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ORIGINALARTICLEDNAfusion-genevaccinationinpatientswithprostatecancerinduceshigh-frequencyCD8+T-cellresponsesandincreasesPSAdoublingtimeLindseyChudleyKatyMcCannAnnManderTorunnTjelleJuanCampos-PerezRosemaryGodesethAntoniaCreakJamesDobbynBernadetteJohnsonPaulBassCatherineHeathPaulKerrIacobMathiesenDavidDearnaleyFredaStevensonChristianOttensmeierReceived:22February2012/Accepted:17April2012/Publishedonline:22May2012TheAuthor(s)2012.
ThisarticleispublishedwithopenaccessatSpringerlink.
comAbstractWereportontheimmunogenicityandclinicaleffectsinaphaseI/IIdoseescalationtrialofaDNAfusionvaccineinpatientswithprostatecancer.
Thevaccineencodesadomain(DOM)fromfragmentCoftetanustoxinlinkedtoanHLA-A2-bindingepitopefromprostate-spe-cicmembraneantigen(PSMA),PSMA27–35.
Weevalu-atedtheeffectofintramuscularvaccinationwithoutorwithelectroporation(EP)onvaccinepotency.
Thirty-twoHLA-A2patientswerevaccinatedandmonitoredforimmuneandclinicalresponsesforafollow-upperiodof72weeks.
Atweek24,cross-overtotheimmunologicallymoreeffectivedeliverymodalitywaspermitted;thiswasshowntobewithEPbasedonearlyantibodydata,andsubse-quently,13/15patientscrossedtotheEParm.
Thirty-twoHLA-A2-controlpatientswereassessedfortimetonexttreatmentandoverallsurvival.
Vaccinationwassafeandwelltolerated.
ThevaccineinducedDOM-specicCD4andPSMA27-specicCD8Tcells,whichweredetectableatsignicantlevelsabovebaselineattheendofthestudy(p=0.
0223andp=0.
00248,respectively).
Of30patients,29hadameasurableCD4T-cellresponseandPSMA27-specicCD8Tcellsweredetectedin16/30patients,withorwithoutEP.
Atweek24,beforecross-over,bothdeliverymethodsledtoincreasedCD4andCD8vaccine-specicTcellswithatrendtoagreatereffectwithEP.
PSAdoublingtimeincreasedsignicantlyfrom11.
97monthspre-treatmentto16.
82monthsoverthe72-weekfollow-up(p=0.
0417),withnocleardifferentialeffectofEP.
ThehighfrequencyofimmunologicalresponsestoDOM-PSMA27vaccinationandtheclinicaleffectsaresufcientlypromisingtowarrantfurther,ran-domizedtesting.
KeywordsImmunotherapyProstatecancerDNAvaccineElectroporationCD8TcellsIntroductionActivatingimmunityagainstcancerinpatientshasbeenadifcultgoal[1]butrandomizedstudiesarenowshowingencouragingresultsinsolidtumors[2,3],includingElectronicsupplementarymaterialTheonlineversionofthisarticle(doi:10.
1007/s00262-012-1270-0)containssupplementarymaterial,whichisavailabletoauthorizedusers.
L.
ChudleyJ.
DobbynC.
Ottensmeier(&)ExperimentalCancerMedicineCentre,CancerSciencesUnit,FacultyofMedicine,UniversityofSouthampton,TremonaRoad,SouthamptonSO166YD,UKe-mail:C.
H.
Ottensmeier@soton.
ac.
ukK.
McCannA.
ManderJ.
Campos-PerezR.
GodesethF.
StevensonC.
OttensmeierCancerSciencesUnit,FacultyofMedicine,UniversityofSouthampton,TremonaRoad,SouthamptonSO166YD,UKT.
TjelleI.
MathiesenInovioPharmaceuticalsInc.
,1787SentryParkwayWest,Building18,Suite400,BlueBell,PA19422,USAA.
CreakB.
JohnsonD.
DearnaleyRoyalMarsdenHospitalandInstituteofCancerResearch,DownsRoad,Sutton,SurreySM25PT,UKP.
BassC.
HeathUniversityHospitalsSouthamptonNHSFoundationTrust,SouthamptonGeneralHospital,TremonaRoad,SouthamptonSO166YD,UKP.
