The development of immunotherapy for human brain tumor using molecular biothechnology

本研究的目的是开发一种先进的方法来澄清肿瘤抗原的肽，从而将修饰的HLA cdna引入人培养的肿瘤细胞系中，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HLA-24的cDNA引入到预后不良的人类脑肿瘤中。将3个修饰后的HLA-A24 cdna全长1098bp，其中1067bp缺失了编码细胞质部分的基因，881bp编码膜外部分的基因插入到带组氨酸标签的载体基因中，克隆后导入5个人胶质母细胞瘤细胞系。基因产物经抗组氨酸包被和抗hla抗体（W6/32） ELISA检测筛选，导入A24 1067bp、A24 881bp的SNB 19和U373MG 4株细胞株培养上清均阳性。其中A24 1067引入了U373MG（命名为U373-A24-1067），反应最高，因此后续的研究重点是U373-A24-1067。免疫组化染色显示U373-A24-1067与抗组氨酸抗体反应阳性，但抗HLA-A24抗体无明显反应，认为引入基因起积极作用。U373-A24-1067的培养上清含有钴亲和柱纯化的蛋白，SDS-PAGE后用western blotting与抗组氨酸和W6/32在42 kDa下反应。亲和纯化片段含有与抗β 2微球蛋白反应的12kda蛋白，因此认为U373-A24-1067产生了与β 2微球蛋白结合的修饰HLA-A24。如上所述，本研究成功建立了通过在培养上清中构建与β 2微球蛋白相关的分泌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，开发肿瘤的免疫治疗方法。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