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

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

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

Abstract Purpose: Despite the great success of HER2 vaccine strategies in animal models, effective clinical results have not yet been obtained. We studied the feasibility of using DNA coding for chimeric rat/human HER2 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 (n ¼ 28) and pancreatic (n ¼ 16) cancer were transfected with DNA plasmids that express human HER2 or heterologous rat sequences in separate plasmids or as chimeric constructs encoding rat/human HER2 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 þ HER2 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/human HER2 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.

Introduction antibodies (mAb) such as trastuzumab, and receptor tyro- The ErbB-2 (neu in rat and HER2 in humans) tyrosine sine kinase inhibitors, that is, lapatinib, are HER2-targeted kinase receptor is an oncoantigen overexpressed by a therapies currently used for the treatment of HER2-over- variety of tumors (1). The driving role of HER2 in epithe- expressing breast cancers (3, 4). Unfortunately, the ther- lial oncogenesis as well as its selective overexpression on apeutic efficacy of both these therapies is abolished malignant tissues makes it an ideal target for immuno- by primary and acquired tumor resistance, probably therapy (2). Passive immunotherapy with monoclonal due to the emergence of compensatory activities via alternative signaling pathways (5, 6). Active immunotherapy against HER2 might thus represent an alternative Authors' Affiliations: Departments of 1Molecular Biotechnology and 2 strategy. Health Sciences, University of Torino, Torino, Italy; Center for Experimen- neu tal Research and Medical Studies (CERMS), AO Citta della Salute e della HER2/ is a self-antigen and thus tolerogenic. On the Scienza di Torino, Torino, Italy; 3Immunogenetic and Transplant Biology basis of HER2-transgenic mouse models, it is now evident 4 Service, AO Citta della Salute e della Scienza Torino, Italy; Division of that this tolerogenicity causes deletion or inactivation of Oncology, Subalpine OncoHematology Cancer Center (COES), AO Citta della Salute e della Scienza di Torino, Torino, Italy; 5Department of Oncol- reactive high-avidity T cells against neu, thereby leading to ogy, University of Turin, Orbassano, Italy; 6Department of Medical self-tolerance (7). Nevertheless, low-avidity self-specific T Sciences, University of Torino, Torino, Italy, 7Department of Molecular Cellular and Animal Biology, University of Camerino, Camerino, Italy cells can be isolated from tolerant hosts and there are reports that such cells when activated and expanded by a vaccine Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). can give an antitumor response (8). Similarly, expansion of specific T cells has been elicited in patients following vac- Corresponding Author: Mirella Giovarelli, Department of Molecular Bio- technology and Health Sciences, University of Torino, via Nizza 52, Torino cination with subdominant (9) or heteroclitic peptides (10, 10126, Italy. Phone: 39-011- 633 5737; Fax: 39-011-6336887; E-mail: 11); however, clinical responses have not yet been obtained [email protected] (12), suggesting that additional mechanisms besides central doi: 10.1158/1078-0432.CCR-13-2663 tolerance contribute to T-cell dysfunction in patients with 2014 American Association for Cancer Research. cancer.

