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10.1245/ASO.2006.03.069
Annals of Surgical Oncology 13:1085-1098 (2006)
© 2006 Society of Surgical Oncology
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Original Article

Investigating the Combination of Trastuzumab and HER2/neu Peptide Vaccines for the Treatment of Breast Cancer

Elizabeth A. Mittendorf, MD1,2, Catherine E. Storrer, BS2, Craig D. Shriver, MD1,2, Sathibalan Ponniah, PhD2 and George E. Peoples, MD1,2

1 Clinical Breast Care Project, Department of Surgery, Walter Reed Army Medical Center, 6900 Georgia Avenue NW, Washington, DC 20307-5001
2 National Naval Medical Center, Henry M. Jackson Foundation for the Advancement of Military Medicine, Clinical Breast Care Project Immunology & Research Center, Building 139, 8901 Wisconsin Avenue, Bethesda, Maryland 20889-5600

Correspondence: Address correspondence and reprint requests to: George E. Peoples, MD; E-mail: george.peoples{at}na.amedd.army.mil.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Trastuzumab, an anti-HER2/neu monoclonal antibody, is thought to promote HER2/neu receptor internalization and/or turnover. This study was designed to investigate the kinetics of trastuzumab treatment on tumor cells with varying levels of HER2/neu expression and to determine the effect of trastuzumab on HER2/neu-specific cytotoxic T lymphocyte–mediated lysis.

Methods: Three cell lines with varying levels of HER2/neu expression were incubated with varying doses of trastuzumab at multiple time points. Trastuzumab binding and HER2/neu expression were determined. Peripheral blood mononuclear cells from three HLA-A2+ healthy donors and four E75 peptide–vaccinated patients were stimulated with HER2/neu-derived peptides and tested in standard chromium release cytotoxicity assays with HER2/neu+ tumor cells pretreated with trastuzumab.

Results: Treatment of tumor cells with 10 µg/mL of trastuzumab in an overnight incubation resulted in saturation of cell-surface HER2/neu receptors. At higher doses, trastuzumab staining and HER2/neu expression decreased in a time-dependent manner. Pretreatment of tumor cells with trastuzumab resulted in increases in specific cytotoxicity by peptide-stimulated cytotoxic T lymphocytes from HLA-A2+ donors over untreated cells by an average of 5.6% and 15.3% (P = .0002) for doses of 10 and 50 µg/mL, respectively. In similar experiments involving peripheral blood mononuclear cells obtained from immunized patients, the average specific cytotoxicity for untreated cells was 34.2% ± 1.3% vs. 40.6% ± 2.5% (P = .035) and 40.7% ± 1.6% (P = .0005) for those treated with 10 and 50 µg/mL, respectively.

Conclusions: Our data suggest that pretreatment of breast cancer cells with trastuzumab induces turnover of the HER2/neu protein and enhanced killing by HER2/neu peptide–stimulated CTLs. This increased lysis occurs regardless of the degree of HER2/neu expression and seems more pronounced in vaccinated patients. These findings support further investigation into the use of combination immunotherapy with trastuzumab and HER2/neu peptide–based vaccines.

Key Words: Breast cancer • HER2/neu • Immunotherapy • Trastuzumab • Peptide vaccine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HER2/neu, a member of the epidermal growth factor receptor family of receptor tyrosine kinases, is an essential mediator of cell proliferation, differentiation, and survival. The protein product of the HER2/neu gene is a 185-kilodalton surface membrane protein normally found in a wide variety of tissues, including epithelia of the breast, ovary, endometrium, lung, kidney, and gastrointestinal tract.1,2 Overproduction of this protein product is seen in many cancers. In particular, overexpression of the HER2/neu protein is observed in 20% to 30% of human breast cancers and correlates with more aggressive tumors and a poorer prognosis.3 Because it is expressed in high levels in a percentage of patients with breast cancer, HER2/neu is an appealing target for anticancer treatment, including immunotherapy. In fact, there are numerous reports of experimental models and clinical trials suggesting that HER2/neu can be immunogenic with the ability to induce antibody-specific, cytolytic T cell–specific, and helper T cell–specific responses in cancer patients with HER2/neu-overexpressing tumors and in healthy individuals.4–6

Several studies have confirmed that HER2/neu is an immune-recognized tumor-associated antigen in breast, ovarian, prostate, lung, and colon cancer patients.7–11 In vitro cultures of peripheral blood lymphocytes from these patients have been shown to contain tumor-reactive cytotoxic T lymphocytes (CTLs) that are able to recognize HER2/neu-derived peptides complexed with major histocompatibility complex (MHC) class I molecules and presented on the surface of tumor cells.12,13 Several clinical trials have therefore investigated anticancer vaccines using HER2/neu-derived peptides to induce peptide-specific CTLs for the elicitation of antitumor immune responses.14–19 Although the generation of CTLs has been demonstrated to correlate well with the prevention or eradication of malignant cells in culture and/or experimental tumors in animal models, results from human trials have been less encouraging in that they usually fail to control tumor growth in metastatic disease but may show promise in the prevention of recurrence in high-risk patients. One hypothesis for the failure in most human trials relates to the expression of antigen by the tumor and poor antigen presentation and immunogenicity.20,21 It has been suggested that, for solid tumors that express a particular tumor antigen, an increase either in the density or in the persistence of CTL epitopes on MHC class I may increase the sensitivity of the tumor to CTL lysis.22 In an experimental model using the HER2/neu-expressing ovarian cancer cell line SKOV3-A2, Castilleja et al.22 demonstrated enhanced sensitivity of tumor cells for CTL lysis after treatment with geldanamycin, an agonist that initiates turnover and enhanced degradation of HER2/neu from the cell surface. Down-modulation of HER2/neu resulted in increased presentation of a HER2/neu-derived peptide at the cell surface, as well as increased lysis by peptide-specific CTLs. Theoretically, degradation of the internalized HER2/neu may result in an increase in the number of HER2/neu-derived peptides available for loading on MHC class I molecules, thus subsequently leading to enhanced tumor lysis by HER2/neu-specific CTLs.

