Chimeric Rat/Human HER2 Efficiently Circumvents HER2 Tolerance in Cancer Patients

13 Pages·2014·1.54 MB·English

Published OnlineFirst March 25, 2014; DOI: 10.1158/1078-0432.CCR-13-2663
Clinical
Cancer
Cancer Therapy: Preclinical
Research
Chimeric Rat/Human HER2 Efficiently Circumvents HER2
Tolerance in Cancer Patients
SergioOcchipinti1,3,LauraSponton1,3,SimonaRolla1,3,CristianaCaorsi4,AnnaNovarino5,MichelaDonadio5,
SaraBustreo5,MariaAntoniettaSatolli6,CarlaPecchioni6,CristinaMarchini7,AugustoAmici7,
FedericaCavallo1,PaolaCappello1,3,DanielePierobon1,3,FrancescoNovelli1,3,andMirellaGiovarelli1,3
Abstract
Purpose:DespitethegreatsuccessofHER2vaccinestrategiesinanimalmodels,effectiveclinicalresults
havenotyetbeenobtained.WestudiedthefeasibilityofusingDNAcodingforchimericrat/humanHER2as
atooltobreaktheunresponsivenessofTcellsfrompatientswithHER2-overexpressingtumors(HER2-CP).
ExperimentalDesign:Dendriticcells(DCs)generatedfrompatientswithHER2-overexpressingbreast
(n¼28)andpancreatic(n¼16)cancerweretransfectedwithDNAplasmidsthatexpresshumanHER2or
heterologousratsequencesinseparateplasmidsoraschimericconstructsencodingrat/humanHER2fusion
proteinsandusedtoactivateautologousTcells.ActivationwasevaluatedbyIFN-gELISPOTassay,perforin
expression,andabilitytohaltHER2þtumorgrowthinvivo.
Results:SpecificsustainedproliferationandIFN-gproductionbyCD4andCD8TcellsfromHER2-CP
wasobservedafterstimulationwithautologousDCstransfectedwithchimericrat/humanHER2plasmids.
Instead,Tcellsfromhealthydonors(n¼22)couldbeeasilystimulatedwithautologousDCstransfected
withanyhuman,rat,orchimericrat/humanHER2plasmid.ChimericHER2-transfectedDCsfromHER2-
CPwerealsoabletoinduceasustainedT-cellresponsethatsignificantlyhinderedtheinvivogrowthof
þ
HER2 tumors.Theefficacyofchimericplasmidsinovercomingtumor-inducedT-celldysfunctionrelieson
theirabilitytocircumventsuppressoreffectsexertedbyregulatoryTcells(Treg)and/orinterleukin(IL)-10
andTGF-b1.
Conclusions:Theseresultsprovidetheproofofconceptthatchimericrat/humanHER2plasmidscanbe
usedaseffectivevaccinesforanyHER2-CPwiththeadvantageofbeingnotlimitedtospecificMHC.Clin
CancerRes;20(11);1–12.(cid:2)2014AACR.
Introduction antibodies(mAb)suchastrastuzumab,andreceptortyro-
The ErbB-2 (neu in rat and HER2 in humans) tyrosine sinekinaseinhibitors,thatis,lapatinib,areHER2-targeted
therapies currently used for the treatment of HER2-over-
kinase receptor is an oncoantigen overexpressed by a
expressing breastcancers(3,4).Unfortunately, thethera-
varietyoftumors(1).ThedrivingroleofHER2inepithe-
peutic efficacy of both these therapies is abolished by
lial oncogenesis as well as its selective overexpression on
primary and acquired tumor resistance, probably due to
malignant tissues makes it an ideal target for immuno-
the emergence of compensatory activities via alternative
therapy (2). Passive immunotherapy with monoclonal
signaling pathways (5, 6). Active immunotherapy against
HER2 might thus represent an alternative strategy.
Authors' Affiliations: Departments of 1Molecular Biotechnology and HER2/neuisaself-antigenandthustolerogenic.Onthe
HealthSciences,UniversityofTorino;2CenterforExperimentalResearch
andMedicalStudies(CERMS)AOCitta(cid:1)dellaSaluteedellaScienzadi basisofHER2-transgenicmousemodels,itisnowevident
Torino, Torino, Italy, 3Immunogenetic and Transplant Biology Service, that this tolerogenicity causes deletion or inactivation of
4DivisionofOncology,SubalpineOncoHematologyCancerCenter(COES),
AOCitta(cid:1)dellaSaluteedellaScienzadiTorino,Torino;5Departmentof reactivehigh-avidityTcellsagainstneu,therebyleadingto
Oncology, University of Turin, Orbassano; 6Department of Medical self-tolerance (7). Nevertheless, low-avidity self-specific T
Sciences, University of Torino, Torino; and 7Department of Molecular cellscanbeisolatedfromtoleranthostsandtherearereports
CellularandAnimalBiology,UniversityofCamerino,Camerino,Italy
thatsuchcellswhenactivatedandexpandedbyavaccine
Note:SupplementarydataforthisarticleareavailableatClinicalCancer cangiveanantitumorresponse(8).Similarly,expansionof
ResearchOnline(http://clincancerres.aacrjournals.org/).
specificTcellshasbeenelicitedinpatientsfollowingvac-
CorrespondingAuthor:MirellaGiovarelli,DepartmentofMolecularBio- cinationwithsubdominant(9)orheterocliticpeptides(10,
technologyandHealthSciences,UniversityofTorino,viaNizza52,Torino
10126,Italy.Phone:[phone];Fax:[phone];E-mail: 11);however,clinicalresponseshavenotyetbeenobtained
[email protected] (12),suggestingthatadditionalmechanismsbesidescentral
doi:10.1158/1078-0432.CCR-13-2663 tolerancecontributetoT-celldysfunctioninpatientswith
(cid:2)2014AmericanAssociationforCancerResearch. cancer.
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Published OnlineFirst March 25, 2014; DOI: 10.1158/1078-0432.CCR-13-2663
Occhipintietal.
morecommonly,aspeptidesinassociationwiththeMHC
Translational Relevance
classIorIImolecules,andtheirapplicationisnotlimitedto
Chimericplasmidscombininghumanandheterolo- oneorfewspecificMHCmolecules(forareview,seerefs.20,
gousHER2sequenceshavebeenshowntobeeffective 21). A first pilot clinical trial from Norell and colleagues
antitumorvaccinesinpreclinicalmodels,butnodataare demonstratedpromisingfeasibility,safety,andtolerability
availableinhumans.Thisworkassessedthefeasibilityof ofvaccinationwithDNAcodingforthefull-lengthHER2
using chimeric rat/human HER2 plasmids as a tool to molecule in combination with granulocyte-macrophage
overcomethetumor-induceddysfunctionofTcellsfrom colony-stimulating factor (GM-CSF) and interleukin (IL)-
patientswithHER2-overexpressingtumors(HER2-CP). 2inpatientswithadvancedbreastcanceralreadyreceiving
While dendritic cells transfected with plasmids coding trastuzumabbutwithlimitedclinicaleffects(22).
forhumanHER2donotactivateTcellsfromHER2-CP, Here,weevaluatedthefeasibilityofusingDNAcodingfor
thosetransfectedwithchimericrat/humanHER2plas- chimeric rat/human HER2 as a tool to counteract the
mids induced antigen-specific perforin and IFN-g pro- dysfunctionofTcellsfromHER2-CP.Wetransfectedmono-
ductionbyHER2-CPTcells.TheseHER2-specificTcells cyte-derived dendritic cells (DCs) from HER2-CP and
wereabletoinhibitinvivothegrowthofHER2þtumors. healthy subjects with DNA plasmids coding for human,
Chimeric plasmids efficacy relied on their ability to rat, or chimeric rat/human HER2. Only DCs transfected
circumventregulatoryTcells(Treg)and/orinterleukin withthechimericplasmidswereabletoelicitaspecificanti-
(IL)-10andTGF-b1suppressoreffects. HER2responsebyTcellsfromHER2-CP.Theirabilityrelies
ThesefindingsandthefactthatDNAvaccinescanbe ontheactivationofasignificantlowernumberofTregcells
administeredtopatientsirrespectiveoftheirMHChap- andlowerproductionofIL-10andTGF-b1thatresultinthe
lotype make chimeric plasmids an appealing tool for rescuefromtumor-inducedimmunedysfunction.
HER2-CPimmunotherapy. In conclusion, these results provide the proof of con-
cept that vaccination with chimeric rat/human HER2
DNA plasmids could be an effective therapeutic option
forallHER2-CP,withtheadvantageofbeingnotlimited
Alargebodyofdatahavebeenaccumulatedinthelast to specific MHC.
decadeabouttheroleofregulatoryTcells(Treg)ininducing
Materials and Methods
T-cellperipheraltoleranceandaboutthe"exhaustion"ofT
cells with expression of inhibitory checkpoint receptors Humanspecimens
typicalofpatientswithchronicinfectionsorcancer(13). Humanperipheralbloodleukocytes(PBL)wereisolated
Several studies have reported increased frequencies of by Ficoll-Hypaque (Lonza) gradient centrifugation from
Treg in blood, draining lymph nodes, and tumor tissues, heparinized venous blood of healthy subjects (n ¼ 22)
associatinganimpairedimmuneresponsetocancerwitha provided by the local Blood Bank (Torino, Italy), and
high frequency and/or hyperactivity of Treg (14). The patientswithcancer(n¼44),notpreviouslytreatedwith
majorityoftheseTregarisefromtumor-inducedconversion radio-orchemotherapy.Patientswithpancreaticadenocar-
of conventional CD4þ T cells and differ from thymus- cinoma(PDAC;n¼16)orbreastcancer(n¼28)recruited
derived natural Treg that play a crucial role in regulating attheCentroOncologicoEmatologicoSubalpino(COES),
theimmuneresponseandinmaintainingimmunehomeo- AOCitt(cid:1)adellaSaluteedellaScienzadiTorino,Torino,Italy,
stasisinhealthyindividuals(forareview,seeref.15). withinformedconsentwereincludedinthestudy.Blood
Toovercometumor-drivenT-celldysfunctioninpatients sampleswereimmediatelyprocessedafterdrawing.Tumors
with cancer, the use of heterologous peptides may be frompatientswithPDACandbreastcancerwereevaluated
advantageous.IncaseofpatientswithHER2-overexpressing for HER2 positivity by immunohistochemistry (IHC).
cancers(HER2-CP),theuseofheterologouspeptideschar- PatientsbearingHER2þtumorsthatwereclassifiedas3þ
acterizedbythepresenceofcriticalaminoacidsubstitutions or2þbyIHCwereclassifiedasHER2-CP(Supplementary
thatmarkedlyimprovetheirimmunogenicitymayinduce TableS1).Patientswithtumorswitha0to1IHCscore,that
activation of nontolerized, nonexhausted, and self-cross- is, HER2-negative (n ¼ 5), were used as a control group
reactive low-affinity T-cell clones (16). These, in turn, (CTRL-CP;SupplementaryTableS2).Todeterminehuman
release cytokines that enhance immune recognition in a leukocyteantigen(HLA)-A2positivity,PBLswereincubated
paracrinewayandeventuallyactivateautoreactiveBcells. withanti-HLA-A2-PEmAb(cloneBB7.2,BDPharmingen),
Chimeric vaccines containing both self-human HER2 andexpressionwasevaluatedbyflowcytometry.
andheterologousratneuDNAsequencesinducedamore
potentcellularandhumoralantitumorimmunitythanself- Cellcultures
sequencealone(17,18).However,nodataontheirpoten- Monocyte-derived DC generation was conducted as
tial efficacy in humans are currently available. Compared previously described (23). TNF-a (50 ng/mL) and IL-1b
with peptide-based vaccines, DNA vaccination has been (50ng/mL,PeproTech)wereaddedforthefinal24hours
shown to be more advantageous (19). Indeed, DNA vac- to induce DC maturation. CD14-depleted PBL were
cinesofferaprecisestrategyfordeliveringantigenstothe storedin liquidnitrogen untiluse.Thawedlymphocytes
immunesystemastheycanbeexpressedoncellsurfacesor, (>80% viability and >50% recovery) were cultured for
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ChimericHER2VaccinesOvercomeCancerPatient'sT-cellDysfunction
7 days with autologous transfected DCs at 20:1 ratio in Flowcytometry
RPMI-1640 medium with 10% heat-inactivated human PBL from healthy subjects and CP were stained with
serumAB(Lonza)at2(cid:2)106/mL.Atday3,onethirdof aCD14-APC (clone M5E2), aHLA-DR-PerCP (clone
supernatantswascollectedandreplacedwithfreshcom- L243; Biolegend), and aIL-4Ra-PE (clone 25463, R&D
þ
plete medium plus IL-7 (10 ng/mL, PeproTech). System) mAb to characterize the phenotype of CD14
ThehumanpancreaticcancercelllineCF-PAC1andthe monocytes. Matched isotype controls were included for
human ovarian carcinoma cell line SKOV-3-A2 (derived eachsample.Changesinmeanfluorescenceintensity(MFI)
from SKOV-3 cells transduced by lentiviral vector with values were calculated by subtracting the fluorescence of
HLA-A2gene),both positive for theexpression of HER2 controlisotypes.
