A Therapeutic Her2/Neu Vaccine Targeting Dendritic Cells Preferentially Inhibits the Growth of Low Her2/Neu–Expressing Tumor I

Published OnlineFirst March 22, 2016; DOI: 10.1158/1078-0432.CCR-16-0044

Cancer Therapy: Preclinical Clinical Cancer Research A Therapeutic Her2/neu Vaccine Targeting Dendritic Cells Preferentially Inhibits the Growth of Low Her2/neu–Expressing Tumor in HLA-A2 Transgenic Mice Thi Tran1,2, Mariana O. Diniz1,2,3, Estelle Dransart4,5,6, Alain Gey7, Nathalie Merillon1, Yu Chun Lone8, Sylvie Godefroy9, Craig Sibley9, Luis CS Ferreira3, Jacques Medioni10, Stephane Oudard1,2,10, Ludger Johannes4,5,6, and Eric Tartour1,2,7

Abstract

þ Purpose: E75, a peptide derived from the Her2/neu protein, is tional and high-avidity E75-specific anti-CD8 T cells resulting in the most clinically advanced vaccine approach against breast a potent tumor protection in HLA-A2 transgenic mice. High cancer. In this study, we aimed to optimize the E75 vaccine using expression of HER2/neu inhibited the expression of HLA-class I a delivery vector targeting dendritic cells, the B-subunit of molecules, leading to a poor recognition of human or murine þ Shiga toxin (STxB), and to assess the role of various parameters tumors by E75-specific cytotoxic CD8 T cells. In line with these (Her2/neu expression, combination with trastuzumab) in the results, STxB-E75 preferentially inhibited the growth of HLA-A2 efficacy of this cancer vaccine in a relevant preclinical model. tumors expressing low levels of Her2/neu. Coadministration of Experimental Design: We compared the differential ability of anti-Her2/neu mAb potentiated this effect. þ the free E75 peptide or the STxB-E75 vaccine to elicit CD8 T cells, Conclusions: STxB-E75 vaccine is a potent candidate to be and the impact of the vaccine on murine HLA-A2 tumors expres- tested in patients with low Her2/neu–expressing tumors. It could sing low or high levels of Her2/neu. also be indicated in patients expressing high levels of Her2/neu Results: STxB-E75 synergized with granulocyte macrophage and low intratumoral T-cell infiltration to boost the recruitment of colony-stimulating factors and CpG and proved to be more T cells—a key parameter in the efficacy of anti-Her2/neu mAb efficient than the free E75 peptide in the induction of multifunc- therapy. Clin Cancer Res; 1–12. 2016 AACR.

Introduction ments and chemotherapy, the progression-free survival in patients with metastatic breast cancer is 18.7 months (1). The Her2/neu proto-oncogene, is a validated target in cancer, as Intrinsic properties of Her2/neu may explain the clinical various anti-Her2/neu antibodies (trastuzumab, pertuzumab, efficacy of these approved drugs and the interest to develop adotrastuzumab emtansine, etc.) or tyrosine kinase inhibitors other therapeutic approaches against this molecule. Indeed, its (lapatinib) demonstrated their clinical efficacy in patients with overexpression is required to maintain the malignant phenotype Her2/neu–overexpressing breast and gastric cancers. Unfortunate- of these cancers and tumor escape by downregulation of Her2/ ly, even after the best combination of Her2/neu–directed treat- neu is thus more difficult to achieve. Cancer stem cells, which contribute to tumor metastasis and treatment resistance, express increased Her2/neu levels (2). Other arguments support 1INSERM U970, Universite Paris Descartes, Sorbonne Paris-Cite, Paris, France. 2Equipe Labellisee Ligue Contre le Cancer, Paris, France. the development of active immunotherapy such as anti-Her2/ 3Institute of Biomedical Sciences, University of Sao~ Paulo, Sao~ Paulo, neu T-cell vaccines to complement and improve the clinical Brazil. 4Institut Curie, PSL Research University, Chemical Biology of 5 6 activity of commercially available anti-Her2/neu antibodies. Membranes and Therapeutic Delivery Unit. INSERM, U 1143. CNRS, – fi UMR 3666, 26 rue d’Ulm, 75248 Paris Cedex 05, France. 7Service Her2/neu is immunogenic and elicits natural Her2/neu speci c d'Immunologie biologique, Hopital Europeen Georges Pompidou- T-cell responses in patients overexpressing this antigen (3, 4). APHP, Paris, France. 8Inserm U-1014, Universite Paris XI, Groupe Hos- Intratumoral T-cell infiltration is associated with a good prog- 9 pitalier Paul-Brousse, France. Immuno Target SAS, Paris, France. nosis in these patients (5) and the presence of high levels of 10Service d'Oncologie Medicale, Hopital Europeen Georges Pompi- dou, Paris, France. tumor-infiltrating lymphocytes (TIL) were associated with fi þ Note: Supplementary data for this article are available at Clinical Cancer increased trastuzumab bene tinHer2/neu breast cancer Research Online (http://clincancerres.aacrjournals.org/). patients (6). The role of T cells in controlling Her2/neu–expressing L. Johannes and E. Tartour are the principal investigators of this article. tumors was also emphasized by the demonstration that intra- Corresponding Author: Eric Tartour, Service d'Immunologie Biologique, Hopital^ tumoral injection of human effector T cells genetically mod- Europeen Georges Pompidou, 20 Rue Leblanc, Paris 75015, France. Phone: 331- ified to express chimeric antigen receptor (CAR) against Her2/ 5609-3942; Fax: 331-5609-2080; E-mail: [email protected] neu completely blocked the growth and metastasis of Her2/ doi: 10.1158/1078-0432.CCR-16-0044 neu–expressing pancreatic adenocarcinoma xenografts in SCID 2016 American Association for Cancer Research. mice (7).

