ONCOLOGY REPORTS 12: 601-607, 2004
Prostate-related antigen-derived new peptides having the capacity of inducing prostate cancer-reactive CTLs in HLA-A2+ prostate cancer patients MAMORUHARADA1, SATOKOMATSUEDA1, AKIHISAYAO 1 , RIKAOGATA 1 , MASANORINOGUCHI2 and KYOGOITOH 1 Departments of 'immunology and Urology, Kurume University School of Medicine, Kurume, Fukuoka, Japan Received March 4, 2004; Accepted May 25, 2004
Abstract. Prostate-related antigens, including prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP), can be targets in specific immunotherapy for prostate cancer. In this study, we attempted to newly identify epitope peptides from these 2 antigens, which are immunogenic in human histocompatibility leukocyte antigen (HLA)-A2+ prostate cancer patients. Twenty-nine peptides (PSMA with 15 and PAP with 14) were prepared based on the HLA-A2 binding motif. Based on our previous finding that antigenic peptides recognized by both cellular and humoral immune systems are useful for peptide-based immunotherapy, peptide candidates were screened first by their ability to be recognized by immunoglobulin G (IgG), and then by their ability to induce peptide-specific cytotoxic T lymphocytes (CTLs). As a result, PSMA441-450 and PAPi 12-120 peptides were found to be frequently recognized by IgG in plasma from prostate cancer patients. These 2 candidates effectively induced HLA-A2-restricted and prostate cancer-reactive CTLs in HLA-A2 + prostate cancer patients with several HLA-A2 subtypes. In addition, their cytotoxicity was mainly dependent on peptide-specific and CD8+ T cells. These results indicate that these PSMA441-450 and PAP112-120 peptides could be promising candidates for peptide-based immunotherapy for HLA-A2+ prostate cancer. Introduction Prostate cancer is one of the most common cancers among elderly men (1). Despite the fact that androgen withdrawal
Correspondence to: Dr Mamoru Harada, Department of Immuno logy, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan E-mail:
[email protected] Abbreviations: CTLs, cytotoxic T lymphocytes; Flu, influenza; Ig, immunoglobulin; IL, interleukin; OD, potical value; PAP, prostatic acid phosphatase; PBMC, peripheral blood mononuclear cell; PSA, prostate-specific antigen; PSMA, prostate-specific membrane antigen Key words: prostate cancer, prostate-related antigens, cytotoxic T lymphocyte, peptide, HLA-A2
therapy is transiently effective for prostate cancer, there is no efficient therapy against recurrent hormone-refractory and metastatic prostate cancer. Therefore, the development of new therapeutic modalities is needed, and specific immuno therapy might be one candidate. Prostate tissue-specific antigens, expressed in normal prostate cells, can be good targets in an anti-cancer vaccine against prostate cancer (2). Indeed, several epitope peptides derived from prostate-related antigens, including prostate-specific antigen (PSA), prostatespecific membrane antigen (PSMA), or prostatic acid phosphatase (PAP), have been identified, and specific immuno therapy targeting them has been carried out (3-7). We also have identified several epitope peptides derived from prostaterelated antigens immunogenic in HLA-A24+ or HLA-A2+ prostate cancer patients (8-11). In our clinical trials involving several types of cancer, several CTL epitope peptides, which had originally been identified by their ability to induce tumor-reactive cytotoxic T lymphocytes (CTLs), were also recognized by immuno globulin g (IgG) (12,13). In addition, clinical trials revealed that the induction of IgG reactive to the administered peptides was positively correlated with the overall survival of patients with several types of cancers (14-17). These observations led us to the idea that peptides that can be recognized by both the humoral and cellular immune systems might be useful in an anti-cancer vaccine. Furthermore, the assay for peptide-specific IgG is much easier than the in vitro sensitization experiment needed to induce peptide-specific CTLs from the peripheral blood mononuclear cells (PBMCs) of cancer patients. Therefore, in this study, we first screened prostate-related antigen-derived peptide candidates according to their ability to be recognized by the humoral immune system, and then determined their potential to induce peptide-specific and prostate cancer-reactive CTLs. As a consequence, we identified 2 new CTL epitope peptides, derived from either PSMA or PAP, that effectively induce prostate cancer-reactive CTLs from prostate cancer patients with several HLA-A2 subtypes. Materials and methods Patients. All prostate cancer patients in this study provided their informed consent before enrollment. None of these participants were infected with HIV. Twenty milliliters of peripheral blood