A second immunotherapeutic strategy used in the treatment of breast cancer involves the use of an antibody directed against HER2/neu-overexpressing tumors. Trastuzumab is a fully humanized anti-HER2/neu monoclonal antibody (MAb) that binds to the extracellular domain of the HER2/neu protein.23 It has been approved for clinical use in patients with metastatic disease from HER2/neu-overexpressing tumors, and in these patients, it has been shown to improve survival.24,25 Trastuzumab has several proposed mechanisms of action, including receptor downmodulation. One possible mode of action is that HER2/neu receptor downmodulation may inhibit HER2/neu-mediated signal transduction and tumor cell growth.26–28 Downmodulation may also enhance tumor lysis by promoting processing of the HER2/neu protein. In an in vitro model, Meyer zum Buschenfelde et al.29 demonstrated that trastuzumab sensitized HER2/neu-overexpressing tumor cells to lysis by HER2/neu-specific CTLs. They proposed that the trastuzumab-induced internalization and degradation was accompanied by the enhanced generation of class I–bound HER2/neu-derived peptides, with subsequent increased susceptibility of HER2/neu-overexpressing tumor cells to CTL-mediated lysis. Additional mechanisms of action for trastuzumab involving the immune system have been proposed. In a murine model, Clynes et al.30 have shown that, via its immunoglobulin (Ig)G1 Fc domain, trastuzumab enhances antibody-dependent cellular cytotoxicity, thus contributing substantially to its antitumor activity. A recent clinical report evaluating patients with HER2/neu+ breast cancer treated with neoadjuvant trastuzumab provided additional evidence that antibody-dependent cellular cytotoxicity is an important mechanism of action.31

In this study, we conducted preclinical studies to investigate a combination-therapy strategy of trastuzumab treatment and HER2/neu peptide vaccination. To assess the potential clinical benefit of using combination therapy, we studied the kinetics of trastuzumab treatment on breast cancer cells with varying degrees of HER2/neu expression. We used peripheral blood mononuclear cells (PBMCs) stimulated with E75 and GP2, two immunogenic, HER2/neu-derived vaccine peptides, to determine the effect of optimal trastuzumab treatment on peptide-specific cytotoxicity. Furthermore, we validated the combination therapy concept by using PBMCs from E75-vaccinated patients to demonstrate enhanced killing of trastuzumab-treated breast cancer cell lines even with low levels of HER2/neu expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient Characteristics and Clinical Trial
A clinical trial evaluating the HER2/neu peptide (E75) vaccine is being conducted under an Investigational New Drug application (No. 9187) approved by the Food and Drug Administration. The study’s principal aim is to determine whether the HER2/neu peptide vaccine, E75, is useful as a preventive vaccine in breast cancer patients—particularly those who are at high risk of tumor recurrence. Patients were accrued to the study through the Walter Reed Army Medical Center’s Comprehensive Breast Center after approval of the protocol by the institution’s Department of Clinical Investigation. All patients had histologically confirmed breast cancer that expressed HER2/neu by standard immunohistochemistry, as well as nodal metastases. Before their enrollment, patients completed a standard course of surgery, chemotherapy, and radiotherapy, as indicated. At the time of enrollment, patients were HLA-typed to determine their HLA-A2 because E75 binds this specific allele, which is found in approximately 40% to 50% of the general population.32 HLA-A2+ patients were vaccinated, whereas HLA-A2– patients were followed up prospectively as matched controls for clinical recurrence.

In the escalation trial, vaccinated patients received a series of intradermal inoculations consisting of 100, 500, or 1000 µg of E75 peptide with 250 µg of granulocyte-macrophage colony-stimulating factor (GM-CSF; Berlex, Seattle, WA) monthly for 6 months. The 1-mL inoculation was split and given intradermally at two sites within 5 cm of each other. The monthly inoculations were all administered into the same extremity. Blood samples were drawn from the vaccinated patients before each inoculation and at 1 and 6 months after completion of the 6-month vaccination series. Healthy donor blood samples were obtained commercially as leukopheresis products from Baltimore Rh Typing (BRT) Laboratories (Baltimore, MD).

E75 Vaccine Peptide
The E75 peptide (HER2/neu, 369-377, KIFGS-LAFL) was produced commercially in good manufacturing practices grade by Multiple Peptide Systems, Inc. (San Diego, CA). The purity of the peptide was verified by high-performance liquid chromatography and mass spectrometry, and the amino acid content was determined by amino acid analysis. The peptide was purified to >95%. Sterility and general safety testing was performed by the manufacturer.