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doses (29), as already observed with other Her2/neu peptide– Translational Relevance based vaccines (30). The E75 peptide derived from the Her2/neu protein has We have developed and validated a nonreplicative vector that been developed as a vaccine and has generated suboptimal targets dendritic cells, the B subunit of the Shiga toxin (STxB). results in clinical trials. Using a delivery vector targeting the When coupled to various tumor antigens, STxB elicits a strong þ E75 peptide to dendritic cells, we improved its potency both in induction of specific CD8 T cells, which can be further enhanced þ terms of induction of CD8 T cells, and tumor protection in by the addition of adjuvants (31, 32). Compared with other relevant preclinical models. We also demonstrated in a series vectors, STxB targets the same glycolipid receptor, gb3 which are of human breast cancer cell lines that high Her2/neu–expres- identical in all species. In addition, we already demonstrated its sing tumor cells are not efficiently recognized by anti-E75 efficacy to favor crosspresentation in human dendritic cells (33). þ CD8 T cells compared with low Her2/neu tumors. These STxB displays intrinsic immunogenicity demonstrated by a weak results were also translated and confirmed in in vivo models. induction of anti-STxB antibodies, but which did not interfere Thus, although Her2/neu is a validated target for various mAbs with repetitive immunization (31). in breast cancer and gastric cancer expressing high levels of this The aim of this study was to improve and optimize the E75 antigen, this study demonstrates that a Her2/neu cancer vac- peptide vaccine using this delivery vector and to assess the role of cine may be preferentially indicated in low Her2/neu–expres- various parameters (Her2/neu expression, combination with sing tumors. A synergistic effect was also observed when cancer trastuzumab) in the efficacy of the cancer vaccine in a relevant vaccine was combined with anti-Her2/neu mAb treatment. preclinical model.

Materials and Methods Mice Various preclinical and clinical studies showed that the com- Humanized HLA-A2 transgenic (TG) mice were obtained from bination of anti-Her2/neu antibodies and vaccines capable to Dr. Y.C. Lone (INSERM U1014, Hopital Paul Brousse, Villejuif, inducing anti-Her2/neu T cells synergized in promoting the France) and from Dr. F. Lemmonier (Institut Pasteur, Paris, France). regression of Her2/neu–expressing tumors (8–10). In mice and To set up an hHer2/neu-HLA-A2 tumor model, we transfected humans, anti-Her2/neu antibodies enhanced the induction of the B16-HLA-A2 melanoma cell line with a cDNA encoding þ Her2/neu–specific CD8 T cells and favor Th1 cell infiltration hHer2/neu. A clone coexpressing HLA-A2 and hHer2 was selected (11–14). In accordance with these results, it has been reported (Supplementary Fig. S1A). We then subcutaneously grafted this that effective Her2/neu antibody treatment also requires an adap- B16-HLA-A2-hHer2/neu clone and monitored the tumor growth þ tive immune response involving CD8 T cells (13). These data in HLA-A2 TG mice. Steady tumor growth was reproducibly argue for boosting T-cell responses against Her2/neu–positive achieved using at least 5 105 cells (Supplementary Fig. S1B). breast cancer and various groups clearly demonstrated the ability All mice were kept under specific pathogen–free conditions at of Her2/neu cancer vaccines based on dendritic cells, peptides, the INSERM U970 animal facility. All experiments have been recombinant proteins, recombinant virus, or DNA to induce reviewed and approved by Paris-Descartes Ethical Committee for þ þ specific CD4 and CD8 T-cell responses in humans (15–17). Animal Experimentation (CEEA34.ET.154.12). However, up until now, no Her2/neu vaccine has been approved for the clinics. Cell lines E75, a nine amino acid peptide derived from the extracellular B16-A2 cells were obtained from Dr. Y.C. Lone (INSERM domain of Her2/neu protein (KIFGSLAFL, Her2/neu residues 369– U1014). B16-A2-Her2/neu cells were generated by stable trans- 377; ref. 18) is the best studied Her2/neu–derived peptide to date. It fection after electroporation with a plasmid encoding human bindstoHLA-A2and HLA-A3expressedin 60%–75% of the general Her2/neu provided by P. Bruhns (Institut Pasteur) and selected population and is processed and presented by tumor cells (19, 20). with 0.5 mg/mL of G418 (Invivogen) and 7 mg/mL of blasticidine Although the exact immunodominant peptide (Her2/neu 369–377 (Invivogen) supplemented medium. or Her2/neu 373–382) presented by tumor cells is debated, anti- þ To obtain the low and high Her2/neu–expressing B16-A2 cells, E75 CD8 T cells recognized these two epitopes (21). stable B16-A2-Her2/neu transfectants were sorted based on the The E75 peptide–based vaccine (nelipepimut-S) has been Her2/neu expression. shown to be immunogenic in humans (22, 23), but induced SKBR3-A2 breast tumor cells were obtained from T. Dubois suboptimal results in clinical trials designed for the prevention of (Institut Curie, Paris, France) and UACC812 cells were bought clinical recurrence in high-risk disease-free breast cancer patients from ATCC. SK-Mel 37 melanoma cells and MCF-7 breast tumor (24). Nevertheless, a phase III clinical trial based on the admin- cells were obtained from B. Maillere (CEA, Saclay). istration of the E75 peptide vaccine combined with GM-CSF is For ELISpot experiments, tumor cell lines were first cultured for ongoing, making it the most clinically advanced Her2/neu target- 48 hours with 100 U/mL of rhIFNg (Peprotech) or Universal IFN ing breast cancer vaccine (NCT01479244). type I (R&D Systems), and then treated with 1 mg/mL mitomycin In parallel, recent impressive clinical results using immuno- C for 1 hour. After washing, cells were used as stimulatory cells in modulators blocking the PD-1/PD-L1 pathway underscore the þ IFNg ELISpot assay (5,000 cells/well). major role of antitumor CD8 T cells in tumor regression (25). þ The E75 peptide vaccine elicited specific CD8 T cells, but their þ levels were weak and the increase of E75-specific CD8 T cells after Flow cytometry vaccine administration was most often lower than 2-fold (26–28). The anti-mouse CD8 (clone 53–6.7) and anti-human Her2/ þ This CD8 T cells waned rapidly and required repetitive booster neu (CD340, clone 24D2) mAb were purchased from Biolegend

