[CANCER RESEARCH 63, 7920 –7925, November 15, 2003] Deoxyribonucleic Acid (DNA) Encoding a Pan-Major Histocompatibility Complex Class II Peptide Analogue Augmented Antigen-specific Cellular Immunity and Suppressive Effects on Tumor Growth Elicited by DNA Vaccine Immunotherapy Koji Teramoto, 1 Keiichi Kontani, 1 Yoshitomo Ozaki, 1 Satoru Sawai, 1 Noriaki Tezuka, 1 Toshi Nagata, 4 Shozo Fujino, 1 Yasushi Itoh, 2 Osamu Taguchi, 5 Yukio Koide, 4 Tohru Asai, 1 Iwao Ohkubo, 3 and Kazumasa Ogasawara 2 Departments of 1 Surgery, 2 Pathology, and 3 Medical Biochemistry, Shiga University of Medical Science, Otsu; 4 Department of Microbiology Immunology, Hamamatsu University School of Medicine, Hamamatsu; and 5 Division of Molecular Pathology, Aichi Cancer Center Research Institute, Nagoya, Japan ABSTRACT Vaccine immunotherapy must induce helper and cytotoxic cell-medi- ated immunity to generate the powerful antitumor immune responses needed to suppress cancer progression. We reported previously that a 16-amino acid peptide analogue derived from pigeon cytochrome c can bind broad ranges of MHC class II types and activate helper T cells in mice. To determine whether DNA encoding the Pan-MHC class II IA peptide (Pan-IA) can increase the efficacy of tumor suppression by DNA vaccine immunotherapy targeting tumor antigens, Pan-IA DNA was ad- ministered with ovalbumin (OVA) DNA to C57BL/6 mice bearing the OVA-expressing tumor cell line E.G7. Specific proliferative responses to and cytotoxic activities against OVA-expressing targets were induced in mice vaccinated with both OVA and Pan-IA DNA but not in those vaccinated with OVA DNA alone or control DNA plus Pan-IA DNA. Growth of E.G7 cells was suppressed only by combined vaccination with OVA and Pan-IA DNA, and tumors in five of the nine mice that received this combined vaccination were eradicated completely. In those mice, the frequency of CD8-positive T cells reactive with OVA 257–264 peptides in the context of H-2K b was significantly increased in the tumor site. Further- more, immunofluorescent study of the inoculated tumors revealed in- creased accumulation of both CD4- and CD8-positive T cells producing IFN- in the tumor only by this vaccine protocol. The data suggest that Pan-IA DNA can augment suppressive effects of DNA vaccines on tumor growth by increasing numbers of antigen-specific CTLs and helper T cells. This is the first study in which established tumors have been eradicated successfully by vaccination with DNA corresponding to CTL epitopes and helper T cell epitopes. Our animal model may contribute to the develop- ment of therapeutic DNA vaccines against cancer. INTRODUCTION To obtain sufficiently powerful antitumor immunity to suppress cancer progression by vaccine immunotherapy, not only cytotoxic but also helper T cell function must be activated (1– 4). Some helper epitopes have been identified in the same or different molecules in which CTL epitopes are located, and they increase the efficacy of cytotoxic activity against tumor cells (5–9). Vaccine immunotherapy for cancer would be ideal if a single helper epitope could enhance any antigen-specific cytotoxic activity of CTLs. We reported previously that a peptide analogue, AEGFSYTVANKAKGIT, which was pre- pared from the pigeon cytochrome c (p43–58) with a two-residue substitution (D to V at 50 and N to A at 54), efficiently stimulated T lymphocytes and that it could be presented by various types of mouse MHC class II molecules (IA b,d,q,s ; Refs. 8 and 9). This peptide analogue should be useful in vaccine immunotherapy for cancer as an adjuvant or a helper T cell activator to efficiently elicit antitumor immunity. Vaccination with DNA encoding tumor antigens enables mainte- nance of a high level of tumor antigen expression at the vaccination site and results in the elicitation of both humoral immunity and cellular immunity specific for DNA-encoding antigens (10 –12). Fur- thermore, DNA vaccines are inexpensive and simple to use once a DNA vector is constructed, and they do not require adjuvants. Thus, DNA vaccines should be more applicable to clinical cancer immuno- therapy than peptide or cancer cell vaccines or adoptive effector cell transfer immunotherapy. We reported previously that DNA vaccines targeting MUC1 tumor antigen could not eradicate MUC1-positive tumors in mice although the vaccine elicited strong anti-MUC1 im- munity (13). This finding suggested that cytotoxic cell-mediated im- mune responses induced by the vaccine should be enhanced. The aim of the present study was to determine whether antitumor immunity induced by DNA vaccines targeting tumor antigens can be sufficiently augmented to suppress tumor growth in vivo by covacci- nation with DNA encoding a Pan-IA peptide analogue. Here, we describe a new DNA vaccine protocol that considerably enhances anticancer immunity in a murine model. MATERIALS AND METHODS Cells and Mice. Female C57BL/6 mice, 6 – 8 weeks of age, were purchased from CLEA Japan Inc. (Tokyo, Japan) and maintained under specific patho- gen-free conditions. Murine lymphoma cell lines EL4 and E.G7, generated by transducing the chicken OVA 6 gene into EL4 cells, were purchased from American Type Culture Collection (Manassas, VA). These cells were maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mML-glutamine, 100 units/ml penicillin G, and 0.1 mg/ml streptomycin (all from Life Technologies, Inc., Tokyo, Japan) in a humidified atmosphere of 5% CO 2 at 37°C. Antibodies. Anti-OVA polyclonal antibody was provided by Cortex Bio- chem, Inc. (San Leandro, CA). Horseradish peroxidase-conjugated antirabbit and antimouse immunoglobulin antibodies were purchased from ICN Pharma- ceutical Inc. (Aurora, OH). Antimouse CD4 (L3T4) and CD8 (Lyt-2.2) mono- clonal antibodies were purified by protein A-Sepharose column chromatogra- phy (Zymed Laboratories, Inc., San Francisco, CA) from the ascites of female severe combined immunodeficient mice (Charles River Inc., Hino, Japan) that had received i.p. inoculations of the hybridoma cell lines GK1.5 and 2.43 (American Type Culture Collection), respectively. Mouse IgG was purchased from DAKO (Kyoto, Japan). FITC-conjugated antimouse CD8 monoclonal antibody, PE-conjugated antimouse CD4 and CD8 monoclonal antibodies, and FITC-conjugated anti-IFN- monoclonal antibodies were purchased from PharMingen (Tokyo, Japan). Received 11/15/02; revised 7/17/03; accepted 9/4/03. Grant support: Grants-in-Aid for Scientific Research (10671249, 13671380, 14571262, and 15591340) from the Ministry of Education, Science, Sports and Culture, Japan. 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. Requests for reprints: Keiichi Kontani, Department of Surgery, Shiga University of Medical Science, Seta-tsukinowa, Otsu 520-2192, Japan. Phone: 81-077-548-2244; Fax: 81-077-544-2901; E-mail: konbat@belle.shiga-med.ac.jp. 6 The abbreviations used are: OVA, ovalbumin; CD, cluster of differentiation; IL, interleukin; PE, phycoerythrin. 7920 Research. on January 26, 2016. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from