Tumor Cell Lines
The HER2/neu-expressing breast cancer cell lines MCF-7 (HLA-A2+, HER2/neu+) and AU565 (HLA-A2–, HER2/neu+) and the ovarian cancer cell line SKOV3 (HLA-A2–, HER2/neu+) were obtained from the American Type Culture Collection (Manassas, VA). AU565 and SKOV3 cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (Gemini BioProducts, Woodland Hills, CA), 1% glutamine, and 1% penicillin-streptomycin (GIBCO-INVITROGEN, Carlsbad, CA). MCF-7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and penicillin-streptomycin, glutamine, and insulin (Sigma Chemicals, St. Louis, MO). The HLA-A2–transfected variant of the SKOV3 parental line, SKOV3-A2 (HLA-A2+, HER2/neu+), was a kind gift from Dr. Constantin Ioannides and was cultured in culture medium in the presence of .2 mg/mL of Geneticin (GIBCO-INVITROGEN).

Monoclonal Antibodies
The humanized anti-HER2/neu antibody trastuzumab (Herceptin; Genentech, San Francisco, CA) was obtained from the Walter Reed Army Medical Center Oncology Pharmacy. The phycoerythrin (PE)-conjugated HER2/neu MAb (clone Neu 24.7) from BD-Pharmingen (San Diego, CA) was used for the surface staining of HER2/neu in both untreated cells and cells that had been incubated with trastuzumab.

Detection of HER2/neu Levels on Tumor Cells
The levels of HER2/neu expressed on the cell surface of the two breast cancer cell lines (MCF-7 and AU565) and the ovarian cancer cell line (SKOV3) were determined by direct staining of 5 x 105 cells per 100 µL in Pharmingen Stain Buffer (BD-Pharmingen) with PE-conjugated anti-HER2/neu MAb for 30 minutes at 4°C. The cells were washed and analyzed by flow cytometry performed by using a FACSCalibur instrument (Becton Dickinson, San Jose, CA). Data were collected on the total cell population, and subsequent analysis was performed by using CellQuest software, version 3.3 (Becton Dickinson). Levels of HER2/neu expression were measured as the mean fluorescence intensity (MFI) of staining obtained for each of the cell populations. Similar experiments were also performed to measure levels of HER2/neu by using an indirect antibody staining method with purified anti-HER2/neu MAb (c-neu, Ab-5; EMD Biosciences Inc., San Diego, CA) and PE- or fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibodies (Caltag Laboratories-INVITROGEN, Carlsbad, CA; data not shown). These experiments gave a pattern of MFI results similar to that shown in Fig. 1Go, which depicts the staining method used in all subsequent investigations.


Figure 1
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  		FIG. 1. Varying levels of HER2/neu expression on tumor cell lines. HER2/neu expression was determined on MCF-7 (breast), SKOV3 (ovarian), and AU565 (breast) tumor cell lines by flow cytometric analysis of staining with a phycoerythrin-conjugated anti-HER2 monoclonal antibody. Results are expressed as mean fluorescence intensities and are representative of replicated experiments.

 
Effect of Increasing Doses of Trastuzumab on the Expression of HER2/neu Levels on Tumor Cell Lines
The effect of increasing doses of trastuzumab on the expression levels of HER2/neu on the cell surface of the three tumor cell lines studied previously was determined as follows. Aliquots of 1 x 106 cells in .5 mL of the respective culture medium were incubated with 0, 1, 10, and 50 µg/mL of trastuzumab in 15-mL tubes overnight in a humidified incubator at 37°C in 5% carbon dioxide. After incubation, the cells were washed, and each sample was split into two aliquots of 100 µL each in Pharmingen Stain Buffer. One of the aliquots was stained directly with PE-conjugated HER2/neu MAb. The second aliquot was subjected to indirect staining with F(ab)2-goat anti-human IgG (Fc)-FITC (Biosource Intl., Camarillo, CA) to detect the presence of bound trastuzumab in the cell populations. All staining procedures with the antibodies were performed at 4°C. The cell populations were then analyzed by flow cytometry, as described in the preceding section, and the data were recorded as MFI values obtained for each sample. For all of the cell lines, respective samples of the cells from all of the tubes were also directly stained with PE-conjugated mouse isotype control antibody or incubated with control human IgG reagent and stained with the secondary antibody. No differences in background levels of antibody staining were observed for any of the cell populations studied.

Determination of the Kinetics for the Turnover of Bound Trastuzumab From the Cell Surface in Tumor Cell Lines
The turnover kinetics of cell surface–bound trastuzumab over a period of time in cancer cells was studied by incubating aliquots of 3 x 106 tumor cells in .5 mL of culture medium with 10 and 50 µg/mL concentrations of trastuzumab at 4°C for 1 hour. After the incubation period, the cells were washed and resuspended in 3 mL of culture medium, which was then split into six aliquots of 5 x 105 cells per .5 mL in 15-mL tubes and placed in a humidified incubator at 37°C in 5% carbon dioxide. One of the tubes was removed immediately, and the cells were washed and resuspended in 200 µL of Pharmingen Stain Buffer and split into two 100-µL aliquots. These aliquots were stained with either PE-conjugated HER2/neu MAb or F(ab)2-goat anti-human IgG (Fc)-FITC antibodies for 15 minutes at 4°C. After incubation, the cells were washed and analyzed by flow cytometry as described in the preceding sections to obtain the zero time point MFI reading for the levels of bound trastuzumab and HER2/neu levels. Subsequently, at the 1-, 4-, 6-, 8-, and 24-hour time points, an aliquot was removed from the incubator and treated in an manner identical to that for the sample from the initial time point to obtain MFI measurements of the HER2/neu and bound trastuzumab levels by flow cytometry.