andHLA-A2,andthehumanlungcancercelllineA549, Fluorescence-activatedcell-sorting(FACS)analysisofcell
positivefortheexpressionofHER2butHLA-A2negative, surfacemoleculesontransfectedDCswascarriedoutusing
were cultured in Dulbecco’s Modified Eagle’s Medium thefollowingmAbs:aCD80-PE(clone2D10),aCD86-PE
(DMEM; Invitrogen) with 10% FBS, penicillin G (50 (clone IT2.2), aCD40-PE (BD Biosciences), aCD83-PE
U/mL), and streptomycin (50 mg/mL). T2 cells, a TAP- (cloneHB15e),andaHLA-DR-PerCP(Biolegend).
deficientB-cell/T-cellhybridcelllinethatexpressHLA-A2 TodetectTregcells,PBLswerestainedwithaCD4-PerCP
butlackantigenicpeptides,wereculturedinRPMI-1640 (cloneOKT4),aCD25-PE(cloneBC96lBiolegend)mAbs
with 20% FBS. onthecellsurface,treatedwithFixationandPermeabiliza-
tion buffer (eBioscience), and stained with aFoxp3-FITC
Plasmidsandnucleofection (clone236A/E7)mAb(eBioscience).
Plasmid pVAX1 was the backbone for all the DNA To detect proliferating cells, PBLs were stained with
constructs used for transfection of DCs. All 4 plasmids aCD4-PerCP and aCD8-PE (clone HIT8a; Biolegend)
codefortheextracellularandtransmembranedomainsof mAbsonthecellsurface,treatedwithFixationandPermea-
HER2aspreviouslydescribed(17).HuHuTcodesforthe bilizationbuffer,andstainedwithaKi-67-APC(cloneKi67)
fully human, and Rat for the fully rat HER2 molecule. mAb(Biolegend).
RHuT codes for the first 2 extracellular domains of rat For intracellular staining, 106 lymphocytes recovered
HER2andtheremainingpartofhumanHER2.Converse- after7daysofco-culturewithtransfectedDCswereresus-
ly, HuRT codes for the first 2 extracellular domains of pended in AIM-V and restimulated with 1 mg/mL coated
humanHER2andtheremainingpartofratHER2.Large- aCD3 (clone OKT3, Biolegend) and 1 mg/mL soluble
scale preparation of the plasmids was carried out using aCD28 (clone CD28.2, Biolegend) in the presence of 10
EndoFree Plasmid Maxi kits (Qiagen). Mature DCs were mg/mLBrefeldinA(Sigma)at37(cid:3)Cfor6hours.Cellswere
harvested on day 6 of culture, resuspended in 100 mL of washed twice and incubated with aCD8-PE and aCD4-
electroporation buffer (DC transfection kit, Amaxa, PerCPmAb(Biolegend)at4(cid:3)Cfor30minutes.Aftertreat-
Lonza) and mixed with 5 mg of plasmid DNA. Electro- mentwithFixationandPermeabilizationbuffer,cellswere
porationwasperformedusingtheNucleofectorprogram stained with aIFNg-FITC (clone B27) and aperforin-APC
U-002 (Amaxa, Lonza). After electroporation, cells were (clonedG9)mAb(Biolegend)for30minutesat4(cid:3)C.
immediatelytransferredto 2mLofcompletemediaand To determine IL-10- and TGF-b–producing cells, lym-
cultured at 37(cid:3)C.Efficiency of transfection was analyzed phocyteswereculturedwithtransfectedDCswithorwith-
by flow cytometry after 6 hours following transfection. outBrefeldinA,respectively.IntracellularstainingwithaIL-
Transfected DCs were fixed, permeabilized, and stained 10-PE(cloneJES3-19F1,Biolegend)orsurfacestainingwith
withmousea-rat-ora-human-HER2mAb(Calbiochem) aLAP1-APC(cloneTW4-2F8,Biolegend)wereperformed.
followed by amouse-PE (BD Biosciences). Stained cells were acquired on a FACSCalibur flow cyt-
ometer equipped with CellQuest software (BD Bios-
ELISPOTassay ciences). Cells were gated according to their light scatter
þ
After7daysofco-culture,HLA-A2–restrictedCD8 T-cell propertiestoexcludecelldebris.
activation was detected by the IFNg ELISPOT assay (BD
Bioscience),followingmanufacturer’sinstruction.T2cells Invitrocytotoxicityassay
were loaded with 10 mg/mL of the HLA-A2þ immunodo- The51Cr-releaseassaywasperformedateffector-to-target
minant p369–377 E75 (KIFGSLAFL) or p654–662 GP2 ratios of 50:1,25:1,12:1, and 6:1. CF-PAC1, SKOV-3-A2,
(IISAVVGIL) peptides (PRIMM) for 6 hours at 37(cid:3)C in andA549targetcellswerelabeledwith50mL51Crsodium
serum-freemedium.Atotalof2.5(cid:2)104recoveredTcells chromate (PerkinElmer) in 5% CO for 1 hour at 37(cid:3)C,
2
wereseededin96-wellELISPOTassayplates(Millipore)at washedtwice,andaddedtowellsof96-wellplates(5(cid:2)103
10:1ratiowithE75-orGP2-loadedorunloadedT2cellsin cellsperwell)witheffectorTcellsrecoveredfrom7-dayco-
AIM-V medium (Invitrogen) for 24 hours. Spots were cultures with transfected DCs. Assays were performed in
counted with a computer-assisted image analysis system, triplicate in a final volume of 200 mL of RPMI-1640 with
Transtec1300ELISPOTReader(AMIBioline).Thenumber 10%heat-inactivatedcertifiedFBS.After4-hourincubation,
ofspecificspotswascalculatedbysubtractingthenumberof 50mLofsupernatantswerecollectedonLumaplate(Perki-
spots produced in the presence of unloaded T2 cells and nElmer),andradioactivitywasmeasuredwithaTopCount
spontaneouslyproducedspots. ScintillationCounter(PackardBiosciences).Thepercentage
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Occhipintietal.
of specific lysis was calculated by [(experimental cpm— Committee for Animal Experimentation of the University
spontaneouscpm)/(maximalcpm—spontaneouscpm)](cid:2) ofTorino,Torino,Italy(Prot.No.4.2/2012).
