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[Cancer Research 64, 8022-8028, November 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Vaccination by Genetically Modified Dendritic Cells Expressing a Truncated neu Oncogene Prevents Development of Breast Cancer in Transgenic Mice

Yoshio Sakai1, Brian J. Morrison1, J. Douglas Burke1, Jong-Myun Park2, Masaki Terabe2, John E. Janik3, Guido Forni4, Jay A. Berzofsky2 and John C. Morris1,3

1 Cancer Gene Therapy Section, Metabolism Branch, 2 Molecular Immunogenetics and Vaccine Research Section, Vaccine Branch, and 3 Clinical Trials, Metabolism Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland; and 4 Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DCs) are powerful antigen-presenting cells that process antigens and present peptide epitopes in the context of the major histocompatibility complex molecules to generate immune responses. DCs are being studied as potential anticancer vaccines because of their ability to present antigens to naïve T cells and to stimulate the expansion of antigen-specific T-cell populations. We investigated an antitumor vaccination using DCs modified by transfer of a nonsignaling neu oncogene, a homologue of human HER-2/neu, in a transgenic model of breast cancer. BALB-neuT mice develop breast cancers as a consequence of mammary gland-specific expression of an activated neu oncogene. We vaccinated BALB-neuT mice with bone marrow-derived DCs transduced with Ad.Neu, a recombinant adenovirus expressing a truncated neu oncoprotein. The vaccine stimulated the production of specific anti-neu antibodies, enhanced interferon-{gamma} expression by T cells, and prevented or delayed the onset of mammary carcinomas in the mice. Over 65% of vaccinated mice remained tumor free at 28 weeks of age, whereas all of the mice in the control groups developed tumors. When challenged with a neu-expressing breast cancer cell line, vaccinated tumor-free animals had delayed tumor growth compared with controls. The antitumor effect of the vaccine was specific for expression of neu. Studies showed that CD4+ T cells were required in order to generate antitumor immunity. Importantly, the effectiveness of the vaccine was not diminished by preexisting immunity to adenovirus, whereas the protection afforded by vaccination that used direct injection of Ad.Neu was markedly reduced in mice with anti-adenovirus antibody titers. DCs modified by recombinant adenoviruses expressing tumor-associated antigens may provide an effective antitumor vaccination strategy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cancer cells express tumor-associated antigens (1 , 2) that can be potentially targeted by the immune system. Dendritic cells (DCs) are powerful antigen-presenting cells that play a central role in generating and directing immune responses through the processing of antigens and presentation of epitopes in the context of surface MHC molecules to interact with T cells. DCs also express a full complement of costimulatory molecules and cytokines that are required to sustain and direct the immune response (3 , 4) . Major efforts at developing antitumor vaccines have focused on harnessing DCs to effectively present tumor antigens to the immune system. A number of clinical trials have examined the efficacy of epitope-modified DCs, most often by "pulsing" DCs ex vivo with synthetic peptides based on antigens (5, 6, 7, 8, 9, 10) , mutant oncogenes (11) , or immunoglobulin idiotypes (12) expressed by tumors. Peptide loading has a number of potential drawbacks including a need for detailed knowledge of the antigen sequence, identification of the recipient MHC molecule, and a requirement for a favorable binding affinity of the peptide for the MHC molecule. Peptide binding half-lives may be brief (13 , 14) , and the ability of a peptide to induce an antigen-specific CTL response correlates with the stability of this complex (15) . An alternative strategy is to load DCs by incubating them with protein lysates manufactured from tumors. Tumor lysates, however, contain many antigens, most of which are not tumor specific.

The introduction of a gene encoding a tumor-associated antigen into DCs may overcome many of these problems. Gene expression and the physiologic processing of intact antigens will likely generate greater numbers of epitopes and more favorable antigen presentation. In addition, constitutive gene expression provides for continuous replenishment of low affinity epitopes bound on MHC molecules. Among gene delivery systems, recombinant adenoviruses (rAds) can efficiently infect DCs and offers the advantage of accepting up to 8-kb cDNA inserts. In addition, viral-associated proteins may provide the "danger signals" required for the activation of DCs and up-regulation of their costimulatory molecules, which results in stronger immune responses (16 , 17) .

The HER-2/neu (ErbB2) oncogene, a member of the epidermal growth factor receptor tyrosine kinase family (18 , 19) , is frequently overexpressed in cancers of the breast (20 , 21) , ovary (22) , uterus (23) , lung (24) , and gastrointestinal tract (25) . Its overexpression in breast cancer is associated with a poorer prognosis (20) . Trastuzumab, a humanized antibody targeting HER-2/neu is approved for the treatment of advanced breast cancer, indicating the ability of this antigen to serve as a target for immunotherapy (26) . Vaccination of patients with advanced breast cancer using HER-2/neu peptides or peptide-pulsed autologous DCs has been examined in clinical trials (8) . Few responses, however, have been reported, although increases in anti-HER-2/neu CTLs were seen in some patients (9) .