KerrRoyalDevonandExeterNHSFoundationTrust,BarrackRoad,Exeter,DevonEX25DW,UK123CancerImmunolImmunother(2012)61:2161–2170DOI10.
1007/s00262-012-1270-0prostatecancer[4].
Prostatecancerimmunotherapyisattractiveatearlybiochemicaldetectionofrecurrencesincerisingprostate-specicantigen(PSA),evenwithoutradiologicallymeasurabledisease,identiespatientsatriskwhohaveverysmallvolumedisease[5].
Vaccinetargets,likeMuc-1[6],PSA[7,8],prostaticacidphosphatase(PAP)[9]orprostate-specicmembraneantigen(PSMA)[10–12],havebeenidentiedaspromisingtargets[13].
ArandomizedphaseIIItrialshowedthatprostate-associatedantigenscanbeeffectivelytargetedbyvaccination[4].
Theimprovedmediansurvivalof4.
1monthsinlate-stagediseasewasnotmirroredbyPSAchanges[4],anobser-vationalsomadeinotherimmunotherapystudies[14].
AlthoughSipuleucel-Tsetsatreatmentparadigm,pro-ducinganewpatient-specicvaccineisatechnical,nancialandlogisticalchallenge.
Overallbenetremainssmall,indicatinganunmetclinicalneedforbetter,ideallynon-toxic,treatmentstoimproveoutcomes[13].
Vaccinationagainstcancerusingexogenouspeptidehasbeentestedwidelyandmayconferclinicaleffectinsomesettings[15–17].
However,CD8T-cellresponsesfollowingvaccinationusingexogenousshortpeptidesappeartransient[18]possiblyduetothelackofT-cellhelp.
Viralvector–basedvaccinesmayovercomethisproblemandhaveshownpromiseinmetastaticdisease[8,19]witheffectsalsoonPSAdoublingtime(PSA-DT)atbiochemicalfailure[6].
However,viralvectorswilleitherfacepre-existingimmunityorinduceitonrepeatinjec-tions.
DNAvaccinesavoidthisproblemandofferanoveldeliveryvehiclefortheinductionofpeptide-specicresponses.
WehavedesignedDNAfusion-genevaccinesabletodelivertumor-derivedpeptides,togetherwithmicrobialgenes,togeneratehighlevelsofT-cellhelp[20].
Ourplatformdesignincludesastronglyimmunogenichelperdomain(DOM),derivedfromfragmentC(FrC)oftetanustoxin,linkedtoatumor-epitopesequenceofchoice[20].
Inpre-clinicalmodels,DOM-epitopevaccinesinducedurabletolerance-breakingepitope-specicCD8T-cellimmunity,abletosuppressarangeoftumors[20].
InmiceexpressingtheHLA-A0201*transgene,theDOM-epitopevaccinedesignincorporatinganepitopefromPSMA(PSMA27VLAGGFFLL)[20,21]inducedhighlevelsofspecicCD8Tcellsabletokilltumorcells[22].
Wehavenowvaccinatedpatientswithbiochemicallyrecurrentprostatecancerand,tooptimizehumantransla-tion,alsoevaluateddeliverywithelectroporation(EP).
EPhasbeenreportedtoincreasethepotencyofDNAvaccinesbyincreasingantigenlevelsandstimulatinglocalinam-mation[23],anditsuseisrapidlyexpandinginbothinfectiousdiseasesandcancervaccination.
Wefoundthatthisapproachwassafe,welltoleratedandsignicantlyincreasedantibodyinduction[24].
WereportheretheeffectofourDOM-epitopevaccineonT-cellimmunityandclinicaloutcome.
ThevaccinereproduciblyinducesT-cellimmunitytoPSMA27andsignicantlyincreasesPSA-DT,andinspiteofthesmallsamplesize,weidentiedatrendtoincreasedtimetonexttreatmentcomparedtoacontrolgroupofunvaccinatedHLA-A2-patients.
Takentogether,thesedatasupportfurtherrandomizedtestingofthevaccine.
PatientsandmethodsPatientpopulationandregulatoryinformationPatientswithbiochemicallyrecurrentprostatecancer,ris-ingPSA(\50ng/mL,PSA-DT[3months)withoutradiologicalevidenceofdistantdiseasebyCTscan,bonescanand/orMRIwereeligible.
Pelvicnodalenlargementupto2cmwasallowed.
TumorPSMAexpressionwasconrmedimmunohistochemicallyatSouthamptonCellu-larPathologyLaboratory.
Otherinclusionandexclusioncriteriahavebeenreportedpreviously[24].
PatientswereHLA-typedinNHSlaboratories.
ThevaccineencodesanHLA-A2-restrictedepitope;onlyHLA-A2patientswerevaccinated.
HLA-A2-patientswhofullledallotherentrycriteriaformedthecontrolgroupandwerefollowedfortheevaluationoftimetonexttreatmentandsurvivalonly.
RegulatoryapprovalforthestudywasgivenbytheUKMedicinesandHealthcareRegulatoryAuthority(MHRA),theGeneTherapyAdvisoryCommitteeandlocalResearchEthicscommittees.
Thestudywasregisteredinthedata-baseofgenetherapytrialsintheUK.
AllpatientsgavewritteninformedconsenttoparticipateinthestudybetweenMarch2005andFebruary2008attheUniversityHospitalsSouthamptonandtheRoyalMarsdenHospital.
StudydesignThestudywasaphaseI/II,open-label,non-randomized,two-center,doseescalationstudy.
DOM-PSMA27vaccine[20,22,24]wasinjectedintothethighmuscle5timesat0,4,8,24and48weeks.
HLA-A2patientswererecruitedtotwostudyarms(Fig.
1).
InarmI,patientsreceivedDNAintra-muscularly(i.
m.
),andinarmII,vaccinewasdeliveredi.
m.
withEPusinganElgenTwinjectordevice[25]asdescribed[24].
Ineacharm,thedosewasescalated,with5patientspergroup:inarmI(withoutEP)—level1:800lg,level2:1,600lg,level3:3,200lgperdoseandinarmII(withEP)—level1:400lg,level2:800lg,level3:1,600lg.
Ifintheabsenceofsafetyconcernstheimmunologicaldatasupportedthis,patientswereallowedtocrossoverbetweenarmsofthestudyaftertherst3vaccinationsandreceivethedoseofthematchedlevelintheoppositearm.
2162CancerImmunolImmunother(2012)61:2161–2170123Follow-uponstudywasatweeks0,2and4followingvaccination,monthlytoweek32andthen2monthlytoweek72.
Ateachvisit,PSAlevelsweremeasured.
Forsafetyevaluation,fullbloodcounts,clotting,serumbio-chemistry,LDH(lactatedehydrogenase),CK(creatinekinase)andautoimmuneprolesofserumweremonitored.
PBMCwerestoredinliquidnitrogenforimmunologicalassessment.
Clinicalfollow-upPSAvalueswereavailableuptothedateofconsent,andduringthe72-weekstudyfollow-upforthevaccinatedcohort;ethicalpermissiontocollecttime-point-matchedPSAvaluesfortheunvaccinatedpatientswasnotobtained.
PSAvaluesusedtocalculatethePSA-DTwereevaluatedbyastudy-independent,blindedreviewer.
UsingonlyevaluablePSAvalues,PSA-DTwascalculatedusinganalgorithmcalculator(http://mskcc.
org/applications/nomograms/prostate/PsaDoublingTime.
aspx).
PSA-DTwascal-culatedforeachpatientfortheperiodpre-study,for6-monthperiodsonstudyandfortheoverall72-weekstudyperiod(calculateduptoweek72oruntiltreatment).
Timetonexttreatmentandsurvival(assessedupto31/12/2010)wererecordedforallpatients.
ImmunologicalevaluationsMIATA(MinimalInformationAboutT-cellAssays;http://www.
miataproject.
org)guidelineswereusedtoreportimmunologicaldataonT-cellresponses[26,27](OnlineResource1).
PBMCwereisolatedfromheparin-izedbloodsamplescollectedateachstudyvisit.
Recoveryandviabilitywerecalculatedusingamanualhemocytom-eterandtrypanblueexclusion.
PBMCwerecryopreservedandstoredinLN2vaporphase(Section1,OnlineResource1).
PBMCwereassessedforimmunologicalresponsesusingassaysvalidatedtoGCPforlaboratories,andlabo-ratorycompliancewasveriedbyexternalaudit[28].
