Abstract B73: Proof of glypican-3 (GPC3) peptide specific CTLs infiltrating into tumor tissue derived from advanced HCC patient vaccinated with GPC3 peptide.
Authors: Yoshikawa Toshiaki , Sakai Mayuko , Ofuji Kazuya , Takahashi Mari , Shimomura Manami , Sawada Yu , Nobuoka Daisuke , Nakatsura Tetsuya 
Published: August 2014

Abstract
Background: Glypican-3 (GPC3) is an onco-fetal antigen which is overexpressed in human hepatocellular carcinoma (HCC), and is only expressed in the placenta and embryonic liver among normal tissues. Previously, we completed a phase I clinical trial of HLA-A2-restricted GPC3 144-152 (FVGEFFTDV) and A24-restricted GPC3 298-306 (EYILSLEEL) peptide vaccine in 33 patients with advanced HCC. This clinical trial of GPC3-derived peptide vaccine showed the vaccination was well tolerated, and indicated many immunological responses. Furthermore, GPC3-specific CTL frequency after vaccination correlated with overall survival. In addition, tumor biopsy was carried out in 7 patients to evaluate the therapeutic effect after vaccination. We evaluated the infiltration of CD8-positive T cells by immunohistochemical staining. In 5 of 7 cases, infiltration of CD8-positive T cells into the tumor was increased after vaccination. However, we did not confirm that the tumor-infiltrating lymphocytes detected after vaccination were GPC3 peptide-specific CTLs. We are currently initiating a pilot study of liver biopsies carried out before and after GPC3 peptide vaccination for advanced HCC to determine whether tumor-infiltrating lymphocytes are indeed GPC3 peptide-specific CTLs. We tried to detect GPC3 peptide-specific CTLs in biopsy specimens from vaccinated patients using GPC3-Dextramer.
Materials and Methods: The trial was designed to assess clinical response, and whether tumor-infiltrating lymphocytes with anti-tumor effect are increased indeed after GPC3 peptide vaccination for advanced HCC (UMIN-CTR number: 000005093). Vaccinations with 3.0 mg of GPC3 peptide, emulsified with incomplete Freund's adjuvant (Montanide ISA-51 VG) were carried out intradermally at 14-day intervals. Peripheral blood was obtained from each patients at times designated in the protocol (before the first vaccination and 2 weeks after each vaccination) and centrifuged using a Ficoll–Paque gradient. Immunological responses were analyzed ex vivo by IFN-γ Enzyme-linked immunospot (ELISPOT) assay using peripheral blood mononuclear cells (PBMCs). Tumor biopsy was carried out after 5-6th vaccination. The cells obtained from biopsy specimens were stained anti-CD3, anti-CD8 antibody and GPC3-Dextramer and analyzed using FACSAria cell sorter.
Results: We could obtain evidence of GPC3 peptide-specific CTLs infiltrating into the HCC tumor from one case. In this case, the frequency of GPC3 peptide specific CTLs after vaccination (290 of 5 x 105 PBMCs) was larger than that before vaccination (0 of 5 x 105 PBMCs) ex vivo in IFN-γ ELISPOT assay. In flow cytometer analysis, CD3+, CD8+, GPC3-Dextramer+ cells were detected in cells obtained from a biopsy specimen of this vaccinated patient. Moreover, we established GPC3 peptide specific CTL clones from these cells by single cell sort using GPC3-Dextramer. These GPC3-Dextramer+ CTL clones showed GPC3 peptide specific IFN-γ secretion.
Conclusion: We could prove that the GPC3 peptide-specific CTLs have been infiltrated into the tumor tissue after peptide vaccination. The evidence serves as a proof-of concept for GPC3 peptide vaccine therapy.
Citation Format: Toshiaki Yoshikawa, Mayuko Sakai, Kazuya Ofuji, Mari Takahashi, Manami Shimomura, Yu Sawada, Daisuke Nobuoka, Tetsuya Nakatsura. Proof of glypican-3 (GPC3) peptide specific CTLs infiltrating into tumor tissue derived from advanced HCC patient vaccinated with GPC3 peptide. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B73.