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(Distributor Ozyme) and the anti-human HLA-A2 (clone BB7.2) Statistical analyses from BD Biosciences. Statistical analyses were performed with GraphPad Prism soft- The HLA-A2-Her2/neu369–377 dextramer was obtained from ware (GraphPad Software Inc.). Data were expressed as means Immudex. For dextramer analysis, cells were incubated with SD and are representative of at least two independent experi- PE-labeled dextramer (45 minutes at 4C in the dark). After ments. Significance was assessed with the Mann–Whitney test to incubation and washes, labeled anti-CD8 mAbs were used to compare two different groups, and Kaplan–Meier curves to com- þ phenotype the positive dextramer CD8 T cells. An irrelevant pare the survival of the different groups of mice. dextramer was used in each experiment to assure the specificity of the reaction against Her2/neu. The background values obtained Results with cells labeled with the irrelevant dextramer were deduced from the percentage shown. STxB-E75 vaccine elicits functional T-cell responses in HLA-A2 Acquisitions were performed on BD LSRII, and data were TG mice analyzed with FlowJo Software (Tree Star). STxB-E75 synergizes with GM-CSF and CpG for the induction of þ antigen-specific CD8 T cells. We have previously demonstrated a ELISpot synergistic effect between the STxB delivery system and the þ CD8 splenic T cells were isolated from immunized HLA-A2 aGalCer adjuvant (31), which binds to CD1d. As HLA-A2 TG TG mice by magnetic beads (Miltenyi Biotec). Specific HLA-A2- mice did not express CD1d, we had to evaluate new adjuvants in þ Her2/neu369–377 CD8 T cells were sorted by cell sorter (FAC- combination with STxB. We showed that HLA-A2 TG mice SAria, Becton Dickinson) after dextramer staining. immunized with STxB-E75 alone did not elicit a significant þ IFNg ELISpot kits were purchased from Diaclone and used anti-E75 CD8 T-cell response, as detected by dextramer stain- according to the manufacturer's recommendations. Briefly, in ing (<0.1%) or ELISpot (Supplementary Fig. S2 and data not þ þ ELISpot IFNg mAb–coated plates, CD8 T cells were cultured in shown). In contrast, 0.2% or 0.75% of anti-E75 CD8 Tcell duplicate with antigen-presenting cells (APC) pulsed with the E75 were detected, when STxB-E75 was coadministered with GM- peptide (10 mg/mL) or with tumor cells (after mitomycin C CSF or CpG at the prime dose, respectively (Supplementary Fig. þ treatment and prestimulated or not with rhIFNg or type I IFN). S2). Interestingly, a synergy for the induction of anti-E75 CD8 Plates were cultured for 18 hours at 37C, and spots were revealed T cell was demonstrated, when GM-CSF and CpG were com- following the manufacturer's instructions. Positive controls bined during the prime with CpG given 24 hours after the included cells stimulated with 100 ng/mL of phorbolmyristate vaccine and GM-CSF administration (Supplementary Fig. S2). þ acetate (PMA) and 500 ng/mL of ionomycin (Sigma Aldrich). Indeed, more than 5% of E75-specificCD8 T cells could be Negative controls included cells cultured in the absence of the E75 observed with the vaccine system comprising STxB-E75 plus peptide. IFNg spot-forming cells were counted on a C.T.L-ELISpot GM-CSF and CpG (Supplementary Fig. S2). system (C.T.L). A response was considered positive if the number of spots in the well stimulated with specific peptide was 2-fold STxB-E75 is more efficient than the non vectorized E75 peptide to þ higher than the number of spots in the well without peptide, with elicit functional CD8 T cells. HLA-A2 TG mice (n ¼ 4) were a cut-off at 10 spot-forming cells. immunized with 0.5 nmol (20 mg) of STxB-E75, or with 0.5 nmol (3 mg) of the E75-derived peptide, as the optimal 9 mer peptide Luminex, in vivo cytotoxicity (Her2/neu369–377), or the optimal 9 mer peptide plus the spacer For details on these techniques, see Supplementary Data. sequence known to favor cleavage (RRAR), also present as a spacer between the Her2/neu369–377 peptide and STxB in the STxB-E75 In vivo tumor protection protein. All the vaccines were combined with GM-CSF and CpG. In the therapeutic setting, HLA-A2 TG mice were subcutane- Immunization of mice with the E75 peptide alone or the E75 ously injected with 1.5 106 B16-HLA-A2-Her2/neu tumor cells peptide flanked with the RRAR sequence did not induce anti-E75- þ in the right flank. Once the tumors were palpable (around 3–4 CD8 T cells, as detected by dextramer analysis (Fig. 1A) or days after challenge), mice were vaccinated by the subcutaneous ELISpot (Fig. 1C). In contrast, vaccination of mice with the route with the STxB-E75 (0.5 nmol) or E75 peptide (0.5 nmol) STxB-E75 fusion protein at the same molarity (0.5 nmol) elicited combined with granulocyte macrophage colony-stimulating fac- high levels of dextramer-positive cells (more than 5%; Fig. 1A and þ tor (GM-CSF; 20 mg.). One day later, CpG (50 mg) was subcuta- B) and significant levels of IFNg-producing CD8 T cells (Fig. 1C). þ neously injected at the same site. Seven days after the first To test for the ability of the E75 peptide to induce specific CD8 immunization, mice were boosted with the vaccine alone. T-cell responses, mice were immunized with 100 mg (25 nmol) of In the prophylactic setting, mice were firstly immunized with the E75 peptide combined with GM-CSF and CpG, which repre- the vaccine at day 0 and day 14 and one week later grafted with the sents a 50-fold molar excess over the dose used for STxB-E75. þ tumor cells. Under these conditions, an induction of anti-E75 CD8 T cells, þ Tumor sizes were measured twice per week by using a caliper reaching a mean value of 0.5% of total CD8 T cells, could be (mm2 ¼ length width). For the survival analysis, a tumor size of observed (Fig. 1A) and they produce IFNg (Fig. 1C). However, at least 300 mm2 was defined as the experimental endpoint. their levels were much lower than those observed in mice immu- For in vivo experiment with anti-Her2/neu mAb (4D5, Bioinvest nized with the STxB-E75 vaccine administered at only 0.5 nmol Int), 100 mg of antibody were injected intraperitoneally at the (Fig. 1). same day as the vaccine. þ þ For in vivo CD8 T-cell depletion, 100 mg of anti-CD8 mAb (YTS Characterization of the anti-E75 specificCD8 T cells induced by þ 1694, Proteogenix) was injected intraperitoneally once per week, E75 or STxB-E75. Multifunctionality of anti-E75 specificCD8 T þ beginning 2 days before the first immunization dose. cells It is well recognized that the quality of CD8 T-cell

