Mimura Kousaku, Kono Koji, Southwood Scott, Fikes John, Takahashi Akihiro, Miyagawa Naoto, Sugai Hidemitsu, Fujii Hideki
First Department of Surgery, University of Yamanashi, 1110 Tamaho, 409-3898 Yamanashi, Japan.
Cancer Immunol Immunother. 2006 Nov;55(11):1358-66. doi: 10.1007/s00262-006-0123-0. Epub 2006 Jan 25.
In order to broaden the possibility for anti-HER-2/neu (HER-2) immune targeting, it is important to identify HLA-A24 restricted peptide epitopes derived from HER-2, since HLA-A24 is one of the most common alleles in Japanese and Asian people. In the present study, we have screened HER-2-derived, HLA-A24 binding peptides for cytotoxic T lymphocyte (CTL) epitopes. A panel of HER-2-derived peptides with HLA-A24 binding motifs and the corresponding analogs designed to enhance HLA-A24 binding affinity were selected. Identification of HER-2-reactive and HLA-A24 restricted CTL epitopes were performed by a reverse immunology approach. To induce HER-2-reactive and HLA-A24 restricted CTLs, PBMCs from healthy donors were repeatedly stimulated with monocytes-derived, mature DCs pulsed with HER-2 peptide. Subsequent peptide-induced T cells were tested for the specificity by enzyme linked immunospot, cytotoxicity and tetramer assays. CTL clones were then obtained from the CTL lines by limiting dilution. Of the peptides containing HLA-A24 binding motifs, 16 peptides (nine mers) including wild type peptides (IC50 <1,000 nM) and substituted analog peptides (IC50 <50 nM) were selected for the present study. Our studies show that an analog peptide, HER-2(905AA), derived from HER-2(905) could efficiently induce HER-2-reactive and HLA-A24 restricted CTLs. The reactivity of the HER-2(905AA)-induced CTL (CTL905AA) was confirmed by different CTL assays. The CTL905AA clones also were able to lyse HER-2(+), HLA-A24(+) tumor cells and cytotoxicity could be significantly reduced in cold target inhibition assays using cold targets pulsed with the HER-2(905) wild type peptide as well as the inducing HER-2(905AA) analog peptide. A newly identified HER-2(905) peptide epitope is naturally processed and presented as a CTL epitope on HER-2 overexpressing tumor cells, and an MHC anchor-substituted analog, HER-2(905AA), can efficiently induce HER-2-specific, HLA-A24 restricted CTLs.
为了拓宽抗HER-2/neu（HER-2）免疫靶向的可能性，识别源自HER-2的HLA-A24限制性肽表位很重要，因为HLA-A24是日本人和亚洲人中最常见的等位基因之一。在本研究中，我们筛选了源自HER-2的、与HLA-A24结合的肽作为细胞毒性T淋巴细胞（CTL）表位。选择了一组具有HLA-A24结合基序的源自HER-2的肽以及为增强HLA-A24结合亲和力而设计的相应类似物。通过反向免疫学方法鉴定HER-2反应性和HLA-A24限制性CTL表位。为了诱导HER-2反应性和HLA-A24限制性CTL，用源自单核细胞的、用HER-2肽脉冲处理的成熟树突状细胞（DC）反复刺激健康供体的外周血单核细胞（PBMC）。随后通过酶联免疫斑点法、细胞毒性和四聚体分析测试肽诱导的T细胞的特异性。然后通过有限稀释从CTL系中获得CTL克隆。在含有HLA-A24结合基序的肽中，选择了16种肽（九聚体），包括野生型肽（IC50<1000 nM）和取代类似物肽（IC50<50 nM）用于本研究。我们的研究表明，源自HER-2(905)的类似物肽HER-2(905AA)能够有效诱导HER-2反应性和HLA-A24限制性CTL。通过不同的CTL分析证实了HER-2(905AA)诱导的CTL（CTL905AA）的反应性。CTL905AA克隆也能够裂解HER-2(+)、HLA-A24(+)肿瘤细胞，并且在使用用HER-2(905)野生型肽以及诱导性HER-2(905AA)类似物肽脉冲处理的冷靶标的冷靶标抑制分析中，细胞毒性可显著降低。一个新鉴定的HER-2(905)肽表位在HER-2过表达肿瘤细胞上被自然加工并呈递为CTL表位，并且一种MHC锚定取代类似物HER-2(905AA)能够有效诱导HER-2特异性、HLA-A24限制性CTL。