100. Spontaneous release was always less than 20% of
maximalrelease. Statisticalanalysis
Statistical analyses were performed using Prism 5.0
GraphPadSoftware,andresultsareexpressedasthemean
Apoptosisassay
(cid:4) SEM. One-way ANOVA was performed, followed by
AnAnnexinV-fluoresceinisothiocyanate(FITC)staining
assay was performed to measure apoptosis in SKOV-3-A2 Dunnett multiple comparison post-test when needed.
cells, seeded in 24-well plates (5 (cid:2) 104 per well), and Kaplan–Meiersurvivalcurveswereevaluatedwithboththe
exposed to different doses of human rIFN-g (PeproTech) log-rank Mantel–Cox and the Gehan–Breslow–Wilcoxon
orsupernatantsderivedfromDC/T-cellco-culturesfor48 test.OnlyP<0.05wasconsideredtobesignificant.
hours.Cellswerethencollectedbytrypsinization,washed
Results
twice with PBS, and stained with Annexin V-FITC and
propidium iodide (PI; BD) for 15 minutes at room tem- OnlychimericRHuT-DCsareabletoelicitaspecific
perature.Positivecellsweredetectedwithflowcytometry. anti-HER2responsebyCD8TcellsfromHER2-CP
MatureDCs(mDCs)weregeneratedinvitrofromCD14þ
Cytokineanalysis monocytesofhealthysubjectsandHER2-CP(Supplemen-
þ
Supernatantscollectedatday3ofDC/T-cellco-cultures tary Table S1), as previously reported (23). CD14 cells
were analyzed by ELISA for the presence of IL-10, TGF-b derivedfromHER2-CPexpresshigheramountsofIL-4Ra
(bothfromeBioscience),andIFN-g (Biolegend)following andlowerlevelsofHLA-DRmoleculesthanfromhealthy
themanufacturer’sinstruction. subjects (Supplementary Fig. S1A). These data are in line
withcurrentnotionsindicatinganexpansionofamonocyte
Mice population with a myeloid-derived suppressor cell–like
NOD-SCID IL2Rgnull (NSG; 6-week-old female) mice phenotypethatcorrelateswithtumorgrowth(24).
werebredundersterileconditionsinouranimalfacilities. Despitethesedifferencesintheirprecursors,mDCsfrom
A total of 1 (cid:2) 106 SKOV-3-A2 tumor cells were injected both HER2-CP and healthy subjects expressed similarly
subcutaneously in the left flank and tumor growth was high levels of the maturation markers and co-stimulatory
measured twice a week with a caliper in 2 perpendicular moleculesCD83,CD80,CD86,CD40,andHLA-DR(Sup-
diameters.Tendaysaftertumorchallenge,107differentlyin plementaryFig.S1BandS1C).
vitro–activated T cells were injected in the tail vein. The Nucleofectionofthe4plasmids(selfHuHuT,chimeric
appearanceofatumordiameterof5mmwasconsideredas HuRT,andRHuT,heterologousRat)alwaysgavearangeof
deathevent. 35%to45%positivemDCsfrombothhealthysubjectsand
HER2-CP (Supplementary Fig. S2A), thus showing high
Immunohistochemistry reproducibility(SupplementaryFig.S2B).DCstransfected
Tumorswereharvestedatnecropsy,fixedin10%forma- with pVAX1 plasmid (empty-DCs) were used as control.
lin,anddehydratedin70%ethanol.Thefixedsampleswere TheseresultsindicatethatmDCsgeneratedfromHER2-CP
thenembeddedinparaffinand4sequentialserialsections displaysimilarfeaturesandpotentialstimulatorycapacity
pertumorwereobtained.SectionswereprocessedforIHC asthosefromhealthysubjects.
usingaCD8(cloneC8/144B,Roche),aCD4(clone4B12), To assess the ability of self versus heterologous and
oraKi-67(cloneMib-1)mAbthatwereappliedusingthe chimericDNAplasmidstoinduceaspecificanti-HER2CD8
Ultra BenchMark automated stainer (Ventana, Roche). T-cellresponse,mDCsgeneratedfromhealthysubjectsand
Images were acquired using 200(cid:2) magnification and 4 HER2-CPweretransfectedwiththedifferentplasmidsspec-
fieldspersamplewerepseudo-randomlyselected.Percent- ifiedaboveandusedtostimulateautologousTcells.After7
age of positive nuclei was quantified by measuring the daysofco-culture,CD8Tcellsfromhealthysubjectsstim-
þ þ þ
percentages of Ki-67 , CD8 , CD4 cells, respectively, ulated with self HuHuT-DCs or chimeric HuRT-DCs and
amongthetotalmononuclearcells. RHuT-DCsdisplayedhigherproliferativeabilitycompared
withthosestimulatedwithempty-DCs,whereasheterolo-
Ethicsstatement gous Rat-DCs had noeffect(Supplementary Fig. S3Aand
The human studies were conducted according to the S3B). In contrast, only chimeric RHuT-DCs induced pro-
Declaration of Helsinki principles. Human investigations liferation of CD8 T cells from HER2-CP (Supplementary
wereperformedafterapprovalofthestudybytheScientific Fig.S4AandS4B).