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A 15 *** B ** *

dextramer dextramer 10 9,98% 1,06% dextramer dextramer 369 − 377 369 − 377 T cells +

/CD8 T cells 5 /CD8 % HLA-A2-Her2 0 CD8 STXB-E75 E75 E75 E75-RRAR % HLA-A2-Her2 STxB-E75 E75 (0.5 nmol/L) (25 nmol/L) (0.5 nmol/L) (0.5 nmol/L) (0.5 nmol/L) (25 nmol/L)

C *** 1,500 *** **

1,000 CD8 T cells

5 500 10 nb IFN γ –producing cells/

0 STXB-E75 E75 E75 E75-RRAR (0.5 nmol/L) (25 nmol/L) (0.5 nmol/L) (0.5 nmol/L)

Figure 1. þ STxB-E75 is more efficient than the E75 peptide to elicit functional anti-Her2369–377–specific CD8 T cells in HLA-A2 TG mice. HLA-A2 TG mice were immunized at day 0 (D0) and D14 by the subcutaneous route with STxB-E75 (0.5 nmol/L) or E75 peptide (Her2369–377; 25 nmol/L or 0.5 nmol/L) or E75 with the flanking sequence (RRAR) present in the STxB vaccine. The vaccines were mixed with GM-CSF at D0 and CpG was injected at day 1. Seven days after the boost, spleen was þ collected and HLA-A2-Her2369–377–specific CD8 T cells were detected by dextramer assay (A and B) and IFNg ELISpot assay (C). A, dextramer analysis of HLA-A2– þ restricted anti-Her2369–377 CD8 T cells induced by the various vaccines. Results are represented as the mean SD of 4 mice/group from five independent þ experiments. B, representative dot plots of spleen HLA-A2-Her2369–377–specific CD8 T cells detected by cytometry. A dextramer control was included in each þ experiment. C, number of IFNg spot-forming cells per 105 CD8 T cells detected by ELISpot. Background obtained with unpulsed cells was subtracted (number of spots was always < 10). Mean SD of 4 mice/group from three independent experiments. , P < 0.05; , P < 0.01; , P < 0.005; , P < 0.0001.

þ response influences the clinical efficacy of antitumor vaccine. and data not shown). Therefore, the anti-E75 CD8 T cells þ More specifically, the presence of multifunctional CD8 T cells induced by STxB-E75 or E75 are bona fide multifunctional is considered as a relevant read-out to predict a positive clinical T cells. þ outcome (34). The E75 peptide vaccine was used at 50-fold We next evaluated the cytotoxic activity of E75-specific CD8 T higher molar concentrations than STxB-E75, as no induction of cells elicited in mice immunized with the different vaccine for- þ specificCD8 T cells was observed at low E75 peptide con- mulations, as this feature is considered a major hallmark of þ þ centrations (Fig. 1). The anti-E75 CD8 T cells detected in mice antitumor CD8 T cells. We demonstrated that the in vivo cyto- þ immunized with STxB-E75 produced more chemokines toxicity effect of anti-E75 CD8 T cells induced by STxB-E75 (Rantes, MIP1a) than those induced by the E75 peptide, but was higher (53% 20%) than that elicited by the E75 peptide we could not conclude from these results about the physiologic (12% 15%; Fig. 2B). consequence of these differences (Fig. 2A). Moreover, except for þ þ the production of IL13, anti-E75 CD8 T cells induced by STxB- High avidity of anti-E75–specific CD8 T cells elicited by STxB-E75 þ E75 or E75 did not produce significant levels (>10 pg/mL) of the Various groups reported that the avidity of CD8 T cells is an main Th2 (IL4, IL5) or Th17 cytokines (IL17; Fig. 2A). Inter- important parameter for their antitumor activity (35). We there- þ estingly, regardless of the vaccine formulation used (E75 pep- fore assessed the avidity of the anti-E75 CD8 T cells induced after þ tide or STxB-E75), E75-specificCD8 T cells also produced a immunization of HLA-A2 TG mice with STxB-E75 or E75. As þ panel of chemokines (i.e., MIP1b, Rantes, eotaxin, IL9) that shown in Fig. 2C, the E75-specific CD8 T cells induced with STxB- play a role in the recruitment of myeloid and T cells, which E75 possess a higher avidity than those induced by the E75 þ could amplify locally the antitumor immune response (Fig. 2A peptide. The anti-E75 CD8 T cells induced by STxB-E75

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A 600 60

400 * Vaccine Pulse 40 Medium * STxB-E75 E75 pepde pg/mL 20 200 Medium * E75 E75 pepde * 0 IL4IL2 IL10 IL12 IL12 IL17 KC RANTES IL13 IL1b TNFa MIP1a MIP1b (p40) (p70)

B Naïve mice STxB-E75 E75 CFSE CFSE CFSE

E75 ** STxB-E75

0 10 20 30 40 50 60 70 80

% Specific lysis

C STXB-E75 * E75 400 * * * 300 *

–specific CD8 cells * 200 * 369 − 377 * * γ –producing cells/1500 100 nb IFN 0 HLA-A2-Her2 10 1 0.3 0.1 0.03 0.01 0.003 0.001 0 Her2 369-377 (mg/mL)

D 8 *** 1,500 *** 6 369 − 377 1,000

4 CD8 T cells 5 500 10

tetramer/CD8 T cells 2 % HLA-A2/Her2 nb IFN γ –producing cells/

0 0 STXB-E75 E75 STXB-E75 E75 (0.5 nmol/L) (25 nmol/L) (0.5 nmol/L) (25 nmol/L)

