chimeric rat/human her2 efﬁciently circumvents her2...

Cancer Therapy: Preclinical

Chimeric Rat/Human HER2 Efficiently Circumvents HER2Tolerance in Cancer Patients

Sergio Occhipinti1,2, Laura Sponton1,2, Simona Rolla1,2, Cristiana Caorsi3, Anna Novarino4, Michela Donadio4,Sara Bustreo4, Maria Antonietta Satolli5, Carla Pecchioni6, Cristina Marchini7, Augusto Amici7,Federica Cavallo1, Paola Cappello1,2, Daniele Pierobon1,2, Francesco Novelli1,2, and Mirella Giovarelli1,2

AbstractPurpose:Despite the great success of HER2 vaccine strategies in animal models, effective clinical results

have not yet been obtained.We studied the feasibility of usingDNA coding for chimeric rat/humanHER2 as

a tool to break the unresponsiveness of T cells from patients with HER2-overexpressing tumors (HER2-CP).

Experimental Design: Dendritic cells (DCs) generated from patients with HER2-overexpressing breast

(n¼ 28) and pancreatic (n¼ 16) cancer were transfected with DNA plasmids that express human HER2 or

heterologous rat sequences in separate plasmids or as chimeric constructs encoding rat/humanHER2 fusion

proteins and used to activate autologous T cells. Activation was evaluated by IFN-g ELISPOT assay, perforin

expression, and ability to halt HER2þ tumor growth in vivo.

Results: Specific sustained proliferation and IFN-g production by CD4 and CD8 T cells from HER2-CP

was observed after stimulation with autologous DCs transfected with chimeric rat/human HER2 plasmids.

Instead, T cells from healthy donors (n ¼ 22) could be easily stimulated with autologous DCs transfected

with any human, rat, or chimeric rat/human HER2 plasmid. Chimeric HER2-transfected DCs from HER2-

CP were also able to induce a sustained T-cell response that significantly hindered the in vivo growth of

HER2þ tumors. The efficacy of chimeric plasmids in overcoming tumor-induced T-cell dysfunction relies on

their ability to circumvent suppressor effects exerted by regulatory T cells (Treg) and/or interleukin (IL)-10

and TGF-b1.Conclusions: These results provide the proof of concept that chimeric rat/humanHER2 plasmids can be

used as effective vaccines for any HER2-CP with the advantage of being not limited to specific MHC. Clin

Cancer Res; 20(11); 2910–21. �2014 AACR.

IntroductionThe ErbB-2 (neu in rat and HER2 in humans) tyrosine

kinase receptor is an oncoantigen overexpressed by avariety of tumors (1). The driving role of HER2 in epithe-lial oncogenesis as well as its selective overexpression onmalignant tissues makes it an ideal target for immuno-therapy (2). Passive immunotherapy with monoclonal

antibodies (mAb) such as trastuzumab, and receptor tyro-sine kinase inhibitors, that is, lapatinib, are HER2-targetedtherapies currently used for the treatment of HER2-over-expressing breast cancers (3, 4). Unfortunately, the ther-apeutic efficacy of both these therapies is abolishedby primary and acquired tumor resistance, probablydue to the emergence of compensatory activities viaalternative signaling pathways (5, 6). Active immuno-therapy against HER2 might thus represent an alternativestrategy.

HER2/neu is a self-antigen and thus tolerogenic. On thebasis of HER2-transgenic mouse models, it is now evidentthat this tolerogenicity causes deletion or inactivation ofreactive high-avidity T cells against neu, thereby leading toself-tolerance (7). Nevertheless, low-avidity self-specific Tcells canbe isolated from tolerant hosts and there are reportsthat such cells when activated and expanded by a vaccinecan give an antitumor response (8). Similarly, expansion ofspecific T cells has been elicited in patients following vac-cination with subdominant (9) or heteroclitic peptides (10,11); however, clinical responses have not yet been obtained(12), suggesting that additionalmechanismsbesides centraltolerance contribute to T-cell dysfunction in patients withcancer.

Authors' Affiliations: Departments of 1Molecular Biotechnology andHealth Sciences, University of Torino, Torino, Italy; 2Center for Experimen-tal Research and Medical Studies (CERMS), AO Citt�a della Salute e dellaScienza di Torino, Torino, Italy; 3Immunogenetic and Transplant BiologyService, AO Citt�a della Salute e della Scienza Torino, Italy; 4Division ofOncology, Subalpine OncoHematology Cancer Center (COES), AO Citt�adella Salute e della Scienza di Torino, Torino, Italy; 5Department of Oncol-ogy, University of Turin, Orbassano, Italy; 6Department of MedicalSciences, University of Torino, Torino, Italy, 7Department of MolecularCellular and Animal Biology, University of Camerino, Camerino, Italy

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

Corresponding Author: Mirella Giovarelli, Department of Molecular Bio-technology and Health Sciences, University of Torino, via Nizza 52, Torino10126, Italy. Phone: 39-011- 633 5737; Fax: 39-011-6336887; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-13-2663

�2014 American Association for Cancer Research.

ClinicalCancer

Research

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Published OnlineFirst March 25, 2014; DOI: 10.1158/1078-0432.CCR-13-2663

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A large body of data have been accumulated in the lastdecade about the role of regulatory T cells (Treg) in inducingT-cell peripheral tolerance and about the "exhaustion" of Tcells with expression of inhibitory checkpoint receptorstypical of patients with chronic infections or cancer (13).Several studies have reported increased frequencies of