ELISPOTPBMCfromallfollow-uptime-pointsfromeachpatientwereassessedforIFNcproductioninresponsetostimu-lationwithrecombinantFrCprotein(20lg/mL)[28]orPSMA27(VLAGGFFLL)peptide(10lg/mL,ProteinPeptideResearch,UK).
ThevalidatedELISPOTmethodusedisdescribedindetailinSection2,OnlineResource1.
CulturedELISPOTPSMA27-specicCD8Tcellswereculturedinvitrofor8days.
Ascellnumberwaslimiting,samplesfromdifferenttime-pointswerepooledandculturedinthefol-lowinggroups:baseline,weeks8,10and12,weeks16,20and24,weeks26,28and32andweeks50,52and60.
Cellswereculturedwith10lg/mLPSMA27peptideorwithapoolofviralpeptidesoracontrolpeptide,HIV.
IL-2wasaddedondays3and6andcellswereharvested,washedandrestedovernightonday8.
Followingre-stim-ulationwith10lg/mLpeptide,IFNcproductionwasmeasuredbyELISPOT.
FulldetailsareprovidedinSec-tion2,OnlineResource1.
StatisticalanalysisMedianvaluesarepresentedthroughout,whereappropriatewith25and75%interquartileboxwithranges.
Signi-cancewasdeterminedbyeitheratwo-sided,nonparametricWilcoxonsignedranktestoraMann–Whitneytest.
Avalueofp\0.
05wasconsideredsignicant.
ResultsPatientdemographicsSixty-fourpatientswereeligibleforthestudy(Fig.
1;Table1).
Thirty-twoHLA-A2patientswerevaccinatedand32HLA-A2-patientsformedthecontrolgroupforclinicalfollow-up.
Twopatientswithadverseevents(AE)aftertwovaccinationswerereplacedperstudyprotocolandincludedinthesafetybutnotintheimmunologicalanalyses(Fig.
1).
Onepatientreceivedthreevaccinationsbeforediseaseprogressionandcommencedandrogensuppressionbutremainedevaluableforimmuneresponses.
Aftertheinitial3vaccinationswitheitherDNAorDNAEP,allbut4patientswentontoreceiveboostervaccineswithEP(weeks24and48).
Twenty-ninepatientscompletedvacci-nationand72-weekstudyfollow-up(Fig.
1).
SafetyandadverseeventsSafetyevaluationinthersttwodosegroupshasbeenreported[24].
FulldataforallpatientsonthestudyandasummaryofAEsrecordedarelistedinOnlineResource2.
Thevaccinewassafeandwelltolerated.
MostAEsweregrade1or2andrangedfrominjectionsitereactionstou-likesymptoms,backpainandnailchanges.
VaccinationwasdiscontinuedduetoAEsintwopatients:oneexperi-encedgrade3worseningofapre-existingpsoriasis,withcausalityassessedaslikelyvaccinerelated,andasecondgrade3AEwasidentiedasworseningofpre-existingParkinson'sdisease,assessedasunlikelytobevaccinerelated.
TwoseriousAEswereobserved:onepatientdevelopedgrade2peripheraledemaandasecondwasCancerImmunolImmunother(2012)61:2161–21702163123admittedforaTURP.
Botheventsresolvedfullyandthepatientscontinuedonstudywithnorecurrence.
Previously,wereportedthesafetyofEPindosegroups1and2bythemeasurementofmuscledamagemarkersCKandLDH[24].
Patientsindosegroup3showednoincrease([twofoldbaseline)ineitherCKorLDH,andanyincreaseobservedatdays1and5aftervaccinationreturnedtobaselinelevelbyday14(datanotshown).
ClinicaloutcomePSA-DTiswidelyusedasanindicatorofoutcome[29]andwasevaluatedforvaccinatedpatients.
PSA-DTshowedsig-nicantincreasesduringthestudyperiod(Fig.
2a),withanincreasefrom11.
98months(range-356.
6to67.
9)pre-studyto17.
26months(range-117.
4to129.
4)forthe24-to48-monthperiodpost-vaccination(p=0.
0020(Fig2a)).
Thiswasaslowincrease,notevidentatthe0-to24-monthperiod.
Overthewholestudyperiod(0–72months),PSA-DTshowedasignicantincrease(p=0.
0417)to16.
82months(range-169.
2to62.
