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Induction of Cellular Immune Responses to Tumor Cells and Peptides in Colorectal Cancer Patients by Vaccination with SART3 Peptides1

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Induction of Cellular Immune Responses to Tumor Cells and Peptides in Colorectal Cancer Patients by Vaccination with SART3 Peptides1 3950 Vol. 7, 3950–3962, December 2001 Clinical Cancer Research Induction of Cellular Immune Responses to Tumor Cells and Peptides in Colorectal Cancer Patients by Vaccination with SART3 Peptides1 Yoshiaki Miyagi,2 Nobue Imai,2 Teruo Sasatomi, eral subsequent clinical studies on melanoma patients have Akira Yamada, Takashi Mine, Kazuko Katagiri, shown increased immune responses to both the vaccinated pep- 4 Masami Nakagawa, Akira Muto, Shinya Okouchi, tides and tumor cells in PBMCs during the postvaccination periods (7–11). To our knowledge, however, there have been no Hiroharu Isomoto, Kazuo Shirouzu, previous reports on peptide-based immunotherapy for colorectal 3 Hideaki Yamana, and Kyogo Itoh cancer patients. Colorectal cancer is one of the most commonly Departments of Surgery [Y. M., T. S., T. M., H. I., K. S., H. Y.] and occurring malignancies in the world, and the prognosis of ad- Immunology [N. I., A. Y., K. K., A. M., S. O., K. I.], Kurume vanced colorectal cancer with distant metastasis is extremely University School of Medicine, and Cancer Vaccine Development Division, Research Center for Innovative Cancer Therapy [A. Y., poor, despite recent clinical trials with chemotherapeutic agents M. N., K. I.], Kurume University, 830-0011 Fukuoka, Japan (12–14). Therefore, the development of new treatment modali- ties, one of which should be specific immunotherapy, is needed. We reported previously that the squamous cell carcinoma anti- ABSTRACT gen recognized by T cells 3 (SART3) is expressed in the The tumor-rejection antigen SART3 possesses two an- majority of colorectal cancers and that two to three SART3- tigenic epitopes (SART3109–118 and SART3315–323) capable derived peptides have the ability to induce CTLs from PBMCs of inducing HLA-A24-restricted and tumor-specific CTLs. of the majority of HLA-A24ϩ and HLA-A2ϩ cancer patients, To determine its safety and ability to generate antitumor including those of colorectal cancer patients (15–17). The immune responses, 12 patients with advanced colorectal SART3 peptides, however, did not induce CTLs from PBMCs cancer were administered s.c. vaccinations of these peptides. of healthy donors. We have also reported recently that SART3 No severe adverse events were associated with the vaccina- plays an important role in the splicing of mRNA, although the tions. Significant levels of increased cellular immune re- physiological function of SART3 is not fully defined (18). In ؉ sponses to both HLA-A24 colon cancer cells and the vac- this report, we describe the cellular and humoral immune re- cinated peptide were observed in the postvaccination sponses in HLA-A24ϩ advanced colorectal cancer patients vac- peripheral blood mononuclear cells in 7 of 11 and 7 of 10 cinated with the two SART3 peptides combined with IFA. patients tested, respectively, and the higher responses were observed in those patients vaccinated with the highest dose (3 mg/injection) of the peptides. These results encourage PATIENTS AND METHODS further development of SART3 peptide vaccine for colorec- Peptide Selection. The peptides used in the present tal cancer patients. study were prepared under conditions of Good Manufactur- ing Practice by the MULTIPLE PEPTIDE SYSTEMS (San INTRODUCTION Diego, CA): SART3-derived peptide at sequence positions Recent advances in the areas of molecular biology and 109–118 (SART3109–118, VYDYNCHVDL) and 315–323 cellular immunology in the field of tumor immunology have (SART3315–323, AYIDFEMKI). These two peptides have the resulted in the identification of a large number of antigens and ability to induce HLA-A24-restricted CTL activity in PBMCs epitopes recognized by HLA class I-restricted CTLs from mel- of the majority of cancer patients tested, as reported elsewhere (15). anomas and epithelial cancers (1–6), thereby opening the door SART3109–118 and SART3315–323 were dissolved in DMSO to new peptide-based specific immunotherapy of cancers. Sev- at 6.7 mg/ml and trifluoracetate at 200 mg/ml, respectively, aseptically aliquoted, and stored at Ϫ80°C. Stock solutions were diluted with saline for SART3109–118 or with 0.12 N NaOH-saline for SART3315–323 just before use. The pH of the Received 6/19/01; revised 8/27/01; accepted 8/28/01. SART3315–323 solution was adjusted from 8.0 to 8.2. Other The costs of publication of this article were defrayed in part by the peptides used for in vitro assays as negative controls were payment of page charges. This article must therefore be hereby marked an HIV-derived peptide with an HLA-A24-binding motif advertisement in accordance with 18 U.S.C. Section 1734 solely to (RYLRQQLLGI) and an Lck-derived peptide at position 488–497 indicate this fact. 1 Supported in part by Grants-in-Aid from the Ministry of Education, Science, Sport, Culture and Technology and Grant H10-genome-003 from the Ministry of Health and Welfare, Japan. 2 These authors contributed equally to this work. 3 To whom requests for reprints should be addressed, at Department of 4 The abbreviations used are: PBMC, peripheral blood mononuclear Immunology, Kurume University School of Medicine, 67 Asahi Machi, cell; IFA, incomplete Freund’s adjuvant; mAb, monoclonal antibody; Kurume 830-0011, Japan. Phone: 81-942-31-7551; Fax: 81-942-31- CEA, carcinoembryonic antigen; PHA, phytohemagglutinin; CT, com- 7699; E-mail: [email protected]. puted tomography; PD, progressive disease. Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2001 American Association for Cancer Research. Clinical Cancer Research 3951 Table 1 Demographics and adverse events of SART3 vaccination Clinical response Time to Overall Group Peptidea Injection Within Within 5 progression survival (patient no.) (dose in mg) timeb Age/Sex PS Stagec Adverse eventsd (grade) 5 weeks months (wk) (wk) e ϩ 1 (001) SART3109–118 (0.3) 24th 47/M 0 IV Local redness/itching (1) SD PD 22 84 ϩ 1 (002) SART3109–118 (0.3) 19th 53/M 0 IV Local redness/itching (1) SD PD 23 83 1 (004) SART3109–118 (0.3) 2nd 44/F 1 IV None 3 SART3315–323 (0.3) 1 (005) SART3109–118 (0.3) 3rd 67/M 1 IV None SD PD 7 12 ϩ 2 (006) SART3109–118 (1) 17th 49/M 0 IIIb None SD SD 39 61 SART3315–323 (1) ϩ 2 (007) SART3109–118 (1) 3rd 64/F 0 IIIb None SD PD 8 50 SART3315–323 (1) ϩ 2 (008) SART3109–118 (1) 8th 76/M 0 IIIa None SD PD 8 42 SART3315–323 (1) 2 (009) SART3109–118 (1) 8th 72/M 1 IV Local redness/itching (1) PD PD 5 16 SART3315–323 (1) 2 (010) SART3109–118 (1) 11th 58/M 0 IIIa None SD PD 6 37 SART3315–323 (1) ϩ 3 (011) SART3109–118 (3) 6th 53/F 0 IIIa Local redness/itching (1) SD PD 6 30 SART3315–323 (3) 3 (012) SART3109–118 (3) 6th 74/F 1 IV Local redness/itching (1) SD PD 6 15 SART3315–323 (3) 3 (013) SART3109–118 (3) 4th 72/M 0 IV Local redness/itching (1) PD PD 5 37 SART3315–323 (3) a Each of the patients received s.c. injections of the SART3 peptides. b Number of vaccination after which the sample was obtained. c Union International Contre Cancer-Tumor Node Metastasis classification of malignant tumors was used for determination of clinical stage. PS, pathological stage. d Toxicity-grading criteria of the Japanese Clinical Oncology Group (22) were used for grading adverse events. e SD, stable disease. (Lck488–497, DYLRSVLEDF), which has been shown to induce injected into the s.c. tissue of the anterior thigh of patients in HLA-A24-restricted and tumor-specific CTLs (19). groups 1 and 2, respectively. For group 3, 2 ml of the peptide Eligibility Criteria and Clinical Protocol. Twelve pa- solution at 2 mg/ml were mixed with an equal volume of IFA tients with advanced colorectal cancer (all adenocarcinomas: and emulsified in the 5-ml glass syringes, and 3 ml were injected inoperable stages IIIa, IIIb, and IV and relapse cases) received into the s.c. tissue. It was intended that all patients would receive the SART3 vaccination, and their characteristics are shown in at least three vaccinations at 2-week intervals; vaccinations after Table 1. All patients, except a patient who did not receive any the first three continued based primarily on patient requests, on chemotherapy, had failed to respond to previous fluorouracil- clinical status evaluated by attending physicians, and on the based chemotherapy treatments. The sites of tumors were the results from immunological analyses. To study the immune ascending colon (patients 008 and 012 in Table 1), sigmoid responses, 30 ml of peripheral blood were withdrawn prior to colon (patient 001), and rectum (the other 9 patients). All the first vaccination and 7 days after every third vaccination, and patients were confirmed to be HLA-A24ϩ both by a conven- PBMCs were isolated and cryopreserved at Ϫ198°C before use. tional serological method and by staining of PBMCs with the This protocol was approved by the Kurume University Review anti-A24 mAb, as reported previously (5, 6). No patient had received any treatments, steroids, or any other immunosuppres- Board and the Independent Ethical Committee (Protocol No. sive drugs for 4 weeks prior to the study. For the skin test, 10 ␮g 9904). Before entry into this trial, all patients gave informed ␮ consent and received a document of the protocol design. of SART3109–118 and 10 g of SART3315–323 were independ- ently injected intradermally with a tuberculin syringe with a Response Evaluation. All known sites of disease were 26-gauge needle. Immediate- and delayed-type hypersensitivity evaluated 7 days after every three vaccinations, and findings reactions were determined at 20 min and 24 h after the skin test, were compared with those obtained before the vaccinations.
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