ARTICLE

Response of Established Human Breast Tumors to Vaccination with Mammaglobin-A cDNA

Kishore Narayanan, Andrés Jaramillo, Nicholas D. Benshoff, Lacey G. Campbell, Timothy P. Fleming, Jill R. Dietz, T. Mohanakumar

Affiliations of authors: Departments of Surgery (KN, AJ, NDB, LGC, TPF, JRD, TM) and Pathology and Immunology (TM), Washington University School of Medicine, St. Louis, MO

Correspondence to: T. Mohanakumar, PhD, Department of Surgery, Washington University School of Medicine, Box 8109-3328, CSRB, 660 S. Euclid Ave., St. Louis, MO, 63110 (e-mail: kumart{at}wustl.edu)


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Background: A novel breast cancer–associated antigen, mammaglobin-A, is expressed in 80% of primary breast tumors. The characterization of immune responses against this highly expressed breast cancer–specific antigen would be of value in the development of new therapeutic strategies for breast cancer. Methods: We developed an in vivo model using human leukocyte antigen-A*0201/human CD8+ (HLA-A2+/hCD8+) double-transgenic mice to define the epitopes and to study the level of protection acquired by mammaglobin-A cDNA vaccination toward mammaglobin-A+/HLA-A2+ breast cancer cell lines. Mammaglobin-A epitopes were identified using an HLA class I peptide binding prediction computer program, and their activity was verified using gamma interferon ELISPOT and cytotoxicity assays. Results: We identified seven mammaglobin-A–derived candidate epitopes that bind the HLA-A*0201 molecule (Mam-A2.1–7). CD8+ cytotoxic T lymphocytes (CTLs) from HLA-A2+/hCD8+ mice reacted to the Mam-A2.1 (amino acids [aa] 83–92, LIYDSSLCDL), Mam-A2.2 (aa 2–10, KLLMVLMLA), Mam-A2.4 (aa 66–74, FLNQTDETL), and Mam-A2.6 (aa 32–40, MQLIYDSSL) epitopes. CD8+ CTLs from breast cancer patients also recognized a similar epitope pattern as did those in the HLA-A2+/hCD8 mice and reacted to the Mam-A2.1, Mam-A2.2, Mam-A2.3, Mam-A2.4, and Mam-A2.7 epitopes. Passive transfer of mammaglobin-A–reactive CTLs into SCID (severe combined immunodeficient) beige mice with actively growing mammaglobin-A+ tumors resulted in statistically significant regression (P<.001) in the growth of the tumors. Conclusions: The HLA-A2+/hCD8+ mouse represents a valuable animal model to characterize the HLA-A*0201–restricted CD8+ CTL immune response to mammaglobin-A in vivo, and the data reported here demonstrate the immunotherapeutic potential of mammaglobin-A for the treatment and/or prevention of breast cancer.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The immune response is a potentially useful tool in cancer prevention and treatment, and developing immunotherapies against proteins expressed on transformed cells remains a major goal of tumor immunology. Cytotoxic T lymphocytes that carry CD8 glycoproteins (CD8+ CTLs) recognize and bind to peptide fragments of the cellular proteins in the class I major histocompatibility complex (MHC) family; in humans, these MHC proteins are known as human leukocyte antigens (HLAs), and in mice they are designated H2 (1). CD8+ CTLs are a critical component of the immune response to tumors; they allow tumor cells to be recognized and killed (2). CD8+ CTLs specific for tumor-associated antigens have been used to characterize peptide targets for vaccines or T-cell therapy. Autologous CD8+ CTLs derived from melanoma patients have been used to identify melanoma-specific antigens such as melanoma antigen (MAGE) (3) and GP100 (4). In addition, CD8+ CTLs have been used to identify peptides from the HER-2/neu protein (5,6), which is overexpressed in a spectrum of malignancies, including breast, ovarian, and lung cancer.

Although it has been well documented that tumor-specific CD8+ CTLs can infiltrate melanoma, renal cell carcinoma, and ovarian carcinoma (79), it has been difficult to consistently identify tumor specificity in the CD8+ CTL response to other types of cancer, including breast cancer (710). A recent study has shown that tumor-infiltrating lymphocytes obtained from metastatic effusions of breast cancer patients contained CD8+ CTLs that recognize autologous tumor cells in a tumor-specific, HLA class I–restricted manner (11). These results strongly suggest that tumor-specific antigens are present in breast cancer cells. Subsequent studies identified the HER-2/neu proto-oncogene and the transmembrane protein mucin (MUC1) (1218), whose expression has been correlated with poor prognosis, as important breast cancer–associated antigens. However, the frequency of expression of these tumor-associated antigens is relatively low in breast cancer (HER-2/neu, 30%; MUC1, 60%) (1921). Other studies have shown that CD8+ CTLs from breast cancer patients recognize the HER-2/neu–derived tumor antigen glycoprotein-2 epitopes (amino acids [aa] 654–662, IISAVVGIL) presented by the HLA-A2 molecule (22,23). Despite the demonstration that in vitro–generated CD8+ CTLs can recognize MUC1, attempts to induce active breast cancer–specific immunotherapy with this antigen has met with little success in clinical trials (24).

Mammaglobin-A, a novel breast cancer–associated antigen, has recently been identified using a differential screening approach (2528). Several properties of mammaglobin-A make it a clinically relevant breast cancer–associated marker (2831). Unlike other genes overexpressed in breast cancer, including HER-2/neu and MUC1 (1921,32), mammaglobin-A is expressed at high levels in most human breast cancer cell lines and primary breast tumors (2631). Interestingly, the levels of mammaglobin-A expression were similar among well-differentiated, moderately differentiated, and poorly differentiated primary breast tumors (28). Due to its exclusive overexpression by breast cancer cells, mammaglobin-A is better than HER-2/neu and cytokeratin 19 for detecting breast cancer cells in peripheral blood, bone marrow, and lymph nodes (2931).

The identification of CD8+ CTL epitopes derived from breast cancer–specific antigens is crucial for the successful development of breast cancer immunotherapies. A clinically feasible vaccine for breast cancer would need to have several CD8+ CTL epitopes presented by HLA class I molecules that are present at a high frequency in the population, such as HLA-A*0201 and HLA-A*0301. To target an immunologic response to mammaglobin-A, we previously identified four endogenous HLA-A*0301–restricted mammaglobin-A–derived epitopes present on breast cancer cells of patients in vivo and showed that a CD8+ CTL line developed in vitro against these epitopes recognizes one of them (aa 23–31, PLLENVISK) on breast cancer cells (33). Studies by other investigators have also shown that CD8+ CTL lines developed in vitro against HLA-A*0201–restricted mammaglobin-A–derived candidate epitopes recognize one of them (aa 80–89, FMQLIYDSSL) on breast cancer cells (34).