To examine the efficacy of antitumor vaccination in a clinically relevant model, we studied the ability of DCs modified by a rAd expressing a truncated neu antigen to suppress the development of mammary carcinomas in a transgenic model of breast cancer in which the neu oncogene is expressed in tissue-specific fashion. We demonstrated that vaccination prevented or delayed the onset of cancers in this aggressive tumor model. Importantly, the efficacy of antitumor vaccination with rAd-infected DCs was unaffected by preexisting immunity to adenovirus.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
TUBO cells derived from a breast cancer of a BALB-neuT mouse (27) , and N202.1A cells isolated from a breast cancer of a FVB-neuN mouse, were the kind gift of Dr. Patrizia Nanni (University of Bologna, Bologna, Italy; refs. 28 , 29 ) and were grown in Dulbecco’s modified Eagle’s medium (DMEM; BioSource, Rockville, MD) supplemented with 10% fetal bovine serum (Gemini, Woodland, CA). Both TUBO and N202.1A cells constitutively express neu. The neu-negative cell line, TS/A (also a gift of Dr. Patrizia Nanni), established from a spontaneous breast cancer from a BALB/c mouse (30) was grown in RPMI 1640 (BioSource) and 10% fetal bovine serum. Human 293 embryonic kidney and A549 lung cancer cells were obtained from American Type Culture Collection (ATCC; Manassas, VA) and were grown in DMEM with 10% fetal bovine serum.

Adenoviral Vectors.
The cDNA encoding the extracellular and transmembrane domains of the rat neu oncogene was provided by Dr. Augusto Amici (University of Camerino, Camerino, Italy; ref. 27 ). Ad.Neu, an E1-, E3-deleted rAd expressing neu, and Ad.null, an "empty" control vector were generated by homologous recombination with the AdMax system (Microbix, Toronto, Canada; ref. 31 ). Ad.GFP, an adenovirus expressing green fluorescent protein (GFP) was obtained from Quantum Biotechnologies (Quebec, Canada). All viruses were double plaque-isolated, expanded on 293 cells, purified on a cesium chloride gradient, titered by serial dilution as plaque forming unit(s) (pfu)/mL and stored at –70°C.

Animals.
All animal studies were approved by the Animal Care and Use Committee of the National Cancer Institute. BALB-neuT mice expressing the rat neu oncogene under the control of a chimeric mouse mammary tumor virus (MMTV) promoter provide an aggressive model of mammary carcinogenesis as the transgene is overexpressed in the rudimentary mammary glands of 3-week-old mice (32, 33, 34) . By 6 weeks, atypical lobular hyperplasia is seen that progresses to multiple in situ carcinomas that enlarge and converge to form rapidly growing, invasive, and metastasizing tumors in all 10 glands by 25 weeks of age (35) . Female BALB/c mice were obtained from the Division of Cancer Treatment, National Cancer Institute (Frederick, MD).

Generation of Dendritic Cells.
DCs were generated with the method of Lutz et al. (36) . Briefly, bone marrow was harvested from femurs of 8- to 10-week-old BALB/c mice. Erythrocytes were lysed with ammonium-chloride-potassium buffer (BioWhittaker, Walkersville, MD) and the nucleated cells were plated in plastic bacteriologic dishes in RPMI 1640 with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.-Invitrogen, Grand Island, NY) and 20 ng/mL murine granulocyte/macrophage-colony stimulating factor (GM-CSF; PeproTec, Rocky Hill, NJ). The cultures were refreshed with 10 mL of medium containing 20 ng/mL GM-CSF on days 3, 6, and 8. On day 8, the nonadherent DCs were collected and infected with rAds. The DCs were used for experiments on day 10 after washing three times in phosphate buffered saline.

Northern Analysis.
On day 8, DCs were infected with Ad.Neu or Ad.null at multiplicities of infection (MOI) 30 pfu/cell. Two days later, total RNA was isolated with Trizol Reagent (Life Technologies, Inc.-Invitrogen) and were treated with DNaseI (Invitrogen). The RNA was subjected to electrophoresis on a 1.2% formaldehyde gel, transferred to nylon membrane, and probed with a 32P-fragment of rat neu cDNA.

