[CANCER RESEARCH 58. 732-736. February 15. 1998] Her-2/neu-derived Peptides Are Tumor-associated Antigens Expressed by Human Renal Cell and Colon Carcinoma Lines and Are Recognized by in Vitro Induced Specific Cytotoxic T Lymphocytes1 Peter Brossart,2 Gernot Stuhler, Thomas Fiad, Stefan Stevanovic, Hans-Georg Rammensee, Lothar Kanz, and Wolfram Brugger Department of Hematology, Oncology and Immunology [P. B., G. S., T. F., L K., W. B.], and Institute for Cell Biology, Department of Immunology ¡S.S., H-C. R.J. University of Tübingen.Tübingen.Germany ABSTRACT The Her-2/neu oncogene encodes a ,U, 185,000 transmembrane protein with homology to the epidermal growth factor receptor. It is overex- pressed in 30-40% of breast and ovarian cancers, and this overexpression was shown to correlate with aggressiveness of malignancy and poor prognosis. Using tumor-associated lymphocytes isolated from patients with ovarian or breast cancer, several HLA-A2-restricted, Her-2/neu- derived peptides were identified. Further studies revealed that these tu mor-associated CTLs can also lyse other tumors, including non-small cell lung and pancreatic cancer cells, suggesting that Her-2/neu epitopes are shared between several distinct types of epithelial tumors. To analyze whether Her-2/neu epitopes are tumor-associated antigens for renal cell carcinoma (RCC) and colon carcinoma, we induced Her-2/neu peptide- specific CTL responses by primary in vitro immunization and used these CTLs to determine the presentation of Her-2/neu epitopes on human tumor lines. Autologous dendritic cells (DCs) generated from peripheral blood monocytes were pulsed with Her-2/neu-derived peptides E75 and GP2 and used as antigen-presenting cells for CTL priming. High CTL activity toward peptide-pulsed targets was obtained after two weekly restimulations. CTLs induced with DCs generated in the presence of IM n elicited a higher cytotoxic activity when they were stimulated with the cognate peptide than did CTLs induced with DCs grown in granulo- cyte macrophage colony-stimulating factor and interleukin 4 alone. The cytotoxicity of induced CTLs was antigen specific and HLA-A2 restricted. Furthermore, these CTLs lysed, in a MHC- and antigen-restricted fash ion, not only breast cancer cells but also colon carcinoma and RCC cell lines expressing Her-2/neu. The cytotoxic activity against tumor cells was blocked by cold HLA-A2-positive targets pulsed with the cognate peptide in cold target inhibition assay and by anti-HLA-A2 monoclonal Ab. These results suggest that epitopes derived from Her-2/neu protein might be attractive candidates for broadly applicable vaccines and may prove useful for adoptive immunothérapies designed for colon carcinoma or RCC. INTRODUCTION Peripheral CD8+ T cells recognize peptide antigens that are pre sented in the groove of MHC class I molecules on APCs.3 Recently, the definition of MHC class I allele-specific motifs allowed the definition of epitopes contained within a given antigen and provided new opportunities for the development of vaccine therapies (1-3). However, to date, with a few exceptions (melanoma-associated anti- Received 9/29/97; accepted 12/16/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported in part by Deutsche Forschungsgemeinschaft Grant SFB 510 and by a grant from Deutsche Krebshilfe. - To whom requests for reprints should be addressed, at Department of Hematology. Oncology, and Immunology, University of Tübingen,Otfried-Müller-Strasse-10, D-72076 Tübingen,Germany. Fax: 49-7071293671. 1The abbreviations used are: ARC, antigen-presenting cell; DC, dendritic cell; RCC, renal cell carcinoma; PBMNC. peripheral blood mononuclear cell; GM-CSF. granulocyte macrophage colony-stimulating factor; IL, interleukin: TNF-o, tumor necrosis factor-a; niAb, monoclonal antibody; IMP, influenza matrix protein. gens), there is only limited information about the identity of CTL epitopes presented by human malignant cells (4). The Her-2/neu oncogene is a Mr 185,000 transmembrane protein with tyrosine kinase activity and extensive homology to the epidermal growth factor receptor (5, 6). It is overexpressed in 30-40% of breast and ovarian cancers, and this overexpression was shown to correlate with aggressiveness of malignancy and poor prognosis (7, 8). Using tumor-associated lymphocytes isolated from patients with ovarian or breast cancers, two HLA-A2 restricted Her-2/neu-derived peptides were identified (E75 and GP2; Refs. 9 and 10). Further studies revealed that these tumor-associated lymphocytes can also lyse other tumors, including non-small cell lung and pancreatic cancer cells, suggesting that Her-2/neu epitopes are shared between several distinct types of epithelial tumors and can be an appropriate candidate for broadly applicable vaccine therapies (11-13). In vitro immunization methods using epitopes derived from self- antigens have often proved unsatisfactory because of the low affinity of the elicited CTLs and, consequently, the lack of a sufficient recognition of naturally processed antigens by these CTLs (14, 15). Presentation of antigens by professional APCs may be critical for the effectiveness of an induced immune response, and the nature of the APCs can determine the outcome, ranging from immunity to tolerance (16). DCs are key regulators in immune responses, being capable of priming naive resting T cells and initiating primary T cell responses when they are pulsed with antigenic peptides or proteins (17-26). Here, we demonstrate that DCs pulsed with Her-2/neu-derived peptides E75 and GP2 elicited a peptide-specific CTL response by primary in vitro immunization in a culture system using peripheral blood from a normal individual. These CTLs showed an antigen- specific and HLA-A2-restricted lysis of target cells coated with the antigenic peptide and established tumor cell lines expressing Her-2/ neu, including colon carcinoma and RCC. These results suggest that epitopes derived from Her-2/neu protein may prove useful for adop tive immunothérapiesdesigned for colon carcinoma or RCC. MATERIALS AND METHODS Tumor Cell Lines. Tumor cell lines used in these experiments were grown in RP10 medium (RPMI 1640 supplemented with 10% heat-inactivated PCS, 2 min L-glutamine, 50¿IM2-mercaptoethanol, and antibiotics). HLA-A2 ex pressing tumors were as follows: MB-MDA-231 (breast cancer), MCF7 (breast cancer), A-498 (RCC), HCT116 (colon carcinoma), MKR (malignant mela noma, kindly provided by U. Keilholz, University of Heidelberg, Heidelberg, Germany), and T2 (174XCEM.T2 hybridoma, TAPI and TAP2 deficient). Croft (HLA-A2) is an EBV-immortalized B-cell line that was kindly donated by O. J. Finn (University of Pittsburgh School of Medicine, Pittsburgh. PA). TW-33 (RCC) was kindly provided by G. Müller(University of Göttingen, Göttingen. Germany). SK-OV-3 (HLA-A3) is an ovarian cell line that over- expresses HER-2/neu. Cell Isolation and Cultures. Generation of DCs from peripheral blood monocytes was performed as described previously (26, 27). In brief, PBMNCs were isolated by Ficoll/Paque (Life Technologies, Inc., Grand Island, NY) 732 on June 1, 2015. © 1998 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from