The development of immunotherapy for human brain tumor using molecular biothechnology

利用分子生物技术开发人脑肿瘤免疫疗法

• 批准号: 14571305
• 负责人: TAKAMI Tsuyoshi
• 金额: $ 2.56万
• 依托单位: Gifu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571305/
• 关键词: peptides of tumor antigen; HLA-24; human glioblastoma cell line; histidiue-tag chimera protein; tumor immunotherapy; 抗体提示; グリオーマ; MHC class Ia; β2ミクログロブリン; ペプチド; 分泌型

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

本研究的目的是建立一种新的肿瘤抗原肽段的纯化方法，并将修饰的HLA cDNA导入人肿瘤细胞系中，从培养上清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HLA-24的cDNA导入到人类脑肿瘤中，这些肿瘤仍然显示出较差的表达。将3个经修饰的HLA-A24 cDNA（全长1098 bp，其中1067 bp缺失胞浆部分编码基因，881 bp缺失膜外部分编码基因）插入带有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。用抗组氨酸包被和抗HLA抗体（W 6/32）检测ELISA筛选基因产物，A24 1067 bp导入、A24 881 bp导入、SNB 19和U373 MG 4株细胞培养上清均呈阳性反应。其中A24 1067引进了U373 MG（命名为U373-A24-1067） ...更多信息 得到最高的反应，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HLA-A24抗体无明显反应，因此认为导入基因正在积极发挥作用。U373-A24-1067的培养上清具有通过钴亲和柱纯化的蛋白，并且在SDS-PAGE之后通过蛋白质印迹与抗组氨酸和W 6/32在42 kDa处反应。亲和纯化的片段具有12 kDa的蛋白质，其与抗β 2微球蛋白反应，因此认为U373-A24-1067产生与β 2微球蛋白结合的修饰的HLA-A24。如上所述，本研究已经成功地建立了能够通过在培养上清液中构建与β 2微球蛋白缔合的分泌型HLA-A24分子来纯化抗原肽的方法。这一结果将有助于肿瘤抗原肽的研究和肿瘤免疫治疗的发展。少