Flow Cytometry.
DCs were incubated with FITC- or phycoerytherin-labeled antimouse CD11c, CD11b, CD40, CD80, CD86, H-2Kd, I-Ad, CD8{alpha} (BD PharMingen, San Diego, CA) and analyzed on a FACSort (Becton Dickinson, San Jose, CA). Forty-eight hours after infection with Ad.Neu or Ad.null, the DCs were incubated with antirat neu monoclonal antibody (Oncogene Research, La Jolla, CA) followed by incubation with secondary FITC-labeled rabbit antimouse immunoglobulin and analyzed by FACSort. To detect cytoplasmic neu, the cells were permeabilized with Cytofix/Cytoperm (BD PharMingen) and similarly stained.

Mixed Lymphocyte Proliferation and CTL Assay.
Lymphocytes obtained from the spleens of 8- to 10-week-old naïve female BALB-neuT mice were incubated with irradiated unmodified DCs or DCs infected with either of the rAds. Four days later, the incorporation of [3H]thymidine was measured as described previously (37) . CTL assays with chromium-51 release were performed as described previously (38) .

Vaccination of Mice with Ad.Neu-Modified Dendritic Cells.
Groups of 5- to 6-week-old female BALB-neuT mice received three weekly subcutaneous injections of 1 x 106 DCs infected with Ad.Neu (DCAd.Neu) or Ad.null (DCAd.null) at MOI 30, or of unmodified DCs. The mice were examined twice weekly for the development of tumors. Tumor volumes were calculated with the estimated volume of a rotational ellipse (39) . Mice free of tumor at 28 weeks were challenged with a subcutaneous injection of 1 x 105 neu+ TUBO cells. Another group of BALB-neuT mice received Ad.null 1 x 108 pfu subcutaneously before anti-neu vaccination and was followed for the development of tumors after undergoing vaccination as described above. This group was compared with mice similarly prevaccinated with Ad.null and then vaccinated against neu by direct subcutaneous injections of Ad.Neu 1 x 108 pfu, weekly for 3 weeks.

In another set of experiments, groups of 5- to 6-week-old BALB/c mice were given injections subcutaneously of Ad.null 1 x 108 pfu weekly for 4-weeks. Two weeks after the last injection of Ad.null, groups of mice were vaccinated either with 1 x 106 DCAd.Neus subcutaneously or directly with 1 x 108 pfu Ad.Neu subcutaneously, weekly for two weeks. One week after the final vaccination, the mice were challenged with 5 x 105 TUBO cells subcutaneously and were monitored for tumor growth.

In another experiment, BALB/c mice vaccinated with DCAd.Neu were challenged with 5 x 105 neu+ TUBO cells subcutaneously. All of the mice remained tumor free, and at 37 days, the mice were divided into two groups and rechallenged with an injection of 5 x 105 neu+ TUBO cells or neu– TS/A cells, and were followed for development of tumor.

Antibody Depletion of Lymphocyte Populations.
Groups of BALB/c mice were given intraperitoneal injections of 200 µg of anti-CD4, or anti-CD8 purified from the supernatants of hybridomas GK1.5 (ATCC) and 2.43 (ATCC), respectively, for 3 consecutive days, then every 3 days, or with anti-asialo-GM1 (WAKO, Richmond, VA). Five days after initiation of antibody treatment, the mice were vaccinated as described above. One week after the last vaccination, the mice were challenged with 5 x 105 TUBO cells and then were monitored for tumor growth.

Detection of serum Anti-neu Antibodies.
Blood was obtained by retro-orbital venipuncture from BALB/c-neuT mice before vaccination and one week after the third vaccination. The blood was allowed to clot, and the sera from each treatment group were pooled. N202.1A cells (neu+) were used to detect and quantify anti-neu antibodies as described previously with slight modification (28) . Briefly, 2 x 105 N202.1A cells were incubated with the sera diluted 1:10 in 2% bovine serum albumin in phosphate buffered saline at 4°C for 1 hour. The cells were washed and incubated with FITC-labeled rabbit antimouse immunoglobulin antibody (DAKO, Carpinteria, CA) and were submitted to flow cytometry. Mean fluorescence intensity was quantified with CellQuest Software (Becton Dickinson).

Detection of Anti-Adenovirus Antibodies.
Sera were obtained pre- and postvaccination from the mice. Anti-adenovirus antibody titers were measured as described previously (40) . Briefly, 5 x 104 A549 cells were seeded in a 24-well plate. Sixteen hours later, 10 µL of 2-fold serially diluted heat-inactivated test sera were incubated with 10 µL of Ad.GFP (2.5 x 106 pfu) at room temperature for 1 hour. The mixture was added to the A549 monolayer (MOI 30). Thirty-six hours later, the A549 cells were trypsinized, washed, and analyzed for GFP expression by flow cytometry. Antibody titers were assessed as the serum dilution that resulted in 50% inhibition of GFP expression compared with controls.