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more commonly, as peptides in association with the MHC Translational Relevance class I or II molecules, and their application is not limited to Chimeric plasmids combining human and heterolo- one or few specific MHC molecules (for a review, see refs. 20, gous HER2 sequences have been shown to be effective 21). A first pilot clinical trial from Norell and colleagues antitumor vaccines in preclinical models, but no data are demonstrated promising feasibility, safety, and tolerability available in humans. This work assessed the feasibility of of vaccination with DNA coding for the full-length HER2 using chimeric rat/human HER2 plasmids as a tool to molecule in combination with granulocyte-macrophage overcome the tumor-induced dysfunction of T cells from colony-stimulating factor (GM-CSF) and interleukin (IL)- patients with HER2-overexpressing tumors (HER2-CP). 2 in patients with advanced breast cancer already receiving While dendritic cells transfected with plasmids coding trastuzumab but with limited clinical effects (22). for human HER2 do not activate T cells from HER2-CP, Here, we evaluated the feasibility of using DNA coding for those transfected with chimeric rat/human HER2 plas- chimeric rat/human HER2 as a tool to counteract the mids induced antigen-specific perforin and IFN-g pro- dysfunction of T cells from HER2-CP. We transfected mono- duction by HER2-CP T cells. These HER2-specific T cells þ cyte-derived dendritic cells (DCs) from HER2-CP and were able to inhibit in vivo the growth of HER2 tumors. 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 circumvent regulatory T cells (Treg) and/or interleukin with the chimeric plasmids were able to elicit a specific anti- (IL)-10 and TGF-b1 suppressor effects. HER2 response by T cells from HER2-CP. Their ability relies These findings and the fact that DNA vaccines can be on the activation of a significant lower number of Treg cells administered to patients irrespective of their MHC hap- and lower production of IL-10 and TGF-b1 that result in the lotype make chimeric plasmids an appealing tool for rescue from tumor-induced immune dysfunction. HER2-CP immunotherapy. In conclusion, these results provide the proof of concept that vaccination with chimeric rat/human HER2 DNA plasmids could be an effective therapeutic option for all HER2-CP, with the advantage of being not limited A large body of data have been accumulated in the last to specific MHC. decade about the role of regulatory T cells (Treg) in inducing T-cell peripheral tolerance and about the "exhaustion" of T Materials and Methods cells with expression of inhibitory checkpoint receptors Human specimens typical of patients with chronic infections or cancer (13). Human peripheral blood leukocytes (PBL) were isolated 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) associating an impaired immune response to cancer with a provided by the local Blood Bank (Torino, Italy), and high frequency and/or hyperactivity of Treg (14). The patients with cancer (n ¼ 44), not previously treated with majority of these Treg arise from tumor-induced conversion radio- or chemotherapy. Patients with pancreatic adenocar- þ of conventional CD4 T cells and differ from thymus- cinoma (PDAC; n ¼ 16) or breast cancer (n ¼ 28) recruited derived natural Treg that play a crucial role in regulating at the Centro Oncologico Ematologico Subalpino (COES), the immune response and in maintaining immune homeo- AO Citta della Salute e della Scienza di Torino, Torino, Italy, stasis in healthy individuals (for a review, see ref. 15). with informed consent were included in the study. Blood To overcome tumor-driven T-cell dysfunction in patients samples were immediately processed after drawing. Tumors with cancer, the use of heterologous peptides may be from patients with PDAC and breast cancer were evaluated advantageous. In case of patients with HER2-overexpressing for HER2 positivity by immunohistochemistry (IHC). þ cancers (HER2-CP), the use of heterologous peptides char- Patients bearing HER2 tumors that were classified as 3þ acterized by the presence of critical amino acid substitutions or 2þ by IHC were classified as HER2-CP (Supplementary that markedly improve their immunogenicity may induce Table S1). Patients with tumors with a 0 to 1 IHC score, 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; Supplementary Table S2). To determine human release cytokines that enhance immune recognition in a leukocyte antigen (HLA)-A2 positivity, PBLs were incubated paracrine way and eventually activate autoreactive B cells. with anti-HLA-A2-PE mAb (clone BB7.2, BD Pharmingen), Chimeric vaccines containing both self-human HER2 and expression was evaluated by flow cytometry. and heterologous rat neu DNA sequences induced a more potent cellular and humoral antitumor immunity than self- Cell cultures sequence alone (17, 18). However, no data on their poten- 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 (50 ng/mL, PeproTech) were added for the final 24 hours shown to be more advantageous (19). Indeed, DNA vac- to induce DC maturation. CD14-depleted PBL were cines offer a precise strategy for delivering antigens to the stored in liquid nitrogen until use. Thawed lymphocytes immune system as they can be expressed on cell surfaces or, (>80% viability and >50% recovery) were cultured for

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7 days with autologous transfected DCs at 20:1 ratio in Flow cytometry RPMI-1640 medium with 10% heat-inactivated human PBL from healthy subjects and CP were stained with serum AB (Lonza) at 2 106/mL. At day 3, one third of aCD14-APC (clone M5E2), aHLA-DR-PerCP (clone supernatants was collected andreplacedwithfreshcom- 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 The human pancreatic cancer cell line CF-PAC1 and the monocytes. Matched isotype controls were included for human ovarian carcinoma cell line SKOV-3-A2 (derived each sample. Changes in mean fluorescence intensity (MFI) from SKOV-3 cells transduced by lentiviral vector with values were calculated by subtracting the fluorescence of HLA-A2 gene), both positive for the expression of HER2 control isotypes. and HLA-A2, and the human lung cancer cell line A549, Fluorescence-activated cell-sorting (FACS) analysis of cell positive for the expression of HER2 but HLA-A2 negative, surface molecules on transfected DCs was carried out using were cultured in Dulbecco’s Modified Eagle’s Medium the following mAbs: aCD80-PE (clone 2D10), 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- (clone HB15e), and aHLA-DR-PerCP (Biolegend). deficient B-cell/T-cell hybrid cell line that express HLA-A2 To detect Treg cells, PBLs were stained with aCD4-PerCP but lack antigenic peptides, were cultured in RPMI-1640 (clone OKT4), aCD25-PE (clone BC96l Biolegend) mAbs with 20% FBS. on the cell surface, treated with Fixation and Permeabiliza- tion buffer (eBioscience), and stained with aFoxp3-FITC Plasmids and nucleofection (clone 236A/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) code for the extracellular and transmembrane domains of mAbs on the cell surface, treated with Fixation and Permea- HER2 as previously described (17). HuHuT codes for the bilization buffer, and stained with aKi-67-APC (clone Ki67) 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 HER2 and the remaining partofhumanHER2.Converse- after 7 days of co-culture with transfected DCs were resus- ly, HuRT codes for the first 2 extracellular domains of pended in AIM-V and restimulated with 1 mg/mL coated human HER2 and the remaining part of rat HER2. 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/mL BrefeldinA (Sigma) at 37C for 6 hours. Cells were harvested on day 6 of culture, resuspended in 100 mLof washed twice and incubated with aCD8-PE and aCD4- electroporation buffer (DC transfection kit, Amaxa, PerCP mAb (Biolegend) at 4C for 30 minutes. After treat- Lonza) and mixed with 5 mg of plasmid DNA. Electro- ment with Fixation and Permeabilization buffer, cells were poration was performed using the Nucleofector program stained with aIFNg-FITC (clone B27) and aperforin-APC U-002 (Amaxa, Lonza). After electroporation, cells were (clone dG9) mAb (Biolegend) for 30 minutes at 4C. immediately transferred to 2 mL of complete media and To determine IL-10- and TGF-b–producing cells, lym- cultured at 37C. Efficiency of transfection was analyzed phocytes were cultured with transfected DCs with or with- by flow cytometry after 6 hours following transfection. out Brefeldin A, respectively. Intracellular staining with aIL- Transfected DCs were fixed, permeabilized, and stained 10-PE (clone JES3-19F1, Biolegend) or surface staining with with mouse a-rat- or a-human-HER2 mAb (Calbiochem) aLAP1-APC (clone TW4-2F8, Biolegend) were performed. followed by amouse-PE (BD Biosciences). Stained cells were acquired on a FACSCalibur flow cyt- ometer equipped with CellQuest software (BD Bios- ELISPOT assay ciences). Cells were gated according to their light scatter þ After 7 days of co-culture, HLA-A2–restricted CD8 T-cell properties to exclude cell debris. activation was detected by the IFNg ELISPOT assay (BD Bioscience), following manufacturer’s instruction. T2 cells In vitro cytotoxicity assay þ were loaded with 10 mg/mL of the HLA-A2 immunodo- The 51Cr-release assay was performed at effector-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 37Cin and A549 target cells were labeled with 50 mL 51Cr sodium 4 serum-free medium. A total of 2.5 10 recovered T cells chromate (PerkinElmer) in 5% CO2 for 1 hour at 37 C, were seeded in 96-well ELISPOT assay plates (Millipore) at washed twice, and added to wells of 96-well plates (5 103 10:1 ratio with E75- or GP2-loaded or unloaded T2 cells in cells per well) with effector T cells recovered from 7-day co- 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 Transtec 1300 ELISPOT Reader (AMI Bioline). The number 10% heat-inactivated certified FBS. After 4-hour incubation, of specific spots was calculated by subtracting the number of 50 mL of supernatants were collected on Lumaplate (Perki- spots produced in the presence of unloaded T2 cells and nElmer), and radioactivity was measured with a TopCount spontaneously produced spots. Scintillation Counter (Packard Biosciences). The percentage