PBMC Isolation
Patients receiving the vaccine had 40 mL of peripheral blood drawn into Vacutainer CPT Tubes (Becton Dickinson) before receiving the initial vaccination and also before the administration of each subsequent vaccine dose. The tubes were centrifuged according to the manufacturer’s instructions for the isolation of PBMC populations. The cells were washed in Hanks’ buffered salt solution and resuspended in complete culture medium consisting of Iscove’s modified Dulbecco’s medium containing 10% human AB serum (Gemini Bioproducts, Calabasis, CA) supplemented with 1 x penicillin/L-glutamine/streptomycin, 1 x sodium pyruvate, and 1x nonessential amino acids (Life Technologies, Rockville, MD). Aliquots of freshly isolated PBMCs were also cryopreserved in 90% fetal calf serum and 10% di-methyl sulfoxide in liquid nitrogen for future testing. Leukopheresis blood products provided by healthy donors and commercially available products from BRT laboratories (Baltimore) were used for the preparation of PBMCs by density-gradient centrifugation with the lymphocyte separation medium Lymphoprep (Accurate Chemical & Scientific Corp., Westbury, NY).

In Vitro Stimulation of PBMC Cultures for Cytotoxicity Assay
The PBMC preparations resuspended in complete culture medium were used for the preparation of dendritic cells (DCs) and purified CD8+ T cells and also as a source of lymphocytes for in vitro stimulation with the E75 and GP2 peptides. Highly pure populations of CD8 T cells were isolated by a magnetic bead separation technique with CD4-, CD14-, and CD19-coated Dynal beads (DYNAL-INVITROGEN, Carlsbad, CA). Highly enriched populations of CD14 monocytes were prepared by magnetic bead depletion of PBMCs that had been incubated with CD2-, CD19-, and CD8-coated beads from Dynal. The CD14 cell population was cultured in Macrophage Serum Free Medium (GIBCO-INVITROGEN, Carlsbad, CA) with 100 µg/mL of GM-CSF and 50 µg/mL of interleukin 4 per milliliter (R&D Systems, Minneapolis, MN) to obtain monocyte-derived DC populations. Recombinant human tumor necrosis factor {alpha} (R&D) was added at 30 ng/mL on day 3 to induce maturation. DCs were then harvested at 6 days, incubated with E75, GP2, or no peptide for 2 hours, and then used to stimulate the purified preparations of CD8 T cells. Cultures were set up in culture medium with 10 ng/mL of interleukin 7, and this was followed by the addition of 25 ng/mL of interleukin 2 on the second day. All cultures were maintained in a humidified incubator at 37°C in 5% carbon dioxide.

Chromium 51 Cytotoxicity Assay
Peptide-specific cytotoxicity in the CD8 T-cell cultures stimulated with peptide-pulsed DCs and in vitro–stimulated PBMC cultures was determined by standard 4-hour chromium release assay. Briefly, targets were labeled with 100 to 150 µCi of sodium chromate (Perkin-Elmer, Boston, MA) for 1.5 hours at 37°C and then were washed twice and plated at 2500 cells per well in 100 µL in 96-well U-bottom plates (Becton Dickinson). EDectors were added at an effector:target (E:T) ratio of 10:1 to 20:1 in 100 µL per well. After 4 hours of incubation, 100 µL of culture supernatant was collected, and radionuclide release was measured on a Microbeta Trilux counter (Perkin-Elmer). All determinants were performed in triplicate. Results are expressed as the percentage-specific lysis, as determined by the following equation:


Formula

where cpm indicates counts per minute. The target cells used in the cytotoxicity assays were MCF-7 cells (HLA-A2+, HER2/neu+) and SKOV3-A2 (HLA-A2+, HER2/neu+) cells. For the experiments to investigate the effects of trastuzumab treatment, the target cells were incubated in the absence or presence of 10 and 50 µg/mL of trastuzumab overnight at 37°C, after which they were washed and prepared for use in the cytotoxicity assay in the usual manner.

Statistical Analysis
Statistical analysis was performed with the SPSS program (SPSS Inc., Chicago, IL). Continuous data were compared by using the paired two-tailed Student’s t-test. Probabilities <.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of HER2/neu Expression on Tumor Cell Lines
We first sought to confirm the levels of HER2/neu expressed in various breast and ovarian cancer cell lines. MCF-7 (HLA-A2+, HER2/neu+), SKOV3 (HLA-A2–, HER2/neu+), and AU565 (HLA-A2–, HER2/neu+) cells were stained with PE-conjugated anti-HER2 MAb, after which flow cytometric analysis was used to determine the MFI of bound antibody. As shown in Fig. 1Go, MCF-7 cells were found to have a low level of expression of HER2/neu, whereas SKOV3 and AU565 were observed to have moderate to high levels of HER2/neu expression on the cell surface. These experiments were repeated multiple times with similar results. Current clinical guidelines applied to breast cancer patients require strong HER2/neu expression on the cell membrane in >10% of the tumor cells for the patient to be eligible for treatment with trastuzumab.33

Effect of Treatment With Trastuzumab on HER2/neu Expression and Trastuzumab Staining After Overnight Incubation
Having shown that varying degrees of HER2/neu expression were present on the three different cell lines, we next investigated the dose of antibody required to achieve maximal binding of trastuzumab to HER2/neu surface receptors. Various doses of trastuzumab, including 0, 1, 10, and 50 µg/mL, were tested. Cells were incubated with the designated dose of trastuzumab overnight, after which they were washed then divided into two aliquots. One of these was stained with fluorescein-conjugated anti-human IgG antibody to determine the extent of trastuzumab binding to HER2/neu receptors on the cell surface. The second aliquot was stained with anti-HER2 MAb to determine the effect of trastuzumab treatment on HER2/neu expression. These experiments were performed multiple times; Fig. 2Go depicts representative results for all three of the cell lines tested. As seen in the figure, regardless of the extent of HER2/neu expression, a 10 µg/mL dose of trastuzumab resulted in maximal binding of the antibody to HER2/neu. This suggests that 10 µg/mL is a saturating dose of antibody for the range of HER2/neu levels expressed on these cells. At the higher dose of 50 µg/mL, all cell lines show a reduction in trastuzumab staining; this suggests that these saturated HER2/neu receptors have begun to undergo internalization.