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þ immunization can be activated by APCs sensitized with as low as tumor protection was lost when the CD8 T cells were depleted 0.001 mg/mL of peptide (Fig. 2C). (Fig. 3E and F). In addition, after vaccination with STxB-E75, we þ could detect intratumoral anti-E75 CD8 T cells (Supplementary þ Persistence of anti-E75–specific CD8 T cells elicited by the anti-E75 Fig. S3). þ vaccines The sustained induction of antitumor CD8 T cells is a prerequisite for the achievement of a clinical antitumor response þ Her2/neu expression levels in tumor cells affects the antitumor and the rapid waning of the antigen-specific CD8 T cells may protective efficacy of the STxB-E75 vaccine explain failure of cancer vaccines (29). To address this point, we þ Preclinical results have pointed out the role of Her2/neu measured the presence of E75 -specific CD8 T cells, 30 days after expression in the downregulation of HLA class I molecules, and the booster dose administration. At this later timepoint, the anti- þ preliminary clinical results suggest that the E75 vaccine is less E75 CD8 T cells that were elicited by STxB-E75 are present at potent in breast cancer patients whose tumors express high levels higher levels and produced more IFNg than those induced by the of Her2/neu (23, 36). On the basis of these evidences, we therefore þ E75 peptides (Fig. 2D). assessed the ability of anti-E75 CD8 T cells generated by the STxB-E75 immunization to recognize tumor cell lines expressing STxB-E75 is more potent to inhibit the growth of hHer2-HLA- variable levels of Her2/neu. þ A2–expressing tumors in HLA-A2 TG mice than the E75 peptide Surprisingly, we found that anti-E75 CD8 T cells were more The efficacy of the STxB-E75 vaccine in controlling hHer2- readily activated by B16-HLA-A2 tumor cells expressing low levels expressing tumors was tested in both prophylactic and therapeutic of Her2/neu, compared with the high Her2/neu HLA-A2 B16 setting in HLA-A2 TG mice. tumors (Fig. 4A). In line with these results, human breast tumor In a therapeutic setting, STxB-E75 clearly delayed tumor growth, cells expressing the highest levels of Her-2 (SKBR-3 and when compared with nonimmunized mice (P < 0.001), or mice UACC812; Supplementary Fig. S4B) were not recognized by the þ vaccinated with the E75 peptide (P < 0.001; Fig. 3A). In agreement anti-E75 CD8 T cells, whereas the SK-Mel37 melanoma cells and with these results, 50% of mice vaccinated with STxB-E75 were still the MCF-7 breast tumor cells which express moderate levels of alive 50 days after the tumor cell engraftment (Fig. 3B). Immuni- Her2/neu were recognized directly, or in the presence of IFNg (Fig. zation with the E75 peptide alone also had a significant albeit 4B). IFNg increases the HLA-A2 expression in the SKBR-3 breast weaker effect on tumor growth, when compared with nonimmu- tumor cells (Supplementary Fig. S4), but when cocultured with þ nized mice (P < 0.05), and 16% of mice immunized with this the anti-E75 CD8 T cells, the number of spots (n ¼ 6) by the IFNg peptide were alive 50 days after tumor graft (Fig. 3B). ELISpot assay remains below the threshold of positivity set at 10 In prophylactic experiments, mice were immunized with STxB- spots (Fig. 4). E75 or the nonvectorized E75 peptide, both in combination with To build on these results, we sorted B16-HLA-A2-Her2/neulow GM-CSF and CpG. A control group (not vaccinated mice) was also or B16-HLA-A2-Her2/neuhigh tumor cells (Supplementary Fig. included. One week after the second immunization, mice were S4). In the therapeutic setting, the STxB-E75 vaccine was more grafted with the HLA-A2-hHer2/neu B16 tumor cells. Immuni- efficient to control the growth of B16-HLA-A2 Her2low tumors zation with STxB-E75 significantly inhibited tumor growth when than the B16-HLA-A2-Her2/neuhigh tumors (Fig. 5A and C). In the compared with nonvaccinated mice (P < 0.05) or mice immu- survival analysis, 35% of the B16-HLA-A2-Her2/neulow tumor- nized with the E75 peptide (P < 0.01; Fig. 3C). The E75 peptide bearing mice treated with the STxB-E75 vaccine were alive at day had no antitumor activity when compared with nonimmunized 50, while none of the B16-HLA-A2-Her2/neulhigh tumor bearing mice (Fig. 3D). These results were confirmed, when using survival mice were alive at the same time point (Fig. 5B and D). As a future as a read out. Indeed, 50% of mice vaccinated with STxB-E75 were cancer vaccine targeting Her2/neu has to position considering the still alive 40 days after tumor grafting, whereas all nonvaccinated current standard therapies, we assessed whether a synergy could mice or those immunized with the E75 peptide had died at day 37 be observed between the STxB-E75 vaccine and the anti-Her2/neu (P < 0.05; Fig. 3D). mAb. Interestingly, the use of anti-Her2/neu mAb alone was þ To confirm the role of CD8 T cells in the clinical efficacy of inefficient to inhibit the growth of tumors at any level of Her2/ the STxB-E75 vaccine, we demonstrated that the therapeutic neu expression (Fig. 5A and C). In combination with the STxB-E75

