HLA-A24 and survivin: possibilities in therapeutic vaccination against cancer
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Recently, it was described that an HLA-A24 restricted peptide derived from the survivin splice variant survivin-2B can be recognized by CD8(+) cytotoxic T-cells. The identification of an HLA-A24 epitope is critical for survivin-based immunotherapy as HLA-24 is the most frequent HLA allele in Asia. Consequently, this survivin-2B epitope is already a target in a clinical study in patients with advanced or recurrent colorectal cancer expressing survivin. However, the splice variant survivin-2B has been described to be pro-apoptotic, and is only expressed at low levels in most malignant tissues. Furthermore, survivin-2B expression are significantly decreased in later tumor stages and inversely correlated with tumor differentiation and invasion. Consequently, survivin is a more general vaccination candidate than the splice variant survivin-2B. Here, we on the basis of spontaneous immune responses in HLA-A24+ cancer patients describes that a HLA-A24-restricted survivin epitopes does indeed exist. Consequently, this epitope is an attractive target for the ongoing survivin-based peptide immunotherapy against cancer.
KeywordsPeripheral Blood Lymphocyte Recurrent Colorectal Cancer Peptide Base Immunotherapy Spontaneous Immune Response Survivin Epitope
The group of Sato recently attempted to identify a HLA-A24-restricted epitope derived from the universal tumor antigen survivin. Although unsuccessful, they reported that an HLA-A24 restricted peptide (AYACNTSTL) derived from the survivin splice variant survivin-2B can be recognized by CD8(+) cytotoxic T-cells [1, 2]. Subsequently, as described in this journal this survivin-2B epitope is the target in a phase I clinical study assessing the efficacy of survivin-2B peptide vaccination in patients with advanced or recurrent colorectal cancer expressing survivin .
As HLA-A24 is a very frequent allele expressed especially in the Asian population a survivin-derived HLA-A24 restricted epitope would be highly interesting in peptide based immunotherapy against cancer. Survivin is an apoptotic inhibitor that is expressed at high levels in a variety of malignancies. At present, four splicing variants are known for survivin (Survivin, Survivin-2B, Survivin-deltaEx3, Survivin-3B, and survivin-2α) [4, 5, 6]. Survivin is over-expressed in almost all cancers including lung, colon, breast, pancreas, stomach, liver, ovaries, and prostate cancer, as well as melanoma, and hematopoetic malignancies [7, 8, 9]. Data from a large analysis of human transcripts revealed survivin as the fourth most highly expressed protein in human cancer tissue compared to normal tissue . In contrast, the expression and function in human cancer of the splice variant the survivin-2B is more indistinct. Thus, most studies describe survivin 2-B as to be pro-apoptotic, and only to be expressed at low levels in most malignant tissues . Furthermore, survivin-2B expression are significantly decreased in later tumor stages and inversely correlated with tumor differentiation and invasion [4, 11]. Likewise, Islam et al (2000) reported that the expression of survivin-2B is predominant in some neuroblastoma with a good prognosis , indicating a possible unfavourable role of survivin-2B in cancer development . In contrast, two recent papers show that high expression of survivin-2B variant is correlated with poor prognosis in cancer patients [14, 15]. However, as survivin-2B is not expressed in many malignancies, survivin is a more attractive candidate as a general vaccination candidate than the splice variant survivin-2B. In the present manuscript, we identified an HLA-A24 restricted epitope derived from the universal tumor antigen survivin.
Materials and methods
Peripheral blood lymphocytes (PBL) from HLA-A24 positive cancer patients were obtained from the University Hospital in Herlev, Denmark. The PBL were isolated using Lymphoprep separation and cryopreserved in FCS with 10% DMSO. Tissue typing was conducted at the Department of Clinical Immunology, The State Hospital, Copenhagen, Denmark. Informed consent was obtained from the patients before any of theses measures.
Interferon-γ and perforin ELISPOT
The interferon-γ and Perforin ELISPOT assays was used to quantify peptide epitope-specific interferon-γ and perforin releasing effector cells, respectively as described previously [16, 17]. Briefly, nitrocellulosebottomed 96-well plates (MultiScreen MAIP N45; Millipore) were coated with 7.5 μg/ml capture anti-human interferon-γ (Pf-80/164, Mabtech, Sweden) or 30 μg/ml coating anti-human perforin (Pf-80/164, Mabtech) in 100 μl sterile PBS per well. The wells were washed and T2-A24 cells (a kind from Yasuto Akiyama, Tokyo) and effector cells were added with or without 10 μM peptide. In some assays isolated, CD8 positive T-cells were used as effector cells using CD8+ MACS microbeads (Miltenyi Biotec GmbH, Gladbach, Germany). After incubation (37°C/5%CO2) medium was discarded and the wells were washed prior to addition of the secondary detection Ab, anti-human biotinylated interferon-γ (Mabtech) at 2 μg/ml in 75 μl PSB/BSA per well or anti-human biotinylated perforin (Pf-344-biotin, Mabtech) at 1 μg/ml in 100 μl PSB, respectively. The plates were incubated at RT for 2 h, washed, and 100 μl of streptavidin-ALP (Mabtech) diluted 1:1000 in PBS, was added to each well and the plates were incubated for one hour at RT and the enzyme substrate NBT/BCIP (Invitrogen Life Technologies) was added to each well. Upon appearance of dark spots, the reaction was terminated by washing with tap water. The spots were counted using the ImmunoSpot Series 2.0 Analyzer (CTL Analyzers).
Results and discussion
Thus, the Sur20–28 reacting cells in the patients PBL are indeed cytotoxic, CD8+ effector cells. Consequently, Sur20–28 is a very attractive target to be included in the ongoing survivin-based peptide immunotherapy against cancer.
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