Enzyme-Linked Immunospot Assay.
Spleens were obtained from BALB-neuT mice 1 week after the last vaccination, and T cells were isolated with the Pan-T Cell Isolation Kit (Miltenyi, Auburn, CA) in accordance with the manufacturer’s protocol. Thirty thousand spleen cells were plated in a 96-well dish coated with antimouse interferon {gamma} (IFN{gamma}) antibody (R&D systems, Minneapolis, MN) and were incubated for 48 hours at 37°C. The plates were developed in accordance with the manufacturer’s protocol, and the spots were counted.

Cytokine Secretion Assay.
One million spleen cells from DCAd.Neu-vaccinated BALB-neuT mice were incubated for 4 hours with an equal number of irradiated DCAd.Neu, DCAd.null, or unmodified DCs. IFN{gamma}-secreting CD4+ and CD8+ T cells were detected with Cytokine Secretion Assay kit (Miltenyi) in accordance with the manufacturer’s protocol. Briefly, restimulated splenocytes were harvested and stained with phycoerytherin-labeled antimouse IFN{gamma} antibody together with peridinin chlorophyll protein–labeled CD45RA/B220 and FITC-labeled anti-CD4 or FITC-labeled antiCD8. Cells were incubated with 7-amino-actinomycin D for 10 minutes. and then analyzed on the FACSort gating out dead and CD45R/B220-positive cells. The fraction of IFN{gamma}-positive CD4 or CD8 cells were calculated and reported as a percentage.

Immunohistochemical Analysis.
Mammary glands, primary breast cancers, and TUBO tumors were removed from BALB-neuT mice, frozen in OCT (optimum cutting temperature) compound (SAKURA-Finetek U.S.A. Inc., Torrance, CA), and were cryostat sectioned. Endogenous peroxidase activity was blocked followed by blocking of avidin and biotin with the Avidin/Biotin Blocking kit (Zymed Laboratories Inc., South San Francisco, CA). After washing with phosphate buffered saline, the tissues were stained with rabbit antimouse CD4 (BD PharMingen) or antimouse CD8 (BD PharMingen). Tumor sections were stained with antirat neu monoclonal antibody (Oncogene Research). The slides were washed and then incubated with biotinylated antirabbit antibody (Vector Laboratory, Burlingame, CA), stained with streptavidin-horseradish peroxidase (DAKO), and counterstained with hematoxylin.

Statistical Analysis.
STATView version 5.01 software (SAS Institute, Cary, NC) was used for log-rank survival analysis and comparisons of tumor multiplicity with the Mann–Whitney U test. Enzyme-linked immunospot (ELISPOT) assay was analyzed with Student’s t test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Adenovirus on Dendritic Cell Phenotype.
DCs generated from 10-day bone marrow cultures expressed CD11c and CD11b, and lacked CD8{alpha}, indicating myeloid differentiation (Fig. 1A)Citation . They also expressed CD80, CD86, CD40, and MHC class I and class II. Compared with unmodified DCs, those infected with Ad.Neu (DCAd.Neu) or Ad.null (DCAd.null) expressed higher levels of CD80, CD86, CD40, and both MHC classes, indicating maturation (Fig. 1B)Citation .



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  		Fig. 1. DC phenotype. In A, surface marker expression of DCs derived from BALB/c mouse bone marrow examined by flow cytometry. Open curves, isotype controls. In B, DC surface marker expression 48 hours after infection with rAds (MOI 30).

 
Dendritic Cells Infected with Ad.Neu-Expressed Truncated neu mRNA and Oncoprotein.
Thirty-six hours after infection with Ad.Neu or Ad.null, Northern analysis of RNA isolated from DCAd.Neu showed the expected 2.1-kb truncated neu mRNA transcript (Fig. 2A)Citation , which was not detected in DCAd.null. The transcript was absent in noninfected BALB-neuT bone marrow-derived DCs, even when analyzed by reverse transcription-PCR, indicating tissue-specific neu expression in the transgenic animals (data not shown). DCs, stained with antirat neu antibody 48 hours after infection with Ad.Neu or Ad.null and examined by flow cytometry, showed neu expression on the cell surface and in the cytoplasm (Fig. 2B)Citation . No expression was seen in DCAd.null.



