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. 2008 Feb 6;99(2):368–375. doi: 10.1111/j.1349-7006.2008.00695.x

Prognostic impact and immunogenicity of a novel osteosarcoma antigen, papillomavirus binding factor, in patients with osteosarcoma

Tomohide Tsukahara 1,2,3, Satoshi Kawaguchi 2,✉, Toshihiko Torigoe 3, Shigeharu Kimura 3, Masaki Murase 2,3, Shingo Ichimiya 3, Takuro Wada 2, Mitsunori Kaya 2, Satoshi Nagoya 2, Takeshi Ishii 4, Shin‐ichiro Tatezaki 4, Toshihiko Yamashita 2, Noriyuki Sato 3
PMCID: PMC11159620  PMID: 18271936

Abstract

To develop peptide‐based immunotherapy for osteosarcoma, we previously identified papillomavirus binding factor (PBF) as a cytotoxic T lymphocytes (CTL)‐defined osteosarcoma antigen in the context of human leukocyte antigen (HLA)‐B55. In the present study, we analyzed the distribution profile of PBF in 83 biopsy specimens of osteosarcomas and also the prognostic impact of PBF expression in 78 patients with osteosarcoma who had completed the standard treatment protocols. Next, we determined the antigenic peptides from PBF that react with peripheral T lymphocytes of HLA‐A24+ patients with osteosarcoma. Immunohistochemical analysis revealed that 92% of biopsy specimens of osteosarcoma expressed PBF. PBF‐positive osteosarcoma conferred significantly poorer prognosis than those with negative expression of PBF (P = 0.025). In accordance with the Bioinformatics and Molecular Analysis Section score, we synthesized 10 peptides from the PBF sequence. Subsequent screening with an HLA class I stabilization assay revealed that peptide PBF A24.2 had the highest affinity to HLA‐A24. CD8+ T cells reacting with a PBF A24.2 peptide were detected in eight of nine HLA‐A24‐positive patients with osteosarcoma at the frequency from 5 × 10−7 to 7 × 10−6 using limiting dilution/mixed lymphocyte peptide culture followed by tetramer‐based frequency analysis. PBF A24.2 peptide induced CTL lines from an HLA‐A24‐positive patient, which specifically killed an osteosarcoma cell line that expresses both PBF and HLA‐A24. These findings suggested prognostic significance and immunodominancy of PBF in patients with osteosarcoma. PBF is the candidate target for immunotherapy in patients with osteosarcoma. (Cancer Sci 2008; 99: 368–375)

Osteosarcoma is the most common primary malignant tumor of bone. The past three decades have witnessed remarkable advances in the treatment of osteosarcoma. These include the introduction of adjuvant chemotherapy, establishment of guidelines for adequate surgical margins, and the development of postexcision reconstruction.( 1 , 2 ) There have also been advances in the field of immunotherapy for osteosarcoma that, unfortunately, have received less attention.( 3 , 4 ) However, the current stagnation in chemotherapy‐based treatments for osteosarcoma has reignited interest in immunotherapeutic approaches.( 5 , 6 )

Recent immunotherapy depends largely on understanding of the molecular interactions between T cell receptors (TCR) on cytotoxic T lymphocytes (CTL) and antigenic peptides on tumor cells. This has led to a variety of vaccination approaches, including those with antigenic peptides( 7 ) recombinant viruses encoding antigenic genes( 8 ) dendritic cells( 9 ) and T lymphocytes, in which the TCR recognizing an antigenic peptide is genetically engineered.( 10 ) Nevertheless, such immunotherapeutic approaches were hampered in osteosarcoma by a lack of defined antigens until we recently identified papillomavirus binding factor (PBF) using an osteosarcoma cell line and an autologous CTL clone restricted by human leukocyte antigen (HLA)‐B*5502.( 11 , 12 ) PBF is a DNA‐binding transcription factor with unknown function.( 13 , 14 ) The oncogenic role of PBF in osteosarcoma and its immunogenicity in patients with common HLA alleles such as HLA‐A2 and HLA‐A24 needs to be disclosed before development of clinically applicable PBF‐based immunotherapy.

In the present study, we analyzed the distribution profile, prognostic impact, and immunogenicity of PBF in patients with osteosarcoma. Immunogenicity analysis focused on frequency and cytotoxicity of T cells in patients with HLA‐A24 allele by using limiting dilution (LD)/mixed lymphocyte peptide culture (MLPC)/tetramer assays.

Materials and Methods

This study was approved under institutional guidelines for the use of human subjects in research. The patients and their families as well as healthy donors gave informed consent for the use of blood samples and tissue specimens in our research.

Generation of anti‐PBF antibody.  A polyclonal antibody against PBF was generated by immunizing rabbits with 100 µg of a 15‐mer peptide, CGDTVDSDQFKREED, once per week for six weeks (SigmaGenosys, Sapporo, Japan). The serum was collected seven days after the last immunization and purified using Protein A column. The specificity of the anti‐PBF antibody was confirmed previously by Western blotting and immunostaining.( 12 )

Immunohistochemistry.  Formalin‐fixed paraffin‐embedded sections were obtained from 83 biopsy specimens of the primary lesion of osteosarcoma (Table 1). The sections were deparaffinized, boiled in a microwave oven, and blocked with 1% non‐fat dry milk before staining with streptavidin‐biotin‐complex (Nichirei, Tokyo, Japan) as previously described.( 15 ) Hematoxylin was used for counter staining. The reactivity of the anti‐PBF antibody was determined by staining of the nuclei of tumor cells.( 12 ) The expression status of PBF was graded semiquantitatively according to the modified classification described by Al‐Batran et al.( 16 , 17 ) negative (positive cells <5%), low (≤5% positive cells ≤50%), and high (positive cells >50%) (Fig. 1). Diffuse expression and heterogeneous expression were regarded as high grade and low grade, respectively. Focal expression was graded as low or negative according to the percentage of positive cells.

Table 1.

The grade of PBF expressioni and the progonosis in 83 patients with osteosarcoma

Patients Age Gender Location Surgical stage Histological type Chemotherapy Histological response grade Operation PBF expression EFS (months) OS (months) Prognosis
1 15 M Femur IIB Osteoblastic T‐12 2 Amputation High  39  46 DOD
2 20 M Humerus IIB Osteoblastic T‐12 0 Amputation Low  12  24 DOD
3  9 F Femur IIB Chondroblastic T‐12 0 WE+FVFG High  14  22 DOD
4 12 M Femur IIB Osteoblastic T‐12 0 Amputation Low   3   9 DOD
5 10 F Humerus IIB Teleangiectatic T‐12 1 WE+FVFG High 103 103 CDF
6 17 F Humerus IIB Osteoblastic T‐12 2 WE+FVFG Low  97  97 CDF
7 14 F Femur IIB Fibroblastic NSH‐7 2 WE+FVFG Negative 119 119 CDF
8 14 M Fibula IIB Chondroblastic NSH‐7 2 WE High  34 102 NED
9 11 F Tibia IIB Osteoblastic NSH‐7 2 WE+FVFG Negative 117 117 CDF
10 42 F Tibia IIB Osteoblastic NSH‐7 2 WE+Prosthesis High  72  72 CDF
11 14 M Tibia IIB Osteoblastic NSH‐7 3 WE+FVFG High  32  96 NED
12 18 M 2nd rib IIB Osteoblastic NSH‐7 0 WE High 106 106 CDF
13 33 M Pelvis IIB Chondroblastic NECO93J 1 WE+FVFG High   8  13 DOD
14 20 M Femur IIB Fibroblastic NECO93J 2 WE+FVFG High  72  72 CDF
15 15 M Femur IIB Osteoblastic NECO93J 2 WE+FVFG High 106 106 CDF
16 15 M Femur IIB Osteoblastic NECO93J 2 WE+FVFG High  20  33 DOD
17 20 F Femur IIB Osteoblastic Not done† (–)† Not done† Negative   0   7 DOD
18 16 M Tibia IIB Chondroblastic NECO93J 2 WE+FVFG High   6   6 DOC‡
19 14 F Femur IIB Osteoblastic NECO93J 2 WE Negative   5   5 DOC‡
20 15 M Fibula IIB Osteoblastic NECO93J 0 Amputation High  13  74 DOD
21 13 M Humerus IIB Chondroblastic NECO93J 0 Amputation High   7   9 DOD
22  7 F Femur IIB Osteoblastic NECO95J 1 WE+FVFG High  85  85 CDF
23 10 F Tibia IIB Osteoblastic NECO95J 2 Amputation Negative  72  72 CDF
24 13 M Femur IIB Osteoblastic NECO95J 0 WE+RP Low   6  11 DOD
25 27 M Tibia IIB Osteoblastic NECO95J 0 WE+FVFG High   8  18 DOD
26 18 F Femur IIIB Fibroblastic NECO95J 1 WE+Prosthesis High   0  16 DOD
27 20 F Humerus IIB Osteoblastic NECO95J 2 WE+Prosthesis Negative  67  67 CDF
28 69 F Femur IIB Fibroblastic NECO95J 0 WE+Prosthesis Low  62  63 NED
29 15 F Femur IIB Osteoblastic NECO95J 3 WE+FVFG High  32  32 CDF
30 46 M Pelvis IIB Fibroblastic NECO95J 0 WE+Fillet Low  47  60 NED
31 40 F Femur IIB Fibroblastic NECO95J 2 Amputation Low  12  20 DOD
32 19 M Tibia IIB Osteoblastic NECO95J 2 Amputation High  41  43 DOD
33 15 F Femur IIB Fibroblastic NECO95J 3 WE+FVFG High  52  52 CDF
34 48 M Femur IIB Osteoblastic NECO95J 2 Amputation High  17  37 DOD
35 15 F Femur IIIB Chondroblastic NECO95J 1 WE+FVFG High   0  17 NED
36 42 F Sacrum IIB Osteoblastic NECO95J (–)§ Not done§ High   0  18 NED
37  7 M Tibia IIB Osteoblastic CCCH2 1 WE+RP Low  13  86 DOD
38 12 F Femur IIB Chondroblastic CCCH2 0 Amputation Low   5  84 DOD
39 17 M Tibia IIB Chondroblastic CCCH2 1 WE+RP High 180 180 CDF
40 14 F Femur IIB Fibroblastic CCCH2 0 WE+Prosthesis High  13 167 NED
41 19 M Tibia IIB Fibroblastic CCCH2 0 WE+RP Low  20  36 DOD
