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

Synovial sarcoma

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

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

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

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

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

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

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

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

HLA-A24-binding motif 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

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

EBVNA24F4.2ABCD Binding affinity to HLA-A24 SYT-SSX peptides

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

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

Synovial sarcoma Other sarcomas Healthy Individuals SYT-SSX A SYT-SSX B SYT-SSX C+D Positive cells (%) Reactivity to circulating T cells

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

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

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

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

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

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

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

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

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

Results

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

ToxicityDTH －－ －－ －－ －－ Fever (Grade1) － Toxicity and DTH Case －－ 6

Before vaccinationAfter 1 st vac. After 6 th vac. HIV tetramer B tetramer Tetramer analysis (Case 4)

Case Pre-vac. 1 st vac. 3 rd vac. 6 th vac N.D N.D Tetramer analysis

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

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

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

Discussion

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

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

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 ）

%Specific Lysis Fuji HS-SYII SW982 K 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

Before vaccinationAfter 1 st vaccination Vaccination of K9I peptide HIV tetramer B tetramer

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