Immunization with recombinant DNA and modified vaccinia virus Ankara (MVA) vectors delivering PSCA and STEAP1 antigens inhibits prostate cancer progression

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Vaccine 29 (2011) 1504–1513

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Immunization with recombinant DNA and modified vaccinia virus Ankara (MVA)vectors delivering PSCA and STEAP1 antigens inhibits prostate cancer progression

Magdalena Krupaa, Marta Canamerob, Carmen E. Gomeza, Jose L. Najeraa, Jesus Gil c, Mariano Estebana,∗

a Department of Molecular and Cellular Biology, National Center of Biotechnology (CNB-CSIC), Campus de Cantoblanco, Darwin 3, 28049 Madrid, Spainb Comparative Pathology Core Unit, National Cancer Research Center (CNIO), Melchor Fernandez Almagro 3, 28029 Madrid, Spainc Cell Proliferation Group, Medical Research Council Clinical Sciences Centre, Faculty of Medicine, Imperial College, Hammersmith Campus, W12 0NN London, United Kingdom

a r t i c l e i n f o

Article history:Received 14 July 2010Received in revised form 1 December 2010Accepted 5 December 2010Available online 21 December 2010

Keywords:TRAMPProstate cancer vaccinePrime/boost immunization

a b s t r a c t

Despite recent advances in early detection and improvement of conventional therapies, there is an urgentneed for development of additional approaches for prevention and/or treatment of prostate cancer, andthe use of immunotherapeutic modalities, such as cancer vaccines, is one of the most promising strategies.In this study, we evaluated the prophylactic efficacy of an active immunization protocol against prostatecancer associated antigens mPSCA and mSTEAP1 in experimental prostate cancer. Two antigen deliveryplatforms, recombinant DNA and MVA vectors, both encoding either mPSCA or mSTEAP1 were used indiversified DNA prime/MVA boost vaccination protocol. Antitumour activity was evaluated in TRAMP-C1subcutaneous syngeneic tumour model and TRAMP mice. DNA prime/MVA boost immunization againsteither mPSCA or mSTEAP1, delayed tumour growth in TRAMP-C1 cells-challenged mice. Furthermore,simultaneous vaccination with both antigens produced a stronger anti-tumour effect against TRAMP-C1tumours than vaccination with either mPSCA or mSTEAP1 alone. Most importantly, concurrent DNAprime/MVA boost vaccination regimen with those antigens significantly decreased primary tumourburden in TRAMP mice without producing any apparent adverse effects. Histopathological analysis ofprostate tumours from vaccinated and control TRAMP mice revealed also that mPSCA/mSTEAP1 based-vaccination was effective at reducing the severity of prostatic lesions and incidence of high-grade poorlydifferentiated prostate cancer. Suppression of the disease progression in TRAMP mice was correlated withdecreased proliferation index and increased infiltration of T-cells in prostate tissue. Active immunizationagainst PSCA and STEAP1 using DNA prime/MVA boost strategy is a promising approach for preventionand/or treatment of prostate cancer.

© 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Prostate cancer is one of the most frequently diagnosed malig-nancies in developed countries and the third leading cause ofmale cancer-related deaths [1]. Currently available treatments forprostate cancer such as radical prostatectomy and radiotherapyare effective in localized disease. Nevertheless, about one thirdof the patients who undergo primary curative attempts experi-ence disease relapse. Therapeutic options for patients who developrecurrent disease or for those who have advanced stage and/ormetastatic disease at the time of diagnosis are limited. Androgenablation therapy and some chemotherapeutic agents lead initiallyto tumour regression in the vast majority of these patients; how-ever, most of them eventually develop refractory disease, which

∗ Corresponding author. Tel.: +34 91 5854553; fax: +34 91 5854506.E-mail address: [email protected] (M. Esteban).

is almost uniformly fatal [1]. Furthermore, current prostate cancertherapies frequently produce significant side effects and long-termcomplications that can have a negative impact on quality of life.Therefore, there is a clear need for development of novel, moreeffective and less toxic therapeutic strategies for management ofpatients with prostate cancer.

The use of immunotherapeutic modalities, in particular cancervaccines that exert an antitumour activity by engaging the exquisiteefficiency and specificity of the immune system to destroy tumourcells, represents a highly attractive approach for cancer preventionand/or treatment. Moreover, induction of systemic immunity tospecific antigen(s) expressed on cancer cells by vaccine is likelyto produce an immunological memory that will prevent diseaserecurrence.

One of the critical factors in tumour vaccine design is thechoice of the antigen. In prostate cancer, the number of anti-gens suitable for immunotherapy remains rather limited and itis still unclear whether any of these will be capable of confer-

0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.vaccine.2010.12.016


M. Krupa et al. / Vaccine 29 (2011) 1504–1513 1505

ring effective antitumour activity. Recently, prostate stem cellantigen (PSCA), a glycosylphosphatidylinositol (GPI)-anchored cellsurface highly glycosylated protein whose biological function atpresent is not clear, was identified in human prostate cancer.Based on sequence homology, it is considered to be a memberof the Ly-6 family and may be involved in signal transductionand cell–cell adhesion. PSCA is overexpressed in 80–90% of pri-mary and 100% of bone-metastatic prostate cancer specimens [2,3].Furthermore, it is present in androgen-dependent and androgen-independent prostate cancer and increased levels of PSCA correlatewith higher Gleason score, advanced stage and the progressionto androgen independence [3,4]. PSCA has been also found to bestrongly expressed in bladder, pancreatic and renal cell carcino-mas [5–7]. These characteristics make PSCA an attractive targetantigen for cancer immunotherapy. Indeed, several PSCA-derivedpeptides have been demonstrated to induce antigen-specific cyto-toxic T lymphocytes (CTLs) in human lymphocyte culture fromhealthy donors and prostate cancer patients, which were able torecognize and destroy prostate cancer cells in vitro [8–10]. Someof these peptides are currently under evaluation in clinical tri-als using autologous dendritic cell-based vaccination [11]. Anotherhighly expressed protein recently identified in advanced humanprostate cancer is six transmembrane epithelial antigen of theprostate 1 (STEAP1). Its function is currently unknown, althoughits localization at cell–cell junction and six transmembrane topol-ogy indicate that this protein might act as a channel or transporterprotein [12,13]. STEAP1 is highly expressed at all stages of prostatecancer and is also present in other tumours (bladder, colon, ovar-ian and Ewing sarcoma) while showing restricted expression innormal human tissues [12]. The favourable expression profilingof STEAP1 in normal and cancer tissues, as well as its cell sur-face localization, suggest its potential use as a target for cancerimmunotherapy. Moreover, STEAP1 peptides have been recentlydemonstrated to induce antigen-specific CTLs that were able to rec-ognize and destroy STEAP1-expressing tumour cells in vitro [14,15].

The murine counterparts are highly homologous to human PSCAand STEAP1 at both nucleotide and amino acid levels. In addi-tion, mPSCA and mSTEAP1 have similar tissue distribution as theirhuman homologues and are highly expressed in primary prostatetumours and metastatic lesions of TRAMP mice as well as in mouseprostate tumour cell lines TRAMP-C1 and TRAMP-C2 providing ananimal system for evaluation of antigen-specific immunotherapeu-tic strategies for prostate cancer [16]. The selection of a vectorfor delivery and expression of targeted tumour associated anti-gen(s) (TAAs) is a critical issue for development of clinically relevantcancer immunotherapy approaches. Viral vectors have been exten-sively used as antigen delivery vehicles due to their high efficiencyof gene transfer and expression, natural cell tropism as well astheir biological characteristics that can significantly enhance theimmunogenicity of antigens carried within them. Among them,poxviruses such as vaccinia virus (VV), canarypox and fowlpox havefound widespread use as vaccine vectors in infectious disease andcancer research (the later reviewed in [17]) due to their good safetyprofile and efficient induction of both cellular and humoral immuneresponses. Concerns about the safety of replication competent VVhave been addressed by development of highly attenuated strainssuch as modified vaccinia Ankara (MVA) [18,19]. Despite its lim-ited replication, MVA provides similar levels of recombinant geneexpression to those of replication-competent VV even in non-permissive cells [18]. Moreover, recombinant MVA vaccines havebeen demonstrated to induce potent immune responses to tar-get antigens that were considerably less affected by pre-existingVV-specific immunity when compared to replication-competentVV vectors [20]. Due to its excellent safety profile along with itshigh immunogenicity, MVA is widely considered as the VV strainof choice for clinical investigation. Indeed, MVA is extensively used

as vector for delivery of many different TAAs in preclinical studiesand clinical trials for the treatment and/or prevention of multipletypes of cancer (reviewed in [21]). Since TAAs are by definitionweakly immunogenic, several strategies using MVA recombinantshave been employed to improve immune response to TAAs includ-ing expression of T cell costimulatory molecules or cytokinesalongside with antigen and diversified prime/boost vaccinationregimens [22,23]. The poor immunogenicity of TAAs together withthe antigenic heterogeneity of tumours calls for vaccine strate-gies to enhance T-cell responses to multiple antigens. Therefore,the aim of this study was to evaluate the effectiveness of diversi-fied prime/boost vaccination regimens with mPSCA and mSTEAP1in mouse prostate cancer models. Our results demonstrate thatDNA prime/MVA boost immunization with both antigens inhibitstumour growth in TRAMP-C1 subcutaneous prostate cancer modelas well as suppresses prostate cancer progression in TRAMP mice.

2. Materials and methods

2.1. Plasmid DNA vectors

The full-length cDNA sequences of mPSCA and mSTEAP1were cloned into mammalian expression vector pCIneo (Promega,Madison, WI) and VV plasmid transfer vector pJR101. Detaileddescription of cloning strategy is provided as supplementarymaterials and methods. The correct open reading frames ofpCI-neo-mPSCA, pCI-neo-mSTEAP1, pJR101-mPSCA and pJR101-mSTEAP1 vectors were confirmed by restriction mapping and DNAsequencing. The plasmid DNA vectors used for vaccination weretransformed into Escherichia coli strain DH5� and purified fromlarge-scale cultures using a Plasmid Mega Kit (Qiagen Iberia SL,Madrid, Spain).

2.2. Generation of MVA recombinants expressing mPSCA andmSTEAP1

MVA recombinants encoding mPSCA or mSTEAP1 under controlof synthetic VV early/late promoter were generated as previouslyreported [24]. Briefly, Syrian hamster kidney BHK-21 cells cul-tured as described elsewhere [25] were infected with the wild-typeMVA, and subsequently transfected with recombinant VV plasmidtransfer vector pJR101-mPSCA or pJR101-mSTEAP1 that directs theinsertion of target antigen into the hemagglutinin (HA) locus withinMVA genome [24]. MVA recombinants were isolated after severalconsecutive rounds of plaque purification and insertion of heterol-ogous genes and absence of parental virus were confirmed by PCRanalysis. For vaccination purposes, the recombinant viruses wereamplified in BHK-21 monolayers and high titer stocks of MVA-mPSCA and MVA-mSTEAP1 were prepared by ultracentrifugationthrough a 45% sucrose cushion.

2.3. RT-PCR

BHK-21 cells were infected with either control vaccinia vec-tor (MVA-wt) or MVA-mPSCA at multiplicity of infection (MOI)of five. Cells infected with MVA-wt were harvested 48 h post-infection, while cells infected with MVA-mPSCA were harvestedat various times after infection indicated in Fig. 1B. Total RNAwas extracted from uninfected and infected BHK-21 cells usingUltraspecTM-II RNA isolation system (Biotexc Laboratories, Hus-ton, TX). Total RNA (5 �g) was subjected to DNase treatmentwith TURBO DNA-free kit (Ambion, Austin, TX). Subsequently, firststrand cDNA was synthesized from 500 ng of total RNA with ran-dom hexamers using SuperScript II RNase H-reverse transcriptase(Invitrogen, Barcelona, Spain) following manufacturer’s protocol


1506 M. Krupa et al. / Vaccine 29 (2011) 1504–1513

Fig. 1. Construction and characterization of recombinant MVA vectors expressing murine prostate cancer associated antigens. (A) Schematic representation of the HA locus ofthe recombinant MVA vectors. �-GUS, �-glucoronidase gene; P 7.5, VV early–late promoter; P E/L, VV synthetic early/late promoter. (B) RT-PCR analysis of mPSCA expression.Total RNA was purified from mock-infected cells and cells infected either with MVA-wt (harvested 48 hpi) or MVA-mPSCA harvested at the indicated time after infection(2, 4, 8, 24 and 48 h). Amplification of cDNA with mPSCA and �-actin primers yielded products of expected size of 371 bp and 350 bp, respectively. NC, PCR negative control.(C) Western blot analysis of mSTEAP1 expression. Whole cell lysates from mock-infected cells and cells infected either with MVA-wt (harvested 48 hpi) or MVA-mSTEAP1harvested at the indicated time after infection (2, 4, 8, 24 and 48 h) were subjected to SDS-PAGE and immunoblot analysis using anti-STEAP1 antibody. Band of approximately38 kDa could be detected only in cells infected with recombinant MVA vector expressing mSTEAP1. (D) Confocal immunofluorescence microscopy images of BHK-21 cellsinfected with 5 PFU per cell of either control vector (MVA-wt) or MVA-mSTEAP1. Six hours after infection, the cells were fixed and stained with rabbit anti-STEAP1 polyclonalantibody (green) and DNA dye ToPro-3 (blue). Open arrowheads indicate DNA-containing viral factories. Closed arrowhead and arrow point to membrane and viral factorylocalization of mSTEAP1 in merged image, respectively.

and cDNA corresponding to 25 ng of RNA was used for PCR ampli-fication. To exclude PCR product amplification from genomic DNAcontamination, for each RNA sample a parallel reaction was run inthe absence of reverse transcriptase. PCR amplification of mPSCAwas performed in 12.5 �L of mixture containing: 1 �L of cDNA, 1×PCR buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.2 �M of eachprimer, and 1.0 unit of Platinum Taq DNA polymerase (Invitrogen).The primers used for amplification of mPSCA cDNA were the sameas for cloning and are described in supplementary materials andmethods. PCR cycling profile consisted of initial denaturation at94 ◦C for 5 min and 25 cycles of 94 ◦C for 30 s, 60 ◦C for 30 s, and 72 ◦Cfor 30 s, followed by a final extension at 72 ◦C for 7 min. Primerssequence and PCR condition for �-actin (internal control) weredescribed previously [25]. PCR products were subjected to elec-trophoresis on a 2% agarose gel, stained with ethidium bromideand visualized under UV light.

2.4. Western blot

BHK-21 cells were infected with either MVA-wt or MVA-mSTEAP1 at 5 MOI. Cells infected with MVA-wt were harvested48 h post-infection, while cells infected with MVA-mSTEAP1 wereharvested at various times after infection indicated in Fig. 1C. Pro-

teins were extracted from uninfected and infected BHK-21 cellsusing RIPA buffer supplemented with complete protease inhibitorcocktail (Roche Diagnostics GmbH, Mannheim, Germany). Proteinconcentration was determined using the Bradford method and20 �g was subjected to SDS-PAGE and immunoblot analysis usingrabbit anti-STEAP1 polyclonal antibody (Abcam, Cambridge, UK).For �-actin detection, blot was stripped, blocked and then incu-bated with monoclonal primary antibody (Sigma, Saint Louis, MO,USA). Detection of primary antibody was performed using HRP-conjugated secondary antibody (Sigma) and ECL Western BlottingDetection System (GE Healthcare, Barcelona, Spain).

2.5. Confocal immunofluorescence microscopy

BHK-21 cells cultured on coverslips uninfected and infected for6 h either with MVA-wt or MVA-mSTEAP1 at 5 MOI were fixedwith 4% paraformaldehyde, washed and quenched with 50 mMammonium chloride. Subsequently, the cells were permeabilizedwith 0.2% Triton-X 100 and after blocking incubated with rab-bit anti-STEAP1 polyclonal antibody for 1 h at 37 ◦C. After severalwashes, cells were incubated with secondary antibody labelledwith Alexa488. ToPro-3 (Molecular Probes, Carlsbad, CA) was usedfor nuclear staining. Coverslips were mounted in ProLong (Molec-



ular Probes) and slides examined using Bio-Rad Radiance 2100confocal laser microscope.

2.6. Immunization protocol and efficacy in murine subcutaneoussyngeneic tumour model

Seven-week-old male C57BL/6 mice (Harlan, Barcelona, Spain)were primed with intramuscular (i.m.) injection of a total doseof 200 �g plasmid DNA vaccine in 100 �L of PBS into quadricepsmuscle. C57BL/6 mice immunized with single plasmid DNA vac-cine received 100 �g either pCI-neo-mPSCA or pCI-neo-mSTEAP1,and 100 �g pCI-neo plasmid lacking insert to equalize the totalamount of injected plasmid DNA. Mice immunized simultane-ously with both pCI-neo-mPSCA and pCI-neo-mSTEAP1 received100 �g of each plasmid DNA. Fifteen days after primary vaccination,mice were boosted intraperitoneally (i.p.) with 2 × 107 PFU/mouseof either MVA-mPSCA or MVA-mSTEAP1 in single-plasmid DNAvaccine groups or with 1 × 107 PFU/mouse of each MVA-mPSCAand MVA-mSTEAP1 in group co-immunized with both plasmidDNA vaccine constructs. The i.p. route was chosen for adminis-tration of MVA recombinants since it has been widely used inmice as an in vivo test system for the evaluation of the effi-cacy of MVA-based vaccines against infectious and malignantdiseases [20,21]. Mice in control group were vaccinated withempty pCI-neo vector and MVA-wt using the same schedule.Ten days after booster vaccination, C57BL/6 mice were chal-lenged subcutaneously (s.c.) with TRAMP-C1 cells as describedbelow.

TRAMP-C1 murine prostate cancer cell line obtained fromAmerican Type Culture Collection was maintained as described pre-viously [16]. Cells reaching 95% confluence were shortly tripsinized,and harvested with serum-containing medium. The cells werewashed and resuspended in serum-free complete medium atconcentration of 5 × 106 viable cells in 1 mL. Half-million TRAMP-C1 cells in 100 �L were implanted subcutaneously into theright flank. Tumour volume was estimated using the formula:m1

2 × m2 × 0.5236, where m1 and m2 represented the short andlong diameter of the tumour, respectively. Tumours were measuredusing a digital calliper twice weekly until day 55 post-implantationor until the tumour burden has met the humane endpoint. Tumoursthat could not be measured with calliper but were found to infiltratesurrounding tissues at the necropsy were assigned as immeasur-able. Tumour-free status was assigned to mice that at the necropsyhave shown lack of residual tumour tissue at the implantationsite.

2.7. Immunization protocol and efficacy in murine transgenicprostate cancer model

TRAMP mice were purchased from Jackson Laboratories andwere maintained in pure C57BL/6 background. Hemizygous TRAMPmales for these studies were routinely obtained by crossing of hem-izygous female transgene carriers with non-transgenic C57BL/6breeder males.

Seven-week-old TRAMP mice were primed with i.m. injec-tion of 100 �g of each pCIneo-mPSCA and pCIneo-mSTEAP1DNA vectors in 100 �L of PBS into quadriceps muscle. Fifteendays after primary vaccination, TRAMP mice were boosted i.p.with 1 × 107 PFU/mouse of each MVA-mPSCA and MVA-mSTEAP1recombinants. Two weeks after booster TRAMP mice received anadditional dose of 100 �g of each plasmid DNA vaccine admin-istrated i.m. followed fifteen days later by i.p. inoculation of2.5 × 107 PFU/mouse of each MVA recombinant. Mice in controlgroup were vaccinated with empty pCI-neo vector and MVA-wt using the same schedule as described above. TRAMP micewere monitored weekly for body weight loss, ruffling of fur,

behaviour and tumour development. Mice that exhibited progres-sive weight loss or signs of substantial morbidity were euthanized.Animals that died or had to be euthanized prior the time pointof sacrifice were excluded from the study. TRAMP mice andage-matched non-transgenic littermates were sacrificed at 24weeks of age and full necropsy was performed including detailedexamination of all organs for gross abnormalities. All animal exper-imental procedures were approved by the Ethical Committee ofAnimal Experimentation of the CNB-CSIC and conducted in com-pliance with institutional guidelines for laboratory animal care anduse.

2.8. Histopathological analyses

The entire genitourinary (GU) tracts of TRAMP mice consist-ing of all the four paired prostate lobes, seminal vesicles, urethra,ampullary gland, and drained bladder were dissected en bloc,weighed, fixed in 4% paraformaldehyde and embedded in paraffin.

For histopathological analysis 5 �m H&E stained sections of GUtract were reviewed in blinded fashion by a certified pathologistand prostate lesions were scored separately for each lobe (anterior,dorsal, lateral and ventral), and the grade of the most advancedlesion observed was assigned according to the criteria proposed bySuttie et al. [26]. Briefly, grade 1 corresponds to simple flat lesions;grade 2 to papillary or cribriform structures, single layered or withcell piling; grade 3 to papillary or cribriform lesions protruding intothe lumen. Grades 1 through 3 are considered hyperplasia. Grade4 (lesions occupying part of the lumen) and grade 5 (lesions fill-ing and expanding the lumen or a distinct epithelial mass insidethe lumen) are consistent with prostate adenoma. Grade 6 corre-sponds to adenocarcinomas, with poorly differentiated epithelialtissue, local invasion or distant metastasis. To determine the high-est overall grade, after examining all four prostatic lobes, a singlescore was assigned for the entire prostate of each animal corre-sponding to the highest grade assigned to any constituent lobe. Theindividual scores of animals within a given treatment group werethen averaged and are expressed as mean ± SEM.

2.9. Immunohistochemical studies

Immunohistochemical staining were performed on 5 �m 4%paraformaldehyde-fixed and paraffin-embedded GU tract sec-tions using the Dako Autostainer (DAKO Corp., Carpinteria, CA)with the following primary antibodies: Ki-67 (Vector Laborato-ries Inc., Burlingame, CA), CD3-e (Santa Cruz Biotechnology, SantaCruz, CA) and F4/80 (BMA Biomedicals, Augst, Switzerland). Afterincubation with the secondary antibodies, positive cells were visu-alized using 3,3-diaminobenzidine tetrahydrochloride plus as achromogen. All sections were counterstained with hematoxylin.Images were taken with a Plan Apochromat 40×/0.95NA objective(0.18 �m/pixel) using a Zeiss MIRAX Scan (Carl Zeiss Microimag-ing GmbH, Gottingen, Germany), converted into standard TIFFformat and analyzed with the AxioVision software (Carl ZeissMicroimaging, GmbH) for quantification of Ki-67, CD3 and F4/80immunostaining.

2.10. Statistics

Statistical analysis was performed using a two-tailed, unpairedStudent’s t-test and p values lower than 0.05 were consideredsignificant. Fisher’s exact test was used to compare incidence ofhyperplasia, adenoma and adenocarcinoma in control-vaccinatedversus mPSCA/mSTEAP1-vaccinated groups.