EthicsCommitteeofAOCitt(cid:1)adellaSaluteedellaScienzadi ActivatedTcellswerethenrestimulatedwithanti-CD3/
Torino (Prot. No. 0085724 and 0012068). Written anti-CD28mAbandanalyzedforIFN-gandperforinexpres-
informedconsentwasreceivedfromeachparticipantbefore sion. A similar increase in the percentage of IFN-
inclusion in the study and specimens were deidentified g–producingCD8Tcellswasobtainedfromco-cultureof
beforeanalysis. healthysubjectsTcellswithHuHuT-DCsorchimericHuRT-
AllanimalstudieswereperformedinaccordancewithEU DCsandRHuT-DCscomparedwiththosewithempty-DCs.
and institutional guidelines approved by the Bioethics Chimeric RuHT-DCs also triggered an increase in the
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ChimericHER2VaccinesOvercomeCancerPatient'sT-cellDysfunction
Figure1. HuHuT-DCs,HuRT-DCs,RHuT-DCs,andRat-DCsfromhealthysubjectselicitanti-HER2CD8response.A,activationofCD8þTcellsfromhealthy
subjectsafter7daysofco-culturewithempty-DCs(*),HuHuT-DCs(&),HuRT-DCs(~),RHuT-DCs(^),orRat-DCs(!).IFN-gELISPOTassayperformed
after7daysofculturewithtransfectedDCsfromhealthysubjects(n¼17).IFN-greleasewasevaluatedinresponsetoT2cellspulsedwithE75
orGP2peptides.Valuesofpeptide-specificspotswerecalculatedbysubtractingthenumberofspotsagainstunloadedT2fromthenumberofspotsagainst
peptide-loadedT2.(cid:5)(cid:5),P<0.001;(cid:5)(cid:5)(cid:5),P<0.0001comparedwithempty-DCs.B,cytotoxicityassay:after7daysco-cultures,recoveredTcellsweretestedin
a4-hour51Crreleaseassayatdifferenteffector:targetratiosagainstCF-PAC1,SKOV-3-A2,orA549cells.Percentageofspecificlysiswasdetermined
asdescribedinMaterialsandMethods.(cid:5),P<0.05;(cid:5)(cid:5),P<0.001;(cid:5)(cid:5)(cid:5),P<0.0001comparedwithempty-DCs.
þ
expression of perforin (Supplementary Fig. S3C). In con- HLA-A2 –matchedT2cellsloadedwithimmunodominant
trast, only chimeric RHuT-DCs from HER2-CP led to the HER2-derived E75 (25) and GP2 (26) peptides. The chi-
expression of both IFN-g and perforin by CD8 T cells; mericHER2constructRHuTcodesforbothE75andGP2
transfectionwiththeotherplasmidshad lowornoeffect peptides whereas HuRT only for E75. Compared with
(SupplementaryFig.S4C).Theconcomitantexpressionof control empty-DCs, self HuHuT-DCs and chimeric
IFN-g and perforin in CD8 T cells implies their potential RHuT-DCs from healthy subjects were able to activate a
cytotoxicability. significantnumberofIFN-g–releasingTcellsinresponseto
ThespecificityoftheCD8T-cellresponseagainsthuman both peptides whereas chimeric HuRT-DCs only in
HER2wasassessedbyIFN-g ELISPOTassay.Lymphocytes responsetotheGP2peptide(Fig.1A).Incontrast,inCP,
þ
from HLA-A2 healthy subjects and HER2-CP recovered onlychimericRHuT-DCswereabletoelicitpeptide-specific
from the different co-cultures were stimulated with IFN-g production(Fig.2A).
Figure2. RHuT-DCsfromHER2-CP
elicitanti-HER2CD8T-cell
response.ActivationofCD8þT
cellsfromCPafter7daysofco-
culturewithempty-DCs(*),
HuHuT-DCs(&),HuRT-DCs(~),
RHuT-DCs(^),orRat-DCs(!).A,
IFN-gELISPOTassayperformed
after7daysofco-culturewith
transfectedDCsfromHER2-CP
(n¼13).IFN-greleasewas
evaluatedinresponsetoT2cells
pulsedwithE75orGP2peptides.
(cid:5)(cid:5)(cid:5),P<0.0001comparedwith
empty-DCs.B,51Crreleaseassay
againstCF-PAC1,SKOV-3-A2,or
A549cells.(cid:5)(cid:5),P<0.001;
(cid:5)(cid:5)(cid:5),P<0.0001comparedwith
empty-DCs.
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Occhipintietal.
Moreover,TcellsactivatedbyHuHuT-DCs,HuRT-DCs, ChimericHER2-transfectedDCsfromHER2-CPelicita
RHuT-DCs,andRat-DCsfromhealthysubjectswereableto T 1response
H
þ
killHER2 CF-PAC1andSKOV-3-A2tumorcells,aseval- To activate a stronger and longer lasting antitumor
uatedbya4-hour51Crreleaseassay(Fig.1B).InTcellsfrom response, vaccines must not only elicit cytotoxic CD8 T
HER2-CP,stimulationwiththechimericRHuT-DCsseems cellsbutalsoThelper(T )1cells(27).Wefirstevaluated
H
tobemoreefficientininducingcytotoxicactivity(Fig.2B). CD4 T cells in in vitro proliferation. All self HuHuT-DCs,
þ (cid:6)
No lysis was observed against A549 (HER2 , HLA-A2 ) chimericHuRT-DCs,andRHuT-DCsandheterologousRat-
controltumorcells. DCsfromhealthysubjectstriggeredproliferationofautol-
Overall,thesedatasuggestthatDCstransfectedwiththe ogous CD4Tcellstosimilar levels,asevaluated byKi-67
chimeric plasmid RHuT were able to overcome tumor- staining(Fig.3AandB).Conversely,onlychimericHuRT-
induceddysfunctionofTcellsfromHER2-CPandtoinduce DCs and RHuT-DCs from HER2-CP stimulated a signifi-
þ
aspecificanti-HER2CD8cytotoxicresponse. cantlyhigherproliferationofCD4 Tcellscomparedwith
Figure3. ChimericHuRT-DCsand
RHuT-DCsfromHER2-CPelicita
TH1response.Proliferationand
IFN-gexpressionofCD4þTcells
after7daysofco-culturewith
transfectedDCs.A,representative
expressionofKi-67onCD4þT
gatedcellsofhealthysubjects(top
row)andHER2-CP(bottomrow).