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

of specific lysis was calculated by [(experimental cpm— Committee for Animal Experimentation of the University spontaneous cpm)/(maximal cpm—spontaneous cpm)] of Torino, Torino, Italy (Prot. No. 4.2/2012). 100. Spontaneous release was always less than 20% of maximal release. Statistical analysis Statistical analyses were performed using Prism 5.0 Apoptosis assay GraphPad Software, and results are expressed as the mean An Annexin V-fluorescein isothiocyanate (FITC) staining SEM. One-way ANOVA was performed, followed by assay was performed to measure apoptosis in SKOV-3-A2 Dunnett multiple comparison post-test when needed. cells, seeded in 24-well plates (5 104 per well), and Kaplan–Meier survival curves were evaluated with both the exposed to different doses of human rIFN-g (PeproTech) log-rank Mantel–Cox and the Gehan–Breslow–Wilcoxon P or supernatants derived from DC/T-cell co-cultures for 48 test. Only < 0.05 was considered to be significant. hours. Cells were then collected by trypsinization, washed twice with PBS, and stained with Annexin V-FITC and Results propidium iodide (PI; BD) for 15 minutes at room tem- Only chimeric RHuT-DCs are able to elicit a specific perature. Positive cells were detected with flow cytometry. anti-HER2 response by CD8 T cells from HER2-CP þ Mature DCs (mDCs) were generated in vitro from CD14 Cytokine analysis monocytes of healthy subjects and HER2-CP (Supplemen- þ Supernatants collected at day 3 of DC/T-cell co-cultures tary Table S1), as previously reported (23). CD14 cells were analyzed by ELISA for the presence of IL-10, TGF-b derived from HER2-CP express higher amounts of IL-4Ra (both from eBioscience), and IFN-g (Biolegend) following and lower levels of HLA-DR molecules than from healthy the manufacturer’s instruction. subjects (Supplementary Fig. S1A). These data are in line with current notions indicating an expansion of a monocyte Mice population with a myeloid-derived suppressor cell–like NOD-SCID IL2Rgnull (NSG; 6-week-old female) mice phenotype that correlates with tumor growth (24). were bred under sterile conditions in our animal facilities. Despite these differences in their precursors, mDCs from A total of 1 106 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 molecules CD83, CD80, CD86, CD40, and HLA-DR (Sup- diameters. Ten days after tumor challenge, 107 differently in plementary Fig. S1 B and S1C). vitro–activated T cells were injected in the tail vein. The Nucleofection of the 4 plasmids (self HuHuT, chimeric appearance of a tumor diameter of 5 mm was considered as HuRT, and RHuT, heterologous Rat) always gave a range of death event. 35% to 45% positive mDCs from both healthy subjects and HER2-CP (Supplementary Fig. S2A), thus showing high Immunohistochemistry reproducibility (Supplementary Fig. S2B). DCs transfected Tumors were harvested at necropsy, fixed in 10% forma- with pVAX1 plasmid (empty-DCs) were used as control. lin, and dehydrated in 70% ethanol. The fixed samples were These results indicate that mDCs generated from HER2-CP then embedded in paraffin and 4 sequential serial sections display similar features and potential stimulatory capacity per tumor were obtained. Sections were processed for IHC as those from healthy subjects. using aCD8 (clone C8/144B, Roche), aCD4 (clone 4B12), To assess the ability of self versus heterologous and or aKi-67 (clone Mib-1) mAb that were applied using the chimeric DNA plasmids to induce a specific anti-HER2 CD8 Ultra BenchMark automated stainer (Ventana, Roche). T-cell response, mDCs generated from healthy subjects and Images were acquired using 200 magnification and 4 HER2-CP were transfected with the different plasmids spec- fields per sample were pseudo-randomly selected. Percent- ified above and used to stimulate autologous T cells. After 7 age of positive nuclei was quantified by measuring the days of co-culture, CD8 T cells from healthy subjects stim- þ þ þ percentages of Ki-67 , CD8 , CD4 cells, respectively, ulated with self HuHuT-DCs or chimeric HuRT-DCs and among the total mononuclear cells. RHuT-DCs displayed higher proliferative ability compared with those stimulated with empty-DCs, whereas heterolo- Ethics statement gous Rat-DCs had no effect (Supplementary Fig. S3A and 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 were performed after approval of the study by the Scientific Fig. S4A and S4B). Ethics Committee of AO Citta della Salute e della Scienza di Activated T cells were then restimulated with anti-CD3/ Torino (Prot. No. 0085724 and 0012068). Written anti-CD28 mAb and analyzed for IFN-g and perforin expres- informed consent was received from each participant before sion. A similar increase in the percentage of IFN- inclusion in the study and specimens were deidentified g–producing CD8 T cells was obtained from co-culture of before analysis. healthy subjects T cells with HuHuT-DCs or chimeric HuRT- All animal studies were performed in accordance with EU DCs and RHuT-DCs compared with those with empty-DCs. and institutional guidelines approved by the Bioethics Chimeric RuHT-DCs also triggered an increase in the