Treg in blood, draining lymph nodes, and tumor tissues,associating an impaired immune response to cancer with ahigh frequency and/or hyperactivity of Treg (14). Themajority of these Treg arise from tumor-induced conversionof conventional CD4þ T cells and differ from thymus-derived natural Treg that play a crucial role in regulatingthe immune response and inmaintaining immune homeo-stasis in healthy individuals (for a review, see ref. 15).To overcome tumor-driven T-cell dysfunction in patients

with cancer, the use of heterologous peptides may beadvantageous. In case of patientswithHER2-overexpressingcancers (HER2-CP), the use of heterologous peptides char-acterized by the presence of critical amino acid substitutionsthat markedly improve their immunogenicity may induceactivation of nontolerized, nonexhausted, and self-cross-reactive low-affinity T-cell clones (16). These, in turn,release cytokines that enhance immune recognition in aparacrine way and eventually activate autoreactive B cells.Chimeric vaccines containing both self-human HER2

and heterologous rat neu DNA sequences induced a morepotent cellular and humoral antitumor immunity than self-sequence alone (17, 18). However, no data on their poten-tial efficacy in humans are currently available. Comparedwith peptide-based vaccines, DNA vaccination has beenshown to be more advantageous (19). Indeed, DNA vac-cines offer a precise strategy for delivering antigens to theimmune system as they can be expressed on cell surfaces or,

more commonly, as peptides in association with the MHCclass I or IImolecules, and their application is not limited tooneor few specificMHCmolecules (for a review, see refs. 20,21). A first pilot clinical trial from Norell and colleaguesdemonstrated promising feasibility, safety, and tolerabilityof vaccination with DNA coding for the full-length HER2molecule in combination with granulocyte-macrophagecolony-stimulating factor (GM-CSF) and interleukin (IL)-2 in patients with advanced breast cancer already receivingtrastuzumab but with limited clinical effects (22).

Here,we evaluated the feasibility of usingDNAcoding forchimeric rat/human HER2 as a tool to counteract thedysfunction of T cells fromHER2-CP.We transfectedmono-cyte-derived dendritic cells (DCs) from HER2-CP andhealthy subjects with DNA plasmids coding for human,rat, or chimeric rat/human HER2. Only DCs transfectedwith the chimeric plasmids were able to elicit a specific anti-HER2 response by T cells fromHER2-CP. Their ability relieson the activation of a significant lower number of Treg cellsand lower production of IL-10 and TGF-b1 that result in therescue from tumor-induced immune dysfunction.

In conclusion, these results provide the proof of con-cept that vaccination with chimeric rat/human HER2DNA plasmids could be an effective therapeutic optionfor all HER2-CP, with the advantage of being not limitedto specific MHC.

Materials and MethodsHuman specimens

Human peripheral blood leukocytes (PBL) were isolatedby Ficoll-Hypaque (Lonza) gradient centrifugation fromheparinized venous blood of healthy subjects (n ¼ 22)provided by the local Blood Bank (Torino, Italy), andpatients with cancer (n ¼ 44), not previously treated withradio- or chemotherapy. Patients with pancreatic adenocar-cinoma (PDAC; n¼ 16) or breast cancer (n¼ 28) recruitedat the Centro Oncologico Ematologico Subalpino (COES),AOCitt�a della Salute e della Scienza di Torino, Torino, Italy,with informed consent were included in the study. Bloodsamples were immediately processed after drawing. Tumorsfrom patients with PDAC and breast cancer were evaluatedfor HER2 positivity by immunohistochemistry (IHC).Patients bearing HER2þ tumors that were classified as 3þor 2þ by IHC were classified as HER2-CP (SupplementaryTable S1). Patients with tumors with a 0 to 1 IHC score, thatis, HER2-negative (n ¼ 5), were used as a control group(CTRL-CP; Supplementary Table S2). To determine humanleukocyte antigen (HLA)-A2positivity, PBLswere incubatedwith anti-HLA-A2-PE mAb (clone BB7.2, BD Pharmingen),and expression was evaluated by flow cytometry.

Cell culturesMonocyte-derived DC generation was conducted as

previously described (23). TNF-a (50 ng/mL) and IL-1b(50 ng/mL, PeproTech) were added for the final 24 hoursto induce DC maturation. CD14-depleted PBL werestored in liquid nitrogen until use. Thawed lymphocytes(>80% viability and >50% recovery) were cultured for

Translational RelevanceChimeric plasmids combining human and heterolo-

gous HER2 sequences have been shown to be effectiveantitumor vaccines in preclinicalmodels, but nodata areavailable in humans. This work assessed the feasibility ofusing chimeric rat/human HER2 plasmids as a tool toovercome the tumor-induced dysfunction of T cells frompatients with HER2-overexpressing tumors (HER2-CP).While dendritic cells transfected with plasmids codingfor human HER2 do not activate T cells from HER2-CP,those transfected with chimeric rat/human HER2 plas-mids induced antigen-specific perforin and IFN-g pro-duction by HER2-CP T cells. These HER2-specific T cellswere able to inhibit in vivo the growth of HER2þ tumors.Chimeric plasmids efficacy relied on their ability tocircumvent regulatory T cells (Treg) and/or interleukin(IL)-10 and TGF-b1 suppressor effects.These findings and the fact that DNA vaccines can be

administered to patients irrespective of their MHC hap-lotype make chimeric plasmids an appealing tool forHER2-CP immunotherapy.

Chimeric HER2 Vaccines Overcome Cancer Patient's T-cell Dysfunction

www.aacrjournals.org Clin Cancer Res; 20(11) June 1, 2014 2911




7 days with autologous transfected DCs at 20:1 ratio inRPMI-1640 medium with 10% heat-inactivated humanserum AB (Lonza) at 2 � 106/mL. At day 3, one third ofsupernatants was collected and replaced with fresh com-plete medium plus IL-7 (10 ng/mL, PeproTech).

The human pancreatic cancer cell line CF-PAC1 and thehuman ovarian carcinoma cell line SKOV-3-A2 (derivedfrom SKOV-3 cells transduced by lentiviral vector withHLA-A2 gene), both positive for the expression of HER2and HLA-A2, and the human lung cancer cell line A549,positive for the expression of HER2 but HLA-A2 negative,were cultured in Dulbecco’s Modified Eagle’s Medium(DMEM; Invitrogen) with 10% FBS, penicillin G (50U/mL), and streptomycin (50 mg/mL). T2 cells, a TAP-deficient B-cell/T-cell hybrid cell line that express HLA-A2but lack antigenic peptides, were cultured in RPMI-1640with 20% FBS.