GraphsshowpercentageofKi-67þ
gatedonCD4þTcellsfrom4
healthysubjects(B)and7HER2-
CP(C).IFN-gintracellular
stainingofCD4Tcells.After7days
ofco-culturewithtransfected
DCs,recoveredTcellswere
restimulatedwithaCD3/CD28
mAb(1mg/mL).Graphsshow
percentageofIFNgþonCD4þT
gatedcellsfrom5healthysubjects
(D)and8HER2-CP(E).(cid:5),P<0.05;
(cid:5)(cid:5),P<0.001;(cid:5)(cid:5)(cid:5),P<0.0001to
empty-DCs.
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Published OnlineFirst March 25, 2014; DOI: 10.1158/1078-0432.CCR-13-2663
ChimericHER2VaccinesOvercomeCancerPatient'sT-cellDysfunction
empty-DCs, heterologous Rat-DCs, and self HuHuT-DCs establishedpalpabletumors,theywereinjectedwith107in
(Fig.3AandC).After7daysofco-culture,activatedTcells vitro–activatedlymphocytesinthetailvein.
wererestimulatedwithanti-CD3/CD28mAbandanalyzed TcellsactivatedbychimericHuRT-DCsandRHuT-DCs
forIFN-g expression.ChimericHuRT-DCsandRHuT-DCs wereabletodelaytumorgrowth,whereasmicetreatedwith
from HER2-CP triggered a higher percentage of IFN- TcellsactivatedbyselfHuHuT-DCsdevelopedtumorswith
g–producingCD4Tcellscomparedwiththeothergroups the same kinetics as mice receiving T cells activated by
(Fig.3E).Incontrast,DCsfromhealthysubjectstransfected empty-DCs orPBS only (Fig.4A). Moreover, T cells from
withthedifferentplasmidsallresultedinasimilarincrease co-cultures with HuRT-DCs and RHuT-DCs significantly
inIFN-g–producingCD4Tcellscomparedwithempty-DCs improvedoverallsurvival(Fig.4B).After50daysfollowing
(Fig.3D). tumorchallenge,57%and20%ofmiceinjectedwithTcells
This evidence suggests that DCs from HER2-CP trans- activated by chimeric RHuT-DCs and HuRT-DCs, respec-
fected with both the chimeric HER2 plasmids are able to tively,werestillalive,comparedwith0%ofmiceinjected
triggeraT 1response. withTcellsactivatedbyempty-DCsorselfHuHuT-DCs.
H
Lowertumorgrowthwasconsistentwith alowerKi-67
TcellsfromHER2-CPactivatedbychimericHuRT-DCs expressionintumorsfrommiceinjectedwithTcellsrecov-
andRHuT-DCsimpedeHER2þtumorgrowthinvivo eredfromco-cultureswithRHuT-DCsandHuRT-DCs(%of
Next,weevaluatedwhetherTcellsfromHER2-CPacti- Ki-67þcells32,3(cid:4)2.6and37.6(cid:4)5.8,respectively,vs.63.9
vatedinvitro,withselforchimericHER2-transfectedDCs, (cid:4) 1.5 in tumorsfrom mice injected with T cells fromco-
wereabletocounteractgrowthofHER2þcancercellsinvivo, cultureswithHuHuT-DCs)andfurthersupportsthenotion
inatherapeuticsetting.ImmunodeficientNSGmicewere thattheseTcellsareabletoimpedetumorgrowth(Fig.4C).
challengedsubcutaneouslyintheleftflankwith106SKOV- Moreover, immunohistochemical analysis showed that
3-A2cells.After10days,whenmicewerealreadydisplaying tumors from mice injected with T cells from co-cultures
Figure4. TcellsfromHER2-CP
activatedinvitrowithHuRT-DCs
andRHuT-DCsareabletoinhibit
HER2þtumorgrowth.Atotalof1(cid:2)
106SKOV-3A2cellswereinjected
s.c.intheleftflankofNSGmice.
Micewereinjectedi.v.with107
invitro–activatedTcellswith
empty-DCs(*,n¼5),HuHuT-DCs
(&,n¼5),HuRT-DCs(~,n¼5),
RHuT-DCs(^,n¼7),orPBS
(*,n¼12)atday10aftertumor
challenge.A,tumorgrowthwas
monitoredweeklyandexpressed
astumorvolume.(cid:5)(cid:5),P<0.001;(cid:5)(cid:5)(cid:5),
P<0.0001.B,Kaplan–Meier
survivalanalysisofuntreatedand
treatedmice.Tumordiameterof5
mmwasconsideredaslethal
event.(cid:5),P<0.05;(cid:5)(cid:5),P<0.001
comparedwithuntreated
groups.C,representative
immunohistochemicalstainingof
tumorsectionsfrommiceinjected
withTcellsrecoveredfromco-
cultureswithtransfectedDCs,for
Ki-67(toprow),CD8(middlerow),
orCD4(bottomrow)expression.
Thepercentageofpositivecellson
totalcellsevaluatedineachsingle
mouseisreportedasmean(cid:4)SEM
(n¼3pergroup).(cid:5),P<0.05;
(cid:5)(cid:5),P<0.001;(cid:5)(cid:5)(cid:5),P<0.0001to
empty-DCs.
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Occhipintietal.
Figure5. HuRT-DCsandRHuT-
DCsfromHER2-CPelicit
enhancedIFN-gproduction.A,
IFN-gproductionanalyzedby
ELISAonsupernatantsofco-
culturesofTcellsfromCPwith
autologoustransfectedDCs
(n¼15)collectedatday3.
(cid:5)(cid:5)(cid:5),P<0.0001.B,representative
AnnexinV/PIassayofSKOV-3-A2
cellsculturedfor48hourswith
supernatantsderivedfromDC
co-cultures.C,graphsshowthe
percentageofAnnexinVþPIþ
SKOV-3-A2cellsculturedfor48
hourswithsupernatantsderived
fromco-culturesof3differentCP.
