The development of immunotherapy for human brain tumor using molecular biothechnology

本研究的目的是建立一种先进的肿瘤抗原肽的分离纯化方法，并将修饰后的HLAcDNAs导入人肿瘤细胞系，从培养上清液中获得并纯化其基因产物。本研究将日本人主要单倍型之一的人类白细胞抗原-24基因导入到预后较差的人脑肿瘤中。将3个全长为1098bp、1067bp、881bp的人类白细胞抗原-A24cNDA基因插入含有组氨酸标签的载体基因中，克隆后导入5株人胶质母细胞瘤细胞系。基因产物经抗组氨酸包被和抗人类白细胞抗原抗体(W6/32)检测，4株细胞培养上清液呈阳性反应，分别为A24 1067bp、A24881bpSNB 19和U373 MG。其中A241067引进U373 MG(命名为U373-A24-1067)…更多的人给出了最高的反应，因此以下研究集中在U373-A24-1067上。免疫组织化学染色显示U373-A24-1067与抗组氨酸抗体呈阳性反应，而抗HLAA24抗体无明显反应，因此认为导入的基因正在发挥作用。U373-A24-1067的培养上清液经钴亲和层析纯化后，与抗组氨酸和W6/32在42 kDa处进行Western blotting反应。亲和纯化的片段含有12 kDa的蛋白，可与抗β2微球蛋白结合，推测U373-A24-1067产生了与β2微球蛋白结合的修饰的人类白细胞抗原-A24。如上所述，本研究成功地建立了一种通过构建与培养上清液中的β2微球蛋白相结合的分泌型HLA-A24分子来纯化抗原肽的方法。这一结果将有助于阐明肿瘤抗原肽，为肿瘤免疫治疗的研究奠定基础。较少

The aim of this research was development of advanced method to clarify the peptides of tumor antigens, and thus modified HLA cDNAs were introduced to human cultured tumor cell lines to obtain and to purify its gene products from the culture supernatants. In this study the cDNA of HLA-24 that is one of the major haplotype in Japanese was introduced into the human brain tumors of which still reveal poor prognoses. Three modified HLA-A24 cNDAs, whole 1098bp, 1067bp of which was deleted the gene coding cytoplasmic portion, and 881bp coding extramembrane part were inserted into the vector gene having histidine-tag and were introduced into the five human glioblastoma cell lines after cloning. The gene products were screened by anti-histidine coating and anti-HLA antibody (W6/32) detecting ELISA, and culture supernatants of four cell lines, A24 1067bp introduced, and A24 881bp introduced SNB 19 and U373MG revealed positive reaction. Among them A24 1067 introduced U373MG (named U373-A24-1067) … More gave highest reaction, and thus following study were focused on the U373-A24-1067. Immunohistochemical staining showed positive reaction of U373-A24-1067 with anti histidine antibody though anti HLA-A24 antibody gave no significant reaction, thus it was considered that introduced gene was actively working. The culture supernatant of U373-A24-1067 had protein of which was purified by cobalt affinity column and was reacted with anti-histidine and W6/32 at 42 kDa by western blotting after SDS-PAGE. Also affinity purified fragment had 12 kDa protein that was reacted with anti beta 2 microglobulin, thus it was considered that U373-A24-1067 produced modified HLA-A24 associating with beta 2 microglobulin. As described above, this study has been succeeded to establish the method that is able to purify the antigenic peptides by constructing secretory HLA-A24 molecule associating with beta 2 microglobulin in culture supernatant. This result will contribute to clarify the tumor antigen peptides and to develop immunotherapy for tumor. Less