Purpose and Experimental Design: We previously reported that glypican-3 (GPC3) was overexpressed, specifically in hepatocellular carcinoma (HCC) and melanoma in humans, and it was useful as a novel tumor marker. We also reported that the preimmunization of BALB/c mice with dendritic cells pulsed with the H-2K d -restricted mouse GPC3 298-306 (EYILSLEEL) peptide prevented the growth of tumor-expressing mouse GPC3. Because of similarities in the peptide binding motifs between H-2K d and HLA-A24 (A*2402), the GPC3 298-306 peptide therefore seemed to be useful for the immunotherapy of HLA-A24 + patients with HCC and melanoma. In this report, we investigated whether the GPC3 298-306 peptide could induce GPC3-reactive CTLs from the peripheral blood mononuclear cells (PBMC) of HLA-A24 (A*2402) + HCC patients. In addition, we used HLA-A2.1 (HHD) transgenic mice to identify the HLA-A2 (A*0201)^restricted GPC3 epitopes to expand the applications of GPC3-based immunotherapy to the HLA-A2 + HCC patients. Results: We found that the GPC3 144-152 (FVGEFFTDV) peptide could induce peptide-reactive CTLs in HLA-A2.1 (HHD) transgenic mice without inducing autoimmunity. In five out of eight HLA-A2 + GPC3 + HCC patients, the GPC3 144-152 peptide-reactive CTLs were generated from PBMCs by in vitro stimulation with the peptide and the GPC3 298-306 peptide-reactive CTLs were also generated from PBMCs in four of six HLA-A24 + GPC3 + HCC patients. The inoculation of these CTLs reduced the human HCC tumor mass implanted into nonobese diabetic/ severe combined immunodeficiency mice. Conclusion: Our study raises the possibility that these GPC3 peptides may therefore be applicable to cancer immunotherapy for a large number of HCC patients.

Purpose: To establish cancer immunotherapy, it is important to identify the tumor-associated antigens (TAA) that are strongly expressed in the tumor cells but not in the normal cells. In this study, to establish an effective anticancer immunotherapy, we tried to identify the useful TAA of pancreatic cancer. Experimental Design: Based on a previous genome-wide cDNA microarray analysis of pancreatic cancer, we focused on cadherin 3 (CDH3)/P-cadherin as a novel candidateTAA for anticancer immunotherapy. To identify the HLA-A2 (A*0201)^restricted CTL epitopes of CDH3, we used HLA-A2.1 (HHD) transgenic mice (Tgm). Furthermore, we examined the cytotoxicity against the tumor cells in vitro and in vivo of CTLs specific to CDH3 induced from HLA-A2p ositive healthy donors and cancer patients. Results: CDH3 was overexpressed in the majority of pancreatic cancer and various other malignancies, including gastric and colorectal cancers, but not in their noncancerous counterparts or in many normal adult tissues. In the experiment using HLA-A2.1 Tgm, we found that the CDH3-4 655-663 (FILPVLGAV) and CDH3-7 757-765 (FIIENLKAA) peptides could induce HLA-A2^restricted CTLs inTgm. In addition, peptides-reactive CTLs were successfully induced from peripheral blood mononuclear cells by in vitro stimulation with these two peptides in HLAA2^positive healthy donors and cancer patients, and these CTLs exhibited cytotoxicity specific to cancer cells expressing both CDH3 and HLA-A2. Furthermore, the adoptive transfer of the CDH3-specific CTLs could inhibit the tumor growth of human cancer cells engrafted into nonobese diabetic/severe combined immunodeficiency mice. Conclusions: These results suggest that CDH3 is a novel TAA useful for immunotherapy against a broad spectrum of cancers, including pancreatic cancer.Pancreatic cancer has a poor prognosis, with an overall 5-year survival rate of f5% (1). A surgical resection remains the only option for a long-term survival, but patients with resectable pancreatic cancer are in the minority (9-22%; refs. 2 -4). Even in these patients, however, the 5-year survival rate remains f20% in spite of surgery with a curative intent (5, 6). Up to 80% of patients present with locally advanced or metastatic disease, and their median survival ranges from 6 to 9 months (7). It is generally thought that the presence of few signs or symptoms in the early stage, lack of an effective screening test, high rate of relapse, and poor response to current therapies contribute to the poor prognosis of this malignancy. Hence, the development of novel therapeutic modalities is an issue of great importance, and immunotherapy may be a potential treatment for pancreatic cancer.To establish an effective antitumor immunotherapy, the identification of tumor-associated antigens (TAA) plays a key role. In the past, many TAAs in various malignancies have been identified using the method of cDNA expression cloning (8 -10) and a serologic analysis of the recombinant cDNA expression library (11 -15). Recently, cDNA microarray...

