Abstract 3508: Zoledronate induce expansion of glypican-3 (GPC3) peptide specific cytotoxic T lLymphocytes sufficient for adoptive cancer immunotherapy
Authors: Yoshikawa Toshiaki , Takahara Masashi , Tomiyama Mai , Kuroda Ayumi , Shirakawa Hirofumi , Nobuoka Daisuke , Sawada Yu , Sakemura Noriko , Nieda Mie , Maekawa Ryuji , Nakatsura Tetsuya 
Published: August 2012

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 GPC3144-152 (FVGEFFTDV) and A24-restricted GPC3298-306 (EYILSLEEL) peptide vaccine in 33 patients with advanced HCC. This clinical trial of GPC3-derived peptide vaccine showed the vaccination to be safe, and indicates many immunological responses. To develop more efficient immunotherapy, we investigated large scale expansion of GPC3 peptide specific CTLs for adoptive immunotherapy. PURPOSE In this study, we investigated the efficiency of new method to induce expansion of GPC3 peptide specific CTLs. METHODS Treatment of human peripheral blood mononuclear cells (PBMCs) with zoledronate is a method that enables large-scale γδ T cell expansion. To induce expansion of GPC3 peptide specific CTLs and γδ T cells, the PBMCs of patients vaccinated with GPC3 peptide were cultured with GPC3 peptide and zoledronate for 14 days. We used CD8+ and CD8− cells that isolated from cultured cells using CD8 microbeads at day 14 as a effector cells. GPC3 peptide specificity and cytotoxic activity of CTLs were analysed by Dextramer assay, cytotoxicity assay and IFN-γ ELISPOT assay. We used human liver cancer cell line SK-Hep-1 (GPC3−, HLA-A*02:01/24:02) and a human GPC3 gene transfectant, SK-Hep-1 [[Unsupported Character - &#8260;]] hGPC3 (GPC3+, HLA-A*02:01/24:02) as target cells. T2 (HLA-A*02:01, TAP−) was pulsed with GPC3 peptide or HIV peptide at room temperature for 1h. To evaluate antitumor activity in vivo, we inoculated SK-Hep-1/hGPC3 and SK-Hep-1/vec cells at the right flank of NOD/SCID mice. We injected the CD8+, CD8− or all cells as effector cells intravenously. RESULTS The expansion of Lymphocytes from a few PBMCs of vaccinated patients with advanced HCC using zoledronate yields cell numbers sufficient for adoptive transfer. The rate of increase of GPC3 specific CTLs was approximate 490 to 170,000-fold, whereas the rate of increase of total cell number was approximate 13 to 1,000-fold. These CD8+ cells including CTLs showed GPC3 specific cytotoxicity against SK-Hep-1/hGPC3 and T2 pulsed with GPC3 peptide, but not against SK-Hep-1/vec and T2 pulsed with HIV peptide, whereas CD8− cells including γδ T cells showed cytotoxicity against SK-Hep-1/hGPC3 and SK-Hep-1/vec while did not show GPC3 specificity. Furthermore, adoptive cell transfer of CD8+ cells, CD8− cells and total cells after expansion significantly inhibited tumor growth in NOD/SCID mouse model. CONCLUSION This study indicates that zoledronate is useful to large-scale expand antigen-specific CTLs and γδ T cells for adoptive immunotherapy.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3508. doi:1538-7445.AM2012-3508