3. Results

3.1. Generation and characterization of DNA and MVA vectorsexpressing mPSCA and mSTEAP1

The recombinant DNA and MVA vectors encoding murineprostate cancer associated antigens mPSCA and mSTEAP1 weregenerated as described in Section 2. Scheme of the HA locus ofrecombinant MVA vectors is shown in Fig. 1A. The expressioncassette containing complementary DNA sequence coding eitherfull-length mPSCA or mSTEAP1 protein under control of strongsynthetic early/late VV promoter alongside with reporter gene �-glucuronidase was inserted by homologous recombination into theHA locus within the MVA genome. The kinetics of mPSCA andmSTEAP1 expression in BHK-21 cells infected with MVA recom-binants was determined by RT-PCR and Western blot, respectively(Fig. 1B and C). RT-PCR analysis using primers spanning the entiremPSCA polypeptide-coding sequence demonstrated the presenceof mPSCA transcript in MVA-mPSCA infected BHK-21 cells at 2 hpost-infection and thereafter; this transcript was not detected inuninfected and MVA-wt infected BHK-21 cells (Fig. 1B). The “no RT”controls for genomic DNA contamination did not yield any amplifi-cation products (data not shown). Western blot analysis revealed arobust expression of mSTEAP1 protein in MVA-mSTEAP1-infectedBHK-21 cells at 2 h post-infection and thereafter, while it was notdetected in uninfected and MVA-wt-infected BHK-21 cells (Fig. 1C).Furthermore, confocal microscopy analysis demonstrated the pres-ence of mSTEAP1 protein in the cytoplasm and plasma membraneof MVA-mSTEAP1-infected cells, as well as accumulation of theantigen in the perinuclear region corresponding to viral factories(Fig. 1D). Expression of gene-specific transcripts from recombinantDNA vectors was confirmed by RT-PCR using the cloning primersdescribed in supplementary materials and methods with RNApreparations from transiently transfected cells (data not shown).

3.2. Prime/boost vaccination with mPSCA and mSTEAP1 delaysTRAMP-C1 tumour growth

To evaluate the efficacy of specific active immunotherapyapproach targeting mPSCA and mSTEAP1, C57BL/6 mice receivedan i.m. inoculation of plasmid DNA constructs expressing mPSCAand mSTEAP1 or admixture of both vectors, followed two weekslater by i.p. booster with MVA recombinants encoding the sameantigen given in priming vaccination (Fig. 2A). Mice in the controlgroup received empty vectors following the same immunizationprotocol as described above. Ten days after booster, vaccinatedmice were challenged subcutaneously with 0.5 × 106 TRAMP-C1cells and the tumour growth was monitored for 55 days (Fig. 2Aand B). As shown in Fig. 2B DNA prime/MVA boost immunizationagainst either mPSCA or mSTEAP1 produced a marked inhibi-tion of tumour growth, however the strongest anti-tumour effectwas observed in the group that was simultaneously vaccinatedagainst both antigens. Animals bearing tumours that could notbe measured accurately due to infiltration of adjacent tissues (2/9and 1/10 mice in control- and mPSCA-vaccinated groups, respec-tively) were excluded from analysis. Partial responses were seenin the majority of mPSCA- and mSTEAP1-vaccinated animals (7/10and 8/10 mice, respectively), while 2 mice in each group did notrespond to the treatment (Fig. 2C). The final mean tumour volume55 days after TRAMP-C1 cells challenge was 442.83 ± 166.03 mm3

and 513.71 ± 51.31 mm3 in the mPSCA- and mSTEAP1-vaccinatedgroups, respectively, while it was 881.57 ± 94.93 mm3 in the con-trol group (Fig. 2A and C). When compared to control animals,DNA prime/MVA boost immunization against single antigen mPSCAand mSTEAP1 considerably reduced tumour volume by 49.8%(p = 0.052) and 41.7% (p = 0.002), respectively. In mPSCA/mSTEAP1-

Fig. 2. DNA prime/MVA boost immunization with mPSCA and mSTEAP1 delaysgrowth of transplantable murine prostate cancer. (A) Schematic representation ofimmunization protocol. Male C57BL/6 mice were immunized at day 0 with recom-binant DNA vectors and boosted 15 days later with MVA recombinants. Mice werevaccinated either with mPSCA and mSTEAP1 alone or with the admixture of bothantigens. In control group, mice received empty DNA vector and MVA-wt as a prim-ing and booster immunization, respectively. Ten days after the last vaccination, micewere challenged subcutaneously with 0.5 × 106 TRAMP-C1 cells. Tumour growthwas evaluated over 55 days. (B) Tumour growth of s.c. transplanted TRAMP-C1 cellsin C57BL/6 mice vaccinated as described above. The data shown represent one oftwo independent experiments. Data are presented as mean tumour volume ± SEMat indicated time points. *Statistically significant difference (p < 0.01) from control-vaccinated animals, Student’s t-test. †Statistically significant difference (p < 0.05)between mSTEAP1- and mPSCA/mSTEAP1-vaccinated groups, Student’s t-test. (C)Scatter plot depicting individual tumour volumes within each group at day 55 afterchallenge with TRAMP-C1 cells. Columns, mean tumour volume; bars, SEM. Numberof animals in each group is indicated in brackets. *Statistically significant difference(p < 0.01) from control-vaccinated animals, Student’s t-test. †Statistically significantdifference (p < 0.05) between mSTEAP1- and mPSCA/mSTEAP1-vaccinated groups,Student’s t-test.

vaccinated group final mean tumour volume was 207.42 ± 106.31and it was significantly decreased by 76.5% (p < 0.001) as comparedwith the control group that received empty vectors (Fig. 2B andC). In this group all mice except one showed partial responses andtwo animals remained tumour-free 55 days after challenge with



Fig. 3. Concurrent vaccination with mPSCA and mSTEAP1 using DNA prime/MVAboost protocol decreases primary tumour burden in TRAMP mice. (A) Schematic rep-resentation of immunization protocol. Seven weeks old TRAMP mice were primedwith admixture of recombinant DNA vectors encoding mPSCA and mSTEAP1 andboosted two weeks later with MVA recombinants expressing the same antigens. Anadditional dose of DNA and MVA recombinant vectors was given at 11 and 13 weeksof age, respectively. In control group, TRAMP mice received empty DNA vector andMVA-wt following the same vaccination protocol as described above. Under thetime line, the different stages of prostate cancer progression in the TRAMP modelare indicated. (B) Scatter plot illustrating the GU tract weight from control- andmPSCA/mSTEAP1-vaccinated TRAMP mice as well as age-matched non-transgeniclittermates at 24 weeks of age. Columns, mean GU tract weight. Data from two inde-pendent experiments with similar results, involving a total number of 11 mice pergroup are shown. The statistical significance of difference between control- andmPSCA/mSTEAP1-vaccinated groups was analyzed by Student’s t-test.