HARADA et al: HLA-A2 BINDING PEPTIDES OF PROSTATE-RELATED ANTIGENS
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Table I. IgG reactive to the prostate-related antigen-derived peptides in plasma of prostate cancer patients. Prostate cancer patients Antigens Peptides PSMA
PAP
663-671 711-719 4-12 27-35 26-34 668-676 707-715 469-477 731-739 35-43 168-177 514-523 441-450 373-382 343-352 18-26 30-38 135-143 379-387 13-21 112-120 390-398 33-41 6-14 383-391 299-307 284-293 196-205 395-404
Sequence MMNDQLMFL ALFDIESKV LLHETDSAV VLAGGFFLL LVLAGGFFL LMFLERAFI GIYDALFDI LMYSLVHNL QIYVAAFTV LGFLFGWFI GMPEGDLVYV KLGSGNDFEV LLQERGVAYI ILGGHRDSWV KMHIHSTNEV
Score 1360 1055 485 400 375 261 251 193 177 67 2605 1412 920 650 176
1
2
3
0.043 0.043 0.086 0.078
-
4
5
0.043 0.071 0.044 0.037
6
7
-
0.064
8 -
9
Detection of peptide10 specific IgG
0.082 0.021 0.035 0.025 0.023
0.049 -
-
0.092 0.048 0.039 -
-
. 0.042 0.077 0.033 0.050 0.030 0.036 0.032 0.106
FLFLLFFWL 22037 ~ VLAKELKFV 1496 ILLWQPrPV 437 QVLKLIFAV 301 SLSLGFLFL 223 0.069 TLMSAMTNL 182 0.050 0.088 CLISAVLMV 160 KELKFVTLV 153 LLLARAASL 134 VIFAVAFCL 107 ALDVYNGLL 0.02. Peptides in italics were previously reported by other reserachers.
was obtained, and the PBMCs were prepared by Ficoll-Conray density gradient centrifugation. The expression of HLA-A2 molecules on the PBMCs of cancer patients and healthy donors was determined by flow cytometry, and the FILA-A2 subtypes were determined using the sequence-specific oligonucleotide probe method. Cell lines. T2 is an HLA-A*0201 -expressing cell line. PC93 is an HLA-A2-negative prostate cancer cell line (HLA0A*6802+) that was established by Dr K. Ohishi (Department of Urology, Kyoto University, Japan). PC93-A2 is a subline, which was stably transfected with the HLA-A'0201 gene (10). The cell lines COLO201 and COLO320 are HLA-A2+ and FJJLA-A2colon carcinomas, respectively. All cell lines were maintained in RPMI-1640 medium (Gibco BRL, Grand Island, NY) supplemented with 10% FCS. Peptides. All prostate-related antigen-derived peptides (listed in Table I) were prepared based on the HLA-A*0201 binding motif (18,19). All peptides were of >90% purity and were
purchased from Biologica Co., Nagoya, Japan. Influenza (Flu) virus-derived (GILGFVFTL), EBV-derived (GLCTLVAML), and HIV-derived peptides (SLYNTYATL) with the HLA-A2 binding motif were used as controls. All peptides were dissolved with dimethyl sulfoxide at a dose of 10 mg/ml. Detection of peptide specific IgG. Peptide-specific IgG levels in the plasma were measured by ELISA using a pre-viously reported method (20,21). In brief, peptide (20 |ig/well)immobilized plates were blocked with Block Ace (Yukijirushi, Tokyo, Japan) and washed with 0.05% Tween-20-PBS, after which 100 |il/well of plasma sample diluted with 0.05% Tween-20-Block Ace was added to the plates. After a 2-h incubation at 37°C, the plates were washed and further incubated for 2 h with a l:1000-diluted rabbit anti-human IgG (y-chain-specific) (Dako, Glostrup, Denmark). The plates were washed, and then 100 |il of l:100-diluted goat anti-rabbit IgG-conjugated horseradish peroxidase (EnVision; Dako) was added to each well, and the plates were incubated at room temperature for 40 min. After the plates were washed once,
ONCOLOGY REPORTS 12: 601-607, 2004
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Figure 1. IgG reactive to peptide candidates derived from prostate-related antigens. (A) The levels of IgG reactive to the indicated peptide candidates in the plasma from patients 4, 5, 9 and 10 were examined by ELISA. The values represent OD. (B) The plasma samples from patients 10 and 5 were cultured in plates that were pre-coated with the indicated peptides, and the levels of IgG reactive to the indicated peptides in the resultant supernatants were determined by ELISA. 'Statistically significant at p