Figure 2. þ þ Anti-Her2369–377–specific CD8 T cells induced by STxB-E75 are multifunctional, of high avidity and persistent. Specific CD8 splenic T cells were sorted by þ magnetic beads from HLA-A2 TG mice vaccinated with STxB-E75 (0.5 nmol) (in black) or E75 (25 nmol) (in grey), then specific HLA-A2/Her2369–377 CD8 T cells were þ sorted by flow cytometry after dextramer staining. A, HLA-A2 restricted Her2369–377 CD8 T cells (5,000 cells/well) were cocultured with APCs (CD3 cells) pulsed with medium (dashed histogram) or Her2369–377 peptide (10 mg/mL; filled histogram). Cytokines and chemokines were measured by Luminex assay performed on the supernatant after 18 hours. Mean SD of 3 mice/group were represented. This experiment is representative of two experiments. , P < 0.05. B, splenocytes from HLA-A2 TG mice were labeled with 5 mmol/L CFSE (CFSEhigh) and pulsed with the E75 peptide at 10 mg/mL or labeled with 1 mmol/L CFSE (CFSElow) and used as nonpulsed target cells. CFSE-labeled cells containing an equal number of cells from each fraction (CFSE high and low) were injected intravenously into mice previously immunized with E75 (25 nmol/L) or STxB-E75 (0.5 nmol/L). After 15–18 hours, single-cell suspensions from spleens were analyzed by flow cytometry. The percentage of specific killing was determined according to the following calculation: [1 [(CFSElow/CFSEhigh of na€ve mice)/ (CFSElow/CFSEhigh of vaccinated mice)] 100. Data are shown as a representative dot plot from each group (top row) or as a bar graph displaying the means SD þ from 9 mice/group. , P < 0.01. C, HLA-A2–restricted Her2369–377 CD8 T cells (1,500 cells/well) were stimulated in the presence of APC with decreasing concentrations of Her2369–377 peptide and the number of IFNg-producing cells was revealed by ELISpot. Mean SD of 3 mice/group were represented. This experiment is representative of two experiments. , P < 0.05. D, mice were vaccinated at D0 and 14 with STxB-E75 (0.5 nmol/L) or E75 peptide (25 nmol/L). The first þ vaccine dose was mixed with GM-CSF at D0 and CpG was injected at day 1. Thirty days after the boost, HLA-A2/Her2369–377–specific CD8 T cells were detected by dextramer assay (left) and IFNg ELISpot assay (right) as in Fig. 1. Means SD of 12 mice/group from 3 independent experiments. , P < 0.001.

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Downloaded from Tumor size n (mm2) 2 Tumor size (mm ) Tumor size (mm2) 100 200 300 400 100 200 300 400 100 200 300 0 0 Vaccine 0 131922263033 7 5 0 21 92 63 33 30 26 23 19 16 12 9 6 (E75 or STxB-E75) 01 62 32 31 27 23 20 16 13 10 7 4 + GM-CSF Anti-CD8

STxB-E75 CpG E75 PBS STXB-E75 +anti-CD8 PBS +anti-CD8 STXB-E75 PBS Published OnlineFirstMarch22,2016;DOI:10.1158/1078-0432.CCR-16-0044

STxB-E75 + GM-CSF StxB-E75 E75 PBS CpG Days aftertumorchallenge clincancerres.aacrjournals.org Vaccine Anti-CD8 STxB-E75 Days aftertumorchallenge Days tumor after challenge Cancer Research. * *** ** on September 25, 2021. © 2016American Association for **** *** ***

Survival (%)

Survival (%) Her2/neu Low Targeting Vaccine Her2/neu Percent survival D 100 100 100 20 40 60 80 20 40 60 80 20 40 60 80 0 0 0 01020304050 10203040500 01 02 03 40 35 30 25 20 15 10 5 0 STXB-E75 +antiCD8 PBS +anti-CD8 STXB-E75 PBS E75 StxB-E75 PBS STxB-E75 E75 PBS Days aftertumorchallenge Days aftertumorchallenge Days aftertumorchallenge lnCne e;2016 Res; Cancer Clin – xrsigTumor Expressing * * * ** ** OF7 Published OnlineFirst March 22, 2016; DOI: 10.1158/1078-0432.CCR-16-0044

Tran et al.

A B 25 25 IFNγ – 20 + ** 20 IFNγ

T cells 15 + 15 T cells + –producing cells/ g –producing cells/ g 10 10 5,000 CD8 5,000 CD8 5 5 Number of IFN

0 Number of IFN 0

low high SKMEL37 MCF-7 SKB-R3 UACC-812 Medium Medium B16-A2-Her2 B16-A2-Her2

Figure 4. Anti-Her2/neu CD8þ T cells preferentially recognize low Her2/neu–expressing tumor cell lines. CD8þ splenic T cells were isolated from HLA-A2 TG mice þ vaccinated with STxB-E75 using magnetic beads and HLA-A2–restricted Her2369–377 CD8 T cells were sorted by ﬂow cytometry after dextramer staining. IFNg þ ELISpot assay was performed after coculturing for 24 hours HLA-A2–restricted Her2369–377 CD8 T cells (5,000 cells/well) with B16-HLA-A2–expressing low or high Her2/neu (A) or with human tumor cell lines expressing variable levels of Her2/neu previously treated or not with IFNg (100 IU) for 48 hours (B). Data are expressed as means of triplicates SD. Experiments are representative of three experiments. Cells treated with IFNg were washed before being transferred in the wells.

vaccine, anti-Her2/neu mAb potentiated the effect of the vaccine support this possible extrapolation, we could show in a pre- on tumor growth in both models, but when survival was analyzed, clinical HLA-A2-hHer2/neu tumor model, that the efficacy of anti-Her2/neu mAb potentiated the efficacy of the vaccine only in STxB-E75 was significantly higher than that of nonvectorized the B16-HLA-A2-Her2/neulow tumors (Fig. 5B). E75 peptide in its capacity to inhibit tumor growth and to improve overall survival. In previous clinical trials, GM-CSF was used in combination with E75 peptide (36). We found that Discussion the addition of CpG to GM-CSF greatly improved the magni- þ In this study, we demonstrated that the STxB-E75 vaccine tude of the anti-E75 CD8 T cells induced by STxB-E75, while efficiently elicits high levels of long-lasting, multifunctional, this effect was less pronounced for the E75 peptide vaccine, þ cytotoxic, and high avidity E75-specificCD8 TcellsinHLA-A2 similar to what has previously been reported for other peptide TG mice, which were able to recognize human tumors expres- vaccines (38). The use of GM-CSF and CpG to recruit dendritic sing Her2/neu. Both in terms of concentrations and quality of cells and induce maturation of dendritic cells, respectively, may þ the induced anti-E75 CD8 T cells, the potency of the STxB-E75 be particularly useful for delivery vectors targeting dendritic vaccine was far greater that of the E75 peptide alone, which is cells such as STxB or anti-Dec205 (39). currently tested in phase III clinical trials in patients with breast To better position the E75-based vaccine from the clinical þ cancer. This difference persisted even when the E75 peptide was perspective, we showed that induced anti-E75 CD8 T cells better used in 50-fold molar excess. This weak immunogenicity of E75 recognized murine tumor cells expressing low Her2/neu levels. in preclinical model was already reported which supports our These results could be explained by the interference of Her2/neu results (37). with T-cell recognition via an inhibition of HLA class I molecules The Peoples group has been able to correlate the magnitude of mediated by an activation of the MAPK pathway (40, 41). An the E75-specific T-cell responses that were elicited by the E75 inverse correlation between Her2/neu and MHC class I antigen- vaccine with clinical benefit (23). This observation suggests that processing machinery (APM) component levels in human mam- the higher potency of the STxB-E75 vaccine to elicit antigen- mary carcinoma lesions has also been reported supporting this þ specific CD8 T cells, may improve the clinical response. To hypothesis (41). Interestingly, patients who benefited most from