In the present study, we used transgenic mice carrying the HLA-A*0201 and human CD8 genes to determine if mammaglobin-A cDNA vaccination could inhibit breast cancer tumor progression. Our goal was to identify HLA-A*0201–restricted mammaglobin-A–derived epitopes recognized by the mice in vivo and compare them with HLA-A*0201–restricted mammaglobin-A–derived epitopes recognized by CD8+ T cells of patients. In addition, we examined whether CD8+ CTLs generated in vivo by mammaglobin-A cDNA vaccination can recognize breast cancer cells in vitro and can induce the regression of established breast cancer tumors in vivo.


    SUBJECTS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Mice

C57BL/6 mice (H-2b) carrying the HLA-A*0201 (HLA-A2+) or human CD8 (hCD8+) genes were kindly provided by Dr. Victor H. Engelhard (University of Virginia, Charlottesville) and Dr. Linda A. Sherman (Scripps Research Institute, La Jolla, CA), respectively (35,36). First-generation (F1) mice from a cross between these two mouse transgenic lines (HLA-A2+/hCD8+) were used in this study. SCID (severe combined immunodeficient) beige mice were obtained from Taconic (Germantown, NY). All protocols followed the institutional guidelines approved by the Animal Studies Committee at Washington University in St. Louis.

Study Subjects

Eight HLA-A2+ patients with a histologic diagnosis of breast carcinoma and five HLA-A2+ healthy female individuals were included in this study. HLAs of the study subjects were typed using sequence-specific oligonucleotide probes that provided low-to-medium resolution for HLA-A genes (Dynal Biotech, Lafayette Hill, PA). Written informed consent was obtained from all the patients, and all investigations were performed after receiving the approval of the Human Studies Review Board of Washington University in St. Louis.

Breast Cancer Cell Lines

Breast cancer cell lines (UACC-812, HBL-100, AU-565, MCF-7, MDA-MB-231, DU-4475, MDA-MB-361, and MDA-MB-415) were obtained from the American Type Culture Collection (Manassas, VA) (Table 1). All breast cancer cell lines were cultured in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% defined fetal bovine serum (HyClone, Logan, UT), 100 µM nonessential amino acids, 2 mM L-glutamine, 25 mM HEPES, 1 mM sodium pyruvate, 100 U of penicillin/mL, and 100 µg of streptomycin/mL (GIBCO) at 37 °C in 5% CO2. The HLA-A of the cell lines was typed as described above; mammaglobin-A expression was determined by reverse transcriptase–polymerase chain reaction as previously described (26).


View this table:
[in this window]
[in a new window]
 
Table 1. Endogenous mammaglobin-A expression in the breast cancer cell lines used in this study*

 
Peptides

Mammaglobin-A–derived peptides that bind the HLA-A*0201 molecule were identified using the HLA class I peptide binding prediction program from the Bioinformatics and Molecular Analysis Section of the National Institutes of Health at http://bimas.dcrt.nih.gov/molbio/hla_bind (last accessed: July 20, 2004) (37). Seven mammaglobin-A–derived peptides with high (Mam-A2.1 [aa 83–92, LIYDSSLCDL], Mam-A2.2 [aa 2–10, KLLMVLMLA]), intermediate (Mam-A2.3 [aa 4–12, LMVLMLAAL], Mam-A2.4 [aa 66–74, FLNQTDETL], Mam-A2.5 [aa 73–81, TLSNVEVFM]), and low (Mam-A2.6 [aa 32–40, MQLIYDSSL], Mam-A2.7 [aa 32–40, TINPQVSKT]) affinities for the HLA-A*0201 molecule as determined from the software program, and one mammaglobin-A derived peptide (Mam-A3.1 [aa 23–31, PLLENVISK]) with high affinity to the HLA-A*0301 molecule, were used in our study. The peptides were synthesized by Research Genetics (Huntsville, AL). The purity of peptides was determined by high-performance liquid chromatography and mass spectrometry by Research Genetics. The peptides were dissolved in dimethyl sulfoxide (Sigma, St. Louis, MO) at a concentration of 10 mg/mL and stored at –70 °C until use.

HLA-A2 Membrane Stabilization Assay

The HLA-A2–binding ability of the peptides was confirmed by cell membrane stabilization of the HLA-A*0201 molecule in transporter associated with antigen processing (TAP)–deficient T2 cells, a human T-B lymphoblastoid hybrid cell line obtained from the American Type Culture Collection. T2 cells (106/mL) were incubated in flat-bottom 96-well plates at 25 °C in the presence of each peptide (40 µg/mL) in 200 µl of RPMI 1640 medium (GIBCO) supplemented with 10% defined fetal bovine serum (HyClone), 100 µM nonessential amino acids, 2 mM L-glutamine, 25 mM HEPES, 1 mM sodium pyruvate, 100 U of penicillin/ml, and 100 µg of streptomycin/mL. Three micrograms of human {beta}2 microglobulin ({beta}2m; Sigma)/mL was also added to the cultures. After 24 hours, the T2 cells were washed three times in phosphate-buffered saline (PBS), and the levels of HLA-A2 expression were determined by flow cytometric analysis. The T2 cells were incubated for 30 minutes at 4 °C with the BB7.2 anti–HLA-A2 monoclonal antibody (10 µg/mL) in PBS supplemented with 2% bovine serum albumin (BSA), 25 mM HEPES, and 0.02% sodium azide. The cells were then washed three times in PBS supplemented with 2% BSA and 25 mM HEPES and incubated for 30 minutes at 4 °C with fluorescein-conjugated goat anti–mouse immunoglobulin G (10 µg/mL; Becton Dickinson, Franklin Lakes, NJ). The cells were then washed three times in PBS, fixed in 1% paraformaldehyde, and used in a single-color flow cytometric analysis in a FACScan flow cytometer (Becton Dickinson). T2 cells cultured in the presence of the HLA-A2–restricted GILGFVFTL influenza–derived epitope (Flu) were used as a positive control (38). T2 cells cultured in the presence of HLA-A3–restricted PLLENVISK (Mam-A3.1) mammaglobin-A–derived epitope were used as a negative control (33). Results are expressed as the mean fluorescence shift, which corresponds to the difference between the mean fluorescence obtained with T2 cells cultured in the presence of the peptide (experimental) and the mean fluorescence obtained with T2 cells cultured in the absence of any peptide (control). The MOPC-11 monoclonal antibody (ISC Bioexpress, Kaysville, UT) was used as an immunoglobulin G1 isotype control (data not shown).

Mammaglobin-A cDNA Construct

Mammaglobin-A cDNA was derived from the mammaglobin-A+ human breast cancer cell line MDA-MB-415 (26). The mammaglobin-A cDNA was modified by polymerase chain reaction to yield EcoRI ends and was cloned into the EcoRI site at the multiple cloning site of the PCI-neo vector (Promega, Madison, WI).

