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: We reported recently the novel tumor marker glypican-3 (GPC3) for hepatocellular carcinoma. In the present study, we investigated the expression of GPC3 in human melanoma cell lines and tissues and asked whether GPC3 could be a novel tumor marker for melanoma.Experimental Design: Expression of GPC3 mRNA and protein was investigated in human melanoma cell lines and tissues using reverse transcription-PCR and immunohistochemical analysis. Secreted GPC3 protein was quantified using ELISA in culture supernatants of melanoma cell lines and in sera from 91 patients with melanoma and 28 diseasefree patients after surgical removal of primary melanoma. All of the subjects were Japanese nationals.Results: In >80% of melanoma and melanocytic nevus, there was evident expression of GPC3 mRNA and protein. Furthermore, GPC3 protein was evidenced in sera of 39.6% (36 of 91) of melanoma patients but not in sera from subjects with large congenital melanocytic nevus (0 of 5) and from healthy donors (0 of 60). Twenty-seven of 36 serum GPC3-positive patients were negative for both serum 5-S-cysteinyldopa and melanoma-inhibitory activity, well-known tumor markers for melanoma. The positive rate of serum GPC3 (39.6%) was significantly higher than that of 5-S-cysteinyldopa (26.7%) and of melanoma-inhibitory activity (20.9%). Surprisingly, we detected serum GPC3 even in patients with stage 0 in situ melanoma. The positive rate of serum GPC3 at stage 0, I, and II (44.4%, 40.0%, and 47.6%) was significantly higher than that of 5-S-cysteinyldopa (0.0%, 8.0%, and 10.0%). Also observed was the disappearance of GPC3 protein in sera from 11 patients after surgical removal of the melanoma.Conclusions: GPC3 is apparently a novel tumor marker useful for the diagnosis of melanoma, especially in early stages of the disorder.

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...

To assess the clinical value of serum biochemical markers, the aminoterminal peptide of type III procollagen, type IV collagen 7S domain, the central triple-helix of type IV collagen and tissue inhibitor of metalloproteinases, as a marker of hepatic fibrosis, we measured these four serum markers in 132 patients with chronic viral liver disease and compared these serum markers with liver histological findings. Serum levels of these markers increased closely with the progress of liver disease, and the abnormal percentages of type III procollagen peptide, type IV collagen 7S domain, central triple-helix of type IV collagen and tissue inhibitor of metalloproteinases in patients with cirrhosis were 97%, 95%, 83% and 48%, respectively. These four serum markers strongly correlated with the histological degree of periportal with or without bridging hepatocellular necrosis and of liver fibrosis and correlated weakly with the degree of intralobular degeneration and focal necrosis and the degree of portal inflammation. The correlation coefficients of serum type IV collagen 7S domain with periportal with or without bridging hepatocellular necrosis and with liver fibrosis were the highest among these four serum markers, suggesting that serum type IV collagen 7S domain is the most valuable diagnostic marker to assess the degree of liver fibrosis in chronic viral liver disease. When we assessed the ability of each serum marker to detect cirrhosis with a receiver operating curve, the best test was type IV collagen 7S domain, and the second best was type III procollagen peptide.(ABSTRACT TRUNCATED AT 250 WORDS)