(cid:5)(cid:5),P<0.001.D,AnnexinV/PIassay
ofSKOV-3-A2cellsculturedfor48
hourswithindicatedconcentration
ofhumanrIFN-g.
with RHuT-DCs displayed high levels of CD4 and CD8 48hourswithsupernatantsderivedfromthedifferentco-
infiltration throughout the tumor mass, whereas those cultures. Supernatants from both chimeric RHuT- and
receivingTcellsfromco-cultureswithHuRT-DCsdisplayed HuRT-DCsco-culturesinducedhigherpercentagesofapo-
onlyhighamountsofCD4,concentratedattheperipheryof ptotic cells in comparison to those from empty-DC co-
thetumorgrowingarea(Fig.4C),suggestingakeyroleof cultures(Fig.5BandC).TheadditionofIFN-gneutralizing
thesecellsincounteractingtumorgrowth.LowornoT-cell mAb to the supernatants abrogated this effect. Moreover,
infiltration was evident in tumors from the other treated when SKOV-3-A2 cells were cultured for 48 hours with
groups. increasing concentrations of recombinant human IFN-g,
Overall,thesedatademonstratethatDCstransfectedwith from0.5to8ng/mL,adoseresponseapoptoticinduction
chimericRHuTandHuRTplasmidsactivateTcellsableto wasobserved(Fig.5D).
impedethegrowthofestablishedtumorsinvivo,andthis Theseresultssuggestthattheantitumorresponseelicited
effectseemstocorrelatewithtumorinfiltrationofCD8and/ by chimeric RHuT-DCs and HuRT-DCs may be, in part,
orCD4Tcells.CD4T-cell–mediatedinhibition oftumor mediated by IFN-g. However, the more potent antitumor
growthisclearlyindependentofperforin,whereascytokine response induced by co-culture with RHuT-DCs seems to
secretion,suchasIFN-g,contributestotheimpairmentof alsoinvolveperforin-expressingCD8Tcells.
tumor growth (28). Higher levels of IFN-g were indeed
detected in supernatants derived from co-cultures with TheinabilityofselfHuHuT-DCstoactivateTcellsfrom
chimeric HuRT-DCs and RHuT-DCs compared with the HER2-CPagainstHER2isdependentonIL-10andTGF-
other co-cultures with self or heterologous HER2 (Fig. b1production
5A),consistentwiththehigherintracytoplasmicexpression Manypublicationshavealreadyreportedanexpansionof
ofIFN-ginbothCD4andCD8Tcells(SupplementaryFig. tumor-induced regulatorycellsintheperipheralbloodof
S4CandS3E). patientswithcancer(29,30).AswestimulatedTcellswith
To verify the role of IFN-g in the inhibition of in vivo transfected DCs, it is conceivable that regulatory cells,
tumor growth, SKOV-3-A2 tumor cells were cultured for already expanded in HER2-CP (Supplementary Fig. S5A)
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ChimericHER2VaccinesOvercomeCancerPatient'sT-cellDysfunction
couldalsobeactivatedandexpanded(31).However,wedid HER2-CPcouldbeattributedtosolublefactorsreleasedby
þ
not observe any differences in the percentage of CD4 immune cells namely IL-10 (32) and TGF-b1 (33). We
þ þ
CD25 FoxP3 Treg cells after 7 days of co-culture with evaluated the presence of these cytokines in the superna-
autologous DCs transfected with the 4 different plasmids tantsofco-cultures.WhilecomparablylowlevelsofIL-10
(Supplementary Fig. S3B). Therefore, we evaluated the weredetectedinco-cultureswithempty-DCs,selfHuHuT-
abilityofTregcellspurifiedfromthePBLofHER2-CPand DCs,chimericRHuT-DCs,andheterologousRat-DCsfrom
cultured with differently transfected DCs to suppress the healthysubjects,selfHuHuT-DCsfromHER2-CPinduceda
þ (cid:6)
activationofCD4 CD25 autologousTcells.Tregcellsco- significantly higher production of IL-10 compared with
culturedwithHuHuT-DCsdisplayedasignificantlyhigher empty-DCs. Interestingly, chimeric HuRT-DCs from both
suppressiveactivitycomparedwiththosewithempty-DCs healthysubjectsandHER2-CPstimulatedhighlevelsofIL-
(SupplementaryFig.S5C). 10secretion(Fig.6A).Incellsfromhealthysubjects,DCs
The inability of DCs transfected with self HuHuT to transfectedwith all4DNAplasmidsinducedtheproduc-
induceaneffectiveresponseofT 1andCD8Tcellsfrom tionofsimilarlevelsofTGF-b1,butincellsfromHER2-CP,
H
Figure6. IL-10andTGF-b1
neutralizationbothrestorethe
abilityofHuHuT-DCstoactivate
anti-HER2T-cellresponsesfrom
HER2-CP.IL-10(A)andTGF-b1(B)
productionanalyzedbyELISAon
supernatantsfromco-culturesofT
cellsfromhealthysubjects(white
dots,n¼9)andCP(graydots,
n¼13)withautologous
transfectedDCs,collectedatday
3.(cid:5),P<0.05;(cid:5)(cid:5)(cid:5),P<0.0001.Cand
D,DCsfromHER2-CPwere
transfectedwithHuHuT(&)or
emptyplasmid(*)andcultured
withautologousTcellsinthe
presenceofneutralizingmAbfor
IL-10and/orTGF-b1orcontrol
isotypes.C,percentageofIFNgþ
andperforinþcellsinCD8þTgated
cells(leftandright).(cid:5),P<0.05.D,
IFNgresponseevaluatedby
ELISPOTassayinresponsetoT2
cellsloadedwithE75andGP2
peptideswascomparedwiththat
elicitedbyRHuT-DCs.(cid:5),P<0.05;
(cid:5)(cid:5),P<0.001comparedwith
empty-DCs.E,percentageof
IFNgþcellsinCD4þTgatedcells.
(cid:5),P<0.05.Percentageof(F)IL-10
and (G) TGF-b–positive cells in
CD4þFoxp3þgatedcells.
(cid:5),P<0.05;(cid:5)(cid:5),P<0.001compared
withempty-DCs.