DNA Vaccination

Five HLA-A2+/hCD8+ mice were injected intramuscularly in the quadriceps with 100 µg of the PCI-neo vector (Promega) containing the mammaglobin-A cDNA or the vector alone (control) respectively. The mice were inoculated four times at 2-week intervals. Eight weeks after the first inoculation, spleens were removed from mice and homogenized in a Petri dish with 5 mL of PBS. The spleen suspension was layered over lympholyte-M (Cedarlane Laboratories, Hornby, Ontario, Canada) and centrifuged at 800g for 20 minutes. The lymphocytes obtained from the interface were tested for CD8+ CTL activity against peptide-loaded T2 cells and breast cancer cells.

Tumor Growth Inhibition Assay

Eight-week-old SCID beige mice (Taconic) were divided into four groups of five so that growth inhibition by mammaglobin-A vaccination of tumors derived from two different breast cancer cell lines could be measured and compared with that of vaccination with the vector control. A total of 107 HBL-100 (HLA-A2+/mammaglobin-A+) or MDA-MB-231 (HLA-A2+/mammaglobin-A) breast cancer cells were resuspended in 300 µL of BD Matrigel basement membrane matrix (BD Biosciences, San Diego CA). The HBL-100 cell suspension was injected subcutaneously into the backs of 10 mice, and the MDA-MB-231 cell suspension was similarly injected into the other 10 mice. A single tumor formed in each mouse. After the tumors reached a volume of approximately 500 mm3, their size was measured. Then, 4 x 107 spleen cells from HLA-A2+/hCD8+ mice vaccinated with either mammaglobin-A cDNA or vector alone were injected intraperitoneally into five mice in each of the two groups of 10. Tumor growth was measured weekly thereafter over a period of 5 weeks. Tumors were measured with calipers by two different investigators who were blinded to experimental treatments.

ELISPOT Assay

MultiScreen 96-well filtration plates (Millipore, Bedford, MA) were coated overnight at 4 °C with 5.0 µg of a capture human gamma interferon (IFN-{gamma})–specific monoclonal antibody (BD Biosciences, Franklin Lakes, NJ)/mL in 0.05 M carbonate–bicarbonate buffer (pH 9.6). The plates were blocked with 1% BSA for 1 hour and washed three times with PBS. Subsequently, 3 x 105 peripheral blood mononuclear cells were cultured in quadruplicate wells in the antibody-coated plates in 200 µL of RPMI 1640 medium supplemented with 10% defined fetal bovine serum, 100 µM nonessential amino acids, 2 mM L-glutamine, 25 mM HEPES, 1 mM sodium pyruvate, 100 U of penicillin/mL, and 100 µg of streptomycin/mL in the presence of individual mammaglobin-A–derived peptides (40 µg/mL). Cells cultured in the presence of the GILGFVFTL influenza–derived epitope (Flu) were used as positive control. Cells cultured in culture medium alone and cells cultured in the presence of the HLA-A3–restricted PLLENVISK (Mam-A3.1) mammaglobin-A–derived epitope were used as negative controls (33). After 24 hours, the plates were washed three times each with PBS and then with PBS supplemented with 0.05% Tween 20, and 2.0 µg/mL of a biotinylated human gamma interferon (IFN-{gamma})–specific monoclonal antibody (BD Biosciences) in PBS–1% BSA–0.05% Tween 20 was added to each well. After an overnight incubation at 4 °C, the plates were washed three times in PBS–BSA–Tween 20, and horseradish peroxidase–labeled streptavidin (BD Biosciences), diluted 1 : 2000 in PBS–1% BSA–0.05% Tween 20, was added to the wells. After 2 hours, 3-amino-9-ethylcarbazole substrate reagent (BD Biosciences) was added to the wells for 5–10 minutes. The plates were washed with tap water to stop the reaction and air-dried.

To test the functional activity of murine CD8+ CTLs from DNA-vaccinated mice, we cultured 5 x 105 spleen cells in quadruplicate wells in MultiScreen 96-well filtration plates coated with a capture mouse IFN-{gamma}–specific monoclonal antibody (BD Biosciences) in 200 µl of RPMI 1640 medium supplemented as described above in the presence of irradiated (10 000 rads) peptide-loaded T2 cells (5 x 105). After 24 hours, the cells were washed and the plates were coated with a biotinylated mouse IFN-{gamma}–specific monoclonal antibody (BD Biosciences), horseradish peroxidase–labeled streptavidin, and substrate reagent as described above.

Spots were analyzed in an ImmunoSpot Series I analyzer (Cellular Technology, Cleveland, OH) that was designed to detect spots with predetermined criteria for spot size, shape, and colorimetric density. For the human studies, a test was considered positive if the number of spots in the experimental cultures was statistically significantly different from the number of spots in the negative control cultures (Student's t test with the {alpha} set at P<0.01). The number of spots in the negative control cultures was subtracted from the number of spots in the experimental cultures. Results are expressed as the number of IFN-{gamma}–positive cells/106 peripheral blood mononuclear cells (human studies) or spleen cells (mouse studies).

Cytotoxicity Assay

Target cells (T2 or breast cancer cells) were labeled with 51Cr (Na51CrO4, 250 µCi; ICN Pharmaceuticals, Costa Mesa, CA) in 100 µL of complete medium. After 1 hour, 5 x 103 labeled cells were plated in quadruplicate cultures in round-bottom 96-well plates in the presence of various numbers of mouse spleen cells (effector-to-target cell ratios of 50, 25, 10, and 5 to 1, respectively) and incubated at 37 °C overnight. Epitope specificity of breast cancer cell lysis was further determined in a cold target inhibition assay by analyzing the capacity of peptide-loaded unlabeled T2 cells to block the lysis of breast cancer cells at an inhibitor-to-target ratio of 20. For antibody blocking experiments, the target cells were incubated with the BB7.2 anti–HLA-A2 monoclonal antibody (10 µg/mL), the GAP-A3 anti–HLA-A3 monoclonal antibody (1 : 50), or the MOPC-11 isotype control monoclonal antibody (10 µg/mL) for 30 minutes before the addition of mouse spleen cells. Control wells for determining spontaneous 51Cr release contained labeled target cells alone. Maximal release was determined by adding 1% Triton X-100 to the target cells. The percent specific lysis was calculated as follows: [(experimental 51Cr release)–(spontaneous 51Cr release)/(maximum 51Cr release)–(spontaneous 51Cr release)] x 100.