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  		Fig. 2. Expression of truncated neu mRNA and antigen in DCs infected with Ad.Neu. In A, on day 8, DCs were infected with Ad.Neu or Ad.null (MOI 30), and RNA was isolated on day 10. Total RNA was probed with a 32P-fragment of rat neu cDNA. In B, DCs were infected with Ad.null (MOI 300) or Ad.Neu (MOI 30 or 300); 48 hours later, the DCs were incubated with an antirat neu primary antibody and were stained with FITC-labeled antimouse immunoglobulin. Open curves, isotype control.

 
Ad.Neu-Transduced Dendritic Cell-Stimulated Proliferation of Naïve BALB-neuT Lymphocytes.
The function of the DCs was examined in mixed lymphocyte culture. Irradiated DCAd.Neu, DCAd.null, or unmodified DCs were cultured in various ratios with lymphocytes from the spleens of nonimmunized BALB-neuT mice and were pulsed with [3H]thymidine. Three to 4-fold greater stimulation of splenocyte labeling was seen with DCAd.Neu compared with DCAd.null or unmodified DCs (Fig. 3)Citation .



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  		Fig. 3. DCs infected with Ad.Neu stimulated the proliferation of naïve BALB-neuT lymphocytes. DCs infected with Ad.Neu (DCAd.Neu) or Ad.null (DCAd.null) or with unmodified DCs (DC) were mixed with naïve BALB-neuT mouse splenic lymphocytes in various ratios, and the incorporation of [3H]thymidine was measured. Error bars, ± SEM.

 
Vaccination of BALB-neuT Mice with DCAd.Neu Prevented Autochthonous Breast Cancers.
Vaccination of BALB-neuT mice with DCAd.Neu significantly improved tumor-free survival compared with mice treated with DCAd.null or unmodified DCs (Fig. 4A)Citation . The median tumor-free survival of the mice treated with unmodified DCs compared with those receiving DCAd.null was not different (19.5 versus 20 weeks, P = 0.438). In contrast, the median tumor-free survival was not reached by 28 weeks in mice receiving DCAd.Neu (P < 0.0001). Furthermore, in the groups of mice treated with the unmodified DCs or DCAd.null, the onset of the first tumor occurred at 14.5 weeks, and all of the mice had developed at least one breast cancer by 23.5 weeks. In mice vaccinated with DCAd.Neu, the first breast lesion occurred at 20 weeks, and 14 (66.7%) of 21 mice were still free of tumor at 28 weeks. In view of the mean tumor multiplicity, DCAd.Neu also conferred significant protection (Fig. 4B)Citation . At 28 weeks, tumor was palpable in all 10 mammary glands of all of the mice treated with unmodified DCs or with DCAd.null. In the group of mice vaccinated with DCAd.Neu, the mean tumor multiplicity was only 2.9 (P < 0.0001). Vaccination of older groups of mice was less effective, and the vaccine had little effect in mice that had developed palpable tumors (data not shown).



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  		Fig. 4. Effect of vaccination on tumors in BALB-neuT transgenic mice. In A, Kaplan–Meier plot of tumor-free survival. BALB-neuT mice (5–6 weeks old) were given injections in the flank with 1 x 106 DCAd.Neu ({square}, n = 21), DCAd.null ({blacksquare}, n = 11), or unmodified DCs ({circ}, n = 10), weekly for 3 weeks. Mice were followed for the development of breast tumors. In B, mean tumor multiplicity (mean number of tumors per mouse) in the treatment groups over time. Error bars, ± SEM; *, P < 0.0001.

 
Vaccination of BALB-neuT Transgenic and BALB/c Mice with DCAd.Neu Inhibited Growth of a Transplantable neu-Expressing Breast Carcinoma.
To test whether DCAd.Neu-vaccinated BALB-neuT mice could successfully reject a challenge with a neu-expressing tumor cell line, mice vaccinated at 5 to 7 weeks of age with DCAd.Neu that were tumor free at 28 weeks (n = 6) were challenged with the neu+ TUBO cells. A small number (n = 3) of surviving similar-age mice that had been vaccinated with unmodified DCs or DCAd.null were also challenged. At a mean of 10 days after injection with 1 x 105 TUBO cells, tumors were evident in the mice receiving unmodified DCs or DCAd.null. The time to the appearance of palpable TUBO tumors was similar (10 ± 1 day) to that of unvaccinated BALB/c or BALB-neuT mice (data not shown). In contrast, in the BALB-neuT mice vaccinated with DCAd.Neu, tumors occurred at a mean of 56 days. Notably, in the DCAd.Neu-vaccinated mice, TUBO tumor growth occurred contemporaneous or subsequent to the development of an autochthonous breast cancer, suggesting a general loss of immunity to neu (Table 1)Citation . Immunohistochemical staining of the TUBO tumors and late-occurring autochthonous breast cancers were positive for neu expression, which indicated that tumor escape was not due to selection and outgrowth of neu-negative tumor cells.