Toward the development of a novel cancer immunotherapy, we have previously identified several tumor-associated antigens (TAAs) and the epitopes recognized by human histocompatibility leukocyte (HLA)-A2/A24-restricted cytotoxic T lymphocyte (CTL). In this study, we tried to identify a TAA of lung cancer (LC) and its HLA-A2 restricted CTL epitopes to provide a target antigen useful for cancer immunotherapy of LC. We identified a novel cancer testis antigen, cell division cycle associated gene 1 (CDCA1), overexpressed in nonsmall cell LC using a cDNA microarray analysis. The expression levels of CDCA1 were also increased in the majority of small cell LC, cholangiocellular cancer, urinary bladder cancer and renal cell cancers. We used HLA-A2.1 transgenic mice to identify the HLA-A2 (A*0201)-restricted CDCA1 epitopes recognized by mouse CTL, and we investigated whether these peptides could induce CDCA1-reactive CTLs from the peripheral blood mononuclear cells (PBMCs) of HLA-A2-positive donors and a NSCLC patient. Consequently, we found that the CDCA1 65-73 (YMMPVNSEV) peptide and CDCA1 [351][352][353][354][355][356][357][358][359] (KLATAQFKI) peptide could induce peptide-reactive CTLs in HLA-A2.1 transgenic mice. In HLA-A2 1 donors, in vitro stimulation of PBMC with these peptides could induce peptide-reactive CTLs which killed tumor cell lines endogenously expressing both HLA-A2 and CDCA1. As a result, CDCA1 is a novel cancer-testis antigen overexpressed in LC, cholangiocellular cancer, urinary bladder cancer and renal cell cancers, and CDCA1 may therefore be an ideal TAA useful for the diagnosis and immunotherapy of these cancers.

Background:Identification of tumour-associated antigens (TAAs) that induce cytotoxic T lymphocytes (CTLs) specific to cancer cells is critical for the development of anticancer immunotherapy. In this study, we aimed at identifying a novel TAA of pancreatic cancer for immunotherapy.Methods:On the basis of the genome-wide cDNA microarray analysis, we focused on KIF20A (also known as RAB6KIFL/MKlp2) as a candidate TAA in pancreatic cancer cells. The HLA-A2 (A*02:01)-restricted CTL epitopes of KIF20A were identified using HLA-A2 transgenic mice (Tgm) and the peptides were examined to check whether they could generate human CTLs exhibiting cytotoxic responses against KIF20A+, HLA-A2+ tumour cells in vitro.Results:KIF20A was overexpressed in pancreatic cancer and in some other malignancies, but not in their non-cancerous counterparts and many normal adult tissues. We found that KIF20A-2 (p12–20, LLSDDDVVV), KIF20A-8 (p809–817, CIAEQYHTV), and KIF20A-28 (p284–293, AQPDTAPLPV) peptides could induce HLA-A2-restricted CTLs in HLA-A2 Tgm without causing autoimmunity. Peptide-reactive human CTLs were generated from peripheral blood mononuclear cells of HLA-A2+ healthy donors by in vitro stimulation with the three peptides, and those CTLs successfully exhibited cytotoxic responses to cancer cells expressing both KIF20A and HLA-A2.Conclusion:KIF20A is a novel promising candidate for anticancer immunotherapeutic target for pancreatic cancers.

The present study attempted to identify a target antigen for immunotherapy for cholangiocarcinoma. Forkhead box M1 (FOXM1) was selected as a candidate antigen based on the data of previous cDNA microarray analysis of clinical samples of cholangiocarcinoma. The level of FOXM1 mRNA was more than 4 times higher in cancer cells in comparison to adjacent normal epithelial cells, in all of 24 samples of cholangiocarcinoma tissues. An immunohistochemical analysis also detected FOXM1 protein in the cancer cells but not in the normal cells. Twenty-three human FOXM1-derived peptides predicted to bind to HLA-A2 were analyzed to determine their ability to induce HLA-A2-restricted T cells in HLA-A2 transgenic mice. FOXM1 362-370 (YLVPIQFPV), FOXM1 373-382 (SLVLQPSVKV), and FOXM1 640-649 (GLMDLSTTPL) peptides primed HLA-A2-restricted cytotoxic T lymphocytes (CTLs) in the HLA-A2 transgenic mice. Human CTL lines reactive to these 3 peptides could also be established from HLA-A2-positive healthy donors and cancer patients. Natural processing of the 3 epitopes from FOXM1 protein was confirmed by specific killing of HLA-A2-positive FOXM1-transfectants by peptide-induced CTLs. FOXM1 is expressed in various types of cancers and it is also functionally involved in oncogenic transformation and the survival of cancer cells. Therefore, FOXM1 may be a suitable target for immunotherapy against various cancers including cholangiocarcinoma.Cholangiocarcinoma is a devastating malignancy, which is difficult to diagnose and treat. Unfortunately, only a few patients are considered suitable for surgery, and chemotherapy and radiotherapy have not yielded substantial improvements in the survival rate. The overall survival rate is poor, with less than 5% of patients surviving upto 5 years. There has been no significant change in this rate over the past 30 years. 1 Recent studies have identified some therapeutic molecular targets in cholangiocarcinoma in preclinical studies, thus supporting the therapeutic potential of selected targeting strategies against cholangiocarcinoma. 2 The results were encouraging, although they were obtained from in vitro studies and tumor cell xenograft models. Therefore, novel therapeutic strategies for cholangiocarcinoma are urgently needed.In cancer immunotherapy, effective induction of cytotoxic T lymphocytes (CTLs) by tumor-associated antigen (TAA) has shown promising results. 3 The utilization of CTLs elicited by TAA is an ideal therapeutic approach, if they specifically attack tumor cells expressing the antigen and reveal little or no adverse effects on normal cells. The development of cDNA microarray technologies, coupled with genome information, has provided comprehensive profiles of the gene expression of malignant cells, which have been compared to those of normal cells. 4,5 TAAs should be expressed excessively and preferentially by the tumor cells but not by the