Statistical Analysis

Results are presented as the means with 95% confidence intervals (CIs) of quadruplicate cultures. Statistical comparisons of tumor size between groups were carried out using two-way analysis of variance. Post-hoc multiple comparisons were made between vaccinated and control groups at each time point. Statistical comparisons of number of IFN-{gamma} spots between the patient group and the healthy individuals were carried out using the Mann–Whitney U test. Statistical comparisons between control and experimental samples were carried out using one-way analysis of variance. (Post-hoc multiple comparisons were made using the Fisher least significant difference method.) P values less than .05 were considered to be statistically significant. All reported P values were two-sided. Post-hoc multiple comparisons between the experimental and control groups were carried out using the Fisher least significant difference method.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Identification of HLA-A*0201–Restricted Mammaglobin-A–Derived CD8+ T-Cell Epitopes

Using an HLA class I peptide binding prediction computer program (37), we screened the mammaglobin-A protein sequence and identified seven candidate epitopes with different binding affinities to the HLA-A*0201 molecule. The actual binding affinity of the peptides was determined using an HLA-A2 membrane stabilization assay in T2 cells. As shown in Fig. 1, all the peptides showed affinity for the HLA-A*0201 molecule similar to that observed for the GILGFVFTL influenza–derived (Flu) epitope (positive control).



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1. Membrane stabilization of the HLA-A2 molecule by mammaglobin-A–derived candidate epitopes. Computer-assisted predicted HLA-A2–binding affinities of the candidate epitopes were confirmed by membrane stabilization of the HLA-A2 molecule in T2 cells, a human T-B lymphoblastoid hybrid cell line deficient in transporter associated with antigen-processing proteins. T2 cells were incubated in the presence of each peptide in complete medium (see "Subjects and Methods"). After 24 hours, HLA-A2 expression was determined with flow cytometric analysis using the BB7.2 anti–HLA-A2 monoclonal antibody (solid bars). T2 cells incubated in the presence of the GILGFVFTL influenza–derived epitope (Flu) were used as a positive control, and T2 cells incubated in the presence of HLA-A3–restricted PLLENVISK (Mam-A3.1) mammaglobin-A–derived epitope were used as negative control. Results are expressed as the mean fluorescence shift, which represents the difference between the mean fluorescence obtained with T2 cells cultured in the presence of peptides and the mean fluorescence obtained with T2 cells cultured in the absence of peptides. Results are presented as the mean of four independent experiments performed in quadruplicate. Error bars represent upper 95% confidence intervals.

 
Immunodominance of HLA-A*0201–Restricted CD8+ CTL Response Against Mammaglobin-A–Derived Epitopes in Mice Vaccinated With Full-Length Mammaglobin-A cDNA

To determine the feasibility of using mammaglobin-A as a breast cancer vaccine, we constructed a DNA expression vector encoding the full-length mammaglobin-A cDNA and evaluated whether intramuscular injection of this plasmid could induce mammaglobin-A–specific immune responses in HLA-A2+/hCD8+ mice. TAP-deficient, T-B lymphoblastoid hybrid T2 cells were individually loaded with the Mam-A2.1, Mam-A2.2, Mam-A2.3, Mam-A2.4, Mam-A2.5, Mam-A2.6, Mam-A2.7, Mam-A3.1, and Flu peptides. The peptide-loaded T2 cells were then used as targets in a standard CTL activity assay. As shown in Fig. 2, A, the resulting CD8+ CTL response in mice immunized with mammaglobin-A showed cytotoxic activity against T2 cells loaded with peptide Mam-A2.1, Mam-A2.2, Mam-A2.4, and Mam-A2.6, when compared with that of mice immunized with empty vector. There was no CTL cytotoxicity toward T2.A2 cells loaded with peptides Mam-A2.3, Mam-A2.5, Mam-A2.7, Mam-A3.1, and Flu peptides. These results indicate that CD8+ CTL lines generated in vivo by means of cDNA vaccination induce the expansion of a multispecific polyclonal CD8+ CTL response.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2. Epitope immunodominance of CD8+ cytotoxic T lymphocytes (CTLs) from mammaglobin-A cDNA–vaccinated HLA-A2+/hCD8+ mice. Five HLA-A2+/hCD8+ mice were each vaccinated with either mammaglobin-A cDNA (solid bars) or empty vector (open bars). A) Immunodominance of the CD8+ CTLs was evaluated using a standard 51Cr release assay against T2 cells, a T-B lymphoblastoid hybrid cell line deficient of transporter associated with antigen processing (TAP) proteins that had been individually loaded with the candidate epitopes Mam-A2.1–7. T2 cells loaded with the GILGFVFTL influenza-derived epitope (Flu) and T2 cells loaded with the HLA-A3–restricted PLLENVISK (Mam-A3.1) mammaglobin-A–derived epitope were used as a negative control. Assay was performed at effector-to-target cell ratios of 50 : 1. Average spontaneous release of Cr51 for all cell lines was 9% of the maximum release. B) Immunodominance of the CD8+ CTLs in the splenocytes was evaluated by means of an interferon gamma (IFN-{gamma}) ELISPOT assay against T2 cells individually loaded with the candidate epitopes Mam-A2.1–7. T2 cells loaded with the GILGFVFTL influenza–derived (Flu) epitope and T2 cells loaded with the HLA-A3–restricted PLLENVISK (Mam-A3.1) mammaglobin-A–derived epitope were used as negative controls. Results are presented as the mean of quadruplicate cultures for each of the five mice. Error bars represent upper 95% confidence intervals.

 
To confirm the reactivity of T cells against mammaglobin-A–derived epitopes, we used an IFN-{gamma} ELISPOT assay to define the profile of CD8+ CTL reactivity in the spleen cells obtained from HLA-A2+/hCD8+ mice immunized with the full-length mammaglobin-A cDNA. As shown in Fig. 2, B, spleen cells from immunized mice reacted to the HLA-A2+ peptides Mam-A2.1, Mam-A2.2, Mam-A2.6, and Mam-A2.4. The results presented in Fig. 2 clearly identify dominant mammaglobin-A–derived epitopes recognized in the context of HLA-A2+ by CD8+ CTLs generated in vivo.