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þ Figure 1. HuHuT-DCs, HuRT-DCs, RHuT-DCs, and Rat-DCs from healthy subjects elicit anti-HER2 CD8 response. A, activation of CD8 T cells from healthy subjects after 7 days of co-culture with empty-DCs (*), HuHuT-DCs (&), HuRT-DCs (~), RHuT-DCs (^), or Rat-DCs (!). IFN-g ELISPOT assay performed after 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 E75 or GP2 peptides. Values of peptide-speciﬁc spots were calculated by subtracting the number of spots against unloaded T2 from the number of spots against peptide-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 in a 4-hour 51Cr release assay at different effector:target ratios against CF-PAC1, SKOV-3-A2, or A549 cells. Percentage of speciﬁc lysis was determined as described in Materials and Methods. , P < 0.05; , P < 0.001; , P < 0.0001 compared with empty-DCs.

þ expression of perforin (Supplementary Fig. S3C). In con- HLA-A2 –matched T2 cells loaded with immunodominant 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; meric HER2 construct RHuT codes for both E75 and GP2 transfection with the other plasmids had low or no effect peptides whereas HuRT only for E75. Compared with (Supplementary Fig. S4C). The concomitant expression of 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 cytotoxic ability. significant number of IFN-g–releasing T cells in response to The specificity of the CD8 T-cell response against human both peptides whereas chimeric HuRT-DCs only in HER2 was assessed by IFN-g ELISPOT assay. Lymphocytes response to the GP2 peptide (Fig. 1A). In contrast, in CP, þ from HLA-A2 healthy subjects and HER2-CP recovered only chimeric RHuT-DCs were able to elicit peptide-specific from the different co-cultures were stimulated with IFN-g production (Fig. 2A).

Figure 2. RHuT-DCs from HER2-CP elicit anti-HER2 CD8 T-cell þ response. Activation of CD8 T cells 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 performed after 7 days of co-culture with transfected DCs from HER2-CP (n ¼ 13). IFN-g release was evaluated in response to T2 cells pulsed with E75 or GP2 peptides. , P < 0.0001 compared with empty-DCs. B, 51Cr release assay against CF-PAC1, SKOV-3-A2, or A549 cells. , P < 0.001; , P < 0.0001 compared with empty-DCs.

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Moreover, T cells activated by HuHuT-DCs, HuRT-DCs, Chimeric HER2-transfected DCs from HER2-CP elicit a RHuT-DCs, and Rat-DCs from healthy subjects were able to TH1 response þ kill HER2 CF-PAC1 and SKOV-3-A2 tumor cells, as eval- To activate a stronger and longer lasting antitumor uated by a 4-hour 51Cr release assay (Fig. 1B). In T cells from response, vaccines must not only elicit cytotoxic CD8 T HER2-CP, stimulation with the chimeric RHuT-DCs seems cells but also T helper (TH)1 cells (27). We first evaluated to be more efficient in inducing cytotoxic activity (Fig. 2B). CD4 T cells in in vitro proliferation. All self HuHuT-DCs, þ No lysis was observed against A549 (HER2 , HLA-A2 ) chimeric HuRT-DCs, and RHuT-DCs and heterologous Rat- control tumor cells. DCs from healthy subjects triggered proliferation of autol- Overall, these data suggest that DCs transfected with the ogous CD4 T cells to similar levels, as evaluated by Ki-67 chimeric plasmid RHuT were able to overcome tumor- staining (Fig. 3A and B). Conversely, only chimeric HuRT- induced dysfunction of T cells from HER2-CP and to induce DCs and RHuT-DCs from HER2-CP stimulated a signifi- þ a specific anti-HER2 CD8 cytotoxic response. cantly higher proliferation of CD4 T cells compared with