42 14 F Tibia IIB Osteoblastic CCCH2 0 WE+Prosthesis Low  19  44 DOD
43 12 M Tibia IIB Osteoblastic CCCH2 2 WE+Prosthesis High 167 167 CDF
44 22 M Femur IIB Fibroblastic NECO93J 3 WE+RP Low 164 164 CDF
45 21 M Femur IIB Chondroblastic NECO93J 1 WE+RP High  34  80 DOD
46 20 M Tibia IIB Osteoblastic NECO93J 1 WE+RP High  37 131 NED
47 17 M Femur IIB Osteoblastic NECO93J 3 WE+FVFG High 127 127 CDF
48  8 F Humerus IIB Osteoblastic NECO93J 3 WE+FVFG Low 126 126 CDF
49 20 F Humerus IIB Fibroblastic NECO93J 1 WE+FVFG High 126 126 CDF
50 16 F Femur IIB Osteoblastic NECO93J 2 WE+RP High 124 124 CDF
51 18 M Tibia IIB Osteoblastic NECO93J 0 WE+RP Low  10  15 DOD
52 13 F Femur IIB Osteoblastic NECO95J 1 WE+Prosthesis Low 100 100 CDF
53 11 M Tibia IIB Chondroblastic NECO93J 2 WE+RP High 121 121 CDF
54 12 F Tibia IIB Osteoblastic NECO95J 1 WE+Prosthesis High 114 114 CDF
55 24 M Femur IIB Osteoblastic NECO95J 1 WE+Prosthesis Negative  79  79 CDF
56 14 F Humerus IIB Chondroblastic NECO95J 1 WE+FVFG High  29  96 NED
57 26 M Tibia IIB Osteoblastic NECO95J 0 WE+Prosthesis High  89  89 CDF
58 17 M Radius IIB Osteoblastic NECO95J 1 WE+FVFG Low  84  84 CDF
59 18 F Femur IIB Chondroblastic NECO95J 1 WE+Prosthesis High  17  69 DOD
60 15 M Femur IIB Osteoblastic NECO95J 2 WE+Prosthesis Low  75  75 CDF
61 17 M Femur IIB Osteoblastic NECO95J 2 WE+Prosthesis Low  74  74 CDF
62 13 M Femur IIB Osteoblastic NECO95J 2 WE+Prosthesis Low  72  72 CDF
63 11 F Femur IIB Osteoblastic NECO95J 3 WE+Prosthesis Low  66  66 CDF
64 13 M Femur IIB Osteoblastic NECO95J 2 WE+Prosthesis High  65  65 CDF
65 13 M Femur IIB Osteoblastic NECO95J 2 WE+Prosthesis Negative  57  57 CDF
66 19 F Femur IIB Osteoblastic NECO95J 1 WE+Prosthesis High  54  54 CDF
67 24 M Tibia IIB Fibroblastic NECO95J 0 WE+Prosthesis Low  45  45 CDF
68 14 F Femur IIB Osteoblastic NECO95J 2 WE+Prosthesis High  48  48 CDF
69 15 F Femur IIB Osteoblastic NECO95J 1 WE+Prosthesis Low   7  18 DOD
70 19 M Radius IIB Osteoblastic NECO95J 1 WE+FVFG Low  27  35 NED
71 16 F Fibula IIB Osteoblastic NECO95J 2 WE+Prosthesis Low  33  33 CDF
72 10 M Tibia IIB Osteoblastic NECO95J 2 Amputation High  12  32 AWD
73 11 M Femur IIB Osteoblastic NECO95J 1 WE+FVFG Low  31  31 CDF
74 29 F Fibula IIB Osteoblastic NECO95J 1 WE High  25  25 CDF
75 10 M Femur IIB Chondroblastic NECO95J 0 WE+Prosthesis High   9  20 DOD
76  8 M Femur IIB Osteoblastic NECO95J 1 WE+RP High   6  19 AWD
77 20 F Tibia IIB Osteoblastic NECO95J 2 Amputation High  16  16 CDF
78 12 M Femur IIB Osteoblastic NECO95J 3 WE+Prosthesis Low  15  15 CDF
79 65 M Tibia IIB Osteoblastic NECO95J (–)§ WE+Prosthesis High   2  13 NED
80 20 M Femur IIB Osteoblastic NECO95J 2 WE+Prosthesis High  12  12 CDF
81 16 M Ilium IIB Osteoblastic NECO95J 1 WE High  13  13 CDF
82 12 M Femur IIB Osteoblastic NECO95J 2 Amputation High   8  13 DOD
83 20 M Femur IIB Osteoblastic NECO95J 2 WE+Prosthesis High  12  12 CDF
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†

Chemotherapy and operation were refused by the patient and her family.

‡

‡Died of acute hepatitis B during postoperative chemotherapy.

§

§Carbon ion radiotherapy was chosen instead of operation.

¶Chemotherapy was instituted only postoperatively. AWD, alive with disease; CDF, continuously disease free; DOC, death of other cause; DOD, death of disease; EFS, event‐free survival; F, female, FVFG, free vascularized fibula graft; M, male; NED, no evidence of disease; OS, overall survival; RP, rotational plasty; PBF, papillomavirus binding factor; WE, wide excision.

Figure 1.

Figure 1

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Immunohistochemical grading of tumor specimens. Representative sections of osteosarcoma specimens stained with an anti‐papillomavirus binding factor antibody are shown (original magnification ×200). Negative indicates that less than 5% of tumor cells were stained positively. Low indicates a positive tumor cell number from 5% to 50%. High indicates a positive tumor cell number of over 50%.

Survivorship analysis.  Survivorship analysis was performed for 78 patients with osteosarcoma who had completed the protocols consisting of pre‐ and postoperative chemotherapy and underwent resection of the primary tumor with wide margin or amputation (Table 1). Five patients excluded were due to refusal (Patient 17) or incompletion of the chemotherapy (Patients 18, 19, and 79) and choice of non‐surgical treatment (Patient 36). There were 43 males and 35 females with an average age of 17.9 years. Primary tumors were located in the femur (41 patients), tibia (19 patients), humerus (8 patients), fibula (4 patients), pelvis (3 patients), radius (2 patients) and rib (1 patient). According to Enneking's surgical staging system,( 18 ) 76 patients were stage IIB and 2 patients were stage IIIB. There were 53 osteoblastic, 12 chondroblastic, 12 fibroblastic and 1 teleangiectatic osteosarcomas. Adjuvant chemotherapy protocols comprised of high‐dose methotrexate‐based multidrug regimen, including T‐12,( 19 ) NSH‐7,( 20 ) CCCH2,( 21 ) NECO93J,( 22 ) and NECO95J.( 23 , 24 ) The responses of the tumors to preoperative chemotherapy were histologically graded according to the classification of the Japanese Orthopaedic Association:( 25 ) Grade 0 (tumor necrosis <50%), 1 (≤50% tumor necrosis <90%), 2 (tumor necrosis ≥90%), 3 (No viable tumor cells in the histological sections). These 78 patients were followed up for an average of 65.0 months (range from 9 to 180 months).

The prognostic significance of the following variables overall and in the event‐free survival of patients with osteosarcoma was determined by univariate analysis using the generalized Wilcoxon test: age ( 15), gender (male or female), stage (IIB or IIIB), histological type (osteoblastic, chondroblastic or fibroblastic), response to chemotherapy (Grades 0 and 1 or Grades 2 and 3), and PBF expression status (negative, low or high). The relationship between each variable and PBF expression status was determined by the chi‐squared test. A probability of less than 0.05 was considered statistically significant.

Cell lines.  An osteosarcoma cell line, OS2000, and an Epstein−Barr virus‐transformed B cell line, LCL‐OS2000, were established previously from a 17‐year‐old patient.( 11 ) Osteosarcoma cell lines HOS and U2OS, and the erythroleukemia cell line K562 were purchased from American Type Culture Collection (Manassas, VA, USA). OS2000, HOS, U2OS and K562 were PBF+ and LCL‐OS2000 was PBF−.( 12 ) The HLA genotypes of osteosarcoma cell lines were as follows: OS2000, A*2402, B*5502, B*4002, Cw*0102; HOS, A*0211, B*5201, Cw*1202; U2OS, A*0201, A*3201, B*4402, Cw*0501, Cw*0704.

Design and synthesis of PBF‐derived peptides.  Based on the entire amino acid sequence of PBF, peptides with the ability to bind to HLA‐A24 class I molecules were searched through the Internet site, Bioinformatics and Molecular Analysis Section (BIMAS) HLA Peptide Binding Predictions (http://bimas.cit.nih.gov/).( 26 ) Based on the binding scores, 10 peptides were selected and synthesized (Table 2).

Table 2.

Sequences and binding affinities of PBF‐derived peptides with HLA‐A*2402 binding motif

Peptide Position Sequence Binding score† HLA‐peptide binding affinity‡ (% MFI increase ± SD)
A24.1  84–93 WYGGQECTGL 200  –3.0 ± 3.2
A24.2 145–153 AYRPVSRNI  84 119.2 ± 7.3
A24.3 409–418 AYQALPSFQI  75  25.3 ± 6.0
A24.4 254–263 GFETDPDPFL  30   2.6 ± 10.3
A24.5 320–328 DFYYTEVQL  20   2.3 ± 9.6
A24.6 118–127 RVEEVWLAEL  15.8  22.5 ± 9.6
A24.7 254–262 GFETDPDPF  15  –1.2 ± 5.9
A24.8  12–20 RSLLGARVL  12  14.2 ± 5.8
A24.9 415–424 SFQIPVSPHI  10.5  11.7 ± 7.9
A24.10 104–113 VTVWlLEQKL   9.5   7.1 ± 1.2
HIV RYLRDQQLLGI  41.2 ± 9.3
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†

Binding score was determined by BIMAS HLA Peptide Binding Predictions.

‡

‡The affinity of each peptide was evaluated by a HLA class I stabilization assay. BIMAS, bioinformatics and molecular analysis section; HLA, human leukocyte antigen; MFI, mean fluorescence intensity; PBF, papillomavirus binding factor; SD, standard deviation.

HLA class I stabilization assay.  The affinity of peptides for HLA‐A24 molecules was evaluated by cell surface HLA class‐I stabilization assay as described previously.( 27 , 28 ) An HLA‐A*2401‐binding HIV peptide (RYLRDQQLLGI) was used for positive control. Assays were performed in triplicate. The affinity of each peptide for HLA‐A*2402 molecules was evaluated by the percent mean fluorescence intensity (%MFI) increase of the HLA‐A*2402 molecules in the calculation: %MFI increase: [(MFI with the given peptide – MFI without peptide)/(MFI without peptide)] × 100.

