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

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

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

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

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

8. Efficacious peptide vaccines for Osteosarcoma Goal

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

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

11. Nabeta et al., J Orthop Sci, 2003. 5016 E/T ratio OS2000 IFN-γ(+) OS2000 IFN-γ(-) ▲ 293EBNA K562 3 OS2000 + TcOScl-303 TcOScl-303 kills OS2000, but not 293EBNA or K562 cells

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

13. Papillomavirus Binding Factor (PBF) as a novel osteosarcoma antigen Clone 1B9.1H4 ↑ 3' UTR (432bps) 704 ΔN-PBF PBF 3371878 ↑ 5' UTR 5' Open reading frame Zn finger -Protein that binds to the E2 binding site of human papillomavirus type 8 Boeckle et al., Virology, 2002

14. Immunostaining with anti-PBF antibody Anti- PBF Osteosarcoma Pancreas Ovary Spleen Osteosarcoma72/78 Ewing’s sarcoma31/34 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 PBF positive: 72 P=0.025 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. Yeast Bait : PBF Prey : cDNA library of OS2000 Yeast Two-hybrid Screening

22. OS2000 BAT3PBFMerge 5’3’ open reading frame1126 Ubiquitin-like domain NLS (1030-1053) HLA-B Associated Transcript 3 (BAT3) 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. HLA-restriction and HLA-limitation A24 A2 HLA in Japanese population B51 B61 B55 OS2000 TCR HLA- B55 TcOScl-303 PBF 16 y.o. F

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

26. Binding Affinity to HLA-A24 AYRPVSRNI

27. ParticipantsAgeStatus ofChemotherapyFrequency tumor bearingamong CD8+ cells 118 (P), M underway 7 x 10 -6 242Pnot done 6 x 10 -7 38(P)underway 6 x 10 -6 412(P)underway 5 x 10 -7 565(P), Munderway < 1 x 10 -6 620(P)underway 2 x 10 -6 716(P)underway 6 x 10 -6 812(P)underway 3 x 10 -6 920Punderway 3 x 10 -6 1018Pdone 9 x 10 -7 Average: 3.2 x10 -6 Frequency of T cells in patients with OS, reacting with the A24.2 peptide

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

29. PBF-derived peptide vaccines HLA-A24HLA-A2 PeptideA24.2 SequenceAYRPVSRNI Publication IRB GMP grade peptide 2008 Approved Delivered A2.2 ALPSFQIPV 2009 Approved Delivered

30. 1w2w3w4w5w6w7w8w 10w11w 12w9w Peptide + IFA 1st 2nd3rd4th5th 6th Vaccination Protocol Peptide (1mg) + IFA (Montanide ISA 51) Case AgePeptide Vaccination Immune response 1 18 A24.2 2ND 2 16 A2.2 2 ND Toxicity Tumor - PD

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

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

33. 1.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. 2.PBF-derived peptides were recognized by peripheral T cells of patients with osteosarcoma and they induced specific cytotoxicity. 3.Safety and efficacy of PBF-derived peptide vaccines are currently in clinical evaluation. Conclusion