38),indicatingaslowingofdiseaseprogression.
IndividualpatientdataareprovidedinOnlineResource3.
Comparedtobaseline,PSA-DTincreasedin24/30patientsatoneormoretime-pointsduringthestudyandin19/30thePSA-DTincreasecontinuedtoweek72.
AnincreaseofC200%inPSA-DTatanypointduringthestudywasobservedin14/24,with4/24patientsretainingthiseffectoutto72weeks.
Figure2bshowstimetonexttreatmentinvaccinatedpatientscomparedtothecontrolgroup.
Withahazardratioof0.
7352(95%condenceinterval0.
37–1.
45),timetonexttreatmentwas243.
3weeksinthevaccinatedgroupand184.
0weeksinthecontrolgroup(p=0.
3785).
TherewasnoobjectivereductioninPSA.
Atamedianof4.
6years'follow-up,5vaccinatedpatientscomparedto6controlgrouppatientshavedied.
NoeffectofDNAdoseonoutcomewasdetected.
ImmunologicalresponsesImmunemonitoringwascarriedoutonallpatients,andresponsesareshowninTable2.
FrC-specicCD4andPSMA27-specicCD8TcellswereevaluatedusingIFNcELISPOT.
ExvivoFrC-specicresponseswereusedtoassessvaccineoperationandtheeffectofdeliveryFig.
1CONSORTdiagram2164CancerImmunolImmunother(2012)61:2161–2170123modality.
Of30patients,29hadasignicantCD4responsetoFrCfollowingvaccination,inkeepingwithanexpansionofamemoryT-cellpopulationdetectableinthebaselinesamples.
ThemedianCD4T-cellresponsemorethandoubledbyweek72comparedtobaseline;baselinemedianIFNcresponsewas34spots/million(range0–153)increasingtoamedianof72spots/million(range1–306)atweek72(p=0.
0208)(Fig.
3a).
T-cellresponsesagainstPSMA27wereassessedincir-culatinglymphocytes.
EffectorCD8Tcellsareunlikelytopersistinblood,andasexpected,wefoundonlylowlevelsexvivo(6/30positiveresponsesdetected,3/6beingobservedat2ormoretime-points).
Todetectcentralmemorycells,weculturedbloodTcellswithpeptide/IL-2for8daysinvitro[30];IFNc-producingPSMA27-specicTcellsweredetectedin55%(16/30)ofpatients.
ThemedianCD8responsehadincreased9.
6-foldbyweek72comparedtobaseline(from27spots/million(range0–2,373)to260spots/million(range0–8,233)(p=0.
0222))(Fig.
3b).
TherewasatrendforpatientswithdetectablePSMA27-specicTcellstohaveanincreasedtimetonexttreatment(Fig.
3c,p=0.
7925);weidentiednolinkbetweenPSA-DTanddetectionofcir-culatingpeptide-specicCD8Tcells.
TherewasnoapparenteffectofDNAdoseonimmunogenicity.
EffectofdeliveryonimmuneresponsesTheeffectofvaccinedeliverycanbeinterpretedfortherst24weeks,duringwhichthe2armsofthestudyremainedindependent.
Thereafter,11/15patientscrossedovertovaccinationwithEPfor2dosesperprotocolbasedonanimprovementinantibodyresponsestoDOMwithEPTable1Patientdemographicsforscreened,eligibleandconsentedpatientsParameterVaccinatedNon-vaccinatedHLA-A2HLA-A2-n=30no.
(%)n=32no.
(%)Medianage,years7175Range58–8466–80PriortreatmentProstatectomy10(30)4(13)Radiationtherapy24(80)29(91)Androgendeprivation25(83)27(84)Gleasonscore\615(50)10(31)714(47)16(50)[81(3)2(6)Unknown4(13)TumorsizeSmall(T1c–T3a)19(63)23(72)Large(T3b–T3c)6(20)4(13)Unknown5(17)5(16)BaselinePSA(ng/mL)Median5.
05.
3Range0.
5–26.
30.
97–48.
0\2.
01(3)1(3)2.
0–5.
017(53)14(44)5.
0–10.
08(27)8(25)[104(13)9(28)Fig.
2Clinicalresponses.
aShowsaboxandwhiskersplotofPSA-DTcalculatedforeachpatientpre-treatment,for6-monthperiodsonstudyandoverallforthewhole72-weekstudyfollow-up.