Plasmids and nucleofectionPlasmid pVAX1 was the backbone for all the DNA

constructs used for transfection of DCs. All 4 plasmidscode for the extracellular and transmembrane domains ofHER2 as previously described (17). HuHuT codes for thefully human, and Rat for the fully rat HER2 molecule.RHuT codes for the first 2 extracellular domains of ratHER2 and the remaining part of human HER2. Converse-ly, HuRT codes for the first 2 extracellular domains ofhuman HER2 and the remaining part of rat HER2. Large-scale preparation of the plasmids was carried out usingEndoFree Plasmid Maxi kits (Qiagen). Mature DCs wereharvested on day 6 of culture, resuspended in 100 mL ofelectroporation buffer (DC transfection kit, Amaxa,Lonza) and mixed with 5 mg of plasmid DNA. Electro-poration was performed using the Nucleofector programU-002 (Amaxa, Lonza). After electroporation, cells wereimmediately transferred to 2 mL of complete media andcultured at 37�C. Efficiency of transfection was analyzedby flow cytometry after 6 hours following transfection.Transfected DCs were fixed, permeabilized, and stainedwith mouse a-rat- or a-human-HER2 mAb (Calbiochem)followed by amouse-PE (BD Biosciences).

ELISPOT assayAfter 7 days of co-culture, HLA-A2–restricted CD8þ T-cell

activation was detected by the IFNg ELISPOT assay (BDBioscience), following manufacturer’s instruction. T2 cellswere loaded with 10 mg/mL of the HLA-A2þ immunodo-minant p369–377 E75 (KIFGSLAFL) or p654–662 GP2(IISAVVGIL) peptides (PRIMM) for 6 hours at 37�C inserum-free medium. A total of 2.5 � 104 recovered T cellswere seeded in 96-well ELISPOT assay plates (Millipore) at10:1 ratio with E75- or GP2-loaded or unloaded T2 cells inAIM-V medium (Invitrogen) for 24 hours. Spots werecounted with a computer-assisted image analysis system,Transtec 1300 ELISPOT Reader (AMI Bioline). The numberof specific spotswas calculated by subtracting the number ofspots produced in the presence of unloaded T2 cells andspontaneously produced spots.

Flow cytometryPBL from healthy subjects and CP were stained with

aCD14-APC (clone M5E2), aHLA-DR-PerCP (cloneL243; Biolegend), and aIL-4Ra-PE (clone 25463, R&DSystem) mAb to characterize the phenotype of CD14þ

monocytes. Matched isotype controls were included foreach sample. Changes inmean fluorescence intensity (MFI)values were calculated by subtracting the fluorescence ofcontrol isotypes.

Fluorescence-activated cell-sorting (FACS) analysis of cellsurface molecules on transfected DCs was carried out usingthe following mAbs: aCD80-PE (clone 2D10), aCD86-PE(clone IT2.2), aCD40-PE (BD Biosciences), aCD83-PE(clone HB15e), and aHLA-DR-PerCP (Biolegend).

To detect Treg cells, PBLs were stained with aCD4-PerCP(clone OKT4), aCD25-PE (clone BC96l Biolegend) mAbson the cell surface, treated with Fixation and Permeabiliza-tion buffer (eBioscience), and stained with aFoxp3-FITC(clone 236A/E7) mAb (eBioscience).

To detect proliferating cells, PBLs were stained withaCD4-PerCP and aCD8-PE (clone HIT8a; Biolegend)mAbs on the cell surface, treated with Fixation and Permea-bilization buffer, and stainedwithaKi-67-APC (cloneKi67)mAb (Biolegend).

For intracellular staining, 106 lymphocytes recoveredafter 7 days of co-culture with transfected DCs were resus-pended in AIM-V and restimulated with 1 mg/mL coatedaCD3 (clone OKT3, Biolegend) and 1 mg/mL solubleaCD28 (clone CD28.2, Biolegend) in the presence of 10mg/mL BrefeldinA (Sigma) at 37�C for 6 hours. Cells werewashed twice and incubated with aCD8-PE and aCD4-PerCP mAb (Biolegend) at 4�C for 30 minutes. After treat-ment with Fixation and Permeabilization buffer, cells werestained with aIFNg-FITC (clone B27) and aperforin-APC(clone dG9) mAb (Biolegend) for 30 minutes at 4�C.

To determine IL-10- and TGF-b–producing cells, lym-phocytes were cultured with transfected DCs with or with-out Brefeldin A, respectively. Intracellular stainingwithaIL-10-PE (clone JES3-19F1, Biolegend) or surface stainingwithaLAP1-APC (clone TW4-2F8, Biolegend) were performed.Stained cells were acquired on a FACSCalibur flow cyt-ometer equipped with CellQuest software (BD Bios-ciences). Cells were gated according to their light scatterproperties to exclude cell debris.

In vitro cytotoxicity assayThe 51Cr-release assay was performed at effector-to-target

ratios of 50:1, 25:1, 12:1, and 6:1. CF-PAC1, SKOV-3-A2,and A549 target cells were labeled with 50 mL 51Cr sodiumchromate (PerkinElmer) in 5% CO2 for 1 hour at 37�C,washed twice, and added to wells of 96-well plates (5� 103

cells per well) with effector T cells recovered from 7-day co-cultures with transfected DCs. Assays were performed intriplicate in a final volume of 200 mL of RPMI-1640 with10%heat-inactivated certified FBS. After 4-hour incubation,50 mL of supernatants were collected on Lumaplate (Perki-nElmer), and radioactivity was measured with a TopCountScintillationCounter (PackardBiosciences). Thepercentage

Occhipinti et al.

Clin Cancer Res; 20(11) June 1, 2014 Clinical Cancer Research2912




of specific lysis was calculated by [(experimental cpm—spontaneous cpm)/(maximal cpm—spontaneous cpm)] �100. Spontaneous release was always less than 20% ofmaximal release.

Apoptosis assayAn Annexin V-fluorescein isothiocyanate (FITC) staining

assay was performed to measure apoptosis in SKOV-3-A2cells, seeded in 24-well plates (5 � 104 per well), andexposed to different doses of human rIFN-g (PeproTech)or supernatants derived from DC/T-cell co-cultures for 48hours. Cells were then collected by trypsinization, washedtwice with PBS, and stained with Annexin V-FITC andpropidium iodide (PI; BD) for 15 minutes at room tem-perature. Positive cells were detected with flow cytometry.

Cytokine analysisSupernatants collected at day 3 of DC/T-cell co-cultures

were analyzed by ELISA for the presence of IL-10, TGF-b(both from eBioscience), and IFN-g (Biolegend) followingthe manufacturer’s instruction.