Figure 2
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  		FIG. 2. Trastuzumab saturation and effect on HER2/neu expression in tumor cell lines. The tumor cell lines (A) MCF-7, (B) SKOV3, and (C) AU565, which express varying levels of HER2/neu, were treated with increasing concentrations of trastuzumab overnight. The saturating concentration of trastuzumab was determined by staining the monoclonal antibody (MAb) with a fluorescence-conjugated anti-human immunoglobulin G secondary antibody. Simultaneously, the effect of the trastuzumab binding on HER2/neu expression was determined by staining the tumor cell lines with a phycoerythrin-conjugated anti-HER2 MAb. Results are expressed as mean fluorescence intensities.

 
As shown in Fig. 2Go, the staining pattern obtained for all three cell lines showed a reduction in cell-surface HER2/neu expression, with doses exceeding 10 µg/mL in overnight experiments. The extent of reduction in cell-surface HER2/neu expression varied with the degree of baseline expression. MCF-7 cells, which had the lowest levels of HER2/neu expression, showed a marginal decrease of 8%, compared with decreases in SKOV3 and AU565 cells of 19.2% (P < .03) and 50.5% (P < .01), respectively. This decrease in HER2/neu expression corresponded with the decline in trastuzumab staining, which supports the concept of receptor internalization and modulation of HER2/neu cell-surface expression being induced in these cells.

Timing of Trastuzumab Effect on HER2/neu-Expressing Tumor Cells
After identifying a dose of trastuzumab ranging from 10 to 50 µg/mL as being sufficient to cause saturation of cell-surface HER2/neu receptors, we sought to determine the time course kinetics and the staining patterns associated with the binding of trastuzumab on HER2/neu-expressing tumor cells. Once again, all three of the cell lines (MCF-7, SKOV3, and AU565) were used for this set of experiments. Cells were dosed with trastuzumab at either 10 or 50 µg/mL and were incubated for varying lengths of time, including 2, 4, 6, and 8 hours, as well as an overnight period. At the designated time point, incubated cells were divided into two aliquots. The first was stained with anti-human IgG antibodies to identify trastuzumab staining, and the second was stained with anti-HER2 MAb to identify HER2/neu expression. Experiments were repeated three times, and representative results are shown in Fig. 3Go. As seen in the figure, regardless of the cell type or dose, all experiments showed a decrease in levels of trastuzumab staining and HER2/neu expression at the 24-hour time point. This provides further evidence that by 24 hours of treatment, trastuzumab has begun to promote HER2/neu receptor internalization. At the earlier time points before the overnight incubation, the data show an overall effect of apparent stabilization of HER2/neu on the cell surface. This is consistent with the well-accepted concept of antibody binding causing initial antigen (i.e., HER2/neu receptor) stabilization, which is then slowly followed by the clustering or capping phenomena that eventually lead to internalization. In the case of the MAb trastuzumab, this stabilization period seems to occur for at least the initial 8 hours, after which receptor internalization seems to be initiated between 12 and 24 hours. Finally, HER2/neu expression begins to decrease at 24 hours in general in these three cell lines tested.


Figure 3
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  		FIG. 3. Time-dependent effect of trastuzumab on the HER2/neu expression on tumor cell lines. The tumor cell lines (A and B) MCF-7, (C and D) SKOV3, and (E and F) AU565, which express varying levels of HER2/neu, were treated with either 10 or 50 µg/mL of trastuzumab. The levels of trastuzumab binding and HER2/neu expression were determined over time. Trastuzumab binding was determined by staining the monoclonal antibody (MAb) with a fluorescence-conjugated anti-human immunoglobulin G secondary antibody. Simultaneously, the effect of the trastuzumab binding on HER2/neu expression was determined by staining the tumor cell lines with a phycoerythrin-conjugated anti-HER2 MAb. Results are expressed as mean fluorescence intensities.

