Purpose: The carcinoembryonic antigen glypican-3 (GPC3) is an ideal target of anticancer immunotherapy against hepatocellular carcinoma (HCC). In this nonrandomized, open-label, phase I clinical trial, we analyzed the safety and efficacy of GPC3 peptide vaccination in patients with advanced HCC.Experimental Design: Thirty-three patients with advanced HCC underwent GPC3 peptide vaccination (intradermal injections on days 1, 15, and 29 with dose escalation). The primary endpoint was the safety of GPC3 peptide vaccination. The secondary endpoints were immune response, as measured by IFN-g ELISPOT assay, and the clinical outcomes tumor response, time to tumor progression, and overall survival (OS).Results: GPC3 vaccination was well-tolerated. One patient showed a partial response, and 19 patients showed stable disease 2 months after initiation of treatment. Four of the 19 patients with stable disease had tumor necrosis or regression that did not meet the criteria for a partial response. Levels of the tumor markers a-fetoprotein and/or des-g-carboxy prothrombin temporarily decreased in nine patients. The GPC3 peptide vaccine induced a GPC3-specific CTL response in 30 patients. Furthermore, GPC3-specific CTL frequency after vaccination correlated with OS. OS was significantly longer in patients with high GPC3-specific CTL frequencies (N ¼ 15) than in those with low frequencies (N ¼ 18; P ¼ 0.033).Conclusions: GPC3-derived peptide vaccination was well-tolerated, and measurable immune responses and antitumor efficacy were noted. This is the first study to show that peptide-specific CTL frequency can be a predictive marker of OS in patients with HCC receiving peptide vaccination.

Abstract. We previously reported that glypican-3 (GPC3) is uniquely overexpressed in human hepatocellular carcinoma and melanoma and that it is an ideal tumor antigen for immunotherapy in mouse models. We recently identified both HLA-A24 (A * 2402) and H-2K d -restricted GPC3 298-306 (EYILSLEEL) and HLA-A2 (A * 0201)-restricted GPC3 144-152 (FVGEFFTDV), both of which can induce GPC3-reactive cytotoxic T cells (CTLs). The present study was a preclinical study in a mouse model that was conducted in order to design an optimal schedule for clinical trial of GPC3-derived peptide vaccine. When BALB/c mice were intradermally vaccinated at the base of the tail with K d -restricted GPC3 298-306 peptide mixed with incomplete Freund's adjuvant (IFA), the peptidespecific CTLs were induced. But the peptide alone could not induce peptide-specific CD8 + T cells. Furthermore, proteomic analyses showed that IFA protected the peptide against degradation in the human serum. Peptide-reactive CTLs were induced by peptide vaccine in a dose-dependent manner. In addition, at least two vaccinations with a single dose >10 μg were needed for the induction of GPC3 298-306 -specific CTLs. But repeated vaccination with a lower dose of GPC3 298-306 did not induce peptide-specific CTLs. Similarly, induction of an Ag-specific immune response by HLA-A2 GPC3 144-152 depended on the dose administered. The results of this study suggested that IFA is one of the indispensable adjuvants for peptide-based immunotherapy, and that the immunological effect of peptide vaccines depends on the dose of peptide injected. IntroductionHepatocellular carcinoma (HCC) is one of the most common tumors worldwide, especially in Asian and Western countries (1). Despite advances in diagnosis and treatment, the overall survival of patients with HCC has not significantly improved in the last two decades (2). The effective treatments currently available are only indicated in a relatively small proportion of early stage cases. When patients presents with clinical manifestations of HCC, the tumor is usually advanced, and there are few treatment options. Many HCC patients have type B or C hepatitis or cirrhosis, so patients treated surgically or by other therapies are also at high risk for recurrence. Furthermore, the liver function of such patients is often very poor, so treatment for recurrence is often restricted. As a result, the prognosis of HCC remains poor and new therapies for cancer development and recurrence, i.e., adjuvant therapy, are urgently needed.We previously reported that glypican-3 (GPC3), glycosylphosphatidylinositol (GPI)-anchored membrane protein, is specifically overexpressed in human HCC and melanoma, and that among normal tissues it is slightly expressed in placenta and embryonic liver (3). We found that GPC3 is useful not only as a novel tumor marker, but also as a target antigen for immunotherapy in several studies with mice (4,5). In addition, we identified CTL epitope peptides: HLA-A24-restricted GPC3 298-306 (EYILSLEEL) and HLA-A2-restricted GPC3...

Abstract. Glypican-3 (GPC3), a carcinoembryonic antigen, is an ideal target for anticancer immunotherapy against hepatocellular carcinoma (HCC). In this study, we attempted to compare the induction of the GPC3-specific T-cell-mediated immune response after locoregional therapies in HCC patients and tumor-bearing mice. Twenty-seven HCC patients treated with locoregional therapies, including radiofrequency ablation (RFA), surgical resection and transcatheter arterial chemoembolization (TACE), were prospectively enrolled in this study. Additionally, we performed RFA experiments using a mouse model. GPC3-specific T-cell response was investigated pretreatment and post-treatment by an interferon-γ enzyme-linked immunospot assay using peripheral blood mononuclear cells from HCC patients and lymph node cells from tumor-bearing mice. Circulating GPC3-specific cytotoxic T lymphocytes (CTLs) were increased in 5 of 9 patients after RFA and in 4 of 9 patients after TACE, but in only 1 of 9 patients after surgical resection. All 7 patients with GPC3-expressing HCCs exhibited an increase in GPC3-specific CTLs after RFA or TACE, whereas none of the 7 patients did after surgical resection. The number of increased GPC3-specific CTLs after RFA was significantly larger than that after surgical resection (P= 0.023). Similarly, the frequency of GPC3-specific CTLs after RFA was significantly greater than that after surgical resection in the mouse model (P=0.049). We validated for the first time the stronger effect on the immune system brought by RFA compared with surgical resection for HCC patients and tumor-bearing mice. Combined treatment of RFA and immunotherapy is a reasonable strategy against HCC. IntroductionHepatocellular carcinoma (HCC) is one of the most common and most serious cancers worldwide (1). Locoregional therapies, including radiofrequency ablation (RFA), surgical resection, and transcatheter arterial chemoembolization (TACE), are recognized as the gold-standard therapies for HCC patients whose cancer lesions are limited to the liver (2). However, the recurrence rate remains quite high despite potentially curative treatment (3,4). The reasons for this are as follows: first, a multicentric new tumor frequently occurs from underlying active hepatitis or cirrhosis and, second, a small tumor undetectable by imaging modalities frequently exists before treatment and would be left untreated (5). Therefore, the establishment of effective adjuvant therapy to prevent recurrence is urgently required, and Radiofrequency ablation for hepatocellular carcinoma induces glypican-3 peptide-specific cytotoxic T lymphocytes

<p>PDF file, 36KB, A 30 mg dose of peptide caused stronger local skin reactions (blushing, induration) at the injection site than did a 3.0 mg dose.</p>

<p>PDF file, 28KB, The levels of AFP or DCP decreased temporarily at least once in nine of the 33 patients. In seven of these nine patients, the levels of DCP fell to < 30% of baseline values.</p>