MiceNOD-SCID IL2Rgnull (NSG; 6-week-old female) mice

were bred under sterile conditions in our animal facilities.A total of 1 � 106 SKOV-3-A2 tumor cells were injectedsubcutaneously in the left flank and tumor growth wasmeasured twice a week with a caliper in 2 perpendiculardiameters. Ten days after tumor challenge, 107 differently invitro–activated T cells were injected in the tail vein. Theappearance of a tumor diameter of 5 mmwas considered asdeath event.

ImmunohistochemistryTumors were harvested at necropsy, fixed in 10% forma-

lin, and dehydrated in 70% ethanol. The fixed samples werethen embedded in paraffin and 4 sequential serial sectionsper tumor were obtained. Sections were processed for IHCusing aCD8 (clone C8/144B, Roche), aCD4 (clone 4B12),or aKi-67 (clone Mib-1) mAb that were applied using theUltra BenchMark automated stainer (Ventana, Roche).Images were acquired using 200� magnification and 4fields per sample were pseudo-randomly selected. Percent-age of positive nuclei was quantified by measuring thepercentages of Ki-67þ, CD8þ, CD4þ cells, respectively,among the total mononuclear cells.

Ethics statementThe human studies were conducted according to the

Declaration of Helsinki principles. Human investigationswere performed after approval of the study by the ScientificEthics Committee of AOCitt�a della Salute e della Scienza diTorino (Prot. No. 0085724 and 0012068). Writteninformed consentwas received fromeachparticipant beforeinclusion in the study and specimens were deidentifiedbefore analysis.All animal studies were performed in accordance with EU

and institutional guidelines approved by the Bioethics

Committee for Animal Experimentation of the Universityof Torino, Torino, Italy (Prot. No. 4.2/2012).

Statistical analysisStatistical analyses were performed using Prism 5.0

GraphPad Software, and results are expressed as the mean� SEM. One-way ANOVA was performed, followed byDunnett multiple comparison post-test when needed.Kaplan–Meier survival curves were evaluated with both thelog-rank Mantel–Cox and the Gehan–Breslow–Wilcoxontest. Only P < 0.05 was considered to be significant.

ResultsOnly chimeric RHuT-DCs are able to elicit a specificanti-HER2 response by CD8 T cells from HER2-CP

Mature DCs (mDCs) were generated in vitro from CD14þ

monocytes of healthy subjects and HER2-CP (Supplemen-tary Table S1), as previously reported (23). CD14þ cellsderived from HER2-CP express higher amounts of IL-4Raand lower levels of HLA-DR molecules than from healthysubjects (Supplementary Fig. S1A). These data are in linewith current notions indicating an expansion of amonocytepopulation with a myeloid-derived suppressor cell–likephenotype that correlates with tumor growth (24).

Despite these differences in their precursors, mDCs fromboth HER2-CP and healthy subjects expressed similarlyhigh levels of the maturation markers and co-stimulatorymolecules CD83, CD80, CD86, CD40, and HLA-DR (Sup-plementary Fig. S1 B and S1C).

Nucleofection of the 4 plasmids (self HuHuT, chimericHuRT, and RHuT, heterologous Rat) always gave a range of35% to 45%positive mDCs from both healthy subjects andHER2-CP (Supplementary Fig. S2A), thus showing highreproducibility (Supplementary Fig. S2B). DCs transfectedwith pVAX1 plasmid (empty-DCs) were used as control.These results indicate that mDCs generated from HER2-CPdisplay similar features and potential stimulatory capacityas those from healthy subjects.

To assess the ability of self versus heterologous andchimeric DNAplasmids to induce a specific anti-HER2CD8T-cell response, mDCs generated from healthy subjects andHER2-CP were transfected with the different plasmids spec-ified above and used to stimulate autologous T cells. After 7days of co-culture, CD8 T cells from healthy subjects stim-ulated with self HuHuT-DCs or chimeric HuRT-DCs andRHuT-DCs displayed higher proliferative ability comparedwith those stimulated with empty-DCs, whereas heterolo-gous Rat-DCs had no effect (Supplementary Fig. S3A andS3B). In contrast, only chimeric RHuT-DCs induced pro-liferation of CD8 T cells from HER2-CP (SupplementaryFig. S4A and S4B).

Activated T cells were then restimulated with anti-CD3/anti-CD28mAbandanalyzed for IFN-g andperforin expres-sion. A similar increase in the percentage of IFN-g–producing CD8 T cells was obtained from co-culture ofhealthy subjects T cellswithHuHuT-DCsor chimericHuRT-DCs and RHuT-DCs comparedwith those with empty-DCs.Chimeric RuHT-DCs also triggered an increase in the






expression of perforin (Supplementary Fig. S3C). In con-trast, only chimeric RHuT-DCs from HER2-CP led to theexpression of both IFN-g and perforin by CD8 T cells;transfection with the other plasmids had low or no effect(Supplementary Fig. S4C). The concomitant expression ofIFN-g and perforin in CD8 T cells implies their potentialcytotoxic ability.

The specificity of the CD8 T-cell response against humanHER2 was assessed by IFN-g ELISPOT assay. Lymphocytesfrom HLA-A2þ healthy subjects and HER2-CP recoveredfrom the different co-cultures were stimulated with

HLA-A2þ–matched T2 cells loadedwith immunodominantHER2-derived E75 (25) and GP2 (26) peptides. The chi-meric HER2 construct RHuT codes for both E75 and GP2peptides whereas HuRT only for E75. Compared withcontrol empty-DCs, self HuHuT-DCs and chimericRHuT-DCs from healthy subjects were able to activate asignificant number of IFN-g–releasing T cells in response toboth peptides whereas chimeric HuRT-DCs only inresponse to the GP2 peptide (Fig. 1A). In contrast, in CP,only chimeric RHuT-DCs were able to elicit peptide-specificIFN-g production (Fig. 2A).