 
Cytotoxicity Experiments Involving Donor PBMCs and Trastuzumab-Treated Tumor Target Cells
Having identified the appropriate dose (10–50 µg/mL) and period of incubation (overnight) at which trastuzumab shows evidence of promoting receptor internalization, we next performed cytotoxicity experiments to determine the effects of trastuzumab treatment on the susceptibility to immune-mediated responses such as lysis of tumor target cells by CTLs. Initial experiments involved the stimulation of purified CD8+ T cells from three HLA-A2+ healthy donors that were stimulated with DCs loaded with the HER2/neu-derived peptide E75. These peptide-stimulated effector cells were then tested in cytotoxicity assays at a ratio of 20:1 with HLA-A2+, HER2/neu+ MCF-7 tumor cells that had been incubated overnight with optimal doses of trastuzumab (0, 10, or 50 µg/mL). These experiments were performed in duplicate, and as shown in Fig. 4AGo, we observed a general increase in the lysis of the tumor cells treated with trastuzumab. Taken together, there was an increase in the percentage change in specific cytotoxicity that ranged from 5% to 12.3% in experiments involving tumor targets treated with 10 µg/mL and from 6.3% to 24.9% in experiments with tumor targets treated with 50 µg/mL of trastuzumab. Combining the data from these experiments, we determined the average specific cytotoxicity associated with each of the three doses of trastuzumab. As depicted in Fig. 4BGo, the average specific cytotoxicity was 47.4% ± 5.0%, 48.3% ± 7.1%, and 54.1% ± 6.4% for doses of 0, 10, and 50 µg/mL, respectively. Although this 15% increase in cytotoxicity of the target cells treated with 50 µg/mL of trastuzumab is modest, it did reach statistical significance (P < .015; Fig. 4BGo). This is consistent with the dosing data depicted previously (Figs. 2Go and 3Go) that suggested that, at a dose of 50 µg/mL, trastuzumab had saturated the cell-surface HER2/neu receptors and promoted receptor internalization, degradation, and peptide generation.


Figure 4
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  		FIG. 4. HER2/neu peptide–stimulated healthy donor purified CD8 T cells recognize and lyse trastuzumab-treated HER2/neu-expressing tumor cell lines. Healthy donor (BRT samples no.) purified CD8 T cells were stimulated with autologous dendritic cells and one of the HER2/neu peptides—(A and B) E75 or (C and D) GP2—and were used in standard 4-hour chromium (51Cr) release assays with trastuzumab-treated MCF-7 (0, 10, or 50 µg/mL overnight) at an effector:target ratio of 20:1. Results are expressed as percentage-specific lysis for individual experiments (A and C), and in combined (n = 6) experiments (B and D) they are presented as the mean ± SE percentage-specific lysis. Asterisks represent statistical differences in the level of cytotoxicity between treated and untreated targets.

 
Similar experiments were performed in duplicate with purified CD8+ T cells from the same three HLA-A2+ healthy donors stimulated with DCs loaded with GP2, a second immunogenic HER2/neu-derived peptide. We have confirmed that GP2 is an immunologically relevant peptide and the subdominant epitope of the HER2/neu protein.13 As shown in Fig. 4CGo, the results from these experiments once again demonstrated that there is a general increase associated with the cytotoxicity of tumor cells treated with trastuzumab. Increases in cytotoxicity were again greatest in experiments where the target cells had been treated with 50 µg/mL of trastuzumab, with increases in specific cytotoxicity ranging from 9.8% to 31.0%. By combining the data from these experiments to determine the average specific cytotoxicity at each of the three doses, we again found a significant overall increase in killing of 20% when the cells were treated with 50 µg/mL (P < .013; Fig. 4DGo).

Cytotoxicity Experiments Involving Patients Enrolled in an E75 Peptide Vaccine Trial and Trastuzumab-Treated Tumor Target Cells
After demonstrating enhanced killing of trastuzumab-treated tumor cells by purified CD8+ T cells from HLA-A2+ healthy donors stimulated with peptide-loaded DCs, we next performed cytotoxicity experiments by using PBMCs obtained from patients enrolled in our ongoing E75 peptide vaccine trial. Our trial involved the administration of a simple vaccine formulation consisting of E75 peptide mixed with GM-CSF in a preventive strategy. All patients had HER2/neu+ tumors and node-positive disease. Because the E75 peptide is HLA-A2 restricted, HLA-A2+ patients were vaccinated, whereas HLA-A2– patients served as controls. All patients were rendered free of disease by conventional therapy; however, given their clinico-pathologic features, all were at high risk for recurrence. Included in the goals of this initial vaccine trial was documentation of the ability to generate E75-specific CD8+ T cells and to assess for effectiveness in preventing recurrence of disease in these patients.

The initial experiments involved PBMCs from immunized patients that were further incubated with the HER2/neu peptides E75 and GP2 ex vivo and then tested in cytotoxicity assays with untreated HLA-A2+, HER2/neu+ MCF-7 cells and cells that had been treated overnight with trastuzumab at either 10 or 50 µg/mL. Cytotoxicity assays were performed at 10:1 and 20:1 ratios. Representative results from one patient are shown in Fig. 5Go, and regardless of the E:T ratio used, treatment of the target cells with trastuzumab enhanced lytic activity. This increase in percentage-specific cytotoxicity was most notable, however, at an E:T ratio of 20:1 and occurred with both the 10 and 50 µg/mL doses.


Figure 5
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  		FIG. 5. Peripheral blood mononuclear cells (PBMCs) from HER2/neu peptide (E75)-vaccinated patients efficiently recognize and lyse trastuzumab-treated HER2/neu-expressing tumor cell lines. PBMCs from patients who were serially inoculated with the E75/granulocyte-macrophage colony-stimulating factor vaccine were used in standard 4-hour chromium-release (51Cr) assays after a single round of ex vivo peptide stimulation with either E75 or GP2 against trastuzumab-treated (0, 10, or 50 µg/mL overnight) MCF-7 (A–C) or SKOV3-A2 (D, E). In (A–C), a representative patient’s results are expressed as percentage-specific lysis at varying effector:target ratios. In (D), results are expressed as percentage-specific lysis for individual patients’ PBMCs stimulated with the designated HER2/neu peptide. In (E), cumulative (n = 7) results are expressed as the mean ± SE percentage-specific lysis. The asterisk represents a statistical difference in the level of cytotoxicity between treated and untreated targets.

