The development of immunotherapy for human brain tumor using molecular biothechnology

2003 Fiscal Year Final Research Report Summary

• Project/Area Number: 14571305
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Cerebral neurosurgery
• Research Institution: Gifu University
• Principal Investigator: TAKAMI Tsuyoshi Gifu University, Immunopathology, Professor, 医学部, 教授 (70136943)
• Co-Investigator(Kenkyū-buntansha): SAIO Masanao Gifu University, Immunopathology, Associate Professor, 医学部, 助教授 (40242721) ; SAKAI Noboru Gifu University, Neurosurgery, Professor, 医学部, 教授 (10021487)
• Project Period (FY): 2002 – 2003
• Keywords: peptides of tumor antigen / HLA-24 / human glioblastoma cell line / histidiue-tag chimera protein / tumor immunotherapy

Research Abstract

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) 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.