To determine whether the GPC3 peptide vaccine could induce a specific immune response, we evaluated the GPC3 peptide-specific immune responses by ex vivo interferon (IFN)-γ Enzyme-Linked ImmunoSpot (ELISPOT) assay using PBMCs obtained from all patients before and after each vaccination. We found that the GPC3 peptide vaccine induced a GPC3-specific CTL response in 30 of the 33 patients (91 %). GPC3-specific CTL frequency increased in a peptide dose-dependent manner (Table 11.1). Generally, CTLs for some tumor antigens cannot be directly detected ex vivo; they can be detected only after expansion by repeated in vitro stimulation with the antigenic peptide on appropriate antigen-presenting cells. This finding can be attributed to the sensitivity of the assay and the low frequency of tumor antigen-specific CTLs. Surprisingly, GPC3-specific CTLs were detected directly ex vivo without in vitro peptide stimulation in almost all patients after GPC3 peptide vaccination.

Vaccination with synthetic peptides occasionally induces ineffective CTL responses due to various underlying mechanisms (Yamshchikov et al. 2001; Matsui et al. 1995; Falo et al. 1992; Amoscato et al. 1998; Chen et al. 1999; Meadows et al. 1997). One possible mechanism is a low affinity of responding T cells such that they recognize only target cells pulsed with high concentrations of the peptide and not tumor cells expressing the relevant epitopes at lower copy numbers. Alternatively, some antigen epitopes were not expressed on the surface of tumor cells (So et al. 2007; Sorensen et al. 2009). When evaluating T cell responses to peptide vaccines, it is important to confirm that responding CTLs lyse human cancer cells. Then, we established several GPC3 peptide-specific CTL clones in PBMCs obtained from vaccinated patients by single cell sorting using a Dextramer^® and CD107a antibody. These CTL clones had high avidity with a recognition efficiency of 10¹⁰ or 10¹¹ M resulting in 50 % cytotoxicity, and could recognize HCC cell lines expressing GPC3 in an HLA-class I-restricted manner. We provided substantial evidence that CTLs displayed not only high avidity, but also natural antigen-specific killing activity against HCC cells that could be induced in HCC patients by the peptide vaccine (Yoshikawa et al. 2011). A tumor biopsy was performed with informed consent in seven patients for evaluation of infiltration of CD8-positive T cells by immunohistochemical staining. In five of seven cases, infiltration of CD8-positive T cells into the tumor was increased after vaccination.

We also examined prognostic factors in the trial. We showed that GPC3-specific CTL frequency could be a predictive marker of GPC3 peptide vaccination effects. We compared patients with GPC3-specfic CTL frequencies ≥50 (n = 15) with those with GPC3-specific CTL frequencies <50 (n = 18) and found no significant difference in clinical background. We found a significant difference (P = 0.004) only for vaccine consumption (≥1.0 vs. <1.0 mg). Analysis of all 33 patients showed that the median OS was 12.2 months (95 % CI 6.5–18.0) in patients with GPC3-specific CTL frequencies ≥50, compared with 8.5 months (95 % CI 3.7–13.1) in those with GPC3-specfic CTL frequencies <50 (P = 0.033). In conclusion, this phase I clinical trial of a GPC3-derived peptide vaccine showed the vaccination to be safe and indicated a plethora of immunologic responses. This study also showed that GPC3-specific CTL frequency was correlated with OS in patients with advanced HCC who received the GPC3 peptide vaccine. These observations suggest that GPC3-derived peptide vaccines represent a novel therapy for patients with HCC, with the potential to improve OS.