 
We next performed cytotoxicity assays by using peptide-stimulated PBMCs from vaccinated patients versus the ovarian cancer cell line SKOV3 transfected with HLA-A2 (SKOV3-A2), an HLA-A2+, HER2/neu+ cell line that expresses higher levels of HER2/neu than MCF-7. As shown in Fig. 5DGo, there was again a general increase in cytotoxicity for cells treated with trastuzumab. In experiments performed with these SKOV3-A2 cells treated with a 10 µg/mL dose of trastuzumab, the average percentage change in specific cytotoxicity was 21.0% ± 2.1%, with a range of 0% to 39.7%. In experiments performed on cells treated with trastuzumab 50 µg/mL, all patients showed increased lytic activity, with an average percentage change in specific cytotoxicity of 19.2% ± 2.8% and a range of 8.3% to 28.5%.

Combining the data from these experiments, cells not treated with trastuzumab had an average specific cytotoxicity of 34.2% ± 1.3%, versus 40.6% ± 2.5% (P < .035) and 40.7% ± 1.6% (P < .0005) for cells treated with 10 and 50 µg/mL, respectively. It is interesting to note that, in contrast to experiments involving healthy donor HLA-A2+ peptide–stimulated effector cells, in which a dose of trastuzumab of 50 µg/mL was required to show a significant increase in cytotoxicity, in experiments involving PBMCs from vaccinated patients, a significant increase in specific cytotoxicity was achieved at the lower dose of 10 µg/mL of trastuzumab. We attribute this increased reactivity to the presence of larger numbers of E75-and GP2-specific CD8+ T cells in the vaccinated patients.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Combination immunotherapy using the MAb trastuzumab and vaccination with HER2/neu-derived peptides would be a novel approach to the treatment of HER2/neu-expressing breast cancer. To assess the clinical utility of this strategy, we investigated the kinetics of trastuzumab therapy as well as the effect of optimal trastuzumab treatment on peptide-specific cytotoxicity. Specifically, we have shown that trastuzumab saturates cell-surface HER2/neu receptors and promotes internalization in both a dose- and time-dependent fashion. This internalization increases the sensitivity of HER2/neu-expressing tumor cells to lysis by PBMCs stimulated with the HER2/neu-derived vaccine peptides E75 and GP2. Furthermore, patients who have been vaccinated with E75 have PBMCs that are even more sensitive to the effects of trastuzumab on breast cancer cells. Of interest, the increased susceptibility of trastuzumab-treated cells to lysis by HER2/neu peptide-stimulated PBMCs occurs even in tumor cells with low HER2/neu expression (i.e., MCF-7). These data suggest that combination therapy with trastuzumab and HER2/neu-derived peptide vaccines has potential clinical utility.

Other investigators have suggested that receptor downmodulation plays an important role in the mechanism of action of MAbs such as trastuzumab. Drebin et al.26 identified a loss of both cell-surface and total cellular HER2/neu when HER2/neu-transformed cells were exposed to inhibitory anti-HER2/neu antibodies. Hurwitz et al.27 showed that this antibody-induced downmodulation was a result of accelerated endocytosis. Collectively, the work of these investigators and others suggested that, after binding trastuzumab, the HER2/neu receptor is internalized and degraded.26,27 In this study, we more thoroughly investigated the kinetics of trastuzumab. Our data suggest that there is a saturating dose of trastuzumab at which staining for the antibody, and, therefore, for trastuzumab binding to cell-surface HER2/neu receptors, reaches a maximum. At higher doses, trastuzumab staining decreases, thus suggesting ongoing trastuzumab-induced receptor internalization. An alternative explanation, such as disassociation of the trastuzumab from the receptor, cannot be completely ruled out; however, maintenance of saturating concentrations of the MAb in the culture media would make this unlikely. Additionally, our concurrent staining of these cells with a HER2/neu MAb also showed downregulation of the cell-surface HER2/neu expression on the same cells. Taken together, these data support the concept of trastuzumab-induced receptor internalization.

Enhanced HER2/neu receptor internalization has been thought to produce trastuzumab’s clinical effect by inhibiting HER2/neu-mediated signal transduction and tumor cell growth. 26,27 We focused on a second potential effect of HER2/neu receptor internalization involving enhanced tumor lysis by HER2/neu-specific CTLs. Our data provide immunological evidence to associate the observed internalization of HER2/neu with increased specific immunogenicity of the trastuzumab-treated cells. This was demonstrated by comparing the susceptibility of trastuzumab-treated and trastuzumab-untreated HER2/neu+ tumor cells to CD8+ T cell–mediated lysis by two different HER2/neu peptide–specific (E75 and GP2) lymphocyte populations. E75- and GP2-stimulated cells from both healthy and breast cancer patients showed significantly enhanced lysis of trastuzumab-treated breast and ovarian cancer cells.

