Annals of Surgical Oncology

, Volume 13, Issue 8, pp 1085–1098 | Cite as

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

  • Elizabeth A. Mittendorf
  • Catherine E. Storrer
  • Craig D. Shriver
  • Sathibalan Ponniah
  • George E. PeoplesEmail author



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.


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.


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.


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.


Breast cancer HER2/neu Immunotherapy Trastuzumab Peptide vaccine 



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.


  1. 1.
    Lemoine NR, Staddon S, Dickson C, Barnes DM, Gullick WJ. Absence of activating transmembrane mutations in the c-erbB-2 proto-oncogene in human breast cancer. Oncogene 1990;5:237–9PubMedGoogle Scholar
  2. 2.
    Natali PG, Nicotra MR, Bigotti A, et al. Expression of the p185 encoded by HER2 oncogene in normal and transformed human tissues. Int J Cancer 1990;45:457–61PubMedGoogle Scholar
  3. 3.
    Slamon DJ, Clark GM, Wong SG, et al. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987;235:177–82.PubMedGoogle Scholar
  4. 4.
    Disis ML, Grabstein KH, Sleath PR, Cheever MA. Generation of immunity to the HER-2/neu oncogenic protein in patients with breast and ovarian cancer using a peptide-based vaccine. Clin Cancer Res 1999;5:1289–97PubMedGoogle Scholar
  5. 5.
    Disis ML, Schiffman K. Cancer vaccines targeting the HER-2/neu oncogenic protein. Semin Oncol 2001;28:12–20PubMedCrossRefGoogle Scholar
  6. 6.
    Anderson BW, Peoples GE, Murray JL, Gillogly MA, Gershenson DM, Ioannides CG. Peptide priming of cytolytic activity to HER-2 epitope 369-377 in healthy individuals. Clin Cancer Res 2000;6:4192–200PubMedGoogle Scholar
  7. 7.
    Ioannides CG, Fisk B, Fan D, Biddison WE, Wharton JT, O’Brian CA. Cytotoxic T cells isolated from ovarian malignant ascites recognize a peptide derived from the HER2/neu proto-oncogene. Cell Immunol 1993;151:225–34PubMedCrossRefGoogle Scholar
  8. 8.
    Yoshino I, Goedegebuure PS, Peoples GE, et al. HER2/neu-derived peptide(s) are shared antigens among human non-small cell lung cancer and ovarian cancer. Cancer Res 1994;54:3387–90PubMedGoogle Scholar
  9. 9.
    Disis ML, Smith JW, Murphy AE, Chen W, Cheever MA. In vitro generation of human cytolytic T cells specific for peptide derived from the HER-2/neu protooncogene protein. Cancer Res 1994;54:1071–6PubMedGoogle Scholar
  10. 10.
    Linehan DC, Goedegebuure PS, Peoples GE, Rogers SO, Eberlein T. Tumor-specific and HLA-A2 restricted cytolysis by tumor-associated lymphocytes in human metastatic breast cancer. J Immunol 1995;155:4486–91PubMedGoogle Scholar
  11. 11.
    Woll MM, Hueman MT, Ryan GB, et al. Preclinical testing of a peptide-based, HER2/neu vaccine for prostate cancer. Int J Oncol 2004;25:1769–80PubMedGoogle Scholar
  12. 12.
    Fisk B, Blevins TL, Wharton JT. Identification of an immunodominant peptide of the HER-2/neu proto-oncogene recognized by ovarian tumor specific CTL lines. J Exp Med 1995;181:2709–17CrossRefGoogle Scholar
  13. 13.
    Peoples GE, Goedegebuure PS, Smith R, Linehan DC, Yoshino I, Eberlein TJ. Breast and ovarian cancer-specific cytotoxic T lymphocytes recognize the same HER2/neu-derived peptide. Proc Natl Acad Sci USA 1995;92:432–6PubMedCrossRefGoogle Scholar
  14. 14.
    Zaks TZ, Rosenberg SA. Immunization with a peptide epitope (P369-377) from HER-2/neu leads to peptide-specific cytotoxic T lymphocytes that fail to recognize HER-2/neu+ tumors. Cancer Res 1998;58:4902–8.PubMedGoogle Scholar
  15. 15.
    Knutson KL, Schiffman K, Cheever MA, Disis ML. Immunization of cancer patients with HER-2/neu, HLA-A2 peptide, p369-377, results in short-lived peptide-specific immunity. Clin Cancer Res 2002;8:1014–8PubMedGoogle Scholar
  16. 16.
    Murray JL, Gillogly ME, Przepiorka D, et al. Toxicity, immunogenicity, and induction of E75-specific tumor lytic CTLs by HER-2 peptide E74 (369-377) combined with granulocyte macrophage colony-stimulating factor in HLA-A2+ patients with metastatic breast and ovarian cancer. Clin Cancer Res 2002;8:3407–18PubMedGoogle Scholar
  17. 17.
    Brossart P, Wirths S, Stuhler G, Reichardt VL, Kanz L, Brugger W. Induction of cytotoxic T-lymphocyte responses in vivo after vaccinations with peptide-pulsed dendritic cells. Blood 2000;96:3102–8PubMedGoogle Scholar
  18. 18.
    Kono K, Takahashi A, Sugai H, et al. Dendritic cells pulsed with HER-2/neu-derived peptides can induce specific T-cell responses in patients with gastric cancer. Clin Cancer Res 2002;8:3394–400PubMedGoogle Scholar
