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