Figure 3. Chimeric HuRT-DCs and RHuT-DCs from HER2-CP elicit a TH1 response. Proliferation and þ IFN-g expression of CD4 T cells after 7 days of co-culture with transfected DCs. A, representative þ expression of Ki-67 on CD4 T gated cells of healthy subjects (top row) and HER2-CP (bottom row). þ Graphs show percentage of Ki-67 þ gated on CD4 T cells from 4 healthy subjects (B) and 7 HER2- CP (C). IFN-g intracellular staining of CD4 T cells. After 7 days of co-culture with transfected DCs, recovered T cells were restimulated with aCD3/CD28 mAb (1 mg/mL). Graphs show þ þ percentage of IFNg on CD4 T gated cells from 5 healthy subjects (D) and 8 HER2-CP (E). , P < 0.05; , P < 0.001; , P < 0.0001 to empty-DCs.

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empty-DCs, heterologous Rat-DCs, and self HuHuT-DCs established palpable tumors, they were injected with 107 in (Fig. 3A and C). After 7 days of co-culture, activated T cells vitro–activated lymphocytes in the tail vein. were restimulated with anti-CD3/CD28 mAb and analyzed T cells activated by chimeric HuRT-DCs and RHuT-DCs for IFN-g expression. Chimeric HuRT-DCs and RHuT-DCs were able to delay tumor growth, whereas mice treated with from HER2-CP triggered a higher percentage of IFN- T cells activated by self HuHuT-DCs developed tumors with g–producing CD4 T cells compared with the other groups the same kinetics as mice receiving T cells activated by (Fig. 3E). In contrast, DCs from healthy subjects transfected empty-DCs or PBS only (Fig. 4A). Moreover, T cells from with the different plasmids all resulted in a similar increase co-cultures with HuRT-DCs and RHuT-DCs significantly in IFN-g–producing CD4 T cells compared with empty-DCs improved overall survival (Fig. 4B). After 50 days following (Fig. 3D). tumor challenge, 57% and 20% of mice injected with T cells 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, were still alive, compared with 0% of mice injected trigger a TH1 response. with T cells activated by empty-DCs or self HuHuT-DCs. Lower tumor growth was consistent with a lower Ki-67 T cells from HER2-CP activated by chimeric HuRT-DCs expression in tumors from mice injected with T cells recov- þ and RHuT-DCs impede HER2 tumor growth in vivo ered from co-cultures with RHuT-DCs and HuRT-DCs (% of þ Next, we evaluated whether T cells from HER2-CP acti- Ki-67 cells 32,3 2.6 and 37.6 5.8, respectively, vs. 63.9 vated in vitro, with self or chimeric HER2-transfected DCs, 1.5 in tumors from mice injected with T cells from co- þ were able to counteract growth of HER2 cancer cells in vivo, cultures with HuHuT-DCs) and further supports the notion in a therapeutic setting. Immunodeficient NSG mice were that these T cells are able to impede tumor growth (Fig. 4C). challenged subcutaneously in the left flank with 106 SKOV- Moreover, immunohistochemical analysis showed that 3-A2 cells. After 10 days, when mice were already displaying tumors from mice injected with T cells from co-cultures

Figure 4. T cells from HER2-CP activated in vitro with HuRT-DCs and RHuT-DCs are able to inhibit þ HER2 tumor growth. A total of 1 106 SKOV-3 A2 cells were injected s.c. in the left ﬂank of NSG mice. Mice were injected i.v. with 107 in vitro–activated T cells with empty-DCs (*, n ¼ 5), HuHuT-DCs (&, n ¼ 5), HuRT-DCs (~, n ¼ 5), RHuT-DCs (^, n ¼ 7), or PBS (*, n ¼ 12) at day 10 after tumor challenge. A, tumor growth was monitored weekly and expressed as tumor volume. , P < 0.001; , P < 0.0001. B, Kaplan–Meier survival analysis of untreated and treated mice. Tumor diameter of 5 mm was considered as lethal event. , P < 0.05; , P < 0.001 compared with untreated groups. C, representative immunohistochemical staining of tumor sections from mice injected with T cells recovered from co- cultures with transfected DCs, for Ki-67 (top row), CD8 (middle row), or CD4 (bottom row) expression. The percentage of positive cells on total cells evaluated in each single mouse is reported as mean SEM (n ¼ 3 per group). , P < 0.05; , P < 0.001; , P < 0.0001 to empty-DCs.

