HLA-A*0201-restricted CTL epitope of a novel osteosarcoma antigen, papillomavirus binding factor.
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 x 10-7 to 5 x 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.


CONCLUSIONS
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.
Tsukahara
Tomohide
T
Department of Orthopaedic Surgery, Sapporo Medical University School of Medicine, South-1, West-16, Chuo-ku, Sapporo, 060-8543, Japan. tukahara@sapmed.ac.jp
Kawaguchi
Satoshi
S
Torigoe
Toshihiko
T
Takahashi
Akari
A
Murase
Masaki
M
Kano
Masanobu
M
Wada
Takuro
T
Kaya
Mitsunori
M
Nagoya
Satoshi
S
Yamashita
Toshihiko
T
Sato
Noriyuki
N
D000951:Antigens, Neoplasm / Q000276:immunology* / Q000378:metabolism; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D000939:Epitopes / Q000276:immunology*; D005838:Genotype; D015234:HLA-A Antigens / Q000096:biosynthesis* / Q000276:immunology*; D015789:HLA-A2 Antigen; D006801:Humans; D007167:Immunotherapy / Q000379:methods; D020014:K562 Cells; D007959:Lymphocyte Culture Test, Mixed; D012516:Osteosarcoma / Q000276:immunology*; D011485:Protein Binding; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
D000951:Antigens, Neoplasm; D000939:Epitopes; D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen