Specific Targeting of a Naturally Presented Osteosarcoma Antigen, Papillomavirus Binding Factor Peptide, Using an Artificial Monoclonal Antibody

Tomohide Tsukahara,Makoto Emori,Kenji Murata,Takahisa Hirano,Norihiro Muroi,Masanori Kyono,Shingo Toji,Kazue Watanabe,Toshihiko Torigoe,Vitaly Kochin,Hiroko Asanuma,Hiroshi Matsumiya,Keiji Yamashita,Tetsuo Himi,Shingo Ichimiya,Takuro Wada,Toshihiko Yamashita,Tadashi Hasegawa,Noriyuki Sato

doi:10.1074/jbc.m114.568725

Abstract

Osteosarcoma is a rare but highly malignant tumor occurring most frequently in adolescents. The prognosis of non-responders to chemotherapy is still poor, and new treatment modalities are needed. To develop peptide-based immunotherapy, we previously identified autologous cytotoxic T lymphocyte-defined osteosarcoma antigen papillomavirus binding factor (PBF) in the context of HLA-B55 and the cytotoxic T lymphocyte epitope (PBF A2.2) presented by HLA-A2. PBF and HLA class I are expressed in ∼90 and 70% of various sarcomas, respectively. However, the expression status of peptide PBF A2.2 presented by HLA-A2 on osteosarcoma cells has remained unknown because it is difficult to generate a specific probe that reacts with the HLA·peptide complex. For detection and qualification of the HLA-A*02:01·PBF A2.2 peptide complex on osteosarcoma cells, we tried to isolate a single chain variable fragment (scFv) antibody directed to the HLA-*A0201·PBF A2.2 complex using a naïve scFv phage display library. As a result, scFv clone D12 with high affinity (KD = 1.53 × 10(-9) M) was isolated. D12 could react with PBF A2.2 peptide-pulsed T2 cells and HLA-A2+PBF+ osteosarcoma cell lines and simultaneously demonstrated that the HLA·peptide complex was expressed on osteosarcoma cells. In conclusion, scFv clone D12 might be useful to select candidate patients for PBF A2.2 peptide-based immunotherapy and develop antibody-based immunotherapy.

Highlights

Peptide vaccine-based immunotherapy targeting tumor-associated antigens can elicit CTL responses
Three scFv Clones Recognized HLA-A21⁄7Osteosarcoma Antigen papillomavirus binding factor (PBF)-derived Peptide Complex—We performed biopanning with a biotinylated HLA-A*02:011⁄7PBF A2.2 peptide complex as the antigen and a rescued library phage
We constructed a naïve scFv phage display library and isolated scFv clone D12, which reacted with the HLA-A*02:011⁄7PBF A2.2 peptide complex with strong affinity

Summary

Background

Peptide vaccine-based immunotherapy targeting tumor-associated antigens can elicit CTL responses. The expression status of peptide PBF A2.2 presented by HLA-A2 on osteosarcoma cells has remained unknown because it is difficult to generate a specific probe that reacts with the HLA1⁄7peptide complex. The introduction of neoadjuvant chemotherapy, establishment of guidelines for adequate surgical margins, and development of postexcision reconstruction have raised the 5-year survival rate to 60 –70% [1, 2] These advances overshadowed the pioneering adjuvant immunotherapy trials using autologous tumor vaccines for patients with osteosarcoma despite their having some therapeutic efficacy [3,4,5]. With the aim to characterize of the expression status of various HLA1⁄7peptide complexes on tumor cells, we constructed a naïve single chain variable fragment (scFv) phage display library and generated an artificial scFv antibody that reacts with an HLA1⁄7peptide complex using the PBF-derived peptide (PBF A2.2) in the context of HLA-A2 as a prototype antigen. We tried to detect the HLA-A21⁄7PBF A2.2 peptide complex on osteosarcoma cells

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION