognition by CTL. However, recognition ofauthentic tumor cells by CTL seems to be less efficient,compared to peptide-pulsed EL4 cells, and this isprobably due to low expression of MHC I molecules onTRAMP-C1 tumor cells.

In other study, STEAP has also been identified as anattractive candidate antigen for treating prostate cancer[13]. In contrast to our study, CD4 T cells play a majorrole in tumor rejection in their study, while CD8 T-cellresponses to STEAP epitope is also involved inprolonged survival of tumor-challenged mice. Thispartial discrepancy might be due to different deliveryvector, different immunization regimen, addition ofgenetic adjuvant such as IL-12m, and/or differentroutes. It remains to be determined whether whichimmunity is necessary and/or sufficient for theprotection from prostate cancer arising. We arecurrently investigating anti-tumor efficacy and mech-anisms of prostate-specific CD8 T cells by adoptivetransfer experiments.

Our study suggests that vaccination in preventivesetting is more effective than in therapeutic setting interms of CTL induction and tumor growth inhibition.This blunted efficacy of therapeutic vaccination mightbe due to the complex immunosuppressive environ-ment often developed by cancer, limiting anti-tumorimmune responses. If this is the case, chemotherapyand/or radiotherapy can be combined with vaccina-tion regimens to enhance their efficacies, since removalof primary tumor can restore immunocompetence ofthe host [14]. Otherwise, vaccination in the early stagesof tumorigenesis could be more effective in inducinganti-tumor response and prolonged survival [15].In conclusion, our results demonstrate that activeimmunization inhibits tumor growth both in preven-tive and therapeutic settings of experimental prostatecancer, possibly by induction of STEAP-specific CTLs.The efficacy of vaccination could be improved by

further refinement and future studies will develop arationale for vaccination as a novel prevention and/ortherapy tools for human prostate cancer.

ACKNOWLEDGMENTS

This work was supported by the Ewha WomansUniversity Research Grant of 2005. We wish toacknowledge technical support of Dr. Won Seo Parkin the Department of Pathology, National CancerCenter.

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