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

Figure 5. HuRT-DCs and RHuT- DCs from HER2-CP elicit enhanced IFN-g production. A, IFN-g production analyzed by ELISA on supernatants of co- cultures of T cells from CP with autologous transfected DCs (n ¼ 15) collected at day 3. , P < 0.0001. B, representative Annexin V/PI assay of SKOV-3-A2 cells cultured for 48 hours with supernatants derived from DC co-cultures. C, graphs show the þ þ percentage of Annexin V PI SKOV-3-A2 cells cultured for 48 hours with supernatants derived from co-cultures of 3 different CP. , P < 0.001. D, Annexin V/PI assay of SKOV-3-A2 cells cultured for 48 hours with indicated concentration of human rIFN-g.

with RHuT-DCs displayed high levels of CD4 and CD8 48 hours with supernatants derived from the different co- infiltration throughout the tumor mass, whereas those cultures. Supernatants from both chimeric RHuT- and receiving T cells from co-cultures with HuRT-DCs displayed HuRT-DCs co-cultures induced higher percentages of apo- only high amounts of CD4, concentrated at the periphery of ptotic cells in comparison to those from empty-DC co- the tumor growing area (Fig. 4C), suggesting a key role of cultures (Fig. 5B and C). The addition of IFN-g neutralizing these cells in counteracting tumor growth. Low or no T-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, these data demonstrate that DCs transfected with from 0.5 to 8 ng/mL, a dose response apoptotic induction chimeric RHuT and HuRT plasmids activate T cells able to was observed (Fig. 5D). impede the growth of established tumors in vivo, and this These results suggest that the antitumor response elicited effect seems to correlate with tumor infiltration of CD8 and/ by chimeric RHuT-DCs and HuRT-DCs may be, in part, or CD4 T cells. CD4 T-cell–mediated inhibition of tumor mediated by IFN-g. However, the more potent antitumor growth is clearly independent of perforin, whereas cytokine response induced by co-culture with RHuT-DCs seems to secretion, such as IFN-g, contributes to the impairment of also involve perforin-expressing CD8 T cells. tumor growth (28). Higher levels of IFN-g were indeed detected in supernatants derived from co-cultures with The inability of self HuHuT-DCs to activate T cells from chimeric HuRT-DCs and RHuT-DCs compared with the HER2-CP against HER2 is dependent on IL-10 and TGF- other co-cultures with self or heterologous HER2 (Fig. b1 production 5A), consistent with the higher intracytoplasmic expression Many publications have already reported an expansion of of IFN-g in both CD4 and CD8 T cells (Supplementary Fig. tumor-induced regulatory cells in the peripheral blood of S4C and S3E). patients with cancer (29, 30). As we stimulated T cells with 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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could also be activated and expanded (31). However, we did HER2-CP could be attributed to soluble factors released by 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 tants of co-cultures. While comparably low levels of IL-10 (Supplementary Fig. S3B). Therefore, we evaluated the were detected in co-cultures with empty-DCs, self HuHuT- ability of Treg cells purified from the PBL of HER2-CP and DCs, chimeric RHuT-DCs, and heterologous Rat-DCs from cultured with differently transfected DCs to suppress the healthy subjects, self HuHuT-DCs from HER2-CP induced a þ activation of CD4 CD25 autologous T cells. Treg cells co- significantly higher production of IL-10 compared with cultured with HuHuT-DCs displayed a significantly higher empty-DCs. Interestingly, chimeric HuRT-DCs from both suppressive activity compared with those with empty-DCs healthy subjects and HER2-CP stimulated high levels of IL- (Supplementary Fig. S5C). 10 secretion (Fig. 6A). In cells from healthy subjects, DCs The inability of DCs transfected with self HuHuT to transfected with all 4 DNA plasmids induced the produc- induce an effective response of TH1 and CD8 T cells from tion of similar levels of TGF-b1, but in cells from HER2-CP,

Figure 6. IL-10 and TGF-b1 neutralization both restore the ability of HuHuT-DCs to activate anti-HER2 T-cell responses from HER2-CP. IL-10 (A) and TGF-b1(B) production analyzed by ELISA on supernatants from co-cultures of T cells from healthy subjects (white dots, n ¼ 9) and CP (gray dots, n ¼ 13) with autologous transfected DCs, collected at day 3. , P < 0.05; , P < 0.0001. C and D, DCs from HER2-CP were transfected with HuHuT (&)or empty plasmid (*)and cultured with autologous T cells in the presence of neutralizing mAb for IL-10 and/or TGF-b1 or control þ isotypes. C, percentage of IFNg þ þ and perforin cells in CD8 T gated cells (left and right). , P < 0.05. D, IFNg response evaluated by ELISPOT assay in response to T2 cells loaded with E75 and GP2 peptides was compared with that elicited by RHuT-DCs. , P < 0.05; , P < 0.001 compared with empty-DCs. E, percentage of þ þ IFNg cells in CD4 T gated cells. , P < 0.05. Percentage of (F) IL-10 and (G) TGF-b–positive cells in þ þ CD4 Foxp3 gated cells. , P < 0.05; , P < 0.001 compared with empty-DCs.