DatarepresentthemedianandrangeforallHLA-A2,vaccinatedpatients(n=30).
PSA-DTatweeks24–48andoverthe72-weekfollow-upperiodissignicantlyincreasedoverpre-treatment(p=0.
0020andp=0.
0417,respectively).
bAKaplan–Meierplotofthetimetonexttreatment.
Grayshadingindicatestheon-studyperiod.
Thesmallverticaltickmarksshowcensoredtimes.
ThedashedlinerepresentsvaccinatedHLA-A2patients(n=30),andsolidlineshowstheunvaccinatedHLA-A2-controlgroup(n=32)CancerImmunolImmunother(2012)61:2161–21702165123[24].
Figures3d,eshowCD4andCD8peakT-cellresponsesuptoweek24,respectively.
ForbothCD4andCD8responses,thedeliveryofvaccine±EPgeneratedasignicantresponsecomparedtobaseline.
TheeffectofaddingEPduringdeliverywasnotdramaticbuttherewasatrendtowardinductionofhigherlevelsofbothCD4andCD8T-cellresponses(p=0.
2128forCD4Tcellsandp=0.
1014forCD8Tcells)(Fig.
3d,e).
Clearly,largernumbersarerequiredbutthisweakeffectcontrastswiththesignicantincreaseinhumoralanti-DOMresponsesbyaddingEP[24].
DiscussionInHLA-A2transgenicmice,pDOM-PSMA27epitopevaccinationstimulatesstrongpeptide-specicCD8T-cellresponses[22].
ThePSMA27epitopeisprocessedfromPSMA,andinducedTcellscankillhumantargetcells,conrmingPSMA27asausefultargetforCD8T-cellattack.
ThephaseI/IIstudywepresentheretakestheseobservationstotheclinic.
InHLA-A2prostatecancerpatientsatbiochemicalfailure,withlowdiseaseburden,vaccinationsignicantlyincreasedPSA-DTcomparedtoTable2SummaryofimmuneresponsesPatientARMDoseAbCD4CD8CD8(cultured)±MaxFoldInc.
Weekofmax.
±Week0–24±Wk0–72MaxFoldInc.
Weekofmax.
±MaxFoldInc.
Weekofmax.
±Wk0–24±Wk0–72MaxFoldInc.
Weekofmax.
2DDDDE1-9.
64-7928–1211DDDDD2-2.
76-2,19226–3213DDDDD2--3.
760-9.
950–6019DDDDE2--()1.
726()752--1DDDEE173.
950860---5DDDEE119.
1521172-12.
550–607DDDEE12.
86066--47350–608DDDEE148.
55220550-82.
350–6015DDDEE23.
6102.
910---17DDDEE2-2.
510201269938–1223DDDEE311.
3522.
550---25DDDEE347.
028-2.
728-22.
38–1227DDDEE321.
1603.
926---29DDDEE3--2.
472---31DDDEE315.
960672---3EEEEE12.
91263127248--4EEEEE126352225227522926–326EEEEE158.
21224601.
78–129EEEEE17.
3283.
54764--10EEEEE142.
81212EEEEE292.
4525.
448()4616-4.
126–3214EEEEE2--4.
450-1,11350–6016EEEEE213.
228556-1,99250–6018EEEEE211.
5202910-5,24716–2420EEEEE215.
250210-1726–3222EEEEE32.
4604.
252---26EEEEE3-4.
616---28EEEEE3-332---30EEE32.
1163.
412---32EEEEE339350410--10.
426–32Datashowwhethertheresponseisnegative(-)(notsignicant),weaklypositive()(\twofoldincreasebutsignicant),positive()([twofoldincreaseandsignicantinonetime-point)orstronglypositive()([twofoldincreaseandsignicantinmorethanonetime-point).
Ifpositive,thefoldincreaseattheweekofmaximumresponseisshown2166CancerImmunolImmunother(2012)61:2161–2170123pre-vaccination.
WecomparedtimetonexttreatmentinvaccinatedpatientswithasynchronousgroupofHLA-A2-patients.
ThedatasuggestthatpDOM-PSMA27vaccinationcouldaffectthenaturalhistoryofprostatecancerandthesuggestionthattimetonexttreatmentcanbeextendedwillneedevaluationinalarger,randomizedstudy.