TRAMP-C1 cells. The final mean tumour volume in the group immu-nized against both antigens was also reduced by 53.2% (p = 0.264)and 59.6% (p = 0.014) when compared to mPSCA- and mSTEAP1-vaccinated animals, respectively.

3.3. Prime/boost vaccination with mPSCA and mSTEAP1decreases primary tumour burden in TRAMP mice

Given the efficacy of concurrent immunization with mPSCA andmSTEAP1 using DNA prime/MVA boost protocol in transplantableTRAMP-C1 tumour model, we examined whether this strategy ofantigen-specific active vaccination could prevent or delay tumourprogression in TRAMP mice, a spontaneous prostate cancer modelthat closely resembles the progression of human prostate cancer[27,28].

TRAMP mice were immunized simultaneously with DNA vec-tors encoding mPSCA and mSTEAP1 at 7 weeks of age, followedtwo weeks later by a booster with admixture of MVA recom-binants expressing the same antigens (Fig. 3A). Having in mindthe aggressiveness of this model, to maximize antigenic challengeat early stages of prostate tumour development the DNA/MVAadministration cycle was repeated at 11 weeks of age (Fig. 3A).It has been described previously that all TRAMP males in a pureC57BL/6 background present prostate tumours by 24 weeks of age

(Fig. 3A) [27]. Therefore, to evaluate anti-tumour effect of DNAprime/MVA boost vaccination with mPSCA and mSTEAP1, TRAMPmice immunized either with both antigens or control vectors weresacrificed at 24 weeks of age and the weight of the entire GU tractwas used as an index of primary tumour burden [28]. The aver-age GU tract weight of mPSCA/mSTEAP1-vaccinated TRAMP mice(0.73 ± 0.05 g) was significantly reduced (p = 0.001) compared tothe control-vaccinated group (1.15 ± 0.08 g) (Fig. 3B). The averageGU tract weight in non-transgenic littermates at 24 weeks of agewas 0.55 ± 0.01 g (Fig. 3B). Upon necropsy, TRAMP mice immu-nized with control vectors displayed enlarged prostate and seminalvesicles compared to age-matched non-transgenic littermates (GUtract weight 2.1-fold higher) and 3/11 mice (27.2%) presented pal-pable, indurate spherical prostate tumours. When compared toage-matched non-transgenic littermates GU tract enlargement inthe group of TRAMP mice that received antigen-specific vaccina-tion was much less pronounced (GU tract weight 1.3-fold higher)and only 1/11 mice (9.1%) demonstrated grossly evident prostatetumour at the time of sacrifice. DNA prime/MVA boost vaccinationagainst mPSCA and mSTEAP1 was safe, since gross examination ofvital organs (heart, lungs, kidney, and liver) upon necropsy did notshow any abnormalities and the mean body weights of the controland vaccinated TRAMP mice as well as non-transgenic littermatesdid not differ significantly throughout experimental protocol (datanot shown).

3.4. Immunization with mPSCA and mSTEAP1 inhibits tumourprogression in TRAMP mice

To assess the severity of prostate lesions in TRAMP mice, the GUtracts were prepared for routine histopathological analysis and thehighest overall grade was assigned for each prostate as describedin Section 2. At 24 weeks of age, there was a significant reductionin severity and extension of prostate lesions in mPSCA/mSTEAP1-vaccinated mice compared to control-vaccinated group (Fig. 4A andB). The mean highest overall grade of tumours from TRAMP miceimmunized against mPSCA and mSTEAP1 was 4.36 ± 0.34, whileit was 5.36 ± 0.24 for TRAMP mice that received control vectors(p = 0.026) (Fig. 4B). In addition, there was a significant difference inthe incidence of hyperplasia and poorly differentiated adenocarci-noma between control- and mPSCA/mSTEAP1-vaccinated animals(p = 0.016; Fig. 4C). In control mice, there was a complete absenceof hyperplasia with 45.5% (5/11) and 54.5% (6/11) incidence ofadenoma and poorly differentiated adenocarcinoma, respectively(Fig. 4C). In general, control-vaccinated mice were distinguishedby a bigger tumorous mass, being possible to recognise vascularembolism in some of the poorly differentiated tumours. On thecontrary, 36.4% (4/11) of mPSCA/mSTEAP1-vaccinated TRAMP micedeveloped hyperplasia and 54.5% (6/11) moderately differentiatedlesions which exhibited most of the prostate ducts incompletelyfilled by proliferating epithelium. A poorly differentiated adeno-carcinoma was observed only in one mouse (9.1%) immunized withmPSCA and mSTEAP1 (Fig. 4C).

Additionally, we determined by immunohistochemistry theexpression of Ki-67 in prostate tissue from mPSCA/mSTEAP1-vaccinated and control TRAMP mice (Fig. 5A). As shown in Fig. 5Bproliferation index was significantly reduced by 51.4% in prostatetumours from mice immunized against mPSCA and mSTEAP1 com-pared to control mice that received empty vectors (p = 0.041).

