[CANCER RESEARCH 59, 4056–4063, August 15, 1999] Identification of a Gene Coding for a Protein Possessing Shared Tumor Epitopes Capable of Inducing HLA-A24-restricted Cytotoxic T Lymphocytes in Cancer Patients1 Damu Yang, Masanobu Nakao, Shigeki Shichijo, Teruo Sasatomi, Hideo Takasu, Hajime Matsumoto, Kazunori Mori, Akihiro Hayashi, Hideaki Yamana, Kazuo Shirouzu, and Kyogo Itoh2 Cancer Vaccine Development Division, Kurume University Research Center for Innovative Cancer Therapy [D. Y., M. N., K. I.], and Departments of Surgery [A. H., H. Y., K. S.], Immunology [S. S., T. S., H. T., H. M., K. I.], and Otolaryngology [K. M.], Kurume University School of Medicine, Kurume, 830-0011, Japan ABSTRACT we report a gene encoding epitopes that are capable of inducing CTLs in PBMCs of patients with SCCs and adenocarcinomas. Genes encoding tumor epitopes that are capable of inducing CTLs against adenocarcinomas and squamous cell carcinomas, two major hu- man cancers histologically observed in various organs, have rarely been MATERIALS AND METHODS identified. Here, we report a new gene from cDNA of esophageal cancer cells that encodes a shared tumor antigen recognized by HLA-A2402- Generation of HLA-A2402-restricted CTLs. HLA-A2402-restricted and restricted and tumor-specific CTLs. The sequence of this gene is almost tumor-specific CTLs were established from the PBMCs of an esophageal identical to that of the KIAA0156 gene, which has been registered in cancer patient (HLA-A2402/A2601) by the standard method of mixed lym- phocyte tumor cell culture, as reported previously (29). Briefly, the patient’s GenBank with an unknown function. This gene encodes a Mr 140,000 protein that is expressed in the nucleus of all of the malignant tumor cell PBMCs were repeatedly stimulated with the autologous tumor cell line (KE4) lines tested and the majority of cancer tissues with various histologies, in the culture medium [45% RPMI 1640, 45% AIM-V medium (Life Tech- including squamous cell carcinomas, adenocarcinomas, melanomas, and nologies, Inc., Grand Island, NY), and 10% FCS (Equitech Bio, Ingram, TX) leukemia cells. However, this protein was undetectable in the nucleus of with 100 units/ml interleukin 2 (Shiongi Pharmaceutical Co., Osaka, Japan)]. any cell lines of nonmalignant cells or normal tissues, except for the testis. The CTLs were tested for cytotoxicity to various cancer cells, EBV-trans- 51 Furthermore, this protein was expressed in the cytosol of all of the formed B cells, and normal cells in a 6-h Cr release assay, as reported proliferating cells, including normal cells and malignant cells, but not in previously (29), at various E:T ratios. normal tissues, except for the testis and fetal liver. Two peptides of this Identification of the Clone 13 Gene. Previously reported expression gene cloning methods were used in this study to identify a gene coding for tumor protein were recognized by HLA-A2402-restricted CTLs and were able to 1 induce HLA-A24-restricted and tumor-specific CTLs from peripheral antigen recognized by CTLs (25). In brief, poly(A) RNA of the KE4 tumor 1 cells was converted to cDNA, ligated to the SalI adapter, and inserted into the blood mononuclear cells of most of HLA-A24 cancer patients tested, but expression vector pSV-SPORT-1 (Life Technologies, Inc., Gaithersburg, MD). not from peripheral blood mononuclear cells of any healthy donors. These 1 cDNA of HLA-A2402 or HLA-A0201 was obtained by reverse transcription- peptides may be useful in specific immunotherapy for HLA-A24 cancer PCR and was cloned into the eukaryotic expression vector pCR3 (Invitrogen, patients with various histological types. San Diego, CA). Both 200 ng of plasmid DNA pools or clones of the KE4 cDNA library and 200 ng of the HLA-A2402 cDNA were mixed with 1 mlof Lipofectin in 70 ml of Opti-MEM (Life Technologies, Inc.) for 15 min. Thirty INTRODUCTION ml of the mixture were then added to the VA13 (2 3 104) cells and incubated for 5 h. Next, 200 ml of RPMI 1640 containing 10% FCS were added and Many genes encoding tumor antigens and peptides that are recog- cultured for 2 days followed by the addition of CTLs (104 cells/well). After an nized by CTLs have been identified from melanoma cDNA (1–15). 