Limiting dilution/mixed lymphocyte peptide culture.  Prior to frequency analysis and cytotoxicity assays, peripheral blood mononuclear cell (PBMC) of patients were subjected to mixed lymphocyte peptide culture under limiting dilution conditions (LD/MLPC) according to the method described by Karanikas et al.( 29 ) with some modifications. For frequency analysis, peripheral blood samples (20 mL) were collected from nine patients with PBF+ osteosarcoma (Patient 26, 36, 76, 78–83) (Table 1). PBMC were suspended in AIM‐V (Invitrogen Crop., Carlsbad, CA, USA) supplemented with 1% human serum (HS) and incubated for 60 min at room temperature with PBF A24.2 peptide (25 µg/mL). Peptide‐pulsed PBMC were seeded at 2 × 105 cells/200 µL/well into round‐bottom 96‐microwell plates in AIM‐V with 10%HS, IL‐2 (20 U/mL; a kind gift from Takeda Chemical Industries Ltd, Osaka, Japan) and IL‐7 (10 ng/mL; R & D Systems, Minneapolis, MN, USA), and incubated. On day 7, half of the medium was replaced by fresh AIM‐V containing IL‐2, IL‐7 and the same peptides. The cell cultures were maintained by adding fresh AIM‐V containing IL‐2. On days 14–21, they were subjected to tetramer‐based frequency analysis.

For cytotoxicity assays, PBMC of Patient 36 were separated into CD8+ cells and CD8− cells using magnetic anti‐CD8 microbeads (Miltenyi Biotec, Gladbach, Germany). CD8− cells were pulsed with the PBF A24.2 peptide for 60 min. Half of the CD8− cells were cryopreserved at –80°C for the second stimulation. CD8+ cells (2.5 × 105/well) and irradiated PBF A24.2 peptide‐pulsed CD8− cells (5 × 105/well) were cocultured in 37 wells of a 48‐well cell culture plate in 500 µL of AIM‐V with 10%HS, IL‐2 and IL‐7. On day 7, the second stimulation was performed by adding irradiated peptide‐pulsed CD8− cells to each culture well in 500 µL of freshly replaced AIM‐V with 10%HS, IL‐2 and IL‐7. On day 14–28, they were subjected to tetramer‐based cytotoxicity assays.

Tetramer‐based frequency analysis.  An fluorescein isothiocyanate‐conjugated HLA‐A24/HIV tetramer (here termed the control tetramer) and a phycoerythrin (PE)‐conjugated HLA‐A24/PBF A24.2 tetramer (A24/PBF A24.2 tetramer) were constructed by Medical & Biological Laboratories Co. Ltd. (Tokyo, Japan). PBMC from patients were stimulated with the PBF A24.2 peptide by LD/MLPC as described above. From each microwell containing 200 µL of the microculture pool, 100 µL was transferred to a V‐bottom microwell and washed. On the spin‐down pellets, the control tetramer and A24/PBF A24.2 tetramer (10 nM in 25 µL of phosphate‐buffered saline (PBS)) were added in combination and incubated for 15 min at room temperature. Then a PE‐Cy5‐conjugated anti‐CD8 antibody (eBioscience, San Diego, CA, USA) was added (dilution of 1:30 in 25 µL of PBS containing the control tetramer and A24/PBF A24.2 tetramer) and incubated for another 15 min. The cells were washed in PBS twice, fixed with 0.5% formaldehyde, and analyzed by flow cytometry using FACScan and CellQuest software (Becton Dickinson, San Jose, CA, USA). CD8+ living cells were gated and the cells labeled with the A24/PBF A24.2 tetramer and non‐labeled cells with the control tetramer were referred to as tetramer‐positive cells. The frequency of anti‐PBF A24.2 CTLs was evaluated using the following calculation: (number of tetramer‐positive wells)/([numbers of total tested wells] × [number of CD8+ cells per well]).

Tetramer‐based cytotoxicity assay.  CTL‐mediated cytolytic activity was measured by a 6 h‐51Cr release assay.( 30 ) Osteosarcoma cell lines (OS2000, HOS and U2OS), EB‐transformed B cell line LCL‐OS2000 and K562 were used as the target cells. OS2000 was treated with and without 100 U/mL interferon‐gamma (R & D Systems, Minneapolis, MN, USA) for 48 h. LCL‐OS2000 was also treated with and without peptides (25 µg/mL) for 2 h at room temperature before assay. Target cells were labeled with 100 µCi of 51Cr for 1 h at 37°C. The labeled target cells were suspended in Dulbecco's modified eagle's medium containing 10% fetal calf serum and seeded to microwells (2 × 103 cells/well). Patient 36‐derived CD8+ CTL lines stimulated with the PBF A24.2 peptide by LD/MLPC were used as the effector cells. Tetramer‐positive CTL lines were transferred to V‐bottom microwells, suspended in AIM‐V and mixed with the labeled target cells. In cold‐target inhibition assays, a 100‐fold excess of unlabeled PBF A24.2‐pulsed target cells was added as cold target cells. After a 6 h incubation period at 37°C, the release of the 51Cr level in the supernatant of the culture was measured by quantification in an automated gamma counter. The percentage of specific cytotoxicity was calculated as the percentage of specific 51Cr release: 100 × (experimental release – spontaneous release)/(maximum release – spontaneous release). The cytotoxicity rate to OS2000 was calculated as (%cytotoxicity to each target cells)/(%cytotoxicity to OS2000).

Results

Expression of PBF protein in osteosarcoma.  To determine the prevalence of the osteosarcoma‐derived antigen PBF protein in osteosarcomas, we stained formalin‐fixed paraffin‐embedded sections of 83 specimens with a polyclonal antibody against PBF (Table 1 and Fig. 1). Of these, 76 specimens (92%) were positively stained with the anti‐PBF antibody, including 49 specimens (59%) with high‐grade staining.

Prognostic impact of PBF expression in patients with osteosarcoma.  We then analyzed the prognostic significance of several variables including expression of PBF, in 78 patients with osteosarcoma who completed chemotherapy protocols and had wide tumor excision (Table 1). As depicted in Table 3, patients with chondroblastic type osteosarcoma showed significantly poorer event‐free survival rate than those with other histological types. Forty‐one patients with osteosarcoma showing a poor response to preoperative chemotherapy (Grades 0 and 1) showed significantly more unfavorable event‐free and overall survival rates than 37 good responders (Grades 2 and 3). With respect to PBF‐expression status, 72 patients with positive expression of PBF in the primary tumor were significantly more unfavorable in event‐free survival than six patients with PBF‐negative osteosarcoma (P = 0.025). This finding was consistent in subgroup analysis with 46 patients with high‐grade PBF expression and 26 patients with low‐grade PBF expression (P = 0.026 and 0.032, respectively). In contrast, age and gender failed to have significant prognostic impacts on event‐free and overall survival rates of the patients.

Table 3.

Univariate analysis of variables in event‐free survial and overall survival

Variables n Event‐free survival (months in average) P‐value Overall survival (months in average) P‐value
Age ≤15 41  49.6  0.215 66.8 0.949
>15 37  54.7 63
Gender Male 46  48.5  0.244 64.4 0.101
Female 32  57.1 70.8
Stage IIB 76  53.4  0.006 66.3 0.062
IIIB  2   0 16.5
Histological type Osteoblastic 53  52.1  0.052* 60.7 0.461*
Chondroblastic 12  38.2 <0.001* 67.7 0.105*
Fibroblastic 12  61.0  0.899* 78.3 0.665*
Teleangiectatic  1 103.4
Response to chemotherapy Grades 0,1 41  40.5 <0.001 60.2 0.007
Grades 2,3 37  64.7 70.3
PBF status Positive 72  49.2  0.025 63.3 0.091
Negative  6  85.2 85.2
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*

P‐value was determined in comparison with the survival of patients with other subtypes.

Subsequently, we analyzed the relationship between the PBF‐expression status in osteosarcoma and other variables. PBF‐expression status was not significantly related to any variables including age (P = 0.472), gender (P = 0.184), stage (P = 0.694), histological type (P = 0.743) and the response to chemotherapy (P = 0.069).

Affinity of PBF‐derived synthetic peptides to HLA‐A*2402 molecules.  To determine the immunogenicity of PBF in patients with HLA‐A24, we synthesized 10 peptides from the PBF sequence in accordance with the BIMAS score for HLA‐A24 affinity (Table 2). Subsequently, we evaluated the affinities of these peptides to HLA‐A24 molecules by the HLA class‐I stabilization assay. As shown in Table 2, peptide PBF A24.2 showed the highest MFI increases in the context of HLA‐A24.

Frequency of anti‐PBF A24.2 CTLs in HLA‐A24+ patients with osteosarcoma.  We then examined the frequency of peripheral CD8+ T‐lymphocytes that recognized the PBF A24.2 peptide in 9 HLA‐A24+ patients with PBF+ osteosarcoma by LD/MLPC/tetramer analysis. As depicted in Table 4 and representatively shown in Fig. 2(a), anti‐PBF A24.2 CTLs were detected as tetramer‐positive cells in eight of the nine patients with osteosarcoma. The frequencies of anti‐PBF A24.2 CTLs were between 5 × 10−7 and 7 × 10−6 (4 × 10−6 in average) in eight tetramer‐positive patients.

Table 4.

Clinical picture and frequency of anti‐PBF A24.2 peptide CTLs in PBMC of patients with PBF‐positive osteosacoma

Participants Status of tumor bearing Chemotherapy Total number of tested wells Number of tetramer‐ positive wells Number of PMBC %CD8 Number of CD8+ cells per pool Frequency †
Patient
26 (P)‡, M underway  62 14 200 000 17 34 000  7 × 10−6
36 P not done 194  6 210 000 23 48 000  6 × 10−7
76 (P) underway  19  2 200 000  9 18 000  6 × 10−6
78 (P) underway  62  1 200 000 15 30 000  5 × 10−7
79 (P), M underway  28  0 200 000 15 30 000 <1 × 10−6
80 (P) underway 160 15 290 000 20 58 000  2 × 10−6
81 (P) underway 149  5 200 000  3   6000  6 × 10−6
82 (P) underway 132  3 200 000  4   8000  3 × 10−6
83 P underway  40  5 200 000 25 50 000  3 × 10−6
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†

Frequency of anti‐PBF A24.2 CTLs among CD8+ cells.

‡

‡Parentheses indicate that the tumor had been present previously but was free at the time blood sample was taken. CTLs, cytotoxic T lymphocytes; M, metastatic tumor; P, primary tumor; PBF, papillomavirus binding factor; PMBC, peripheral blood mononuclear cell.

Figure 2.

Figure 2

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Tetramer‐based detection of anti‐PBF A24.2 peptide CTLs in peripheral blood of patients with osteosarcoma. (a) peripheral blood mononuclear cell of Patient 26 were seeded into 62 microwells and stimulated with the papillomavirus binding factor (PBF) A24.2 peptide by mixed lymphocyte peptide culture under limiting dilution conditions (LD/MLPC). The resultant cytotoxic T lymphocytes (CTL) pools were stained with the phycoerythrin (PE)‐conjugated A24/PBF A24.2 tetramer, fluorescein isothiocyanate‐conjugated control tetramer, and a PE‐Cy5‐conjugated anti‐CD8 mAb. Cells reacting with the anti‐CD8 mAb were gated. The reactivity of gated cells with the A24/PBF A24.2 tetramer and the control tetramer are shown. The upper and middle columns display six representative pools with positive reactivity to the A24/PBF A24.2 tetramer. The cells labeled with the A24/PBF A24.2 tetramer and non‐labeled cells with the control tetramer were considered to be tetramer‐positive cells and are boxed to show their proportion among CD8+ cells. The bottom row shows three negative pools. (b) CD8+ cells (2.5 × 105 cells/well) of Patient 36 were seeded into 37 wells of a 48‐well culture plate and stimulated with irradiated peptide‐pulsed CD8− cells (5 × 105 cells/well) by LD/MLPC. Tetramer analysis was performed on day 14. The results of one of the four tetramer‐positive CTL line (CTL #034) in the 37 pools are shown. Cells reacting with the anti‐CD8 mAb were gated. The tetramer‐positive cells are boxed to show their proportion among CD8+ cells. (c) The cytotoxicity of CTL #034 against allogeneic osteosarcoma cell lines (OS2000, HOS, U2OS), LCL‐OS2000 and K562 was assessed by a 6 h standard 51Cr release assay at the indicated effector:target ratios. OS2000 was assayed in the presence and absence of 48 h‐interferon‐gamma pretreatment. (d) The cytotoxicity of CTL #034 against peptide‐pulsed LCL‐OS2000 and K562 was assessed by a 6 h standard 51Cr release assay at the indicated effector:target ratios. LCL‐OS2000 was pulsed with 25 µg/mL of PBF A24.2 peptide or HIV control peptide for 2 h at room temperature before labeling with 51Cr. (e) Cold target inhibition assay. The cytotoxicity of CTL #034 against interferon‐gamma‐treated, 51Cr‐labeled OS2000 was assessed in the presence and absence of a 100‐fold excess of PBF A24.2 peptide‐pulsed, cold LCL‐OS2000 at a 10:1 effector‐target ratio. 51Cr‐labeled LCL‐OS2000 cells pulsed with the PBF A24.2 peptide or HIV peptide were also used as control target cells.

Tetramer‐based cytotoxicity of anti‐PBF A24.2 CTLs against osteosarcoma cell lines.  Finally we assessed the cytotoxic activity of tetramer‐positive cells against allogeneic osteosarcoma cell lines. We induced tetramer‐positive anti‐PBF A24.2 CTLs from 1 × 107 CD8+ cells of Patient 36 by LD/MLPC using 48‐well culture plates. Irradiated peptide‐pulsed CD8− cells were used as stimulator cells. As a result, 4 of 37 tetramer‐positive CTLs were detected by tetramer analysis on day 14. Four tetramer‐positive CTLs contained 3.47%, 0.03%, 15.26% and 20.19%. One of four CTL lines (CTL #034) was shown in Fig. 2(b).

The cytotoxicity against OS2000 (PBF+, A24+), LCL‐OS2000 (PBF−, A24+), HOS (PBF+, A24−), U2OS (PBF+, A24−) and K562 (PBF+, HLA class I loss) was comparatively assessed by 51Cr release assay. As depicted in Fig. 2(c), CTL #034 showed specific cytotoxicity against OS2000 and the cytotoxicity was enhanced by interferon‐gamma pretreatment. In contrast, none of these CTL lines exhibited cytotoxic activity against LCL‐OS2000, HOS, U2OS, or K562 cells. The other three tetramer‐positive CTL lines also showed specific cytotoxicity against OS2000 (data not shown).

We subsequently determined the specificity of cytotoxicity with the PBF A24.2 peptide by using peptide‐pulsed LCL‐OS2000. As shown in Fig. 2(d), CTL #034 lyzed PBF A24.2 peptide‐pulsed LCL‐OS2000 more than control peptide (HIV)‐pulsed LCL‐OS2000 or K562 cells. Peptide‐specific cytotoxicity of CTL #034 was also assessed by cold‐target inhibition assay (Fig. 2e). Cytotoxicity against OS2000 pretreated with interferon‐gamma was inhibited by adding a 100‐fold excess of cold LCL‐OS2000 pulsed with PBF A24.2.

Discussion

In the present study, we examined the distribution profile, prognostic impact, and immunogenicity of the novel tumor‐associated antigen PBF in osteosarcoma. We found: (i) that 92% of 83 osteosarcoma specimens expressed PBF protein; (ii) that PBF‐positive osteosarcomas conferred a significantly poorer prognosis than those with negative expression of PBF in event‐free survival of 78 patients who completed the standard treatment; (iii) that CD8+ T cells reacting with a PBF‐derived HLA‐A24‐binding peptide (PBF A24.2 peptide) were detected in eight out of nine HLA‐A24‐positive patients with osteosarcoma at the frequency from 5 × 10−7 to 7 × 10−6; and (iv) that PBF A24.2 peptide induced CTL lines from an HLA‐A24‐positive patient, which specifically killed an osteosarcoma cell line that expresses both PBF and HLA‐A24. These findings suggest the oncogenic and antigenic role of PBF in patients with osteosarcoma, especially those with HLA‐A24. The proof of immunogenicity of PBF has been limited to an HLA‐B55‐positive patient with osteosarcoma.( 12 ) Wide distribution of PBF in osteosarcoma and the immunogenicity of PBF seen in patients with HLA‐A24 extended the possibility of PBF‐targeted immunotherapy against osteosarcoma. Patients with negative expression of PBF in osteosarcoma can be treated successfully by the current chemotherapy‐based treatment protocols.

To date, peptide‐based immunotherapy in patients with bone and soft tissue sarcomas has been reported only with the use of fusion gene‐derived peptides.( 31 , 32 ) This approach has been available for tumors in which specific chromosomal translocations have been identified, including synovial sarcoma and Ewing sarcoma. However, for other sarcomas including osteosarcoma, where chromosomal translocation and a resultant fusion gene have not been identified, novel tumor‐associated antigens need to be defined. With this aim, we developed autologous pairs of tumor cells and CTLs from patients with sarcomas. Consequently, PBF was identified from an autologous osteosarcoma‐CTL pair. PBF protein was defined in 89% of the various bone and soft tissue sarcomas (Tsukahara et al. 2004, unpub. data). Therefore, the PBF A24.2 peptide might be applicable to immunotherapy against bone and soft tissue sarcomas without known chromosomal translocations, other than osteosarcoma.

Our univariate analysis revealed prognostic significance of PBF in event‐free survival. Such prognostic values need to be verified by multivariate analysis. In this regard, all of the six patients with PBF‐negative osteosarcoma analyzed are continuously disease‐free. Unfortunately, the disproportional profile of these patients caused failure in multivariate analysis. In contrast, poor response to chemotherapy and chondroblastic subtype remained significant in the multivariate analysis (Tsukahara et al. 2007, unpub. data), indicating the validity of the patient population in the present analysis. The unfavorable prognostic value of PBF was also seen in our analysis with 20 patients with Ewing sarcoma.( 33 )

The frequency of anti‐PBF CTL precursor was determined between 5 × 10−7 and 7 × 10−6. In melanoma patients, the anti‐MAGE3.A1 CTL precursor frequency was estimated to be <10−7 in normal donors and prevaccinated patients, and 10−6 in postvaccinated patients.( 34 ) Although the anti‐PBF A24.2 peptide CTL precursor frequency defined in the present study was relatively higher than the anti‐MAGE3.A1 CTL frequency, it was still under the detection level of the standard tetramer analysis and thus required the LD/MLPC/tetramer procedure for detection. The LD/MLPC/tetramer procedure was advantageous with its high sensitivity for the frequency analysis of peptide‐specific CTLs in preclinical studies and clinical trials.( 35 , 36 ) Also, this procedure served as prescreening of CTLs for subsequent cytotoxicity analysis. However, the multistep procedure of LD/MLPC/tetramer analysis requires labor‐intensive laboratory work and long‐term cell culture.( 37 , 38 ) This gives rise to a concern about possible changes in the effector function and differentiation status of CTLs during the analysis.( 39 , 40 )

Cytotoxicity of CTL induced with PBF A24.2 peptide was proved only in an osteosarcoma cell line that is positive for PBF and HLA‐A24. It would be ideal to conduct cytotoxicity assays with more number of cell lines. However, limited cell numbers of CTL lines expanded after LD/MLPC/tetramer analysis made it difficult to carry out. Instead, we examined the specificity of the cytotoxicity by peptide‐pulsation as well as cold inhibition assays.

In conclusion, the present study demonstrated the feasibility and population of candidates for PBF‐targeted immunotherapy for osteosarcoma. The combination of LD/MLPC with tetramer labeling as well as 51Cr release cytotoxicity assay enables us to concurrently determine the frequency and function of CTL precursors, and thus serves as a useful tool for identification of novel antigenic peptides and immunomonitoring in clinical immunotherapy trials.

Acknowledgments

We thank Drs Pierre G. Coulie and Tomoko So for their kind advice about the LD/MLPC/tetramer procedure, Dr Hideo Takasu for the kind donation of synthetic peptides and Drs Naoki Hatakeyama and Takeshi Terui for their clinical support with chemotherapy and donation of blood samples.

Grant support: This work was supported by Grants‐in‐Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant no. 16209013 to N. Sato), Practical Application Research from the Japan Science and Technology Agency (Grant No. H14‐2 to N. Sato), the Ministry of Health, Labor and Welfare (Grant No. H17‐Gann‐Rinsyo‐006 to T. Wada), Postdoctoral Fellowship of the Japan Society for the Promotion of Science (Grant no. 02568 to T. Tsukahara) and Northern Advancement Center for Science and Technology (Grant No. H18‐Waka‐075 to T. Tsukahara).

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