Vaccination With Full-Length Mammaglobin-A cDNA and HLA-A*0201–Restricted CD8+ CTL Response to Breast Cancer Cells

We next determined the ability of spleen-derived CD8+ CTLs from mice immunized with mammaglobin-A to lyse breast cancer cells in an HLA-A*0201–restricted and mammaglobin-A–specific manner. As shown in Fig. 3, A, CD8+ CTLs from immunized mice showed cytotoxic activity at an effector-to-target cell ratio of 50 : 1, against three HLA-A2+/mammaglobin-A+ breast cancer cell lines (AU-565, HBL-100, and UACC-812) but not against HLA-A2+/mammaglobin-A (MCF-7 and MDA-MB-231) and HLA-A2/mammaglobin-A+ (DU-4475, MDA-MB-361, and MDA-MB-415) breast cancer cell lines (Fig. 3).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3. Induction of CD8+ cytotoxic T lymphocyte (CTL) activity against human breast cancer cells in mammaglobin-A cDNA–vaccinated HLA-A2+/hCD8+ mice. Five HLA-A2+/hCD8+ mice were each vaccinated with either mammaglobin-A cDNA (solid bars) or empty vector (open bars). A) Mammaglobin-A specificity and HLA-A*0201 restriction of the CD8+ CTLs were then evaluated against the HLA-A+/mammaglobin-A+ (UACC-812, HBL-100, AU-565), HLA-A+/mammaglobin-A (MCF-7 and MDA-MB-231), and HLA-A/mammaglobin-A+ (DU-4475, MDA-MB-361, and MDA-MB-415) breast cancer cell lines by means of a standard 51Cr release assay. Assay was performed at effector-to-target (E : T) ratios of 50 : 1. Cytotoxicity of CD8+ CTLs of immunized mice was evaluated against HLA-A+/mammaglobin-A+ AU565 (B), HBL-100 (C), and UACC-812 (D) breast cancer cell lines by means of a standard 51Cr release assay at E : T cell ratios of 50, 25, 10, and 5 to 1. Average spontaneous release of Cr51 for all cell lines was 10% of the maximum release. Results are presented as the mean of quadruplicate cultures for each of five mice. Error bars represent upper 95% confidence intervals.

 
The cytotoxic activity of the anti–mammaglobin-A CD8+ CTLs was determined in the presence of T2 cells pulsed with mammaglobin-A–derived epitopes Mam-A2.1–7. As shown in Fig. 4, the addition of T2 cells pulsed with mammaglobin-A–derived epitopes Mam-A2.1 and Mam-A2.2 greatly inhibited the cytotoxic effect in two HLA-A2+/mammaglobin-A+ breast cancer cell lines (60% and 50% inhibition in the HBL-100 cell line and 57.5% and 50% inhibition in the AU-565 cell line, respectively, compared with the no-peptide, Flu, and Mam-A3.1 controls). However, the addition of T2 cells pulsed with the mammaglobin-A–derived epitopes Mam-A2.4 and Mam-A2.6 resulted in the inhibition of cytotoxic activity to a lesser extent (30% and 30% inhibition in the HBL-100 cell line and 37.5% and 25% inhibition in the AU-565 cell line, respectively, compared with the corresponding controls).



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4. Recognition of the Mam-A2.1 and Mam-A2.2 peptides on human breast cancer cells by CD8+ cytotoxic T lymphocytes (CTLs) from mammaglobin-A cDNA–vaccinated HLA-A2+/hCD8+ mice. Five HLA-A2+/hCD8+ mice were vaccinated with either mammaglobin-A cDNA (solid bars) or empty vector (open bars). Peptide specificity of the CD8+ CTL was then evaluated against the HBL-100 (top) and the AU-565 (bottom) (both HLA-A2+/mammaglobin-A+) breast cancer cell lines in the presence of a 20-fold excess of unlabeled transporter associated with antigen processing (TAP)–deficient T2 cells individually loaded with the Mam-A2.1–7, GILGFVFTL influenza–derived epitope (Flu), or HLA-A3–restricted PLLENVISK Mam-A3.1 peptides. Cytotoxicity assay was performed at an effector-to-target cell ratio of 50 : 1. Average spontaneous release of Cr51 for all cell lines was 10% of the maximum release. Results are presented as the mean of quadruplicate cultures for each of five mice (*, P<.001 compared with no-peptide control). Error bars represent upper 95% confidence intervals.

 
Inhibition of Cytotoxic Activity of HLA-A2–Restricted Mammaglobin-A–Reactive CD8+ CTL by Anti–HLA-A2 Antibodies

To confirm the HLA-A2 restriction of the CD8+ CTLs from the vaccinated mice, we determined the cytotoxic activity of the anti–mammaglobin-A CD8+ T cells in the presence of either anti–HLA-A2 or anti–HLA-A3 monoclonal antibodies. As shown in Fig. 5, the addition of the BB7.2 anti–HLA-A2 monoclonal antibodies inhibited the lysis of the mammaglobin-A+/HLA-*0201+ breast cancer cell lines (HBL-100 and AU565), whereas the addition of the GAP-A3 anti–HLA-A3 monoclonal antibodies or the control MOPC-11 antibodies had no effect on the cytotoxic activity against these breast cancer cell lines. These results further confirm the HLA-A2 restriction of the Mam-A2.1 and Mam A2.2 peptide recognition by anti–mammaglobin-A CD8+ CTLs.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5. Inhibition of the cytotoxic activity of the mammaglobin-A–reactive CD8+ cytotoxic T lymphocytes (CTLs) from mammaglobin-A cDNA–vaccinated HLA-A2+/hCD8+) mice by anti–HLA-A2 antibodies. Five HLA-A2+/hCD8+ mice were vaccinated with mammaglobin-A cDNA. HLA class I restriction of the CD8+ CTLs was then evaluated against the HBL-100 (solid bars) and AU-565 (open bars) (both HLA-A2+/mammaglobin-A+) breast cancer cell lines in the presence of the BB7.2 anti–HLA-A2 monoclonal antibody, the GAP-A3 anti–HLA-A3 monoclonal antibody, or the MOPC-11 isotype control monoclonal antibody. Cytotoxicity assay was performed at an effector-to-target cell ratio of 50 : 1. Average spontaneous release of Cr51 for all cell lines was 11% of the maximum release. Results are presented as the mean of quadruplicate cultures for each of the five mice (*, P<.001 compared with no-antibody control). Error bars represent upper 95% confidence intervals.

 
Inhibition of Breast Cancer Tumor Growth In Vivo by Mammaglobin-A–Reactive CD8+ CTLs

To determine the ability of HLA-A2–restricted mammaglobin-A–reactive CD8+ CTLs to inhibit tumor growth in vivo, we inoculated the CD8+ CTLs from mammaglobin-A–vaccinated mice to SCID beige mice with tumors derived from either HBL-100 (HLA-A2+/mammaglobin-A+) or MDA-MB-231 (HLA-A2+/mammaglobin-A) breast cancer cell lines. As shown in Fig. 6, the CD8+ CTLs obtained from HLA-A2+/hCD8+ mice caused regression of HBL-100–derived tumor growth over 5 weeks. However, the same CD8+ CTLs were inefficient in regressing the growth of the MDA-MB-231–derived tumors. The MDA-MB-231–derived tumors showed a continual increase in size with time. The CD8+ CTLs from vector alone–treated HLA-A2+/hCD8+ mice had no effect on the growth of either tumor type over the same 5-week period. The results thus indicate that CD8+ CTLs generated by the vaccination with full-length mammaglobin-A cDNA are capable of specifically inducing the regression of mammaglobin-A+/HLA-A2+ breast cancer tumor growth in vivo.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 6. Regression of tumor growth in severe combined immunodeficient (SCID) beige mice by passive transfer of mammaglobin-A–reactive CD8+ cytotoxic T lymphocytes (CTLs) from mammaglobin-A cDNA–vaccinated HLA-A2+/hCD8+ mice. Tumors derived from HBL-100 (HLA-A2+/mammaglobin-A+, top) or MDA-MB-231 (HLA-A2+/mammaglobin-A-, bottom) breast cancer cell lines were developed in 20 SCID beige mice. Ability of the CD8+ CTLs from HLA-A2+/hCD8+ mice vaccinated with mammaglobin-A cDNA (triangles) or empty vector (squares) to cause regression of tumor growth was evaluated by inoculation into the SCID beige mice bearing the tumors. Tumor volume was analyzed weekly for 5 weeks, and the results are presented as the mean of five tumors, one per mouse (*, P<.001). Error bars represent upper 95% confidence intervals.

