1. PHASE I VACCINATION TRIAL OF SYT-SSX JUNCTION PEPTIDE AND ITS HLA-A*2402 ANCHOR SUBSTITUTE IN PATIENTS WITH DISSEMINATED SYNOVIAL SARCOMA Kawaguchi S, Wada T, Nagoya S, Ida K, Sato Y, Torigoe T, Sato N, Ishii T, Tatezaki S, Yamashita T Sapporo Medical University Chiba Cancer Center Hospital

2. Synovial sarcoma

3. Survival rate Guillou L et al., J Clin Oncol, 2004

4. Peptide vaccination (MAGE-3) Peptide immunotherapy for malignant melanoma Coulie PG, Universite de Louvain, Brussels

5. Antigenic peptide Tumor cell T cell Dendritic cell Patient T cell Injection T cell Peptide immunotherapy

6. Gene T cell Protein Antigenic peptide Tumor cell Antigenic peptide HLA TCR Activation

7. Desirable antigenic peptide 1.Tumor specific expression 2. High affinity to HLA 3. Immunogenicity

8. Desirable antigenic peptide 1.Tumor specific expression 2. High affinity to HLA 3. Immunogenicity

9. TumorFusion gene Frequency (%) Synovial sarcoma SYT-SSX1 65 SYT-SSX2 35 Ewing sarcoma EWS-FLI1 85 EWS-ERG 5-10 Liposarcoma TLS-CHOP >95 RMS PAX3-FKHR >75 Clear cell sarcoma EWS-ATF1 >80 DFSP COL1A1-PDGFb >99 Specific fusion gene

10. Desirable antigenic peptide 1.Tumor specific expression 2. High affinity to HLA 3. Immunogenicity

11. HLA-A24-binding motif 2 2 9 9 10 1 3 4 5 6 7 8 2 nd Portion Y, F, W, or M 9 th or 10 th Portion F, L, I, W, R, or K HLA-A24 Tumor cell T cell receptor T cell

12. SYTSSX (1, 2) ……… PQQRPYGYDQIMPKKPAEHAWTHRLRERK Y, W, K, R: HLA-A24 binding motif A : PYGYDQIMPK C : AWTHRLRER B : GYDQIMPKK D : AWTHRLRERK Peptides SYT-SSX peptides

13. EBVNA24F4.2ABCD 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 Binding affinity to HLA-A24 SYT-SSX peptides

14. Desirable antigenic peptide 1.Tumor specific expression 2. High affinity to HLA 3. Immunogenicity

15. Reactivity to circulating T cells ◇◇ ◇◇ T cell (CD8) HLA/Peptide Tetramer FACS 1.17% HLA-A24-positive Synovial sarcoma patients: 16 Other sarcoma patients: 10 Healthy individuals: 10

16. Synovial sarcoma Other sarcomas Healthy Individuals -0.1 0 0.1 0.4 0.5 0.6 0.25 SYT-SSX A SYT-SSX B SYT-SSX C+D Positive cells (%) Reactivity to circulating T cells

17. Induction of cytotoxic T lymphocytes (CTL) Peptide stimulation (SYT-SSX B) Mixed Culture Cr release assay 51 T cells Synovial sarcoma Cell line

18. HLA-A24 SYT-SSX ●Fuji (+) (+) ○HS-SY-II (+) (+) ■SW982-A24 (+) (  ) □K562 (  ) (  ) E/T ratio 30 20 10 0 40 30103 %Specific Lysis CTL induction Sato et al., J Immunol, 2002

19. SYT-SSX B peptide 1.Tumor specific expression 2. High affinity to HLA-A24 3. Immunogenicity

20. SYT-SSX SYTSSX T cell HLA-A24 SYT-SSX B Rationale ・・ PQQRPYGYDQIMPKKPA ・・・

21. SYT-SSX B peptide Synovial sarcoma cell T cell Dendritic cell Patient T cell Injection T cell Phase I clinical trial

22. To evaluate (i)Toxicity (ii) Immunological property (iii) Anti-tumor effect of SYT-SSX B peptide vaccine in patients with disseminated synovial sarcoma Purpose