Figure 1. HuHuT-DCs, HuRT-DCs, RHuT-DCs, and Rat-DCs from healthy subjects elicit anti-HER2 CD8 response. A, activation of CD8þ T cells from healthysubjects after 7 days of co-culture with empty-DCs (*), HuHuT-DCs (&), HuRT-DCs (~), RHuT-DCs (^), or Rat-DCs (!). IFN-g ELISPOT assay performedafter 7 days of culture with transfected DCs from healthy subjects (n ¼ 17). IFN-g release was evaluated in response to T2 cells pulsed with E75or GP2 peptides. Values of peptide-specific spots were calculated by subtracting the number of spots against unloaded T2 from the number of spots againstpeptide-loaded T2. ��, P < 0.001; ��, P < 0.0001 compared with empty-DCs. B, cytotoxicity assay: after 7 days co-cultures, recovered T cells were tested ina 4-hour 51Cr release assay at different effector:target ratios against CF-PAC1, SKOV-3-A2, or A549 cells. Percentage of specific lysis was determinedas described in Materials and Methods. �, P < 0.05; ��, P < 0.001; ��, P < 0.0001 compared with empty-DCs.

Figure2. RHuT-DCs fromHER2-CPelicit anti-HER2 CD8 T-cellresponse. Activation of CD8þ Tcells from CP after 7 days of co-culture with empty-DCs (*),HuHuT-DCs (&), HuRT-DCs (~),RHuT-DCs (^), or Rat-DCs (!). A,IFN-g ELISPOT assay performedafter 7 days of co-culture withtransfected DCs from HER2-CP(n ¼ 13). IFN-g release wasevaluated in response to T2 cellspulsed with E75 or GP2 peptides.��, P < 0.0001 compared withempty-DCs. B, 51Cr release assayagainst CF-PAC1, SKOV-3-A2, orA549 cells. ��, P < 0.001;��, P < 0.0001 compared withempty-DCs.

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Moreover, T cells activated by HuHuT-DCs, HuRT-DCs,RHuT-DCs, and Rat-DCs from healthy subjects were able tokill HER2þ CF-PAC1 and SKOV-3-A2 tumor cells, as eval-uated by a 4-hour 51Cr release assay (Fig. 1B). In T cells fromHER2-CP, stimulation with the chimeric RHuT-DCs seemsto be more efficient in inducing cytotoxic activity (Fig. 2B).No lysis was observed against A549 (HER2þ, HLA-A2�)control tumor cells.Overall, these data suggest that DCs transfected with the

chimeric plasmid RHuT were able to overcome tumor-induced dysfunction of T cells fromHER2-CP and to inducea specific anti-HER2 CD8 cytotoxic response.

Chimeric HER2-transfected DCs from HER2-CP elicit aTH1 response

To activate a stronger and longer lasting antitumorresponse, vaccines must not only elicit cytotoxic CD8 Tcells but also T helper (TH)1 cells (27). We first evaluatedCD4 T cells in in vitro proliferation. All self HuHuT-DCs,chimeric HuRT-DCs, and RHuT-DCs and heterologous Rat-DCs from healthy subjects triggered proliferation of autol-ogous CD4 T cells to similar levels, as evaluated by Ki-67staining (Fig. 3A and B). Conversely, only chimeric HuRT-DCs and RHuT-DCs from HER2-CP stimulated a signifi-cantly higher proliferation of CD4þ T cells compared with

Figure 3. Chimeric HuRT-DCs andRHuT-DCs from HER2-CP elicit aTH1 response. Proliferation andIFN-g expression of CD4þ T cellsafter 7 days of co-culture withtransfected DCs. A, representativeexpression of Ki-67 on CD4þ Tgated cells of healthy subjects (toprow) and HER2-CP (bottom row).Graphs show percentage of Ki-67þ

gated on CD4þ T cells from 4healthy subjects (B) and 7 HER2-CP (C). IFN-g intracellularstaining of CD4 T cells. After 7 daysof co-culture with transfectedDCs, recovered T cells wererestimulated with aCD3/CD28mAb (1 mg/mL). Graphs showpercentage of IFNgþ on CD4þ Tgated cells from 5 healthy subjects(D) and 8 HER2-CP (E). �, P < 0.05;��, P < 0.001; ��, P < 0.0001 toempty-DCs.






empty-DCs, heterologous Rat-DCs, and self HuHuT-DCs(Fig. 3A and C). After 7 days of co-culture, activated T cellswere restimulated with anti-CD3/CD28 mAb and analyzedfor IFN-g expression. Chimeric HuRT-DCs and RHuT-DCsfrom HER2-CP triggered a higher percentage of IFN-g–producing CD4 T cells compared with the other groups(Fig. 3E). In contrast, DCs from healthy subjects transfectedwith the different plasmids all resulted in a similar increasein IFN-g–producing CD4 T cells comparedwith empty-DCs(Fig. 3D).

This evidence suggests that DCs from HER2-CP trans-fected with both the chimeric HER2 plasmids are able totrigger a TH1 response.

T cells from HER2-CP activated by chimeric HuRT-DCsand RHuT-DCs impede HER2þ tumor growth in vivo

Next, we evaluated whether T cells from HER2-CP acti-vated in vitro, with self or chimeric HER2-transfected DCs,were able to counteract growth ofHER2þ cancer cells in vivo,in a therapeutic setting. Immunodeficient NSG mice werechallenged subcutaneously in the left flank with 106 SKOV-3-A2 cells. After 10 days, whenmice were already displaying

established palpable tumors, they were injected with 107 invitro–activated lymphocytes in the tail vein.

T cells activated by chimeric HuRT-DCs and RHuT-DCswere able to delay tumor growth, whereasmice treated withT cells activated by self HuHuT-DCs developed tumors withthe same kinetics as mice receiving T cells activated byempty-DCs or PBS only (Fig. 4A). Moreover, T cells fromco-cultures with HuRT-DCs and RHuT-DCs significantlyimproved overall survival (Fig. 4B). After 50 days followingtumor challenge, 57% and 20%ofmice injected with T cellsactivated by chimeric RHuT-DCs and HuRT-DCs, respec-tively, were still alive, compared with 0% of mice injectedwith T cells activated by empty-DCs or self HuHuT-DCs.