This enhanced lytic activity is likely attributable to increased HER2/neu receptor internalization and turnover, which may even occur without a substantial decrease in cell-surface expression of HER2/neu, depending on the rate of turnover. Theoretically, receptor internalization would lead to enhanced proteolytic processing and degradation of the protein, thus leading to a subsequent increase in the number of HER2/neu-derived peptides available to be complexed with MHC class I molecules. This would lead to increased lysis by cytotoxic T cells recognizing the peptide complexed to the MHC class I molecule on the cell surface. This theory is supported by the finding that trastuzumab enhances ubiquitination of HER2/neu. Proteins tagged to ubiquitin are targeted to the proteasome machinery that cleaves proteins such as HER2/neu into small peptides. These peptides are then processed through the endoplasmic reticulum, loaded on MHC class I molecules, and then represented on the cell surface, where they can be recognized by peptide-specific CTLs.28 A recent study by Kono et al.18 using CTLs generated from tumor-associated lymphocytes in the ascitic fluid of a patient with a HER2/neu-overexpressing gastric cancer demonstrated similar results: trastuzumab treatment sensitized HER2/neu-overexpressing tumors to CTL lysis. They investigated the mechanism of this enhanced lytic activity and identified enhanced class I–restricted presentation of endogenous HER2/neu antigen via the proteasome step.

In another study, Meyer zum Buschenfelde et al.29 showed that trastuzumab sensitized HER2/neu+ SKOV3-A2 tumor cells to lysis by HER2-specific CTLs in vitro. These investigators, however, used only a single dose and duration of trastuzumab treatment on a single tumor cell line with moderate to high levels of HER2/neu expression. In our study, we confirmed the preliminary results of these other investigators, and, more importantly, we extended these findings by showing increased lysis by HER2/neu-specific CTLs against MCF-7 breast cancer cells, which have low levels of HER2/neu expression. Patients with overexpressing HER2/neu+ tumors have a more rapid protein turnover, which leads to a higher number of peptides complexed with MHC class I molecules on the cell surface.34 In contrast, patients with low levels of HER2/neu expression have fewer HER2/neu-derived peptides/MHC class I complexes on the cell surface as a result of less cell-surface HER2/neu protein and slower internalization and processing. Therefore, the ability to promote receptor internalization, HER2/neu protein degradation, and subsequent cell-surface peptide presentation by treatment with trastuzumab may be most beneficial in patients with lower levels of HER2/neu expression.

We have also shown that PBMC samples from breast cancer patients who are receiving the HER2/neu E75 peptide vaccine in our ongoing clinical trial show increased recognition and lysis of both breast and ovarian cancer cells treated with trastuzumab. This represents the first direct clinical evidence supporting the potential benefits that may be derived from our proposed combination immunotherapy.

It is interesting to note that we observed a difference in cytotoxicity at different doses of trastuzumab when we compared killing by HER2/neu peptide–stimulated effector cells from HLA-A2+ healthy donors with killing by PBMCs generated from patients treated for HER2/neu+, node-positive breast cancer and subsequently administered our E75 peptide vaccine. For experiments involving healthy donors, we did not observe a significant increase in lytic activity when the tumor target cells were treated overnight with the saturating dose of trastuzumab. However, a significant increase in tumor target killing was seen when cells were treated overnight with higher doses. In contrast, when cytotoxicity assays were performed by using peptide-stimulated PBMCs generated from patients who had received our E75 peptide vaccine, there was a significant increase in tumor target killing when cells were treated with either the saturating dose or the higher dose. We have shown an increase in the number of E75-specific CD8+ T cells in our vaccinated patients by direct staining with the HLA-A2:Ig dimer molecule.35 Therefore, we attribute this difference in tumor target killing to a higher precursor frequency or number of T cells that can recognize HER2/neu-derived peptides in our vaccinated patients as compared with a lower precursor frequency of naive T cells present in the PBMCs of healthy donors.

Therefore, the results of our study suggest that patients may benefit from combination immunotherapy. E75 and GP2 are already in clinical trials as cancer vaccines. Treatment of patients with the MAb trastuzumab may increase the amount of peptide complexed and presented on MHC class I molecules that can be recognized by CTLs. Combination immunotherapy may, therefore, result in increased lysis of tumor targets by both vaccine-induced increases in the number of precursor T cells and MAb-induced increases in the number of targets displaying peptide in conjunction with MHC class I molecules. The combination may be more effective than treatment with either agent alone.

If proven effective, this combination immunotherapy may have significant treatment implications for HER2/neu+ breast cancer. Current clinical guidelines require strong HER2/neu expression on the cell membrane in >10% of tumor cells for patients to be eligible for treatment with trastuzumab.33 Preclinical work with trastuzumab suggested that its effects were seen only in cell lines that expressed the highest levels of HER2/neu. In contrast, we have shown that trastuzumab is effective in enhancing the cytolytic activity of HER2/neu peptide-specific effector cells against HER2/neu tumor targets regardless of the degree of HER2/neu expression. Furthermore, we have provided direct evidence that even breast cancer cells expressing low levels of HER2/neu (MCF-7 cells) can be significantly sensitized by trastuzumab treatment to E75- and GP2-stimulated lymphocytes from patients currently receiving the E75 peptide vaccine. This finding may dramatically increase the clinical utility and potential therapeutic benefit of trastuzumab when used in combination with HER2/neu peptide vaccines.


   ACKNOWLEDGMENTS
 
Supported by funds from the Department of Defense to the Henry M. Jackson Foundation for the Advancement of Military Medicine (Rockville, MD) for the Clinical Breast Care Project, by the United States Army Medical Research and Materiel Command, and by the Department of Clinical Investigation at the Walter Reed Army Medical Center. The authors thank Diane Papay and Stacy O’Neill of the Clinical Breast Care Project, who provided excellent patient care and administration of the clinical trial. We also thank the staff of the Clinical Breast Care Project Immunology & Research Center for their clinical and administrative assistance.


   FOOTNOTES
 
The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense.

Received for publication March 10, 2005. Accepted for publication January 18, 2006.


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 RESULTS
 DISCUSSION
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