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

self HuHuT-DCs stimulated higher secretion of TGF-b1 Anti-HER2 vaccines consisting of MHC class I–restricted compared with the empty-DCs (Fig. 6B). In conclusion, an peptides demonstrated the ability to elicit immunologic increase of both IL-10 and TGF-b1 was detected in co- responses and some clinical benefits in disease-free patients cultures of T cells from HER2-CP with HuHuT-DCs. with breast cancer (34). However, the efficacy of the To assess whether IL-10 and TGF-b1 production had a immune response required for antigen-specific tumor inhi- role in inhibiting the CD8 and CD4 T-cell response against bition depends not only on correct antigen presentation by human HER2, lymphocytes from HER2-CP were activated DCs and activation of cytotoxic CD8 T cells but also on the with self HuHuT-DCs in the presence of anti-IL-10 and/or magnitude of CD4 TH reactivity (35, 36). Indeed, vaccina- anti-TGF-b1 neutralizing mAb. Neutralization of both cyto- tion of patients with cancer with both TH epitopes and MHC kines increased the ability of HuHuT-DCs to induce IFN-g class I–binding motifs elicited enhanced HER2 peptide– and perforin expression by CD8 T cells (Fig. 6C) and also a specific cytotoxic T lymphocyte (CTL) expansion and pro- specific response against the immunodominant E75 and vided durable responses detectable more than 1 year after GP2 peptides (Fig. 6D) as well as RHuT-DCs (Fig. 2A). In the final vaccination (37). addition, the ability of CD4 T cells to produce IFN-g was Nevertheless, HLA restriction limits the potential number also increased (Fig. 6E). Overall, these data strongly suggest of patients who can receive these vaccines, and the use of that the presentation of self HER2 could promote suppres- DNA plasmids coding for tumor antigens has therefore sive mechanisms such as IL-10 and TGF-b1 production that been shown to be advantageous (19). Vaccines able to impair antigen-specific CD8 and CD4 T-cell activation. induce both CD8 and CD4 responses, and hence CTL and On the basis of our results, we hypothesized that in humoral immunity, are considered better than vaccines HER2-CP, HER2-specific tumor-induced regulatory cells are able to induce just one response. expanded and that self HuHuT-DCs could stimulate these Here, we demonstrated that different combinations of cells to produce IL-10 and TGF-b1. To verify this hypothesis, rat/human HER2 sequences induce anti-HER2 immune we evaluated whether transfected DCs stimulated Treg cells 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– overcoming tolerance to HER2 or exhaustion of CD4 and þ þ associated LAP (Fig. 6G) in CD4 Foxp3 T cells compared CD8 T cells from HER2-CP. with empty-DCs and DCs transfected with the other con- CD4 TH cells provide critical signals for priming and þ structs. To further clarify this point, DCs generated from maintenance of effector T cells (35). Moreover, CD4 TH CTRL-CP with breast cancer and PDAC negative for HER2 1 cells can directly mediate tumor inhibition through cyto- expression (Supplementary Table S2) were transfected and kine secretion, such as IFN-g, which may induce cytotoxic co-cultured with autologous lymphocytes. In this case, self and cytostatic effects on tumor cells (38) as well as their HuHuT-DCs did not suppress the production of IFN-g and senescence (39). Indeed, chimeric-transfected DCs from perforin by CD8 T cells (Supplementary Fig. S6A) similarly HER2-CP elicited enhanced T-cell IFN-g secretion that to what was observed in co-cultures from healthy subjects induced apoptosis of cancer cells. (Supplementary Fig. S3C). Moreover, self HuHuT-DCs were In recent years, a number of reports have identified Treg also able to expand CD4 T cells expressing IFN-g, as for cells specific for a range of different tumor antigens in chimeric HuRT-DCs and RHuT-DCs and heterologous Rat- human cancer, including HER2 (40). The presence of these DCs (Supplementary Fig. S6B). Notably, self HuHuT-DCs cells in patients with cancer raises serious concerns about from CTRL-CP did not induce suppressive mechanisms the potential of cancer vaccines to expand not only effector such as IL-10 (Supplementary Fig. S6C) and TGF-b1 pro- but also regulatory cells. Many cancer vaccines have failed to duction (Supplementary Fig. S6D). induce significant clinical benefits, despite the induction of These data indicate an increase of HER2-specific regula- seemingly potent tumor antigen–specific responses (41, tory cells in HER2-CP as a result of antigen overexpression 42). that can be restimulated by the total self-sequence of HER2. Vaccination with a xenogeneic antigen has been reported to be effective in overcoming the immunologic tolerance to Discussion self-proteins (43). Results obtained from transgenic mouse In the current study, we demonstrated, for the first time, models demonstrated that vaccination with DNA plasmids that DNA plasmids coding for chimeric rat/human HER2 coding for xenogeneic HER2 elicited a strong immunologic are able to elicit an effective immune response by T cells response without cross-reaction (16). Chimeric rat/human from HER2-CP and efficiently circumvent T-cell dysfunc- HER2 plasmids were most effective in blunting immune tion. No T-cell response against HER2 was induced by tolerance to both rat and human HER2, suggesting that the autologous DCs transfected with DNA plasmids coding presence of heterologous regions enhances immunogenic- for self or fully heterologous HER2. In contrast, both self ity against the antigen (17, 18). Thus, the self-sequence HuHuT-DCs and chimeric RHuT-DCs from healthy sub- ensures the specificity of the immune response, whereas the jects, as well as those from CTRL-CP, in which there are xenogeneic part circumvents immune tolerance. no HER2-specific negative regulatory mechanisms, Increased levels of Treg cells were observed both in our showed a similar induction of HER2-specific CD8 T-cell cohort of patients and in patients with different malignan- response. cies and are often associated with worse outcomes (44). Treg

