development of hla-a2- and hla-a24- restricted peptide vaccines from a novel osteosarcoma antigen,...
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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
Osteosarcoma
Introduced by Alexis Boyer in 1814.
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
Amputation
Autologous tumor cell vaccination as adjuvant therapy
Marcove et al, CA, 1973
T cell
Tumor cell
Current immunotherapy
TCR
Vaccination
Antigenic peptide
Peptide vaccines
Tumor cell
T cell
T cell
T cell
T cell
T cellT cell
T cell
T cell
Dendritic cell
Patients
T cell
T cell
T cell
Injection
T cell
Active immunotherapy (vaccination)
MAGE-3
Peptide vaccination for malignant melanoma
Coulie PG, Universite de Louvain , Brussels
Efficacious peptide vaccinesfor Osteosarcoma
Goal
To identify tumor antigens and antigenic peptides
Tumor
T cellTCR HLA
class I
16 y.o. F, Osteosarcoma
TcOScl-303
OS2000
Nabeta et al., J Orthop Sci, 2003.
0
5
10
15
20
25
30
35
40
%sp
ecif
ic ly
sis
5050 1616E/T ratioE/T ratio
OS2000 IFN-γ(+)
OS2000 IFN-γ(-)
▲ 293EBNAK562
33
OS2000+
TcOScl-303
TcOScl-303 kills OS2000, but not 293EBNA or K562 cells
OS2000
Antigenic peptide
Expression cloning
Cloning of a gene encoding the antigenic peptide
TCR HLA class I
TcOScl-303
Papillomavirus Binding Factor (PBF)as a novel osteosarcoma antigen
Clone 1B9.1H4 ↑3' UTR (432bps)
704 PolyAΔN-PBF
PBF
337 1878↑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
Immunostaining with anti-PBF antibody
Anti-PBF
Osteosarcoma Pancreas Ovary Spleen
Osteosarcoma 72/78Ewing’s sarcoma 31/34Synovial sarcoma 18/20
Tsukahara et al., Cancer Res, 2004
Stages of PBF Project
I
II
III
IV
Identification
Function
Regulation
Application
Boeckle et al., 2002Tsukahara et al., 2004
Stages of PBF Project
I
II
III
IV
Identification
Function
Regulation
Application
Boeckle et al., 2002Tsukahara et al., 2004
Prognostic significance, Targeting/Overexpression
Associated proteins,Expression of HLA class I
Development of peptide vaccines
Event-free Survival of OS Pts.
0
10
20
30
40
50
60
70
80
90
100
0 50 100 150 200months
Eve
nt-f
ree
surv
ival
rate
(%)
PBF negative: 6
PBF positive: 72
P=0.025
Tsukahara et al., Cancer Sci, 2008
Stages of PBF Project
I
II
III
IV
Identification
Function
Regulation
Application
Boeckle et al., 2002Tsukahara et al., 2004
Prognostic significance, Targeting/Overexpression
Associated proteins,Expression of HLA class I
Development of peptide vaccines
PBF knock-out mice
PBF (+/-)
Stages of PBF Project
I
II
III
IV
Identification
Function
Regulation
Application
Boeckle et al., 2002Tsukahara et al., 2004
Prognostic significance, Targeting/Overexpression
Associated proteins,Expression of HLA class I
Development of peptide vaccines
YeastBait : PBF
Prey : cDNA library of OS2000
Yeast Two-hybrid Screening
OS2000
BAT3PBF Merge
5’ 3’
open reading frame 1126
Ubiquitin-like domain NLS(1030-1053)
HLA-B Associated Transcript 3 (BAT3)
Tsukahara et al., Cancer Sci, 2009
Stages of PBF Project
I
II
III
IV
Identification
Function
Regulation
Application
Boeckle et al., 2002Tsukahara et al., 2004
Prognostic significance, Targeting/Overexpression
Associated proteins,Expression of HLA class I
Development of peptide vaccines
HLA-restriction and HLA-limitation
A24A2
HLA in Japanese population
B51
B61B55
OS2000
TCR HLA-B55
TcOScl-303
PBF
16 y.o. F
PBF-peptides w/ HLA-24 binding motif
PBF
Binding Affinity to HLA-A24
AYRPVSRNI
Participants Age Status of Chemotherapy Frequencytumor bearing among CD8+ cells
1 18 (P), M underway 7 x 10 -6
2 42 P not done 6 x 10 -7
3 8 (P) underway 6 x 10 -6
4 12 (P) underway 5 x 10 -7
5 65 (P), M underway < 1 x 10 -6
6 20 (P) underway 2 x 10 -6
7 16 (P) underway 6 x 10 -6
8 12 (P) underway 3 x 10 -6
9 20 P underway 3 x 10 -6
10 18 P done 9 x 10 -7
Average: 3.2 x10-6
Frequency of T cells in patients with OS, reacting with the A24.2 peptide
Tsukahara et al., Cancer Sci, 2008Tsukahara et al., J Transl Med, 2009
-5 0 5 10 15 20
10:13:11:1
OS2000OS2000+IFNγLCL-OS2000
HOSU2OSK562
CTL #034
20.2%
Cytotoxicity of anti-A24.2 CTLs
PBF-derived peptide vaccines
HLA-A24 HLA-A2
Peptide A24.2Sequence AYRPVSRNIPublicationIRBGMP grade peptide
2008Approved Delivered
A2.2ALPSFQIPV2009ApprovedDelivered
1w 2w 3w 4w 5w 6w 7w 8w 10w 11w 12w9wPeptide
+ IFA 1st 2nd 3rd 4th 5th 6th
Vaccination ProtocolPeptide (1mg) + IFA (Montanide ISA 51)
Case Age Peptide Vaccination Immune response
1 18 A24.2 2 ND 2 16 A2.2 2 ND
Toxicity Tumor - PD - PD
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
1970s High-dose MTX Rosen G
A clue
2000s High-dose Rosenberg SA cell transfer
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