WhetherHLA-A2initsownrightisanadverseprognosticfactorhasnotbeenanswereddenitively,thoughthereisasug-gestionoflinktoprostatecancerincidence[31],increasedproportionoflargetumors(T3b–T3c)andhigherpost-operativeGleasonsumscomparedtotheHLA-A2-controlgroup[32].
AnadverseeffectofHLA-A2onoutcomewouldstrengthenaclinicaleffectofvaccination.
TheincreaseinPSA-DTbecamevisibleafter[24weeksafterrstvaccination,andin14/30patients,theincreasewas200%orgreater.
Fromabaselineof12months,PSA-DTincreasedto17months.
Whilecautionisneededintheabsenceofrandomizedcontrols[33],aconsistentstorysupportinganeffectofvaccinationatbiochemicalrecur-renceisemerging,wherevaccinationsignicantlyincreasesPSA-DT[6,9,34–36].
Withinthelimitsofcomparabilitybetweenstudies,itappearsthatourDNAvaccine,targetingasinglePSMAepitope,isatleastaseffectiveasothermorecomplexDNA-orpeptide-basedvaccines.
TcellsagainsttheDOMhelpersequenceexpandedinalmostall(29/30,97%)patients,demonstratingpatients'immunocompetenceandtheimmunologicalperformanceofthevaccine.
pDOM-PSMA27inducedCD8T-cellresponsesin16/30(55%)ofpatients,usingpre-denedassaycriteriaandasingleroundofinvitroculture.
Fig.
3Immuneresponses.
aandbRepresentdatafromallpatientswhocompletedvaccination(n=29)displayedasaboxandwhiskersplotandshowthemedianandrangeofCD4andCD8IFNcresponsestoFrCproteinandPSMA27peptideatbaseline(solid)andpost-nalvaccination(checked).
cShowsthetimetonexttreatmentfor14patientsthatrequiredadditionaltreatmentandcomparespatientswhomadeaPSMA27-specicCD8response(responder,n=7)withthosethatdidnothaveadetectableresponse(non-responder,n=7).
dande,Scatterplots,representpatientswhomadeasignicantresponseuptoweek24,comparingpatientsreceivingvaccinationwithout(-EP)orwith(EP)EP.
dComparesCD4responsestoFrCatbaselineandatweekofmaxresponse(n=11andn=13for-EPandEP,respectively).
eComparesCD8responsestoPSMA27atbaseline(solid)andatweekofmaxresponse(n=7andn=6for-EPandEP,respectively).
AllCD8responseshavebeenassessedaftershortterminvitrocultureCancerImmunolImmunother(2012)61:2161–21702167123Comparisonofimmunogenicitybetweentrialsishamperedbywidelyvaryingassaysystemsusedforimmunemoni-toring,andadditionally,onlyfewstudiesareavailablethatreportthisdataincomparableclinicalsettings[6,9].
ThedatasetbyMcNeeletal.
[9]withafull-lengthDNAvac-cineencodingPAPismostsimilartoourown,andinthisstudy,3/22patientshadmeasurableCD8T-cellresponsescomparedto6/30patientsinourdatasetexvivo.
Incorporationoffull-lengthantigensequenceintotheDNAvaccineseemsattractivesinceitwouldallowvac-cinationofallratherthantothe40%ofpatientswhocarryHLA-A2[9].
However,therearecogentreasonsforusingapeptide-focusedvaccinesincetheinductivepoweroftherepositionedpeptideisgenerallyconsiderablyhigherthanfromfull-lengthsequence[37].
CD8Tcellsspecicforasingleepitopeareclearlycapableofsuppressingevenanacuteviralinfection[38].
Shouldescapefromfocusedattackoccur,asecondvaccineagainstadifferentepitopecouldbeused[39],andweareexploringdoubleattackinourcurrentclinicaltrialagainsttheWT-1antigen[40].
Althoughourvaccinedesigncouldreadilyincorporatetumor-derivedMHCclassII-bindingepitopes,thereisnoclearevidencethatthesearerequiredforthemaintenanceofcytotoxicTcellsandthereisadangerthatregulatoryTcellsmightbeinduced[13,41].
Viralvector–basedvaccineshavetheproblemofpre-existingorinducedantiviralimmunity.