 
Immunodominance of HLA-A2–Restricted T Cells From Breast Cancer Patients Against Mammaglobin-A–Derived Epitopes In Vitro

To determine the immunoreactivity of T cells from breast cancer patients toward mammaglobin-A–derived epitopes, we used all the peptides in an IFN-{gamma} ELISPOT assay to examine the profile of T-cell reactivity in eight HLA-A2–positive breast cancer patients and five healthy individuals. As shown in Table 2, T-cell reactivity was observed against peptides Mam-A2.1, Mam-A2.2, Mam-A2.3, Mam-A2.4, and Mam-A2.7. Six patients (75%) showed CD8+ CTL reactivity against peptide Mam-A2.2; five patients (62.5%) showed T-cell reactivity against peptides MamA-2.1, Mam-A2.3, and Mam-A2.7; and four patients (50%) showed reactivity against the Mam-A2.4 peptide. Interestingly, all healthy control individuals displayed reactivity against peptide Mam-A2.2.


View this table:
[in this window]
[in a new window]
 
Table 2. Recognition of HLA-A2–restricted mammaglobin-A–derived epitopes by T cells from breast cancer patients*

 

    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
In this article, we have demonstrated, using a transgenic mouse model expressing human HLA-A2 and human CD8, that vaccination with mammaglobin-A cDNA results in the development of a CD8+ CTL response against mammaglobin-A+ tumors. In addition, CD8+ CTLs from vaccinated mice were able to induce the regression of established breast cancer tumors in vivo. Furthermore, we have identified four HLA-A2–restricted CD8+ CTL epitopes from a breast cancer–specific antigen, mammaglobin-A, that are recognized by the CD8+ CTLs of both breast cancer patients and mammaglobin-A cDNA–vaccinated HLA-A2+/hCD8+ mice. These results show that mammaglobin-A can serve as a breast cancer–specific antigen and may be useful for designing new immunotherapy protocols for the treatment and prevention of breast cancer.

Several reports have used HLA-A2.1 transgenic mice as an in vivo model to identify peptides presented by HLA-A2.1 molecules (39,40). However, one problem with using CTLs from these mice is the inability of murine CD8 to interact effectively with human HLA molecules, which results in suboptimal recognition of peptide class I MHC complexes in human cell lines (41,42). However, Lustgarten et al. (35) showed that CTLs from HLA-A2+/hCD8 mice immunized with A2.1-binding peptides from HER-2/neu were able to recognize and lyse human tumor cells that process and present HER-2/neu epitopes on HLA-A2.1 (35). In our in vivo model, we used a computer program that predicts HLA class I peptide binding (37) to identify the highest affinity epitopes and found that four of these HLA-*0201–restricted mammaglobin-A–derived epitopes, Mam A2.1, Mam A2.2, Mam A2.4, and Mam A2.6, showed high affinity for the HLA-A2 molecule, similar to that observed for an influenza-derived epitope. HLA-A2+/HCD8 mice immunized with full-length mammaglobin-A cDNA generated a significant CD8+ CTL response against only two of the four HLA-*0201–restricted mammaglobin-A–derived epitopes (Mam A2.1 and Mam A2.2).

The discrepancies between the predicted affinities of the peptides and the actual membrane stabilization abilities of the HLA-A*0201 molecules may reflect the fact that the binding affinity of a given peptide is determined by its amino acid sequence (binding motifs) as well as the three-dimensional structure of the peptide in the MHC class I groove (43,44). In this regard, it has been previously shown that the affinity of a given peptide to an MHC class I molecule does not necessarily coincide with its ability to generate CD8+ CTLs (45,46).

The immunodominant peptide Mam-A2.2 is derived from the signal sequence of mammaglobin-A, which is identical to that of mammaglobin-B. Because mammaglobin-B is expressed in normal uterine tissue and salivary glands, there is a potential for the development of autoreactive T cells following vaccination with mammaglobin-A cDNA. Studies to address such concerns need to be carried out prior to using this strategy as an effective peptide vaccine.

CD8+ CTLs from the immunized mice showed cytotoxic activity against three HLA-A2+/mammaglobin-A+ breast cancer cell lines (AU-565, HBL-100, UACC-812) but not against HLA-A2+/mammaglobin-A (MCF-7, MDA-MB-231) and HLA-A2/mammaglobin-A+ (DU-4475, MDA-MB-361, MDA-MB-415) breast cancer cell lines. However, there was a clear gradation in the observed cytotoxicity toward the three HLA-A2+/mammaglobin-A+ breast cancer cell lines. This gradation may be due to the fact that the breast cancer cell lines differentially express mammaglobin-A–derived epitopes as a result of differential enzyme cleavage of the mammaglobin-A protein in the proteasome (47) and/or selective loss of the membrane expression of a given HLA class I allele (48).

Passive transfer of splenocytes containing mammaglobin-A–specific, HLA-A2+–restricted CD8+ CTLs from vaccinated mice to SCID beige mice with established HLA-A2+/mammaglobin-A+ tumors resulted in tumor regression. These results thus indicate that mammaglobin-A cDNA vaccination was able to generate an antitumor CD8+ CTL response against mammaglobin-A+ tumors in this in vivo humanized mouse model, and this activity was maintained even when the spleen cells from vaccinated mice were passively transferred to mice with actively growing tumors.

