IDENTIFICATION OF HLA-A*0201-RESTRICTED CTL EPITOPES ENCODED BY THE TUMOR-SPECIFIC MAGE-2 GENE PRODUCT Marjan J.W. VISSEREN 1 , Sjoerd H. VAN DER BURG 1 , Ellen I.H. VAN DER V OORT 1 , Remco M.P. BRANDT 1 , Peter I. SCHRIER 2 , Pierre VAN DER BRUGGEN 3 , Thierry BOON 3 , Cornelis J.M. MELIEF 1 * and W. Martin KAST 4 1 Department of Immuno-hematology and Blood Bank, Leiden University Hospital, the Netherlands 2 Department of Clinical Oncology, Leiden University Hospital, the Netherlands 3 Ludwig Institute for Cancer Research, Brussels, Belgium 4 Cardinal Bernardin Cancer Center, Loyola University of Chicago, Maywood, IL, USA M AGE-2 isexpressed in many tumors, including melanoma, laryngeal tumors, lung tumors and sarcomas, but not in healthy tissue, with the exception of testis. Thus, M AGE-2- derived peptides that bind to H LA class I molecules and elicit cytotoxic T lymphocyte (CT L) responses could be of signifi- cant therapeutic importance. In this study, we show that several M AGE-2-derived peptides bind with high affinity to HLA-A*0201. Three of them form complexes with HLA- A*0201 that are stable at 37°C and are immunogenic in HLA-A*0201K b transgenic mice. Moreover, CT Ls against 2 of them (M2 112-120, and M2 157-166) specifically recognize cells that express both the M AGE-2 protein and HLA- A*0201K b . These 2 peptides are processed and presented in the context of HLA-A*0201. Therefore, these peptides are candidate components in peptide-based vaccines for the treatment and prevention of several types of M AGE-2- expressing cancers. Int. J. Cancer 73:125–130, 1997.  1997 Wiley-Liss, Inc. Cytotoxic T lymphocyte (CTL) responses are important in the defense against viruses and tumors (Melief and Kast, 1992). CTLs recognize small peptides that are derived from cellular proteins and are presented by MHC class I molecules at the cell surface (Townsend et al., 1986). Tumor cells do express tumor-specific proteins that can potentially be recognized by CTLs capable of eliminating the tumor cells, but such a CTL response is not always elicited for a number of reasons. Tumor cells can use several mechanisms to evade a CTL response, such as down-modulation of MHC class I molecules (Ruiter et al., 1984), secretion of cytokines that frustrate CTL activation (Torre-Amione et al., 1990) and down-modulation of the CTL -chain (Mizoguchi et al., 1992). Suboptimal expression of tumor-specific peptides also precludes induction of a CTL response. This can be circumvented by forced induction of a CTL-mediated anti-tumor response, for example by vaccination with killed tumor cells or tumor specific peptides, or by other modes of specific vaccination, either with or without added cytokines. Once insufficient immunogenicity of tumor cells has been overcome by vaccination, effective CTL-mediated immunity can be demonstrated (Visseren et al., 1994). Peptide-based vaccines preferably contain peptides derived from tumor-specific proteins that are processed and presented by MHC class I molecules on the cell surface of the tumor cells. One example of a possibly useful tumor-specific protein is the MAGE-2 gene product. It is expressed in 70% of metastatic melanomas but also in other types of tumors such as 46% of laryngeal tumors and 35% of lung tumors and sarcomas (De Smet et al., 1994). Moreover, normal fetal and adult tissues do not express it, with the exception of testis tissue (De Plaen et al., 1994). Furthermore, other members of the MAGE gene family, most of which are expressed less frequently, have been shown to give rise to peptides that are processed and presented in MHC class I (Traversari et al., 1992). It is therefore conceivable that MAGE-2 derived peptides are also presented in HLA class I molecules. Since HLA-A*0201 is a very common HLA type (Imanishi et al., 1992), a vaccine based on MAGE-2-derived HLA-A*0201 binding peptides could be of therapeutic importance. Immunogenicity of HLA-A*0201 binding peptides, and their natural presence in cells expressing both antigen and appropriate HLA, can be studied in HLA-A*0201K b transgenic mice (Vitiello et al., 1991). These mice express the chimeric HLA-A*0201K b molecule that consists of the human 1 and 2 domains of HLA-A*0201, forming the peptide-binding groove, and the 3 domain of H2-K b , allowing binding of murine CD8. This chimeric molecule enables murine CD8 + CTL to interact in a physiological way with HLA-A*0201-peptide complexes. Murine peptide- specific CTL can subsequently be tested on cells expressing both HLA-A*0201K b and antigen, to study natural processing of the peptides investigated. In this study, we have identified 7 MAGE-2-derived peptides that bind with sufficient affinity to HLA-A*0201. Three of them are able to form stable complexes with this MHC class I molecule at physiological temperature, which is an important feature in vivo. These peptides were capable of eliciting a CTL response in HLA-A*0201K b transgenic mice. Furthermore, we observed that at least 2 of these peptides are processed and presented by HLA- A*0201. Therefore, these are candidate peptides in vaccines for treatment and prevention of several types of MAGE-2-expressing cancers. MATERIAL AND METHODS Cell culture The EBV-transformed B cell line JY, homozygous for HLA- A*0201, was cultured in RPMI 1640 (Life Technologies, Paisley, UK) supplemented with penicillin (100 IU/ml, Brocades Pharma, Leiderdorp, the Netherlands), L-glutamine (2 mM, ICN Biochemi- cals, Costa Mesa, CA), and 8% heat-inactivated FCS (Hyclone, Logan, UT). Jurkat A*0201K b cells are derived from the human T cell line Jurkat upon transfection with the chimeric HLA-A*0201K b gene (Vitiello et al., 1991). Jurkat A*0201K b and COS-7 cells were cultured in Iscove’s modified Dulbecco’s medium with Glutamax I (IMDM; Life Technologies) supplemented with penicillin, 8% heat-inactivated FCS and 20 μM 2-ME (Merck, Darmstadt, Ger- many). WEHI 164 clone 13 cells were cultured in RPMI 1640 supplemented with 8% heat-inactivated FCS, penicillin, L- glutamine (216 mg/ml), L-asparagine (36 mg/ml) and L-arginine- HCl (116 mg/ml). The MAGE-3-specific CTL clone LB705-CTL 297/22 (van der Bruggen et al., 1994) was cultured using standard CTL culture procedures. Synthetic peptides Peptides were made by Fmoc chemistry with the multiple peptide synthesizer AMS 422 (Abimed, Langenfeld, Germany). Upon synthesis peptides were analyzed by reversed phase HPLC, Contract grant sponsor: Macropa Stichting; Contract grant sponsor: Dutch Cancer Society; Contract grant number: NKB 91-15. *Correspondence to: Department of Immunohematology and Blood Bank, Building 1, E3-Q, Leiden University Hospital, PO Box 9600, 2300 RC Leiden, the Netherlands. Fax: +31 5216751. E-mail: ihbsecr@euronet.nl Received 6 March 1997; Revised 24 May 1997 Int. J. Cancer: 73, 125–130 (1997)  1997 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer