Osteosarcoma

1. Development of HLA-A2- and HLA-A24-restricted peptide vaccines from a novel osteosarcoma antigen, papillomavirus binding factor S Kawaguchi, T Tsukahara, T Wada, S Nagoya, M Kaya, T Torigoe, N Sato, T Ishii, S Tatezaki, T Yamashita Sapporo Medical University, Chiba Cancer Center Hospital

2. Osteosarcoma Introduced by Alexis Boyer in 1814.

3. Immunotherapy for osteosarcoma Campbell CJ, Cohen J, Enneking WF: New therapies for osteogenic sarcoma. J Bone Joint Surg Am 57:143-144, 1975. - Immunotherapy - Methotrexate - Adriamycin

4. Autologous tumor cell vaccination as adjuvant therapy Amputation Marcove et al, CA, 1973

5. Current immunotherapy Antigenic peptide TCR T cell Tumor cell Vaccination

6. T cell T cell T cell Active immunotherapy (vaccination) Patients Peptide vaccines Injection T cell T cell Dendritic cell T cell T cell T cell T cell T cell T cell T cell Tumor cell

7. Peptide vaccination for malignant melanoma MAGE-3 Coulie PG, Universite de Louvain ,Brussels

8. Goal Efficacious peptide vaccines for Osteosarcoma

9. To identify tumor antigens and antigenic peptides T cell TCR HLA class I Tumor

10. 16 y.o. F, Osteosarcoma OS2000 TcOScl-303

11. TcOScl-303 kills OS2000, but not 293EBNA or K562 cells OS2000 IFN-γ(+) OS2000 + TcOScl-303 OS2000 IFN-γ(-) ▲293EBNA K562 3 50 16 E/T ratio Nabeta et al., J Orthop Sci, 2003.

12. Cloning of a gene encoding the antigenic peptide TcOScl-303 TCR HLA class I OS2000 Expression cloning Antigenic peptide

13. Papillomavirus Binding Factor (PBF) as a novel osteosarcoma antigen PBF Open reading frame Zn finger 5' 337 1878 ↑ 5' UTR Clone 1B9.1H4 ΔN-PBF 704 ↑ 3' UTR (432bps) • Protein that binds to the E2 binding site of human • papillomavirus type 8 Boeckle et al., Virology, 2002

14. Osteosarcoma Pancreas Ovary Spleen Anti- PBF Immunostaining with anti-PBF antibody Osteosarcoma 72/78 Ewing’s sarcoma 31/34 Synovial sarcoma 18/20 Tsukahara et al., Cancer Res, 2004

15. Stages of PBF Project I II III IV Identification Function Regulation Application Boeckle et al., 2002 Tsukahara et al., 2004

16. Stages of PBF Project I II III IV Identification Function Regulation Application Boeckle et al., 2002 Tsukahara et al., 2004 Prognostic significance, Targeting/Overexpression Associated proteins, Expression of HLA class I Development of peptide vaccines

17. Event-free Survival of OS Pts. PBF negative: 6 P=0.025 PBF positive: 72 Tsukahara et al., Cancer Sci, 2008

18. Stages of PBF Project I II III IV Identification Function Regulation Application Boeckle et al., 2002 Tsukahara et al., 2004 Prognostic significance, Targeting/Overexpression Associated proteins, Expression of HLA class I Development of peptide vaccines

19. PBF knock-out mice PBF (+/-)

20. Stages of PBF Project I II III IV Identification Function Regulation Application Boeckle et al., 2002 Tsukahara et al., 2004 Prognostic significance, Targeting/Overexpression Associated proteins, Expression of HLA class I Development of peptide vaccines

21. Bait : PBF Yeast Two-hybrid Screening Yeast Prey : cDNA library of OS2000

22. open reading frame 1126 5’ 3’ Ubiquitin-like domain NLS (1030-1053) HLA-B Associated Transcript 3 (BAT3) OS2000 PBF BAT3 Merge Tsukahara et al., Cancer Sci, 2009

23. Stages of PBF Project I II III IV Identification Function Regulation Application Boeckle et al., 2002 Tsukahara et al., 2004 Prognostic significance, Targeting/Overexpression Associated proteins, Expression of HLA class I Development of peptide vaccines

24. PBF TcOScl-303 TCR HLA-B55 OS2000 16 y.o. F HLA in Japanese population B55 A24 B61 A2 B51 HLA-restriction and HLA-limitation

25. PBF-peptides w/ HLA-24 binding motif PBF

26. Binding Affinity to HLA-A24 AYRPVSRNI

27. Frequency of T cells in patients with OS, reacting with the A24.2 peptide Participants Age Status of Chemotherapy Frequency tumor bearing among CD8+ cells -6 (P) , M 7 x 10 1 18 underway -7 6 x 10 2 42 P not done -6 6 x 10 3 8 (P) underway -7 5 x 10 4 12 (P) underway -6 < 1 x 10 5 65 (P), M underway 2 x 10 6 20 (P) underway -6 6 x 10 7 16 (P) underway -6 3 x 10 8 12 (P) underway -6 3 x 10 9 20 P underway -6 9 x 10 10 18 P done -7 Average: 3.2 x10-6

28. 20.2% OS2000 OS2000+IFNγ LCL-OS2000 HOS U2OS K562 CTL #034 Cytotoxicity of anti-A24.2 CTLs Tsukahara et al., Cancer Sci, 2008 Tsukahara et al., J Transl Med, 2009

29. A2.2 ALPSFQIPV 2009 Approved Delivered PBF-derived peptide vaccines HLA-A24 HLA-A2 Peptide A24.2 Sequence AYRPVSRNI Publication IRB GMP grade peptide 2008 Approved Delivered

30. Case Age Peptide Vaccination Immune response 1 18 A24.2 2 ND 2 16 A2.2 2 ND 10w 11w 1w 2w 3w 4w 5w 6w 7w 8w 9w 12w Peptide + IFA 2nd 3rd 4th 5th 1st 6th Toxicity Tumor - PD - PD Vaccination Protocol Peptide (1mg) + IFA (Montanide ISA 51)

31. A giant step forward – If…. For many years osteogenic sarcoma has been the “bete noire” of the chemotherapeutist. Burchenal JH, New Engl J Med:1974

32. A clue 1970s High-dose MTX Rosen G 2000s High-dose Rosenberg SA cell transfer

33. Conclusion • In the staged translational research, we found that papillomavirus binding factor (PBF) serves as a novel antigen, a poor prognostic factor, and an apoptosis regulator with BAT3, of osteosarcoma. • PBF-derived peptides were recognized by peripheral T cells of patients with osteosarcoma and they induced specific cytotoxicity. • Safety and efficacy of PBF-derived peptide vaccines are currently in clinical evaluation.