However,anMVA-MUC-1vaccineinducedanIFNcT-cellresponsetoMUC-1aftershort-termculturein7/34patientswithprostatecancer[6].
PoxviraldeliveryinmetastaticdiseasealsogeneratedPSApeptide-specicCD8T-cellrespon-sesin13/29patientsfollowingPSA-TRICOMvaccination[42]andin9/24patientsfollowingMVA-Trovaxvacci-nation[43].
Itappearsthatourapproachhasatleastcomparableimmunogenicity.
Wewouldcontend,however,thatavoidingblockingimmunity,likelytoarisefromMVA[44],willbeimportantforrepeatedvaccinationsrequiredtomaintainattackoncancer.
AconcernattheoutsetofourstudyhadbeenwhetherT-cellresponseswouldbedurable,aswithsomevaccinesapproachesCD8T-cellresponsescanbelostrapidlyandthennotre-expandafterrepeatedinjection[18].
OurdataarguethatwithDNAvaccinationthisisnotaproblemwithT-cellresponsesmaintainedtotheendofthefollow-upperiod.
ToexaminewhetherourDNAvaccinehadsufcientpotencytobescaledfrommousetohuman,weexaminedthedeliveryofourDNAvaccineusingtheInovioElgen100deviceforthersttimeintheclinic.
Wehadfoundpre-clinically[22,45]thatEPincreasedantibodyresponses,withlesserincreaseinCD8T-cellresponsestoourDNAfusionvaccine.
Intheclinic,thisdichotomyisalsoevidentwithclearincreasesinantibody[24]butonlyatrendforincreaseinbothCD4andCD8T-cellresponseswithEP.
Aftercross-overof11/15patientstoEPboosting,thereisasignicantanddurableincreasetotheendofthestudybutwecannolongerassesstheimpactoftheindividualdeliverymodalities.
Itisintriguingtospec-ulatewhyEPhasanapparentlysmallereffectonT-cellresponsescomparedtohumoralresponses.
Inthetrial,thismaysimplybeareectionofverysmallpatientnumberstreatedwithoutelectroporation,andarandomizeddatasetneedstoevaluatethecomparativequestionfurther.
ApossibleexplanationforboththemurineandhumandatacouldbethatunlikeB-cellresponses,wheretheincreasedmuscularantigenexpressionafterelectroporationleadstohigherhumoralresponses[24],forTcellstheremaynotbesuchastrictcorrelationwiththequantityofantigenexpressedbythemusclecells.
Insummary,thepDOM-PSMA27vaccineissafe,gener-atesanti-PSMAresponsesinthemajorityofpatientsandisassociatedwithanincreaseinPSA-DT.
UseofEPwaswelltoleratedandmayincreaseT-cellularvaccineefcacy.
Thesendingsmeritfurthertestinginarandomizedsetting.
Examiningthevaccine-inducedTcellsfortheirabilitytohometothetumorwillbeacriticalcomponentoffurtherevaluationandmayofferthetooltobetteridentifyalinkbetweenvaccine-inducedimmunityandclinicaloutcome.
AcknowledgmentsThisworkwassupportedbytheNIHRSouth-amptonExperimentalCancerMedicineCentre,fundedbyCancerResearchUKandtheDepartmentofHealth,UK.
ClinicalworkwasundertakenintheUniversityHospitalsSouthamptonandattheRoyalMarsdenNHSFoundationTrustwhoreceivedaproportionofitsfundingfromtheNHSExecutive.
TheworkwasfurthersupportedbytheAlanWillettFoundation,anunrestrictededucationalgrantfromInovioPharmaceuticals,theInstituteofCancerResearch,theBobChampionCancerTrustandCancerResearchUKSectionofRadiotherapy[CRUK]grantnumberC46/A2131.
WeacknowledgeNHSfundingtotheNIHRBiomedicalResearchCentreandsupportbytheWelcomeTrustClinicalResearchFacility,Southampton.
AssaydevelopmentandvalidationwasinpartsupportedbytheWallaceCoulterFoundation.
ConictofinterestTheauthorsdeclarethattheyhavenoconictofinterest.
OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttributionLicensewhichpermitsanyuse,dis-tribution,andreproductioninanymedium,providedtheoriginalauthor(s)andthesourcearecredited.
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