Identification Of HLA-A～*0201-resticted Cytotoxic T Lymphocyte Epitope From CML28

The therapeutic options for treatment of patients with carcinomas are limited to three fundamental modalities: (a) surgical resection; (b) chemotherapy; and (c) radiation therapy. However, these modalities do not yield good results, especially against advanced carcinomas. Over the past few years, the analysis of spontaneous immune responses to autologous tumors in cancer patients has allowed the identification of several categories of tumor-associated antigens that can be the target of tumor-specific immune responses based on the recognition of tumor antigens by CTL in an MHC-clas I /peptide complex-restricted manner[1,2]. Therefore, cancer-specific immunotherapy has became a very attractive fourth-modality therapeutic approach against carcinomas. Among them, one of the most relevant for the development of tumor immunotherapy is peptide-based, cancer-specific immunotherapy using the group of so-called CT antigen.The cancer-testis (CT) antigen family has been an attractive target for tumor immunotherapy, because these antigens can be recognized by human CTLs [3,4]and are expressed in a variety of malignant tumors from many kind of histological origins but not in normal tissues, with the exception of the testis and placenta[5,6]. The antigenic peptides derived from CT antigens such as MAGE-1[7], MAGE-3[8] and NY-ESO-1[9] have been proven to be elicit CTL responses in the context of MHC class I molecules. Moreover, immunization with these peptides-pulsed dendritic cells vaccine, as a modality of specific immunotherapy, has been applied to melanoma patients and other malignant patients and proven to have some clinical effectiveness [8,10]. However, expression of CT antigens is heterogeneous among tumors of different histological origins, different patients, and between individual lesions. These characteristics of CT antigen expression suggest that the development of specific immunotherapy based on as many antigens as possible maybe clinically beneficial.The CML28 antigen was a newly discovered CT antigen, which was initially identified by Xiao-Feng Yang [11] by SEREX (serological identification of recombinant cDNA expressing cloning). This gene is found highly expressed in a variety of hematopoietic and epithelial tumor cell lines, but not in normal tissues or other normal tissue, with the exception of testis. Because HLA-A*0201 is the most common HLA-A allele in Asian populations, especially in the Chinese, with an estimated frequency of >50%[12]. Therefore, if CML28-derived HLA-A*0201-restricted CTL epitopes are identified, these peptides may be widely applied for specific immunotherapy against CML28-postive tumor in the HLA-A*0201+ population.In this study, a HLA-A*0201-CTL epitope was identified by using the following four-step procedure: (a) computer-based epitope prediction from the amino acid sequence of CML28 antigen; (b) peptide-binding assay to determine the affinity of the predicted peptides with HLA-A*0201 molecule; (c) stimulation of primary T-cell response against the predicted peptides in vitro and testing of the induced CTLs towards LB373-Mel cells expressing CML28 antigen and HLA-A*0201; (d) stimulation of primary T-cell response by immunization of HLA-A*0201 transgenic mice with PCI/CML28 plasmid, and testing of the induced CTLs towards Jurket Kb/A2 pulsed with predicted peptides.As a result, we first identified four CML28-derived peptides with sound affinities to the HLA-A*0201 molecule verified with MHC peptide-binding assay and epitope computer algorithms. We then induced CML28-specific CTLs from HLA-A*0201-postive peripheral blood mononuclear cells (PBMCs) of four healthy donors by in vitro stimulation with a synthetic peptdie and from spleen cells of HLA-A*0201 transgenic mice immunized with PCI/CML28.