Figure 3. STxB-E75 inhibits the growth of B16-HLA-A2/Her2 tumor in a therapeutic and prophylactic setting via a CD8-dependent mechanism. Mice were grafted with B16-HLA-A2-Her2/neu (1.5 106 cells) and then treated or not with STxB-E75 (0.5 nmol/L) or E75 (0.5 nmol/L) by the subcutaneous route at day 3 and day 10. The ﬁrst vaccine dose was combined with GM-CSF (20 mg) and CpG (50 mg) was administered at day 4. The growth (A) and survival (B) of mice were monitored. Mice (n ¼ 6) were immunized or not by the subcutaneous route with STxB-E75 (0.5 nmol/L) or E75 (0.5 nmol/L) at day 0 and 14 combined with GM-CSF (20 mg) at day 0 and CpG (50 mg) at day 1. At day 21, mice were grafted or not with B16-HLA-A2-Her2 (1.5.106 cells) and the growth (C) and survival (D) of mice were monitored. Mice were grafted with B16-HLA-A2-Her2/neu (1.5 106 cells) and then treated or not with STxB-E75 (0.5 nmol/L) by the subcutaneous route at day 3 and 10 combined with GM-CSF (20 mg) at day 3 and CpG (50 mg) at day 4. For in vivo depletion, anti-mCD8 mAb (100 mg) was injected intraperitoneally once a week, beginning 2 days before the ﬁrst vaccination. The growth (E) and survival (F) of mice were monitored. All of these experiments have been reproduced at least two times and the growth and survival of mice were monitored twice per week. Each group included 6 mice. , P < 0.05; , P < 0.01; , P < 0.001.

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A B 400 PBS 4D5 100

) STxB-E75 PBS 2 300 STxB-E75 + 4D5 80 4D5 **** STxB-E75 60 STxB-E75 + 4D5 200 **** ** 40 Tumor size (mm size Tumor 100 Survival (%) ** 20

0 0 7 3530282522171310 0 10 20 30 40 50 Days after tumor challenge Days after tumor challenge C D 600 PBS 4D5 ) 100 2 STXB-E75 PBS STXB-E75+4D5 4D5 400 STXB STXB-E75+4D5 ns 50 200 **

ns Survival (%) Tumors size (mm size Tumors ns 0 60 3835272114 0 0 5040302010 Days after tumor challenge Days after tumor challenge

Figure 5. þ Anti-Her2/neu CD8 T cells preferentially recognize low Her2/neu–expressing tumor cell lines. Mice were grafted with B16-HLA-A2-Her2/neuLow (1.5 106 cells; A and B) or B16-HLA-A2-Her/neuhigh (C and D) and then treated or not with STxB-E75 (0.5 nmol, s.c.) and/or anti-Her2 mAb (4D5; 100 mg, i.p.) at day 3 and 10. The first vaccine dose was combined with GM-CSF (20 mg; day 3) and CpG (50 mg) was administered at day 4. The growth (A and C) and survival (B and D) of mice were monitored. Each group included 6 mice and results shown are representative of two experiments. , P < 0.05; , P < 0.01; , P < 0.0001. vaccination with the E75 peptide included those who did not In humans, combination of trastuzumab with Her2/neu–based express high levels of Her2/neu (3þ), but rather low or moderate vaccines showed a synergistic effect in the induction of potent levels (1þ,2þ; ref. 23). Beyond the Her2/neu cancer vaccine, high T-cell responses and encouraging clinical results were observed Her2/neu–expressing tumors from breast cancer patients will be (8). Two phase II clinical trials are ongoing based on these associated with impaired antigen presentation leading to a poor preliminary results (NCT02297698 and NCT01570036). A mech- þ recognition of tumor antigen by CD8 T cells and this group of anism that could account for this synergy is the ability of the anti- patients is unlikely to respond to cancer vaccines and immuno- Her2/neu antibody to induce Her2/neu internalization and deg- therapy (40). In line with these results, we observed that the STxB- radation, leading to the generation of Her2/neu–derived peptides E75 vaccine was efficient in a therapeutic setting to inhibit presented by MHC class I molecules to correspondingly specific the growth of low-Her2/neu expressing HLA-A2-B16 tumors in lymphocytes (42). Indeed, trastuzumab-pretreated breast cancer HLA-A2 mice, while it showed no significant effect in the control cells were lyzed more efficiently by E75 primed CTL, when of high-Her2/neu-expressing HLA-A2-B16 tumors both in term compared with untreated breast cancer cells (43). In the current þ of tumor growth and survival analysis. Thus unexpectedly, low study, we did not observe increased levels of anti-E75 CD8 T cells Her2–expressing tumors are more responsive to the vaccine than when the STxB-E75 vaccine was combined with anti-Her2/neu high Her2/neu–expressing tumors. mAb (data not shown). When the STxB-E75 vaccine was combined with anti-Her2/neu A possible limitation of the current study resides in the fact that mAb, a clear synergy was observed on the control of growth of hHer2/neu is not a self-antigen in humanized HLA-A2 TG mice. tumors expressing low or high levels of Her2/neu. However, an To the best of our knowledge, HLA-A2-hHer2/neu TG mice are not impact on survival was mainly observed with the low Her2/neu– available. However, we have already demonstrated the ability of expressing tumor model. The lack of effect with the anti-Her2/neu STxB coupled to murine Her2/neu–derived peptide to break mAb when used alone could be explained by the fact that in this tolerance against Her2/neu as self-antigen (44). In addition, model, Her2/neu is not a natural driver of the tumor, which spontaneous Her2/neu–specific T-cell responses and antibody þ expresses this antigen only after transfection. responses have been documented in patients with Her2/neu