Although mammaglobin-A cDNA vaccination of HLA-A2+/hCD8+ mice resulted in the generation of antitumor activity, we wanted to further confirm that a similar response would indeed be observed in breast cancer patients. The T cells from both the breast cancer patients and the HLA-A2+/hCD8+ mice reacted with the mammaglobin-A–derived epitopes Mam-A2.1, Mam-A2.2, and Mam-A2.4, thus indicating that there is considerable similarity in the antitumor reactivity between humans and humanized mice. Interestingly, however, the T cells of healthy individuals also recognize one of these peptides (Mam-A2.2). It has previously been shown that CD8+ CTL lines generated from healthy individuals and directed against this candidate epitope were not able to recognize breast cancer cell lines in vitro (34). These results show that Mam-A2.2–specific CD8+ CTLs are normally expanded in humans. This recognition may be due to cross-reaction between the Mam-A2.2 peptide and a naturally presented peptide derived from a cross-reacting environmental pathogen recognized by the T cells of HLA-A2+ individuals. Given the high frequency of expression of mammaglobin-A in breast tumors (80%) and its total lack of or low level of expression in normal tissues, this protein seems to be an important breast cancer–specific antigen that could be utilized for in vitro expansion of breast cancer–specific CD8+ CTLs for autologous transfer and/or as a systemic vaccine to induce a protective immune response in high-risk patients. However, the low-level expression of mammaglobin-A in normal breast tissue might also lead to the development of autoreactive T cells toward mammaglobin-A–expressing breast tissue. Further studies need to be carried out to address such concerns before using this strategy for effective immunotherapy. The data presented here demonstrate for the first time, to our knowledge, that cDNA vaccination with mammaglobin-A could represent a novel therapeutic alternative for breast cancer.


    NOTES
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Supported by a grant from the Susan G. Komen Breast Cancer Foundation.

We thank Dr. Richard Schuessler for his assistance with statistics.

Timothy P. Fleming owns stock in Corixa (Seattle, WA), which owns the license for mammaglobin, including its use as a tumor vaccine.


    REFERENCES
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

1 Townsend A, Bodmer H. Antigen recognition by class I-restricted T lymphocytes. Annu Rev Immunol 1989;7:601–24.[CrossRef][ISI][Medline]

2 Linehan DC, Goedegebuure PS, Eberlein TJ. Vaccine therapy for cancer. Ann Surg Oncol 1996;3:219–28.[Abstract]

3 van der BP, Traversari C, Chomez P, Lurquin C, De Plaen E, Van den EB, et al. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 1991;254:1643–7.[ISI][Medline]

4 Bakker AB, Schreurs MW, de Boer AJ, Kawakami Y, Rosenberg SA, Adema GJ, et al. Melanocyte lineage-specific antigen gp100 is recognized by melanoma-derived tumor-infiltrating lymphocytes. J Exp Med 1994;179:1005–9.[Abstract]

5 Disis ML, Smith JW, Murphy AE, Chen W, Cheever MA. In vitro generation of human cytolytic T-cells specific for peptides derived from the HER-2/neu protooncogene protein. Cancer Res 1994;54:1071–6.[Abstract]

6 Fisk B, Blevins TL, Wharton JT, Ioannides CG. Identification of an immunodominant peptide of HER-2/neu protooncogene recognized by ovarian tumor-specific cytotoxic T lymphocyte lines. J Exp Med 1995;181:2109–17.[Abstract]

7 Finke JH, Rayman P, Alexander J, Edinger M, Tubbs RR, Connelly R, et al. Characterization of the cytolytic activity of CD4+ and CD8+ tumor-infiltrating lymphocytes in human renal cell carcinoma. Cancer Res 1990;50:2363–70.[Abstract]

8 Peoples GE, Schoof DD, Andrews JV, Goedegebuure PS, Eberlein TJ. T-cell recognition of ovarian cancer. Surgery 1993;114:227–34.[ISI][Medline]

9 Topalian SL, Solomon D, Rosenberg SA. Tumor-specific cytolysis by lymphocytes infiltrating human melanomas. J Immunol 1989;142:3714–25.[Abstract/Free Full Text]

10 Baxevanis CN, Dedoussis GV, Papadopoulos NG, Missitzis I, Stathopoulos GP, Papamichail M. Tumor specific cytolysis by tumor infiltrating lymphocytes in breast cancer. Cancer 1994;74:1275–82.[ISI][Medline]

11 Linehan DC, Goedegebuure PS, Peoples GE, Rogers SO, Eberlein TJ. Tumor-specific and HLA-A2-restricted cytolysis by tumor-associated lymphocytes in human metastatic breast cancer. J Immunol 1995;155:4486–91.[Abstract]

12 Apostolopoulos V, Sandrin MS, McKenzie IF. Carbohydrate/peptide mimics: effect on MUC1 cancer immunotherapy. J Mol Med 1999;77:427–36.[CrossRef][ISI][Medline]

13 Boon T, van der Bruggen P. Human tumor antigens recognized by T lymphocytes. J Exp Med 1996;183:725–9.[ISI][Medline]

14 Boon T, Coulie PG, Van den Eynde B. Tumor antigens recognized by T cells. Immunol Today 1997;18:267–8.[CrossRef][ISI][Medline]

15 Brossart P, Heinrich KS, Stuhler G, Behnke L, Reichardt VL, Stevanovic S, et al. Identification of HLA-A2-restricted T-cell epitopes derived from the MUC1 tumor antigen for broadly applicable vaccine therapies. Blood 1999;93:4309–17.[Abstract/Free Full Text]

16 Domenech N, Henderson RA, Finn OJ. Identification of an HLA-A11-restricted epitope from the tandem repeat domain of the epithelial tumor antigen mucin. J Immunol 1995;155:4766–74.[Abstract]

17 Finn OJ, Jerome KR, Henderson RA, Pecher G, Domenech N, Magarian-Blander J, et al. MUC-1 epithelial tumor mucin-based immunity and cancer vaccines. Immunol Rev 1995;145:61–89.[ISI][Medline]

18 Peoples GE, Goedegebuure PS, Smith R, Linehan DC, Yoshino I, Eberlein TJ. Breast and ovarian cancer-specific cytotoxic T lymphocytes recognize the same HER-2/neu-derived peptide. Proc Natl Acad Sci U S A 1995;92:432–6.[Abstract]

19 Graham RA, Burchell JM, Taylor-Papadimitriou J. The polymorphic epithelial mucin: potential as an immunogen for a cancer vaccine. Cancer Immunol Immunother 1996;42:71–80.[CrossRef][ISI][Medline]

20 Lohrisch C, Piccart M. HER-2/neu as a predictive factor in breast cancer. Clin Breast Cancer 2001;2:129–35.[Medline]

21 Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989;244:707–12.[ISI][Medline]

22 Brossart P, Bevan MJ. Selective activation of Fas/Fas ligand-mediated cytotoxicity by a self peptide. J Exp Med 1996;183:2449–58.[Abstract]

23 Houbiers JG, Nijman HW, van der Burg SH, Drijfhout JW, Kenemans P, van de Velde CJ, et al. In vitro induction of human cytotoxic T lymphocyte responses against peptides of mutant and wild-type p53. Eur J Immunol 1993;23:2072–7.[ISI][Medline]