Furthermore, to assess whether antigen-specific vaccina-tion induced infiltration of immune cells, such as T-cells andmacrophages, at the tumour site, we stained the sections ofprostate tissue from mPSCA/mSTEAP1- and control-vaccinatedmice using anti-CD3 (a marker of T-cells) and anti-F4/80 (a markerof macrophages) antibodies. There was an increase of about 25%in T-cell infiltration within the tumours from mPSCA/mSTEAP1-



Fig. 4. Concurrent vaccination with mPSCA and mSTEAP1 using DNA prime/MVA boost protocol inhibits prostate cancer progression in TRAMP mice. (A) Representativephotomicrographs of H&E stained GU tract sections of two 24 weeks old animals from control- and mPSCA/mSTEAP1-vaccinated groups. A higher magnification of the areabordered by the black rectangle in the low power image is shown on the right. (B) The histopathological grading was performed on all four prostate lobes for 11 animalsper group. Sections were scored on a scale of 1–6 as described in Section 2, and the grade of most advanced lesion was assigned for each lobe. The highest overall grade forthe entire prostate equivalent to the highest grade assigned to any one constituent lobe was designated and used to calculate the mean for control- and mPSCA/mSTEAP1-vaccinated groups. For additional results on histopathological scoring for each prostate lobe refer to supplementary Fig. 1. Columns, mean highest overall grade (n = 11); bars,SEM. The statistical significance of difference between control- and mPSCA/mSTEAP1-vaccinated groups was analyzed by Student’s t-test. (C) The highest overall grade foreach prostate was assigned as described above and distribution of each histopathological grade in control- and mPSCA/mSTEAP1-vaccinated TRAMP mice at 24 weeks of age

is shown. Numbers inside the columns correspond to the histopathological grade. Grades 1–3 represent hyperplasia ( ), grade 4 and 5 adenoma ( ), and grade 6 poorlydifferentiated adenocarcinoma ( ). Fisher’s exact test was used to compare control- and mPSCA/mSTEAP1-vaccinated groups for the frequency of hyperplasia, adenomaand adenocarcinoma.

immunized mice when compared to the control group thatcould be associated with the observed inhibition of prostate car-cinogenesis after vaccination, although it was not statisticallysignificant (Fig. 5C). No difference was observed in macrophagestaining between control- and antigens-vaccinated groups (datanot shown).

4. Discussion

Recent advances in the field of tumour immunology, a betterunderstanding of the immune system surveillance and tolerancemechanisms as well as the progress in defining TAAs, have providedthe rationale for the development of numerous immunologi-cal strategies for prevention and/or treatment of human cancersincluding cancer vaccines. Because TAAs are by definition poorlyimmunogenic, the challenge within the field of cancer vaccines isto find conditions to break a possible tolerance towards them. Thus,antigen delivery platform that effectively express and facilitate pro-

cessing as well as presentation of tumour derived-immunogensis a critical requirement for the development of cancer vaccines.Various approaches are currently used to enhance immunogenic-ity of TAAs including diversified prime/boost strategies that consistof sequential vaccination using different antigen delivery systemsencoding the same antigen.

The induction of tumour-protective immunity against prostatecancer by using PSCA and STEAP1 as a targets is a promisingstrategy. The human PSCA and STEAP1 are attractive targets forimmunotherapy since they are highly expressed at all stagesof the disease progression while showing restricted expressionin normal tissues. The murine PSCA and STEAP1 are highlyhomologous to human counterparts and are upregulated in theTRAMP-C1 mouse prostate cancer cell line as well as in prostatetumours in the TRAMP model. Therefore, different immunother-apeutic approaches targeting these antigens including diversifiedprime/boost strategies consisting of either DNA vectors followedby Venezuelan equine encephalitis virus replicons (VEE VRP) orrecombinant adenoviruses followed by dendritic cell-based vaccine



Fig. 5. Immunohistochemical analysis of tumour cell proliferation and T lymphocytes infiltration in prostate tissue from control- and mPSCA/mSTEAP1-vaccinated TRAMPmice. GU tract sections were stained with either Ki-67 or CD3-e antibodies and counterstained with hematoxylin. (A) Ki-67 expression in representative prostate tissue of 24weeks old control- and mPSCA/mSTEAP1-vaccinated TRAMP mice. (B) Bar graph representing the average percentage of prostate area that scored positive for Ki-67. Columns,mean Ki-67-positive area (n = 5); bars, SEM. The statistical significance of difference between control- and mPSCA/mSTEAP1-vaccinated groups was analyzed by Student’st-test. (C) Bar graph depicting the average percentage of prostate area that scored positive for CD3. Columns, mean CD3-positive area (n = 5); bars, SEM.

were already under investigation in experimental prostate cancermodels [29–31].

In the present study, we chose the highly attenuated poxvirusvector MVA to evaluate the efficacy of active immunization againstmPSCA and mSTEAP1 since it has several characteristics that makesit an attractive candidate for use as a vaccine vector [21]. Indeed,recombinant MVA vectors have been extensively used in preclin-ical and clinical trials as gene delivery systems for vaccinationagainst a broad range of infectious diseases and malignancies [21].The capacity of a viral vector such as MVA, which has impairedreplication ability, to elicit high-frequency immune responsesdepends critically on the efficient presentation of antigens to Tlymphocytes. MVA has a pronounced tropism not only for mono-cytes/macrophages and B cells, all of which can act as professionalantigen presenting cells (APCs), but also efficiently infects den-dritic cells (DCs), key professional APCs essential for generationof immune responses [32,33]. Due to its ability to infect and effi-ciently produce viral and recombinant antigens in both professionaland non-professional APCs, MVA has characteristics that enabledirect priming as well as cross priming, the later being likely themajor mechanism of primary induction of immune responses uponMVA vaccination [34,35]. Furthermore, MVA induces activation andmaturation of DCs as well as enhances expression of various proin-flammatory cytokines, such as MCP-1, TNF, INF-�, IL-2, IL-6 andIL-12 [33,35–37]. Recently, MVA has been shown in macrophages

to activate TLR2-TLR6-MyD88 and MDA-5-IPS-1 pathways leadingto the production of several chemokines and type I interferon aswell as NALP3 inflammasome that controls the processing and mat-uration of IL-1� and IL-18 [38]. In vivo MVA tropism, in particularits capacity to infect macrophages and DCs, as well as activation ofinnate immunity leading to the production of supportive cytokinesare of crucial importance, since it is likely to improve antigen pre-sentation and subsequent T cell priming and therefore enhance thegeneration of tumour-specific immunity and antitumour activity.