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  		Table 1 DCAd.Neu vaccination protects BALB-neuT mice from challenge with neu-expressing TUBO breast cancer cells

 
To examine the specificity of protection derived from vaccination with DCAd.Neu, BALB/c mice (n = 8) were vaccinated with 1 x 106 DCAd.Neu weekly for 2 weeks, and were then subcutaneously challenged with 5 x 105 TUBO cells a week later. All of the mice remained free of tumor at 37 days. These mice were rechallenged with 5 x 105 of either TUBO cells (n = 4) or neu-negative TS/A cells (n = 4). All of the vaccinated mice that received injections of TS/A cells developed tumors by day 10, whereas those receiving TUBO cells remained free of disease up to 121 days later (Table 2)Citation .


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  		Table 2 Vaccination induced specific immunity to neu-expressing tumors

 
DCAd.Neu-Vaccination Induced Serum Anti-neu Antibodies and IFN{gamma} Secretion by T Cells.
We measured the induction of anti-neu antibodies in groups of BALB-neuT mice pre-vaccination and one week after the final vaccination. Major increases of anti-neu antibody titers were detected in the serum of mice vaccinated with DCAd.Neu. Minor changes were seen in the mice receiving DCAd.null, and no titers were detected in mice vaccinated with the unmodified DCs (Fig. 5A)Citation .



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  		Fig. 5. Antibody and cellular responses to vaccination. In A, vaccination with DCAd.Neu induced the production of anti-neu antibodies. Serum collected from BALB-neuT mice before vaccination and 1 week after the last (third) vaccination were analyzed for anti-neu antibodies, as described in Materials and Methods. The results represent the mean titers of five mice in each group. Error bars, ± SEM. In B, DCAd.Neu vaccination increased the secretion of IFN{gamma} from CD4+ and CD8+ T cells. Splenic T cells isolated from BALB-neuT mice 1 week after completion of the DCAd.Neu vaccination were assayed for IFN{gamma} secretion after restimulation by DCAd.Neu, DCAd.null, or unmodified DCs by fluorescent antibody capture flow cytometry.

 
Tumor-specific CTLs could not be demonstrated by chromium-release assay in the vaccinated mice; however, IFN{gamma} production by splenocytes was seen on ELISPOT (Table 3)Citation . The numbers of IFN{gamma}-expressing T cells were increased in DCAd.Neu vaccinated mice compared with the mice vaccinated with DCAd.null (P < 0.0001), or unmodified DCs (P < 0.0001). These results were confirmed by cytokine secretion assay. A more than 3.5-fold increase in the numbers of IFN{gamma}-expressing CD4+ lymphocytes was detected in the spleens of DCAd.Neu-vaccinated BALB-neuT mice that were restimulated with DCAd.Neu, compared with restimulation with unmodified DCs; and a greater than 2-fold increase was noted compared with restimulation with DCAd.null, indicating a neu-specific response (Fig. 5B)Citation . The frequency of IFN{gamma}-secreting CD8+ T lymphocytes was increased by 4-fold compared with stimulation with DCAd.null or with unmodified DCs.


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  		Table 3 Interferon {gamma} ELISPOT

 
Infiltration of Mammary Glands with CD4+ and CD8+ T Cells.
Immunohistochemical staining of mammary tissue from BALB-neuT mice vaccinated with DCAd.Neu showed modestly increased numbers of CD4+ and CD8+ cells infiltrating the periphery of hyperplastic and dysplastic mammary glands (data not shown). The breast tissue of mice receiving the control vaccines showed little cellular infiltrate.

CD4+ T Cells Are Required for Generation of Antitumor Immunity with DCAd.Neu.
To explore the mechanism of DCAd.Neu antitumor vaccination, we administered anti-CD4, anti-CD8, or anti-asialo-GM1 antibodies to groups of BALB/c mice before and during DCAd.Neu vaccination to deplete these lineages. One week after the last vaccination, the mice were challenged with 5 x 105 TUBO cells. The control animals, vaccinated with DCAd.Neu but not treated with antibodies, were protected from tumor growth (Fig. 6)Citation . Mice treated with anti-CD8 or anti-asialo GM1 antibodies also failed to form tumors; however, mice depleted of CD4+ cells developed tumors, indicating that CD4+ cells are required for the generation of antitumor immunity after vaccination with DCAd.Neu. Treatment with anti-CD4 after vaccination had no effect (data not shown).