24 Musselli C, Ragupathi G, Gilewski T, Panageas KS, Spinat Y, Livingston PO. Reevaluation of the cellular immune response in breast cancer patients vaccinated with MUC1. Int J Cancer 2002;97:660–7.[CrossRef][ISI][Medline]

25 Watson MA, Fleming TP. Isolation of differentially expressed sequence tags from human breast cancer. Cancer Res 1994;54:4598–602.[Abstract]

26 Watson MA, Fleming TP. Mammaglobin, a mammary-specific member of the uteroglobin gene family, is overexpressed in human breast cancer. Cancer Res 1996;56:860–5.[Abstract]

27 Watson MA, Darrow C, Zimonjic DB, Popescu NC, Fleming TP. Structure and transcriptional regulation of the human mammaglobin gene, a breast cancer associated member of the uteroglobin gene family localized to chromosome 11q13. Oncogene 1998;16:817–24.[CrossRef][ISI][Medline]

28 Watson MA, Dintzis S, Darrow CM, Voss LE, DiPersio J, Jensen R, et al. Mammaglobin expression in primary, metastatic, and occult breast cancer. Cancer Res 1999;59:3028–31.[Abstract/Free Full Text]

29 Fleming TP, Watson MA. Mammaglobin, a breast-specific gene, and its utility as a marker for breast cancer. Ann N Y Acad Sci 2000;923:78–89.[Abstract/Free Full Text]

30 Grunewald K, Haun M, Urbanek M, Fiegl M, Muller-Holzner E, Gunsilius E, et al. Mammaglobin gene expression: a superior marker of breast cancer cells in peripheral blood in comparison to epidermal-growth-factor receptor and cytokeratin-19. Lab Invest 2000;80:1071–7.[ISI][Medline]

31 Marchetti A, Buttitta F, Bertacca G, Zavaglia K, Bevilacqua G, Angelucci D, et al. mRNA markers of breast cancer nodal metastases: comparison between mammaglobin and carcinoembryonic antigen in 248 patients. J Pathol 2001;195:186–90.[CrossRef][ISI][Medline]

32 Disis ML, Schiffman K. Cancer vaccines targeting the HER-2/neu oncogenic protein. Semin Oncol 2001;28:12–20.

33 Jaramillo A, Majumder K, Manna PP, Fleming TP, Doherty G, Dipersio JF, et al. Identification of HLA-A3-restricted CD8+ T cell epitopes derived from mammaglobin-A, a tumor-associated antigen of human breast cancer. Int J Cancer 2002;102:499–506.[CrossRef][ISI][Medline]

34 Tanaka Y, Amos KD, Fleming TP, Eberlein TJ, Goedegebuure PS. Mammaglobin-A is a tumor-associated antigen in human breast carcinoma. Surgery 2003;133:74–80.[CrossRef][ISI][Medline]

35 Lustgarten J, Theobald M, Labadie C, LaFace D, Peterson P, Disis ML, et al. Identification of Her-2/Neu CTL epitopes using double transgenic mice expressing HLA-A2.1 and human CD.8. Hum Immunol 1997;52:109–18.[CrossRef][ISI][Medline]

36 Man S, Newberg MH, Crotzer VL, Luckey CJ, Williams NS, Chen Y, et al. Definition of a human T cell epitope from influenza A non-structural protein 1 using HLA-A2.1 transgenic mice. Int Immunol 1995;7:597–605.[Abstract]

37 Parker KC, Bednarek MA, Coligan JE. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J Immunol 1994;152:163–75.[Abstract/Free Full Text]

38 Bednarek MA, Sauma SY, Gammon MC, Porter G, Tamhankar S, Williamson AR, et al. The minimum peptide epitope from the influenza virus matrix protein. Extra and intracellular loading of HLA-A2. J Immunol 1991;147:4047–53.[Abstract/Free Full Text]

39 Theobald M, Biggs J, Dittmer D, Levine AJ, Sherman LA. Targeting p53 as a general tumor antigen. Proc Natl Acad Sci U S A 1995;92:11993–7.[Abstract]

40 Wentworth PA, Vitiello A, Sidney J, Keogh E, Chesnut RW, Grey H, et al. Differences and similarities in the A2.1-restricted cytotoxic T cell repertoire in humans and human leukocyte antigen-transgenic mice. Eur J Immunol 1996;26:97–101.[ISI][Medline]

41 Irwin MJ, Heath WR, Sherman LA. Species-restricted interactions between CD8 and the alpha 3 domain of class I influence the magnitude of the xenogeneic response. J Exp Med 1989;170:1091–101.[Abstract]

42 Kalinke U, Arnold B, Hammerling GJ. Strong xenogeneic HLA response in transgenic mice after introducing an alpha 3 domain into HLA B27. Nature 1990;348:642–4.[CrossRef][ISI][Medline]

43 Kuhns JJ, Batalia MA, Yan S, Collins EJ. Poor binding of a HER-2/neu epitope (GP2) to HLA-A2.1 is due to a lack of interactions with the center of the peptide. J Biol Chem 1999;274:36422–7.[Abstract/Free Full Text]

44 Tanaka Y, Amos KD, Joo HG, Eberlein TJ, Goedegebuure PS. Modification of the HER-2/NEU-derived tumor antigen GP2 improves induction of GP2-reactive cytotoxic T lymphocytes. Int J Cancer 2001;94:540–4.[CrossRef][ISI][Medline]

45 Bullock TN, Mullins DW, Colella TA, Engelhard VH. Manipulation of avidity to improve effectiveness of adoptively transferred CD8(+) T cells for melanoma immunotherapy in human MHC class I-transgenic mice. J Immunol 2001;167:5824–31.[Abstract/Free Full Text]

46 Nijman HW, Houbiers JG, van der Burg SH, Vierboom MP, Kenemans P, Kast WM, et al. Characterization of cytotoxic T lymphocyte epitopes of a self-protein, p53, and a non-self-protein, influenza matrix: relationship between major histocompatibility complex peptide binding affinity and immune responsiveness to peptides. J Immunother 1993;14:121–6.[ISI][Medline]

47 Luckey CJ, Marto JA, Partridge M, Hall E, White FM, Lippolis JD, et al. Differences in the expression of human class I MHC alleles and their associated peptides in the presence of proteasome inhibitors. J Immunol 2001;167:1212–21.[Abstract/Free Full Text]

48 Luckey CJ, King GM, Marto JA, Venketeswaran S, Maier BF, Crotzer VL, et al. Proteasomes can either generate or destroy MHC class I epitopes: evidence for nonproteasomal epitope generation in the cytosol. J Immunol 1998;161:112–21.[Abstract/Free Full Text]

Manuscript received January 30, 2004; revised July 12, 2004; accepted July 14, 2004.



             
Copyright © 2004 Oxford University Press (unless otherwise stated)
Oxford University Press Privacy Policy and Legal Statement