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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.

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.

Glypican-3 (GPC3) is an onco-fetal antigen that is overexpressed in human hepatocellular carcinoma (HCC), and is only expressed in the placenta and embryonic liver among normal tissues. Previously, we identified an HLA-A2-restricted GPC3 [144][145][146][147][148][149][150][151][152] (FVGEFFTDV) peptide that can induce GPC3-reactive CTLs without inducing autoimmunity in HLA-A2 transgenic mice. In this study, we carried out a phase I clinical trial of HLA-A2-restricted GPC3 144-152 peptide vaccine in 14 patients with advanced HCC. Immunological responses were analyzed by ex vivo c-interferon enzyme-linked immunospot assay. The frequency of GPC3 144-152 peptide-specific CTLs after vaccination (mean, 96; range, 5-441) was significantly larger than that before vaccination (mean, 6.5; range, 0-43) (P < 0.01). An increase in the GPC3 144-152 peptide-specific CTL frequency was observed in 12 (86%) of 14 patients after vaccination. Additionally, there was a significant correlation between the maximum value of GPC3 144-152 peptide-specific CTLs after vaccination and the dose of the peptide injected (P = 0.0166, r = 0.665). Moreover, we established several GPC3 144-152 peptide-specific CTL clones from PBMCs of patients vaccinated with GPC3 144-152 peptide by single cell sorting using Dextramer and CD107a antibody. These CTL clones had high avidity (the recognition efficiency showing 50% cytotoxicity was 10 )10 or 10 )11 M) and could recognize HCC cell lines expressing GPC3 in an HLA-class I-restricted manner. These results suggest that GPC3 144-152 peptide vaccine can induce high avidity CTLs capable of killing HCC cells expressing GPC3. This trial was registered with University Hospital Medical Information Network number 000001395. (Cancer Sci 2011; 102: 918-925)

We have recently established a method to generate dendritic cells from mouse embryonic stem cells. By introducing exogenous genes into embryonic stem cells and subsequently inducing differentiation to dendritic cells (ES-DC), we can now readily generate transfectant ES-DC expressing the transgenes. A previous study revealed that the transfer of genetically modified ES-DC expressing a model antigen, ovalbumin, protected the recipient mice from a challenge with an ovalbumin-expressing tumor. In the present study, we examined the capacity of ES-DC expressing mouse homologue of human glypican-3, a recently identified oncofetal antigen expressed in human melanoma and hepatocellular carcinoma, to elicit protective immunity against glypican-3-expressing mouse tumors. CTLs specific to multiple glypican-3 epitopes were primed by the in vivo transfer of glypican-3-transfectant ES-DC (ES-DC-GPC3). The transfer of ES-DC-GPC3 protected the recipient mice from subsequent challenge with B16-F10 melanoma, naturally expressing glypican-3, and with glypican-3-transfectant MCA205 sarcoma. The treatment with ES-DC-GPC3 was also highly effective against i.v. injected B16-F10. No harmful side effects, such as autoimmunity, were observed for these treatments. The depletion experiments and immunohistochemical analyses suggest that both CD8 + and CD4 + T cells contributed to the observed antitumor effect. In conclusion, the usefulness of glypican-3 as a target antigen for antimelanoma immunotherapy was thus shown in the mouse model using the ES-DC system. Human dendritic cells expressing glypican-3 would be a promising means for therapy of melanoma and hepatocellular carcinoma. (Cancer Res 2006; 66(4): 2414-22)