Lower tumor growth was consistent with a lower Ki-67expression in tumors from mice injected with T cells recov-ered from co-cultures with RHuT-DCs andHuRT-DCs (%ofKi-67þ cells 32,3� 2.6 and 37.6� 5.8, respectively, vs. 63.9� 1.5 in tumors from mice injected with T cells from co-cultures with HuHuT-DCs) and further supports the notionthat these T cells are able to impede tumor growth (Fig. 4C).Moreover, immunohistochemical analysis showed thattumors from mice injected with T cells from co-cultures

Figure 4. T cells from HER2-CPactivated in vitro with HuRT-DCsand RHuT-DCs are able to inhibitHER2þ tumor growth. A total of 1�106 SKOV-3 A2 cells were injecteds.c. in the left flank of NSG mice.Mice were injected i.v. with 107

in vitro–activated T cells withempty-DCs (*,n¼5), HuHuT-DCs(&, n ¼ 5), HuRT-DCs (~, n ¼ 5),RHuT-DCs (^, n ¼ 7), or PBS(*, n ¼ 12) at day 10 after tumorchallenge. A, tumor growth wasmonitored weekly and expressedas tumor volume. ��, P < 0.001; ��,P < 0.0001. B, Kaplan–Meiersurvival analysis of untreated andtreated mice. Tumor diameter of 5mm was considered as lethalevent. �, P < 0.05; ��, P < 0.001compared with untreatedgroups. C, representativeimmunohistochemical staining oftumor sections from mice injectedwith T cells recovered from co-cultures with transfected DCs, forKi-67 (top row), CD8 (middle row),or CD4 (bottom row) expression.The percentage of positive cells ontotal cells evaluated in each singlemouse is reported as mean� SEM(n ¼ 3 per group). �, P < 0.05;��, P < 0.001; ��, P < 0.0001 toempty-DCs.

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with RHuT-DCs displayed high levels of CD4 and CD8infiltration throughout the tumor mass, whereas thosereceiving T cells from co-cultures with HuRT-DCs displayedonly high amounts of CD4, concentrated at the periphery ofthe tumor growing area (Fig. 4C), suggesting a key role ofthese cells in counteracting tumor growth. Low or no T-cellinfiltration was evident in tumors from the other treatedgroups.Overall, these data demonstrate thatDCs transfectedwith

chimeric RHuT and HuRT plasmids activate T cells able toimpede the growth of established tumors in vivo, and thiseffect seems to correlatewith tumor infiltration ofCD8and/or CD4 T cells. CD4 T-cell–mediated inhibition of tumorgrowth is clearly independent of perforin, whereas cytokinesecretion, such as IFN-g , contributes to the impairment oftumor growth (28). Higher levels of IFN-g were indeeddetected in supernatants derived from co-cultures withchimeric HuRT-DCs and RHuT-DCs compared with theother co-cultures with self or heterologous HER2 (Fig.5A), consistent with the higher intracytoplasmic expressionof IFN-g in both CD4 and CD8 T cells (Supplementary Fig.S4C and S3E).To verify the role of IFN-g in the inhibition of in vivo

tumor growth, SKOV-3-A2 tumor cells were cultured for

48 hours with supernatants derived from the different co-cultures. Supernatants from both chimeric RHuT- andHuRT-DCs co-cultures induced higher percentages of apo-ptotic cells in comparison to those from empty-DC co-cultures (Fig. 5B and C). The addition of IFN-g neutralizingmAb to the supernatants abrogated this effect. Moreover,when SKOV-3-A2 cells were cultured for 48 hours withincreasing concentrations of recombinant human IFN-g ,from 0.5 to 8 ng/mL, a dose response apoptotic inductionwas observed (Fig. 5D).

These results suggest that the antitumor response elicitedby chimeric RHuT-DCs and HuRT-DCs may be, in part,mediated by IFN-g . However, the more potent antitumorresponse induced by co-culture with RHuT-DCs seems toalso involve perforin-expressing CD8 T cells.

The inability of self HuHuT-DCs to activate T cells fromHER2-CP against HER2 is dependent on IL-10 and TGF-b1 production

Manypublications have already reported an expansion oftumor-induced regulatory cells in the peripheral blood ofpatients with cancer (29, 30). As we stimulated T cells withtransfected DCs, it is conceivable that regulatory cells,already expanded in HER2-CP (Supplementary Fig. S5A)

Figure 5. HuRT-DCs and RHuT-DCs from HER2-CP elicitenhanced IFN-g production. A,IFN-g production analyzed byELISA on supernatants of co-cultures of T cells from CP withautologous transfected DCs(n ¼ 15) collected at day 3.��, P < 0.0001. B, representativeAnnexin V/PI assay of SKOV-3-A2cells cultured for 48 hours withsupernatants derived from DCco-cultures. C, graphs show thepercentage of Annexin VþPIþ

SKOV-3-A2 cells cultured for 48hours with supernatants derivedfrom co-cultures of 3 different CP.��,P < 0.001. D, Annexin V/PI assayof SKOV-3-A2 cells cultured for 48hours with indicated concentrationof human rIFN-g .






could alsobe activated andexpanded (31).However,wedidnot observe any differences in the percentage of CD4þ

CD25þFoxP3þ Treg cells after 7 days of co-culture withautologous DCs transfected with the 4 different plasmids(Supplementary Fig. S3B). Therefore, we evaluated theability of Treg cells purified from the PBL of HER2-CP andcultured with differently transfected DCs to suppress theactivation of CD4þCD25� autologous T cells. Treg cells co-cultured with HuHuT-DCs displayed a significantly highersuppressive activity compared with those with empty-DCs(Supplementary Fig. S5C).