  19. 19.
    Disis ML, Gooley TA, Rinn K, et al. Generation of T-cell immunity to the HER-2/neu peptide-based vaccines. J Clin Oncol 2002;20:2624–32PubMedCrossRefGoogle Scholar
  20. 20.
    Kono K, Halapi E, Hising C, et al. Mechanisms of escape from CD8+ T cell clones specific for the HER-2/neu proto-oncogene expressed in ovarian carcinomas: related and unrelated to decreased MHC class I expression. Int J Cancer 1997;70:112–9PubMedCrossRefGoogle Scholar
  21. 21.
    Maeurer MJ, Gollin SM, Martin D, et al. Tumor escape from immune recognition: lethal recurrent melanoma in a patients associated with downregulation of the peptide transporter protein TAP-1 and loss of expression of the immunodominant MART-1/Melan-A antigen. J Clin Invest 1996;98:1633–41PubMedCrossRefGoogle Scholar
  22. 22.
    Castilleja A, Ward NE, O’Brian CA, et al. Accelerated HER-2 degradation enhances ovarian tumor recognition by CTL: implications for tumor immunogenicity. Mol Cell Biochem 2001;217:21–33PubMedCrossRefGoogle Scholar
  23. 23.
    Sliwkowski MX, Lofgren JA, Lewis GD, Hotaling TE, Fendly BM, Fox JA. Nonclinical studies addressing the mechanism of action of trastuzumab (Herceptin). Semin Oncol 1999;26:60–70PubMedGoogle Scholar
  24. 24.
    Baselga J, Tripathy D, Mendelsohn J, et al. Phase II study of weekly intravenous recombinant humanized anti-p185 HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer. J Clin Oncol 1996;14:737–44PubMedGoogle Scholar
  25. 25.
    Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001;344:783–92PubMedCrossRefGoogle Scholar
  26. 26.
    Drebin JA, Link VC, Stern DF, Winberg RA, Greene M. Down-modulation of an oncogene protein product and reversion of the transformed phenotype by monoclonal antibodies. Cell 1985;41:697–706PubMedCrossRefGoogle Scholar
  27. 27.
    Hurwitz E, Stancovski I, Sela M, Yarden Y. Suppression and promotion of tumor growth by monoclonal antibodies to ErbB-2 differentially correlate with cellular uptake. Proc Natl Acad Sci USA 1995;92:3352–7Google Scholar
  28. 28.
    Klapper LN, Waterman H, Sela M, Yarden Y. Tumor-inhibitory antibodies to HER-2/erbB-2 may act by recruiting c-cbl and enhancing ubiquitination of HER-2. Cancer Res 2000;60:3384–8PubMedGoogle Scholar
  29. 29.
    Meyer zum Buschenfelde C, Hermann C, Schmidt B, Peschel C, Bernhard H. Antihuman epidermal growth factor receptor 2 (HER2) monoclonal antibody trastuzumab enhances cytolytic activity of class I-restricted HER2-specific T lymphocytes against HER2-overexpressing tumor cells. Cancer Res 2002;62:2244–7Google Scholar
  30. 30.
    Clynes RS, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nat Med 2000;6:443–6PubMedCrossRefGoogle Scholar
  31. 31.
    Gennari R, Menard S, Fagnoni F, et al. Pilot study of the mechanism of action of preoperative trastuzumab in patients with primary operable breast tumors overexpressing HER2. Clin Cancer Res 2004;10:5650–5PubMedCrossRefGoogle Scholar
  32. 32.
    Lee TD. Distribution of HLA antigens in North American Caucasians, North American Blacks and Orientals. In: Lee J, ed. The HLA System. Secaucus, NJ: Springer-Verlag, 1990Google Scholar
  33. 33.
    Brown RE, Bernath AM, Lewis GO. HER-2/neu protein-receptor-positive breast carcinoma: an immunologic perspective. Ann Clin Lab Sci 2000;30:249–58PubMedGoogle Scholar
  34. 34.
    Schubert U, Anton LC, Gibbs J, Norbury CC, Yewdell JW, Bennick JR. Rapid degradation of a large fraction of newly synthesized proteins by proteasomes. Nature 2000;404:770–3PubMedCrossRefGoogle Scholar
  35. 35.
    Woll MM, Fisher CM, Ryan GB, et al. Direct measurement of peptide-specific CD8+ T cells using HLA-A2:Ig dimer for monitoring the in vivo immune response to a HER2/neu vaccine in breast and prostate cancer patients. J Clin Immunol 2004;24:449–61PubMedCrossRefGoogle Scholar

Copyright information

© The Society of Surgical Oncology, Inc. 2006

Authors and Affiliations

  • Elizabeth A. Mittendorf
    • 1
    • 2
  • Catherine E. Storrer
    • 2
  • Craig D. Shriver
    • 1
    • 2
  • Sathibalan Ponniah
    • 2
  • George E. Peoples
    • 1
    • 2
    Email author
  1. 1.Clinical Breast Care Project, Department of SurgeryWalter Reed Army Medical CenterWashingtonUSA
  2. 2.National Naval Medical Center, Henry M. Jackson Foundation for the Advancement of Military MedicineClinical Breast Care Project Immunology & Research CenterBethesdaUSA

Personalised recommendations