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tumors (3), and patients treated with peptide-based vaccines have T-cell infiltration and ideally combined with a blockade of PD-1/ mounted HER2/neu-specific immune responses (23), implying PD-L1 pathway (54). In addition, in low Her2/neu–expressing that overcoming immunologic tolerance against Her2/neu in tumors, cancer vaccines alone may overcome the clinical resis- patients with breast cancer is feasible. tance to anti-Her2/neu mAb likely because T cells could be It might also be argued that the use of a single CD8 peptide activated by a low amount of the HLA-peptide complex, while exerts less immune pressure than polyepitopic vaccines, and that high levels of Her2/neu are required for the efficacy of antibodies. the absence of CD4-associated peptides as in the case of STxB-E75, þ usually does not favor the generation of persistent CD8 T cells. Conclusion However, encouraging clinical responses have already been The body of experiments presented in this work clearly demon- obtained with the E75 peptide alone, as well as with other CD8 strates that the STxB-E75 vaccine is a potent candidate to be tested peptide-based vaccine strategies (45). Although the use of long in the clinics especially in patients with tumors expressing low peptides incorporating CD4 and CD8 epitopes is often privileged þ levels of Her2/neu. In association with an anti-Her2/neu mAb, the to achieve significant CD8 T-cell responses (17, 46), this study Her2/neu cancer vaccine may be particularly useful in patients and previously published results from our group clearly docu- with low intratumoral immune infiltrates, either alone, or in ment that the STxB delivery vector coupled to various CD8 þ combination with blockers of checkpoint inhibitors. peptides induces functional and persistent CD8 T-cell responses, thereby bypassing the requirement of CD4 helper signal (44). In Disclosure of Potential Conflicts of Interest addition, epitope spreading has been reported after Her2/neu C. Sibley reports receiving commercial research grants from ImmunoTargets vaccines administration and the secondary recruitment of a broad SAS. No potential conflicts of interest were disclosed by the other authors. antitumor T-cell repertoire may participate in the clinical response of the monoepitopic vaccines (8, 47, 48). Authors' Contributions Cardiac toxicity has initially represented a clinical concern for Conception and design: T. Tran, M.O. Diniz, J. Medioni, E. Tartour the targeting of Her2/neu by mAb and a serious adverse event was Development of methodology: T. Tran, M.O. Diniz, A. Gey, J. Medioni, reported following the administration of chimeric antigen recep- E. Tartour tor T-cell recognizing Her2/neu in a patient with colorectal cancer Acquisition of data (provided animals, acquired and managed patients, (49). However, all Her2/neu vaccines that were tested in humans provided facilities, etc.): T. Tran, M.O. Diniz, E. Dransart, A. Gey, N. Merillon, alone or in combination with trastuzumab were devoid of sig- Y.C. Lone, E. Tartour Analysis and interpretation of data (e.g., statistical analysis, biostatistics, nificant clinical toxicity. computational analysis): T. Tran, M.O. Diniz, A. Gey, N. Merillon, S. Oudard, Regarding the toxicity of STxB, in vitro STxB did not exhibit E. Tartour cytotoxic effect on various normal cell lines and after repetitive Writing, review, and/or revision of the manuscript: T. Tran, M.O. Diniz, administration of high dose of STxB, no toxicity was observed in S. Godefroy, C. Sibley, J. Medioni, S. Oudard, L. Johannes, L.C.S. Ferreira, mice (50). E. Tartour The analysis of the dynamics of spontaneous or vaccine- Study supervision: S. Godefroy, L. Johannes, E. Tartour þ Other (financial support for research conducted leading to this manuscript): induced antitumor CD8 T cells clearly showed that in the course C. Sibley of tumor progression, they are anergized via the upregulation of checkpoint inhibitors (PD-1, CTLA-4) or blocked by various Grant Support immunosuppressive mechanisms including regulatory T cells fi This work was supported by grant from Institut National du Cancer (INCA) (51). Reversal of these blocking factors improved the ef cacy of (PLBIO11-022- IDF-JOHANNES; to L. Johannes and E. Tartour), Immuno a Her2/neu cancer vaccine by reactivating intratumoral T cells Target SAS (to E. Tartour and L. Johannes), Ligue contre le Cancer (to leading to improved clinical results (52). E. Tartour), Universite Sorbonne Paris Cite (to E. Tartour), Labex Immuno- As from a clinical perspective, Her2/neu cancer vaccines would Oncology (to E. Tartour), SIRIC CARPEM (to E. Tartour), and ERC advanced be combined with anti-Her2/neu mAb, especially breast cancers grant (project 340485; to L. Johannes). The costs of publication of this article were defrayed in part by the payment of expressing high levels of Her2/neu, it is important to notice that fi page charges. This article must therefore be hereby marked advertisement in T cells and immune signatures are crucial for the ef cacy of anti- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Her2/neu mAb treatments (13, 53). Patients with low intratumoral immune infiltrates will thus likely benefit from a combi- Received January 6, 2016; revised February 16, 2016; accepted March 6, 2016; nation of Her2/neu mAb with Her2/neu cancer vaccine to favor published OnlineFirst March 22, 2016.

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OF12 Clin Cancer Res; 2016 Clinical Cancer Research

A Therapeutic Her2/neu Vaccine Targeting Dendritic Cells Preferentially Inhibits the Growth of Low Her2/neu− Expressing Tumor in HLA-A2 Transgenic Mice

Thi Tran, Mariana O. Diniz, Estelle Dransart, et al.

Clin Cancer Res Published OnlineFirst March 22, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-16-0044

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