The inability of DCs transfected with self HuHuT toinduce an effective response of TH1 and CD8 T cells from

HER2-CP could be attributed to soluble factors released byimmune cells namely IL-10 (32) and TGF-b1 (33). Weevaluated the presence of these cytokines in the superna-tants of co-cultures. While comparably low levels of IL-10were detected in co-cultures with empty-DCs, self HuHuT-DCs, chimeric RHuT-DCs, and heterologous Rat-DCs fromhealthy subjects, self HuHuT-DCs fromHER2-CP induced asignificantly higher production of IL-10 compared withempty-DCs. Interestingly, chimeric HuRT-DCs from bothhealthy subjects and HER2-CP stimulated high levels of IL-10 secretion (Fig. 6A). In cells from healthy subjects, DCstransfected with all 4 DNA plasmids induced the produc-tion of similar levels of TGF-b1, but in cells fromHER2-CP,

Figure 6. IL-10 and TGF-b1neutralization both restore theability of HuHuT-DCs to activateanti-HER2 T-cell responses fromHER2-CP. IL-10 (A) and TGF-b1 (B)production analyzed by ELISA onsupernatants from co-cultures of Tcells from healthy subjects (whitedots, n ¼ 9) and CP (gray dots,n ¼ 13) with autologoustransfected DCs, collected at day3. �,P < 0.05; ��,P < 0.0001. C andD, DCs from HER2-CP weretransfected with HuHuT (&) orempty plasmid (*)and culturedwith autologous T cells in thepresence of neutralizing mAb forIL-10 and/or TGF-b1 or controlisotypes. C, percentage of IFNgþ

and perforinþ cells in CD8þ T gatedcells (left and right). �, P < 0.05. D,IFNg response evaluated byELISPOT assay in response to T2cells loaded with E75 and GP2peptides was compared with thatelicited by RHuT-DCs. �, P < 0.05;��, P < 0.001 compared withempty-DCs. E, percentage ofIFNgþ cells in CD4þ T gated cells.�, P < 0.05. Percentage of (F) IL-10and (G) TGF-b–positive cells inCD4þFoxp3þ gated cells.�, P < 0.05; ��, P < 0.001 comparedwith empty-DCs.

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self HuHuT-DCs stimulated higher secretion of TGF-b1compared with the empty-DCs (Fig. 6B). In conclusion, anincrease of both IL-10 and TGF-b1 was detected in co-cultures of T cells from HER2-CP with HuHuT-DCs.To assess whether IL-10 and TGF-b1 production had a

role in inhibiting the CD8 and CD4 T-cell response againsthuman HER2, lymphocytes from HER2-CP were activatedwith self HuHuT-DCs in the presence of anti-IL-10 and/oranti-TGF-b1 neutralizingmAb. Neutralization of both cyto-kines increased the ability of HuHuT-DCs to induce IFN-gand perforin expression by CD8 T cells (Fig. 6C) and also aspecific response against the immunodominant E75 andGP2 peptides (Fig. 6D) as well as RHuT-DCs (Fig. 2A). Inaddition, the ability of CD4 T cells to produce IFN-g wasalso increased (Fig. 6E). Overall, these data strongly suggestthat the presentation of self HER2 could promote suppres-sive mechanisms such as IL-10 and TGF-b1 production thatimpair antigen-specific CD8 and CD4 T-cell activation.On the basis of our results, we hypothesized that in

HER2-CP,HER2-specific tumor-induced regulatory cells areexpanded and that self HuHuT-DCs could stimulate thesecells to produce IL-10 and TGF-b1. To verify this hypothesis,we evaluated whether transfected DCs stimulated Treg cellsto secrete these cytokines. Interestingly, HuHuT-DCs eli-cited higher expression of IL-10 (Fig. 6F) and TGF-b1–associated LAP (Fig. 6G) in CD4þFoxp3þ T cells comparedwith empty-DCs and DCs transfected with the other con-structs. To further clarify this point, DCs generated fromCTRL-CP with breast cancer and PDAC negative for HER2expression (Supplementary Table S2) were transfected andco-cultured with autologous lymphocytes. In this case, selfHuHuT-DCs did not suppress the production of IFN-g andperforin by CD8 T cells (Supplementary Fig. S6A) similarlyto what was observed in co-cultures from healthy subjects(Supplementary Fig. S3C).Moreover, selfHuHuT-DCswerealso able to expand CD4 T cells expressing IFN-g , as forchimeric HuRT-DCs and RHuT-DCs and heterologous Rat-DCs (Supplementary Fig. S6B). Notably, self HuHuT-DCsfrom CTRL-CP did not induce suppressive mechanismssuch as IL-10 (Supplementary Fig. S6C) and TGF-b1 pro-duction (Supplementary Fig. S6D).These data indicate an increase of HER2-specific regula-

tory cells in HER2-CP as a result of antigen overexpressionthat can be restimulated by the total self-sequence of HER2.

DiscussionIn the current study, we demonstrated, for the first time,

that DNA plasmids coding for chimeric rat/human HER2are able to elicit an effective immune response by T cellsfrom HER2-CP and efficiently circumvent T-cell dysfunc-tion. No T-cell response against HER2 was induced byautologous DCs transfected with DNA plasmids codingfor self or fully heterologous HER2. In contrast, both selfHuHuT-DCs and chimeric RHuT-DCs from healthy sub-jects, as well as those from CTRL-CP, in which there areno HER2-specific negative regulatory mechanisms,showed a similar induction of HER2-specific CD8 T-cellresponse.

Anti-HER2 vaccines consisting of MHC class I–restrictedpeptides demonstrated the ability to elicit immunologicresponses and some clinical benefits in disease-free patientswith breast cancer (34). However, the efficacy of theimmune response required for antigen-specific tumor inhi-bition depends not only on correct antigen presentation byDCs and activation of cytotoxic CD8 T cells but also on themagnitude of CD4 TH reactivity (35, 36). Indeed, vaccina-tion of patients with cancerwith both TH epitopes andMHCclass I–binding motifs elicited enhanced HER2 peptide–specific cytotoxic T lymphocyte (CTL) expansion and pro-vided durable responses detectable more than 1 year afterthe final vaccination (37).

Nevertheless, HLA restriction limits the potential numberof patients who can receive these vaccines, and the use ofDNA plasmids coding for tumor antigens has thereforebeen shown to be advantageous (19). Vaccines able toinduce both CD8 and CD4 responses, and hence CTL andhumoral immunity, are considered better than vaccinesable to induce just one response.

Here, we demonstrated that different combinations ofrat/human HER2 sequences induce anti-HER2 immuneresponses through different mechanisms, suggesting thatthe position of heterologous moieties is determinant forovercoming tolerance to HER2 or exhaustion of CD4 andCD8 T cells from HER2-CP.

CD4 TH cells provide critical signals for priming andmaintenance of effector T cells (35). Moreover, CD4þ TH1 cells can directly mediate tumor inhibition through cyto-kine secretion, such as IFN-g , which may induce cytotoxicand cytostatic effects on tumor cells (38) as well as theirsenescence (39). Indeed, chimeric-transfected DCs fromHER2-CP elicited enhanced T-cell IFN-g secretion thatinduced apoptosis of cancer cells.