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cells inhibit primary T-cell activation and are paradoxically efficient anti-HER2 response that avoids the activation of expanded by tumor vaccines coding for self-sequences (45– regulatory mechanisms. Therefore, chimeric HER2 DNA 48). Indeed, we show that HuHuT-DCs did not affect Treg plasmids, or DCs transfected with these plasmids, could expansion, but elicited their stronger suppressive ability, represent a novel therapeutic approach for all patients with probably due to their increased production of IL-10 and HER2-overexpressing cancer and introduce a new concept TGF-b1. It is possible that DCs transfected with self-HuHuT for designing anti-cancer vaccines. present the immunodominant peptides recognized by Treg cells or by exhausted T cells. In contrast, the combination of Disclosure of Potential Conflicts of Interest heterologous sequences, as present in chimeric RHuT and No potential conflicts of interest were disclosed. HuRT, counteracts this phenomenon by presenting addi- Authors' Contributions tional non-self-peptides, activating new TH cells able to Conception and design: S. Occhipinti, S. Rolla, P. Cappello, F. Novelli, release cytokines that rescue bystander dysfunctional M. Giovarelli þ CD8 T and B lymphocytes specific for autologous HER2 Development of methodology: S. Occhipinti, L. Sponton, C. Caorsi, F. Cavallo, C. Marchini, A. Amici, F. Cavallo, M. Giovarelli epitopes. The chimeric benefit is even more evident con- Acquisition of data (provided animals, acquired and managed patients, sidering that the heterologous rat sequence alone was not provided facilities, etc.): S. Occhipinti, L. Sponton, A. Novarino, sufficient to circumvent tumor-induced unresponsiveness M. Donadio, S. Bustreo, M.A. Satolli, D. Pierobon, M. Giovarelli Analysis and interpretation of data (e.g. statistical analysis, biostatis- to HER2. This may be due to the repertoire of peptides tics, computational analysis): S. Occhipinti, L. Sponton, C. Caorsi, generated by the processing of the complete rat protein. A. Amici, D. Pierobon, F. Novelli, M. Giovarelli Writing, review, and/or revision of the manuscript: S. Occhipinti, Most of these peptides are not shared with the HER2 S. Rolla, P. Cappello, F. Cavallo, F. Novelli, M. Giovarelli sequence but are predicted to have a binding affinity to Administrative, technical or material support (i.e. reporting or orga- HLA-I superior or comparable to that of HER2 peptides and nizing data, constructing databases): S. Occhipinti, L. Sponton, þ A. Novarino, C. Pecchioni, C. Marchini, M. Giovarelli thus compete with them for presentation to CD8 T cells, Study supervision: M. Giovarelli indeed reducing the anti-HER2 response. The weakly induced suppressive machinery, such as IL-10 Grant Support and TGF-b1 secretion, seemed to represent the success of This work was supported by grants from the Associazione Italiana per la chimeric variants in activating antitumor responses. We Ricerca sul Cancro to M. Giovarelli (AIRC IG, n. 9366), to F. Cavallo (AIRC IG, n. 5377 and 11675); to F. Novelli [AIRC 5 1000 (no. 12182) and IG observed a higher production of the suppressive cytokines (no. 5548 and 11643)]; European Community, Seventh Framework Pro- IL-10 and TGF-b1 in HuHuT-DC co-cultures from HER2- gram European Pancreatic Cancer-Tumor-Microenvironment Network (EPC-TM-Net, no. 256974); Regione PIEMONTE: Ricerca Industriale "Con- CP, but not from CTRL-CP or healthy subjects. Moreover, verging Technologies" (BIOTHER), Progetti strategici su tematiche di inter- when we blocked the effects of these cytokines by adding esse regionale o sovra regionale (IMMONC); Ministero dell’Istruzione e della neutralizing antibodies, the ability of self-sequences to Ricerca (MIUR), Progetti di Rilevante Interesse Nazionale (PRIN 2009); University of Torino-Progetti di Ateneo 2011: Mechanisms of REsistance to activate both CD4 and CD8 responses was restored. These anti-angiogenesis regimens THErapy (grant Rethe-ORTO11RKTW); Fonda- results further confirm the key role of IL-10 (49) and TGF-b1 zione Ricerca Molinette Onlus, the University of Torino and the Compagnia (50) in suppressing an antigen-specific CD8 T-cell response di San Paolo (Progetti di Ricerca Ateneo/CSP). The costs of publication of this article were defrayed in part by the in patients with cancer and in inhibiting antitumor immune payment of page charges. This article must therefore be hereby marked responses. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Our results provide the proof of concept that chimeric rat/ this fact. human HER2 DNA constructs are able to overcome tumor- Received September 27, 2013; revised February 6, 2014; accepted February induced dysfunction of T cells from HER2-CP and elicit an 17, 2014; published OnlineFirst March 25, 2014.

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

Sergio Occhipinti, Laura Sponton, Simona Rolla, et al.

Clin Cancer Res 2014;20:2910-2921. Published OnlineFirst March 25, 2014.

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