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Occhipintietal.
self HuHuT-DCs stimulated higher secretion of TGF-b1 Anti-HER2vaccinesconsistingofMHCclassI–restricted
comparedwiththeempty-DCs(Fig.6B).Inconclusion,an peptides demonstrated the ability to elicit immunologic
increase of both IL-10 and TGF-b1 was detected in co- responsesandsomeclinicalbenefitsindisease-freepatients
culturesofTcellsfromHER2-CPwithHuHuT-DCs. with breast cancer (34). However, the efficacy of the
To assess whether IL-10 and TGF-b1 production had a immuneresponserequiredforantigen-specifictumorinhi-
roleininhibitingtheCD8andCD4T-cellresponseagainst bitiondependsnotonlyoncorrectantigenpresentationby
humanHER2,lymphocytesfromHER2-CPwereactivated DCsandactivationofcytotoxicCD8Tcellsbutalsoonthe
withselfHuHuT-DCsinthepresenceofanti-IL-10and/or magnitudeofCD4T reactivity(35,36).Indeed,vaccina-
H
anti-TGF-b1neutralizingmAb.Neutralizationofbothcyto- tionofpatientswithcancerwithbothT epitopesandMHC
H
kinesincreasedtheabilityofHuHuT-DCstoinduceIFN-g class I–binding motifs elicited enhanced HER2 peptide–
andperforinexpressionbyCD8Tcells(Fig.6C)andalsoa specificcytotoxicTlymphocyte(CTL)expansionandpro-
specific response against the immunodominant E75 and videddurableresponsesdetectablemorethan1yearafter
GP2peptides(Fig.6D)aswellasRHuT-DCs(Fig.2A).In thefinalvaccination(37).
addition, the ability of CD4 T cells to produce IFN-g was Nevertheless,HLArestrictionlimitsthepotentialnumber
alsoincreased(Fig.6E).Overall,thesedatastronglysuggest ofpatientswhocanreceivethesevaccines,andtheuseof
thatthepresentationofselfHER2couldpromotesuppres- DNA plasmids coding for tumor antigens has therefore
sivemechanismssuchasIL-10andTGF-b1productionthat been shown to be advantageous (19). Vaccines able to
impairantigen-specificCD8andCD4T-cellactivation. inducebothCD8andCD4responses,andhenceCTLand
On the basis of our results, we hypothesized that in humoral immunity, are considered better than vaccines
HER2-CP,HER2-specifictumor-inducedregulatorycellsare abletoinducejustoneresponse.
expandedandthatselfHuHuT-DCscouldstimulatethese Here, we demonstrated that different combinations of
cellstoproduceIL-10andTGF-b1.Toverifythishypothesis, rat/human HER2 sequences induce anti-HER2 immune
weevaluatedwhethertransfectedDCsstimulatedTregcells responses through different mechanisms, suggesting that
to secrete these cytokines. Interestingly, HuHuT-DCs eli- the position of heterologous moieties is determinant for
cited higher expression of IL-10 (Fig. 6F) and TGF-b1– overcomingtolerancetoHER2orexhaustionofCD4and
þ þ
associatedLAP(Fig.6G)inCD4 Foxp3 Tcellscompared CD8TcellsfromHER2-CP.
with empty-DCs andDCs transfectedwith the othercon- CD4 T cells provide critical signals for priming and
H
þ
structs. To further clarify this point, DCs generated from maintenance of effector T cells (35). Moreover, CD4 T
H
CTRL-CPwithbreastcancerandPDACnegativeforHER2 1cellscandirectlymediatetumorinhibitionthroughcyto-
expression(SupplementaryTableS2)weretransfectedand kinesecretion,suchasIFN-g,whichmayinducecytotoxic
co-culturedwithautologouslymphocytes.Inthiscase,self and cytostatic effects on tumor cells (38) as well as their
HuHuT-DCsdidnotsuppresstheproductionofIFN-gand senescence (39). Indeed, chimeric-transfected DCs from
perforinbyCD8Tcells(SupplementaryFig.S6A)similarly HER2-CP elicited enhanced T-cell IFN-g secretion that
towhatwasobservedinco-culturesfromhealthysubjects inducedapoptosisofcancercells.
(SupplementaryFig.S3C).Moreover,selfHuHuT-DCswere Inrecentyears,anumberofreportshaveidentifiedTreg
also able to expand CD4 T cells expressing IFN-g, as for cells specific for a range of different tumor antigens in
chimericHuRT-DCsandRHuT-DCsandheterologousRat- humancancer,includingHER2(40).Thepresenceofthese
DCs(SupplementaryFig.S6B).Notably,selfHuHuT-DCs cellsinpatientswithcancerraisesseriousconcernsabout
from CTRL-CP did not induce suppressive mechanisms thepotentialofcancervaccinestoexpandnotonlyeffector
such as IL-10 (Supplementary Fig. S6C) and TGF-b1 pro- butalsoregulatorycells.Manycancervaccineshavefailedto
duction(SupplementaryFig.S6D). inducesignificantclinicalbenefits,despitetheinductionof
ThesedataindicateanincreaseofHER2-specificregula- seemingly potent tumor antigen–specific responses (41,
torycellsinHER2-CPasaresultofantigenoverexpression 42).
thatcanberestimulatedbythetotalself-sequenceofHER2. Vaccinationwithaxenogeneicantigenhasbeenreported
tobeeffectiveinovercomingtheimmunologictoleranceto
Discussion
self-proteins(43).Resultsobtainedfromtransgenicmouse
Inthecurrentstudy,wedemonstrated,forthefirsttime, modelsdemonstratedthatvaccinationwithDNAplasmids
thatDNAplasmidscodingforchimericrat/humanHER2 codingforxenogeneicHER2elicitedastrongimmunologic
areabletoelicitaneffectiveimmuneresponsebyTcells responsewithoutcross-reaction(16).Chimericrat/human
fromHER2-CPandefficientlycircumventT-celldysfunc- HER2 plasmids were most effective in blunting immune
tion. No T-cell response against HER2 was induced by tolerancetobothratandhumanHER2,suggestingthatthe
autologous DCs transfected with DNA plasmids coding presenceofheterologousregionsenhancesimmunogenic-
forselforfullyheterologousHER2.Incontrast,bothself ity against the antigen (17, 18). Thus, the self-sequence
HuHuT-DCs and chimeric RHuT-DCs from healthy sub- ensuresthespecificityoftheimmuneresponse,whereasthe
jects, as well as those from CTRL-CP, in which there are xenogeneicpartcircumventsimmunetolerance.
no HER2-specific negative regulatory mechanisms, IncreasedlevelsofTregcellswereobservedbothinour
showed a similar induction of HER2-specific CD8 T-cell cohortofpatientsandinpatientswithdifferentmalignan-
response. ciesandareoftenassociatedwithworseoutcomes(44).Treg
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