Research

HLA-A*0201-restricted CTL epitope of a novel osteosarcoma antigen, papillomavirus binding factor

Tomohide Tsukahara^1,2 , Satoshi Kawaguchi¹ , Toshihiko Torigoe² , Akari Takahashi² , Masaki Murase^1,2 , Masanobu Kano^1,2 , Takuro Wada¹ , Mitsunori Kaya¹ , Satoshi Nagoya¹ , Toshihiko Yamashita¹ and Noriyuki Sato²

¹  Department of Orthopaedic Surgery, Sapporo Medical University School of Medicine, South-1, West-16, Chuo-ku, Sapporo, 060-8543, Japan

²  Department of Pathology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo, 060-8556, Japan

author email corresponding author email

Journal of Translational Medicine 2009, 7:44doi:10.1186/1479-5876-7-44

Published:	12 June 2009

Abstract

Background

To develop peptide-based immunotherapy for osteosarcoma, we previously identified papillomavirus binding factor (PBF) as a CTL-defined osteosarcoma antigen in the context of HLA-B55. However, clinical application of PBF-based immunotherapy requires identification of naturally presented CTL epitopes in osteosarcoma cells in the context of more common HLA molecules such as HLA-A2.

Methods

Ten peptides with the HLA-A*0201 binding motif were synthesized from the amino acid sequence of PBF according to the BIMAS score and screened with an HLA class I stabilization assay. The frequency of CTLs recognizing the selected PBF-derived peptide was determined in peripheral blood of five HLA-A*0201⁺ patients with osteosarcoma using limiting dilution (LD)/mixed lymphocyte peptide culture (MLPC) followed by tetramer-based frequency analysis. Attempts were made to establish PBF-specific CTL clones from the tetramer-positive CTL pool by a combination of limiting dilution and single-cell sorting. The cytotoxicity of CTLs was assessed by ⁵¹Cr release assay.

Results

Peptide PBF A2.2 showed the highest affinity to HLA-A*0201. CD8+ T cells reacting with the PBF A2.2 peptide were detected in three of five patients at frequencies from 2 × 10^-7 to 5 × 10^-6. A tetramer-positive PBF A2.2-specific CTL line, 5A9, specifically lysed allogeneic osteosarcoma cell lines that expressed both PBF and either HLA-A*0201 or HLA-A*0206, autologous tumor cells, and T2 pulsed with PBF A2.2. Five of 12 tetramer-positive CTL clones also lysed allogeneic osteosarcoma cell lines expressing both PBF and either HLA-A*0201 or HLA-A*0206 and T2 pulsed with PBF A2.2.

Conclusion

These findings indicate that PBF A2.2 serves as a CTL epitope on osteosarcoma cells in the context of HLA-A*0201, and potentially, HLA-A*0206. This extends the availability of PBF-derived therapeutic peptide vaccines for patients with osteosarcoma.