Furthermore, MVA recombinants used in this study express full-length mPSCA and mSTEAP1 antigens providing multiple potentialMHC class I- and MHC class II-restricted epitopes as well as astrategy to optimally target the dominant antigen presentationpathway. These vaccines together with DNA vectors encoding thesame full-length antigens were consecutively administered in aDNA prime/MVA boost strategy, since it is well established thatthe use of poxvirus based vaccines in diversified prime/boost reg-imens increases the generation of T cell immunity to recombinantantigen and may also improve therapeutic responses [23,39–44].

Our results demonstrate that DNA prime/MVA boost vacci-nation of C57BL/6 mice against mPSCA, delayed tumour growthin mice challenged with TRAMP-C1 cells compared with tumourgrowth in control-vaccinated mice. Similarly, immunization ofC57BL/6 mice with vectors expressing mSTEAP1 significantly inhib-ited transplantable TRAMP-C1 tumour growth. Recently, it has been



shown also that prophylactic prime/boost immunization againsteither mPSCA or mSTEAP1 using DNA vectors followed by VEE VRPinduced immune response against these antigens and had anti-tumour effect in TRAMP-C2 cells-challenged mice [29,30].

In view of the fact that tumours express antigens heteroge-neously, targeting two distinct TAAs in a single vaccination regimenis likely to generate polyclonal T cell responses that could play animportant role in preventing tumour escape through antigen loss aswell as have better anti-tumour effect, since the concomitant tar-geting of more than one antigen may elicit additive or synergisticimmune responses. To address this issue, we set up a protocol basedon the concurrent inoculation of DNA and MVA vectors expressingmPSCA and mSTEAP1 in the same vaccine formulation. Indeed, DNAprime/MVA boost immunization with a combination of vectorsencoding both antigens was more effective at inducing protectiveimmunity leading to a stronger inhibition of TRAMP-C1 tumourgrowth than vaccination against either antigen alone. We demon-strated here for the first time the advantage of combining these twoantigens in the same vaccine formulation using DNA prime/MVAboost protocol, since as previously reported, immunization withadmixture of recombinant adenovirus vectors expressing threemurine prostate cancer-associated antigens (mPSCA, mSTEAP andmPSMA) followed by dendritic cell-based vaccine did not improvethe efficacy of any single-antigen vaccine in TRAMP-C1 tumourmodel [31].

Our results in subcutaneous model prompted us to evaluatethe DNA prime/MVA boost schedule using admixture of the vec-tors expressing both antigens in a more physiologically relevantmodel such as the TRAMP mice that develop spontaneously pro-gressive forms of prostate cancer over time. Immunization ofTRAMP mice against mPSCA and mSTEAP1 produced a significantreduction of primary tumour burden as compared to control-immunized mice that presented large tumours at the time ofsacrifice. Furthermore, histopathological analysis revealed that theprostate tumours present at 24 weeks of age in mPSCA/mSTEAP1-vaccinated mice exhibited lower histological grade than those ofTRAMP mice that received control vectors. To evaluate whetherthe reduction in histological grade in treated mice was relatedto a decrease in cell proliferation, we performed Ki-67 immuno-histochemistry in tumour tissue of mPSCA/mSTEAP1-vaccinatedand control-vaccinated TRAMP mice. Our results showed a markeddecrease in the proliferation index in vaccinated mice as comparedto control mice. Therefore, it is likely that the observed reduc-tion in histological grade is due to a less proliferative status ofthe tumours in mPSCA/mSTEAP1-vaccinated mice. Moreover DNAprime/MVA boost vaccination against these antigens significantlyreduced the incidence of poorly differentiated adenocarcinoma, themost aggressive form of prostate cancer.

Although our immunization protocol did not completely pre-vent prostate cancer development in TRAMP mice, the reductionin both histological tumour grade and tumour incidence indi-cates that DNA prime/MVA boost vaccination against mPSCA andmSTEAP1 was sufficient to inhibit disease progression. Consider-ing the aggressiveness of TRAMP model where malignant cellsarise continuously over time in entire organ, this vaccinationprotocol may show a greater efficacy against cancer in humansince the vast majority of tumours are of clonal origin and malig-nant transformation is likely less frequent than that observed inTRAMP mice. Furthermore, combination of this basic vaccinationprotocol in multi-nodal strategies with other immunotherapeuticagents, in particular those that aim at blocking immunosuppres-sive mechanisms, and/or with conventional therapeutic modalitiesmay further improve its efficacy.

Currently, several prostate cancer vaccines including recom-binant vaccinia and fowlpox viruses, employed in a diversifiedprime/boost strategy, as well as DNA or MVA vectors, used as

a single immunizing agents, are under investigation in clinicaltrials showing encouraging results and in some cases clinical ben-efit [44–49]. In the present study we demonstrated that DNAprime/MVA boost vaccination strategy targeting two prostatecancer associated antigens, PSCA and STEAP1 inhibits tumour pro-gression in experimental prostate cancer models and thereforecould be considered as a feasible approach for the preventionand/or treatment of prostate cancer.

Acknowledgements

We are thankful to Dr. Jose M. Arencibia and Dr. Artur Plonowskifor suggestions and comments on the manuscript and to VictoriaJimenez, Virginia Alvarez and Natalia Matesanz for expert techni-cal assistance. This work was supported by grants from the SpanishMinistry of Science and Innovation (SAF2008-02348; M. Esteban),the Fundación Marcelino Botín (M. Esteban) and the Spanish Min-istry of Education (AP2003-1722; M. Krupa). This work was for thefulfilment of the PhD degree by M.K. at the Autónoma University ofMadrid.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.vaccine.2010.12.016.

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Immunization with recombinant DNA and modified vaccinia virus Ankara (MVA) vectors delivering PSCA and STEAP1 antigens inhibits prostate cancer progression

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