Phase I trial of a glypican-3-derived peptide vaccine for advanced hepatocellular carcinoma: immunologic evidence and potential for improving overall survival.

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-γ 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 α-fetoprotein and/or des-γ-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 = 15) than in those with low frequencies (N = 18; P = 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.

Radiofrequency ablation for hepatocellular carcinoma induces glypican-3 peptide-specific cytotoxic T lymphocytes.

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 chemo-embolization (TACE), were prospectively enrolled in this study. Additionally, we performed RFA experiments using a mouse model. GPC3-specific T-cell response was investigated pre-treatment 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.

Glypican-3 is a useful diagnostic marker for a component of hepatocellular carcinoma in human liver cancer.

Primary liver cancers are classified into three types based on their morphology and cytogenetic characteristics hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular and cholangiocarcinoma (CHC). It is often difficult to distinguish these liver tumors. Glypican-3 (GPC3) is serological and histochemical marker of hepatocellular carcinoma. In order to separate these three types of liver cancers, we analyzed the GPC3 expression in 85 liver resection specimens, including 46 HCCs, 28 ICCs and 11 CHCs. GPC3 immunohistochemical staining was used to distinguish HCC from ICC by comparing with the conventional biomarker, alpha-fetoprotein (AFP). The immunostaining of GPC3 was identified in 78.3% (36/46) of HCCs, 60% (9/15) of well differentiated, 88.9% (16/18) of moderately differentiated and 84.6% (11/13) of poorly differentiated HCCs. It was negative in the ICCs. We confirmed that GPC3 expression is specific to HCC component (8/11, 72.7%) but few samples also showed weakly in ICC component (2/11, 18.2%) of CHC sections among 11 cases compared with HCC biomarkers including AFP and hepatocyto paraffin 1 (HepPar1), and ICC biomarkers cytokeratin (CK) 7 and CK19. Three cases in which the macroscopic features resembled ICC did not express GPC3 even in the pathological HCC component. Most (10/11, 91%) of the pathological cholangiocarcinoma components in CHC showed positive staining for CK7 and CK19. The results of this study suggest that GPC3 is a biomarker that is sensitive and specific to HCC component of CHC, and CK7 and CK19 are markers for pathological cholangiocarcinoma component of CHC.

HLA-A2 and -A24-restricted glypican-3-derived peptide vaccine induces specific CTLs: preclinical study using mice.

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-2Kd-restricted GPC3298-306 (EYILSLEEL) and HLA-A2 (A*0201)-restricted GPC3144-152 (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 Kd-restricted GPC3298-306 peptide mixed with incomplete Freund's adjuvant (IFA), the peptide-specific 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 microg were needed for the induction of GPC3298-306-specific CTLs. But repeated vaccination with a lower dose of GPC3298-306 did not induce peptide-specific CTLs. Similarly, induction of an Ag-specific immune response by HLA-A2 GPC3144-152 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.