In recent years, a number of reports have identified Tregcells specific for a range of different tumor antigens inhuman cancer, including HER2 (40). The presence of thesecells in patients with cancer raises serious concerns aboutthe potential of cancer vaccines to expand not only effectorbut also regulatory cells.Many cancer vaccines have failed toinduce significant clinical benefits, despite the induction ofseemingly potent tumor antigen–specific responses (41,42).

Vaccination with a xenogeneic antigen has been reportedto be effective in overcoming the immunologic tolerance toself-proteins (43). Results obtained from transgenic mousemodels demonstrated that vaccination with DNA plasmidscoding for xenogeneic HER2 elicited a strong immunologicresponse without cross-reaction (16). Chimeric rat/humanHER2 plasmids were most effective in blunting immunetolerance to both rat and human HER2, suggesting that thepresence of heterologous regions enhances immunogenic-ity against the antigen (17, 18). Thus, the self-sequenceensures the specificity of the immune response, whereas thexenogeneic part circumvents immune tolerance.

Increased levels of Treg cells were observed both in ourcohort of patients and in patients with different malignan-cies andare often associatedwithworse outcomes (44). Treg






cells inhibit primary T-cell activation and are paradoxicallyexpanded by tumor vaccines coding for self-sequences (45–48). Indeed, we show that HuHuT-DCs did not affect Tregexpansion, but elicited their stronger suppressive ability,probably due to their increased production of IL-10 andTGF-b1. It is possible that DCs transfected with self-HuHuTpresent the immunodominant peptides recognized by Tregcells or by exhausted T cells. In contrast, the combination ofheterologous sequences, as present in chimeric RHuT andHuRT, counteracts this phenomenon by presenting addi-tional non-self-peptides, activating new TH cells able torelease cytokines that rescue bystander dysfunctionalCD8þ T and B lymphocytes specific for autologous HER2epitopes. The chimeric benefit is even more evident con-sidering that the heterologous rat sequence alone was notsufficient to circumvent tumor-induced unresponsivenessto HER2. This may be due to the repertoire of peptidesgenerated by the processing of the complete rat protein.Most of these peptides are not shared with the HER2sequence but are predicted to have a binding affinity toHLA-I superior or comparable to that of HER2 peptides andthus compete with them for presentation to CD8þ T cells,indeed reducing the anti-HER2 response.

Theweakly induced suppressivemachinery, such as IL-10and TGF-b1 secretion, seemed to represent the success ofchimeric variants in activating antitumor responses. Weobserved a higher production of the suppressive cytokinesIL-10 and TGF-b1 in HuHuT-DC co-cultures from HER2-CP, but not from CTRL-CP or healthy subjects. Moreover,when we blocked the effects of these cytokines by addingneutralizing antibodies, the ability of self-sequences toactivate both CD4 and CD8 responses was restored. Theseresults further confirm the key role of IL-10 (49) andTGF-b1(50) in suppressing an antigen-specific CD8 T-cell responsein patientswith cancer and in inhibiting antitumor immuneresponses.

Our results provide theproof of concept that chimeric rat/human HER2 DNA constructs are able to overcome tumor-induced dysfunction of T cells from HER2-CP and elicit an

efficient anti-HER2 response that avoids the activation ofregulatory mechanisms. Therefore, chimeric HER2 DNAplasmids, or DCs transfected with these plasmids, couldrepresent a novel therapeutic approach for all patients withHER2-overexpressing cancer and introduce a new conceptfor designing anti-cancer vaccines.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S. Occhipinti, S. Rolla, P. Cappello, F. Novelli,M. GiovarelliDevelopment of methodology: S. Occhipinti, L. Sponton, C. Caorsi,F. Cavallo, C. Marchini, A. Amici, F. Cavallo, M. GiovarelliAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): S. Occhipinti, L. Sponton, A. Novarino,M. Donadio, S. Bustreo, M.A. Satolli, D. Pierobon, M. GiovarelliAnalysis and interpretation of data (e.g. statistical analysis, biostatis-tics, computational analysis): S. Occhipinti, L. Sponton, C. Caorsi,A. Amici, D. Pierobon, F. Novelli, M. GiovarelliWriting, review, and/or revision of the manuscript: S. Occhipinti,S. Rolla, P. Cappello, F. Cavallo, F. Novelli, M. GiovarelliAdministrative, technical or material support (i.e. reporting or orga-nizing data, constructing databases): S. Occhipinti, L. Sponton,A. Novarino, C. Pecchioni, C. Marchini, M. GiovarelliStudy supervision: M. Giovarelli

Grant SupportThis work was supported by grants from the Associazione Italiana per la

Ricerca sul Cancro to M. Giovarelli (AIRC IG, n. 9366), to F. Cavallo (AIRCIG, n. 5377 and 11675); to F. Novelli [AIRC 5 � 1000 (no. 12182) and IG(no. 5548 and 11643)]; European Community, Seventh Framework Pro-gram European Pancreatic Cancer-Tumor-Microenvironment Network(EPC-TM-Net, no. 256974); Regione PIEMONTE: Ricerca Industriale "Con-verging Technologies" (BIOTHER), Progetti strategici su tematiche di inter-esse regionale o sovra regionale (IMMONC);Ministero dell’Istruzione e dellaRicerca (MIUR), Progetti di Rilevante Interesse Nazionale (PRIN 2009);University of Torino-Progetti di Ateneo 2011: Mechanisms of REsistance toanti-angiogenesis regimens THErapy (grant Rethe-ORTO11RKTW); Fonda-zione Ricerca Molinette Onlus, the University of Torino and the Compagniadi San Paolo (Progetti di Ricerca Ateneo/CSP).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 27, 2013; revised February 6, 2014; accepted February17, 2014; published OnlineFirst March 25, 2014.

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2014;20:2910-2921. Published OnlineFirst March 25, 2014.Clin Cancer Res Sergio Occhipinti, Laura Sponton, Simona Rolla, et al. in Cancer PatientsChimeric Rat/Human HER2 Efficiently Circumvents HER2 Tolerance

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