18-h incubation, 100 ml of supernatant were collected to measure IFN-g by an Some of these peptides can induce CTLs that recognize tumor cells ELISA kit in a duplicate assay, as reported previously (25). This concentration from PBMCs3 of melanoma patients (16–20). Immunotherapy with (100 ng/ml) of HLA-A2402 cDNA was chosen based on the fact that expres- some of the peptides that are capable of inducing HLA class I- sion levels of HLA-A24 antigens on the surface of VA13 cells transfected with restricted CTLs has been shown to result in tumor regression in 50, 100, 200, and 400 ng/ml of HLA-A2402 cDNA were 35, 42, 40, and 20% 1 by FACScan analysis with anti-HLA-A24 mAb, respectively. DNA sequenc- HLA-A0201 melanoma patients (21, 22). These results indicate that ing was performed with dideoxynucleotide sequencing method using a DNA identification of the peptides capable of inducing CTLs may provide Sequence kit (Perkin-Elmer Corp., Foster, CA), and the sequence was analyzed a new modality of cancer therapy. However, only a few tumor by the ABI PRISM 377 DNA Sequencer (Perkin-Elmer). rejection antigen genes have been identified from the adenocarcino- Northern Blot Analysis. Nylon membranes (Hybond-N1; Amersham, mas and SCCs, which are, histologically, the most frequently ob- Buckinghamshire, United Kingdom) with UV-fixed total RNA (5 mg/lane) served cancers in various organs (23–28). In addition, peptides capa- from various tissues were prehybridized for 20 min and hybridized overnight ble of inducing CTLs against these cancers have not yet been fully at 65°C in the same solution [7% SDS, 1 mM EDTA, and 0.5 M Na2HPO4 (pH 32 identified. We have recently reported a SART1 gene coding for tumor 7.2)], containing the P-labeled clone 13 as a probe. The membranes were washed three times at 65°C in a washing buffer [1% SDS and 40 mM Na HPO antigens and peptides from the cDNA of SCCs (25). Here, we inves- 2 4 (pH 7.2)] and then autoradiographed. We tentatively named this full-length tigated new genes encoding CTL-directed antigens from SCCs, and gene encoding a tumor antigen recognized by HLA-A2402 restricted CTLs the SART3 gene. The relative expression level of the SART3 mRNA was calculated Received 3/22/99; accepted 6/18/99. by the following formula: index 5 (SART3 density of a sample/b-actin density The costs of publication of this article were defrayed in part by the payment of page of a sample) 3 (b-actin density of the KE4 tumor/SART3 density of the KE4 charges. This article must therefore be hereby marked advertisement in accordance with tumor). 18 U.S.C. Section 1734 solely to indicate this fact. Cloning of the SART3 Gene. The SART3 gene was obtained from the 1 This study was supported in part by Ministry of Education, Science, Sport and Culture (Japan) Grants-in-Aid 08266266, 09470271, 10153265, 09770985, 10671230, cDNA libraries of both KE4 and PBMCs of healthy donors by the standard 32 and 09671401 and Ministry of Health and Welfare (Japan) Grant H10-genome-003. colony hybridization method using the P-labeled clone 13 as a probe, as 2 To whom requests for reprints should be addressed, at Department of Immunology, reported previously (25). The differences in sequence at nucleotide positions Kurume University School of Medicine, 67 Asahi-machi Kurume, Fukuoka 830-0011, 88 and 547 of SART3 between the PBMCs and KE4 were further analyzed by Japan. Phone: 81-942-31-7551; Fax: 81-942-31-7699; E-mail: [email protected] treatment of the PCR products with restriction enzyme DdeI and AluI, respec- u.ac.jp. 9 3 The abbreviations used are: PBMC, peripheral blood mononuclear cell; SCC, squa- tively. The primers used for the amplification were: 5 -CGAAACCTCGGCT- mous cell carcinoma; SART, SCC antigen recognized by T cells; ORF, open reading TCAGAAC-39 (forward)/59-GGTCTTGTATGTCGCAGCGG-39 (reverse) frame; Ab, antibody; mAb, monoclonal Ab; PHA, phytohemagglutinin; aa, amino acid. and 59-GATGGCCTGGACAGAGAGC-39 (forward) and 59-CCACCAACT- 4056 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1999 American Association for Cancer Research. A GENE CODING FOR A SHARED TUMOR ANTIGEN GAGTACTGGCC-39 (reverse), which covered the regions around positions 88 the expanded cells were transferred to 24-well plate and incubated in the and 547, respectively. The PCR was carried out for 45 cycles of 0.5 min at absence of either a peptide or feeder cells for 14–20 days. Their CTL activity 94°C and 1 min at 60°C using the AmpliTaq Gold DNA polymerase (Perkin- was rechecked by an IFN-g production assay, and the cells were then tested for Elmer). their cytotoxicity by a 6-h 51Cr release assay at different E:T ratios. Preparation of the SART3-tag Fusion Protein in Expression Vector Constructs. For preparation of SART3/myc, SART3 cDNA was digested with RESULTS EcoRI and BstEII. The SART3 gene at positions 2390–2895 was amplified by reverse transcription-PCR using 59-GGTGTTCAGGTACAGCACTTCC-39 Establishment of HLA-A24-restricted CTLs. A HLA-A2402- 9 9 (forward) and 5 -GCTTGGCAAACTCGAGATTGGACATC-3 (reverse), and restricted and tumor-specific CTL line was established from PBMCs the amplified product was digested with BstEII and XhoI. The EcoRI-BstEII of an esophageal cancer patient (KE4, HLA-A2402/2601) by repeated and BstEII-XhoI fragments were then together ligated to the EcoRI and XhoI stimulation with KE4 autologous tumor cells, as reported previously sites of pcDNA3.1/Myc-His A vector (Invitrogen, San Diego, CA).