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  		Fig. 6. Protection by DCAd.Neu vaccination requires CD4+ T cells. BALB/c mice were depleted of specific cell populations using anti-CD4, anti-CD8, or anti-asialo-GM1 before and during DCAd.Neu vaccination. One week after completion of vaccination, the mice were given subcutaneous injections with 1 x 105 TUBO cells. Tumor volumes at day 63 are shown.

 
Efficacy of DCAd.Neu Vaccination Is Unaffected by Preexisting Immunity to Adenovirus.
To assess the effect of preexisting immunity to adenovirus on the efficacy of DCAd.Neu vaccination, 4-week-old BALB-neuT mice were given subcutaneous injections of Ad.null, 1 x 108 pfu, to generate anti-adenovirus immunity. Three weeks later, mice were divided into two groups and were vaccinated subcutaneously directly with Ad.Neu 1 x 108 pfu or with 1 x 106 DCAd.Neu. Direct injection of Ad.Neu is protective in this model.5 At 28 weeks, four of seven mice receiving DCAd.Neu remained tumor-free. This is similar to mice treated with DCAd.Neu and not undergoing prevaccination with Ad.null (Fig. 7ACitation ; Fig. 4ACitation ). All of the mice vaccinated with Ad.null and then treated with Ad.Neu developed tumors by 24.5 weeks (Fig. 7A)Citation .



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  		Fig. 7. Effect of preexisting immunity to adenovirus on the efficacy of DCAd.Neu vaccination. In A, the protective effect of vaccination with DCAd.Neu in BALB-neuT mice was not affected by preexisting immunity to adenovirus, and the benefit of direct vaccination using Ad.Neu was lost. BALB-neuT mice were injected with Ad.null 1 x 108 pfu s.c. Two weeks later, mice were vaccinated with either 1 x 106 DCAd.Neu s.c. (n = 7), or directly with 1 x 108 pfu Ad.Neu s.c. (n = 7). BALB-neuT mice (n = 7), not previously treated with Ad.null and given injections of Ad.Neu 1 x 108 pfu subcutaneously, served as controls. In B, DCAd.Neu vaccination was effective in BALB/c mice with preexisting immunity to adenovirus in protecting against TUBO breast cancer, whereas the protection from direct vaccination with Ad.Neu was lost. BALB/c mice were injected weekly for 4 weeks with Ad.null 1 x 108 pfu subcutaneously, and serum anti-adenovirus antibody titers were measured. The mice were then vaccinated with 1 x 106 DCAd.Neu or with direct injection of 1 x 108 pfu Ad.Neu subcutaneously weekly for 2 weeks. Mice vaccinated with Ad.Neu or DCAd.Neu alone served as controls. The mice were challenged with 5 x 105 TUBO cells subcutaneously 1 week later. Twenty-one days after injection of the TUBO cells, the tumor volumes were compared.

 
To confirm these results, BALB/c mice were given subcutaneous injections weekly for 4 weeks with Ad.null 1 x 108 pfu. All of the mice receiving Ad.null showed significant increases in serum anti-adenovirus antibody titers, from a mean of less than 1:2 to a mean greater than 1:256, 1 week after the last injection. Untreated mice had a mean baseline titer of less than 1:2 that remained unchanged over 6 weeks (data not shown). Two weeks later, the mice were vaccinated with either 1 x 108 pfu Ad.Neu, or 1 x 106 DCAd.Neu. One week later, the mice were challenged with 5 x 105 TUBO cells. Twenty-one days later, all of the mice treated directly with Ad.Neu had developed tumors, whereas six of seven mice receiving DCAd.Neu remained tumor-free (Fig. 7B)Citation , which suggested that immunity to adenovirus abrogated the protection afforded by direct injection of Ad.Neu.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We showed that vaccination of BALB neuT-transgenic mice with bone marrow-derived DCs modified by a rAd-expressing truncated neu oncoprotein prevented the development, or significantly delayed the onset, of breast cancer in the mice. Antitumor vaccination with DCs modified by adenoviral-mediated transfer of genes encoding tumor-associated antigens offers potential advantages over antigen-loading strategies that use peptides or tumor lysates. These include long-term antigen expression, activation of the DCs by the vector, and induction of DC maturation with enhanced expression of MHC and costimulatory molecules. DCs modified by adenovirus are able to stimulate naïve splenocytes to a greater degree than are unmodified DCs, possibly because of activation by viral proteins (41) . This may enhance the vaccine’s efficacy, because mature DCs are more effective at generating immune responses (42) .

In our model, the induction of anti-neu antibodies and the inability to demonstrate specific CTLs suggests that the antibody may be the primary protective mechanism of the vaccine; however, a T-cell response is not excluded. Increased numbers of neu-specific IFN{gamma}-expressing CD4+ and CD8+ T cells were seen in the vaccinated mice, and studies that depleted T-cell subsets demonstrated a critical role for CD4+ cells in the generation of antitumor immunity in the vaccinated animals. The activation of CD4+ helper T cells by DCs is required for the complete activation of B cells by many antigens (43) . Although the infection of DCs with rAds induced maturation and increased the expression of costimulatory molecules, the direct interaction of CD8+ T cells with DCs may not be sufficient for the induction of tumoricidal activity. Preconditioning by CD4+ helper T cells may be required for CD8+ cells to be fully activated (44 , 45) .

In humans, a significant fraction of the population has been exposed to adenovirus; therefore, preexisting immunity to the vector might hinder the effectiveness of adenovirus-based vaccines (46, 47, 48) . We found no attenuation of the DCAd.Neu vaccine in animals preimmunized with Ad.null. In contrast, the effectiveness of direct vaccination with Ad.Neu was diminished by prior adenovirus exposure, which suggested that vaccination with rAds to modify DC ex vivo can overcome the inhibition of gene expression seen in individuals previously exposed to adenovirus (49 , 50) .

Y. Chen et al. (51) and Z. Chen et al. (52) reported that vaccination with DCs modified with a rAd to express HER-2/neu, offered partial protection in a transplantable HER-2/neu-expressing tumor model. In the transplantable TUBO cell model, DCAd.Neu vaccination was fully effective (data not shown); we, therefore, focused efforts on the transgenic BALB-neuT model. We found that a series of vaccinations before the development of in situ carcinomas in the animals was effective in preventing or delaying the onset of breast tumors. The age at which the mice were vaccinated was critical. Vaccination of 5-to-6-week-old BALB-neuT mice resulted in 66.7% of the mice being tumor-free at 28 weeks, whereas all of the control animals developed tumor. Vaccination of older groups of transgenic mice was less efficacious, and little effect was noted in mice that had already developed tumors, which suggests that other factors such as immunologic escape or tolerance may play a role. DCAd.Neu-vaccinated BALB/c mice rejected repeated challenges with neu-expressing TUBO cells but were not protected when challenged with neu-negative TS/A cells, indicating that DCAd.Neu vaccination generated specific immunity. Vaccinated mice, challenged with neu-expressing TUBO cells, were protected as long as they did not develop a primary breast cancer, indicating immunologic memory against neu antigen. Once a primary breast cancer was detected, growth of the implanted TUBO cells followed. Immunohistochemical studies found that autochthonous tumors and TUBO lesions continued to express neu antigen, which indicated that the relapse likely involved the loss of immunity and not the development of antigen-negative escape mutants.

BALB-neuT mice develop breast cancers in a genetically inherited manner. The fact that these lesions were controlled by vaccination with gene-engineered DCs in a transgenic animal model that displays an early and diffuse overexpression of the neu antigen and relentlessly develops cancers, suggests that a new and rational approach exists to secure immunologic control of HER-2/neu-expressing lesions. Vaccination with DCs that are modified by rAds encoding nonfunctional tumor antigens, such as nonsignaling HER-2/neu, may offer a preventative strategy for patients at risk of metastases after primary treatment of a tumor, or for those at high risk of developing cancer.


   ACKNOWLEDGMENTS
 
We wish to thank Dr. Leon van den Broeke for his help developing dendritic cell culture in our laboratory, Dr. Raya Mandler and Dr. Thomas A. Waldmann for their many helpful discussions, and Poonam Mannan for her tissue immunohistochemistry expertise.


   FOOTNOTES
 
Grant support: Supported by National Cancer Institute Project Z-01SC-010294, and, in part, by a grant from the Italian Association for Cancer Research (G. Forni).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Y. Sakai is a research fellow of Japan Society for the Promotion of Science.

Requests for reprints: John C. Morris, Metabolism Branch, Center for Cancer Research, National Cancer Institute, NIH, Building 10, Room 4N115, 10 Center Drive, Bethesda, MD 20892-1374. Phone: (301) 402-2912; Fax: (301) 402-1001; E-mail: jmorris{at}mail.nih.gov

5 J-M. Park, M. Terabe, Y. Sakai, J. Munasinghe, G. Forni, J. C. Morris, J. A. Berzofsky. Role of CD4+ cells and antibodies in HER-2 vaccine protection against autochthonous mammary carcinomas, manuscript in preparation. Back

Received 11/ 4/03. Revised 7/ 6/04. Accepted 8/30/04.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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