23. Eligibility 1. Histologically diagnosed synovial sarcoma 2. SYT-SSX positive 3. HLA-A*2402 positive 4. Unresectable tumors 5. One month or more after chemotherapy

24. Protocol 2108640 Peptide: SYT-SSX B: GYDQIMPKK Schedule: Every two weeks Dose: 0.1mg in 3 patients 1.0mg in 3 patients

25. 1. Toxicity National Cancer Institute of Common Toxicity Criteria 2. Immunological property (i)Delayed-type hypersensitivity (DTH) (ii)HLA-A*2402/peptide tetramer (iii) In vitro CTL induction 3. Anti-tumor effect CT scan Evaluation

26. Results

27. CaseAgeGenderDose No. of immunization 169M0.1mg1 232M0.1mg3 321F0.1mg6 421M1mg6 539F1mg6 Participants 626M1mg4

28. ToxicityDTH －－ －－ －－ －－ Fever (Grade1) － Toxicity and DTH Case 1 2 3 4 5 －－ 6

29. Before vaccinationAfter 1 st vac. After 6 th vac. HIV tetramer B tetramer 0.06 0.41 0.47 0.010.00 0.01 Tetramer analysis (Case 4)

30. Case Pre-vac. 1 st vac. 3 rd vac. 6 th vac. 2 0.02 0.02 3.05 N.D 3 0.42 0.49 0.52 0.62 4 0.06 0.41 0.36 0.47 5 0.50 0.52 0.09 0.03 6 0.02 0.15 0.08 N.D Tetramer analysis

31. Case Pre-vac. 1 st vac. 3 rd vac. 6 th vac. 2 FailureFailureSuccess ND 3 SuccessSuccessSuccess Failure 4 FailureSuccessSuccess ND 5 FailureSuccessFailure Failure 6 FailureFailureFailure ND CTL induction

32. May 15 June 18 Anti-tumor effect (Case 3)

33. Anti-tumor effect (Case 5) July 8July 22 Sept 16 Aug 19

34. Discussion

35. Clinical trials of fusion-gene peptides Tumor Peptide Adjuvant Remission SSClass I None0/6 ES Class IIL-2 (9x10 6 IU/m 2 ) 1/12 RMS Class I IL-2 (9x10 6 IU/m 2 ) 0/4 CMLClass I QS-21 5/14 Class II

36. Modification of peptide and protocol 1.Anchor motif substitution 2. Adjuvant and cytokine 3. Class II peptides 4. Protocol at the adjuvant setting

37. Tumor G D Q I M P K K Y HLA-A24 Anchor motif substitution SYT-SSX B peptide W （ tryptophan ） L （ leucine ） F （ phenylalanine ） I （ isoleucine ）

38. 0 10 20 30 40 %Specific Lysis Fuji HS-SYII SW982 K562 0 10 20 30 40 E/T=30 E/T=10 E/T=3 Fuji HS-SYII SW982 K562 B peptide K9I peptide CTL induction Ida et al., J Immunol, 2004

39. Before vaccinationAfter 1 st vaccination Vaccination of K9I peptide HIV tetramer B tetramer 0.02 0.00 0.41 0.01

40. 1.The safety and efficacy of SYT-SSX B peptide were evaluated in 6 patients with disseminated synovial sarcoma. 2.A total of 16 vaccinations were carried out, resulting in (i) no serious adverse effects or DTH reactions, (ii) tumor dormancy in 1, (iii) increases in CTL in 3, and (iv) CTL induction from 4 patients. 3.The present trial demonstrated the safety and immunogenicity of the peptide. Subsequent trial using a modified peptide and adjuvants is currently underway. Conclusion