Development for new transduction of gene by using mouse monoclonal antibody or its humanized antibody

使用小鼠单克隆抗体或其人源化抗体进行新的基因转导的开发

• 批准号: 08457363
• 负责人: SHIMIZU Keiji
• 金额: $ 4.42万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457363/
• 关键词: Monoclonal antibody; Humanized antibody; Single-chained antibody; Gene therapy; Gene transfer; HVJ-liposome; Immunoliposome; Intemalization; HVJ-lioposome; Intermalization

The glioma-associated antigen, which mouse monoclonal antibody ONS-M21 (Br J Cancer, 1999) recognized, was integrin alpha3. The expression of integrin alpha3 in the normal brain has been reported to be very low or even undetectable. It appears that the level of this antigen expressed in glioma is correlated with the grade of malignancy and invasion. It is, therefore, very important to investigate integrin alpha3 in glioma cells in order to expect the prognosis of their patients. On the other hand, in the experimental gene therapy of glioma with retroviral vectors reconstructed brain-specific promoter, myelin basic protein (MBP) promoter (Hum Gene Ther, 1997 ; Gene Ther, 1998), it is demonstrated that mouse glioma models were completely recovered with intraperitoneal administration of ganciclovir (GCV), if herpes simplex thymidine kinase (HTK) could be introduced into about 25 % of glioma cells. To obtain high-titer recombinant retroviruses, we constructed plasmid pDL^#, which carries t … More he extended Psi region and the polyomavirus early region, including the replication origin and the early gene. Then we modified the packaging cell lines, PA317, by stably introducing the polyomavirus early gene, and established PAMP5 1 from PA317 cells (Hum Gene Ther, 1998). In vivo experiments by using these high-titer recombinant retroviruses, 80 % of mouse glioma models were completely recovered (Hum Gene Ther, in submitted). In this way we could get very effective vectors, but we could not get therapeutic effects at all by these vectors, if tumor does not have retrovirus receptors. For the tumors that did not have these receptors, we investigated the delivery of HTK genes into glioma cells in vivo using hemagglutinating virus of Japan (HVJ)-liposomes, which are coated by Sendai virus envelope protein. Highly efficient delivery was observed in disseminated glioma cells of MG mice, and 8O0/c of these mice expressing the HTK gene were cured by administration of GCV (Gene Ther, 1997). And moreover, we will thoroughly investigate more specific gene therapy for glioma by HVJ liposome-binding humanized (Mol Immunol, 1995) or single-chain antibodies (Anticancer Res, 1998). When humans medulloblastoma ONS-76 cells were co-cultured with ONS-M21 antibodies, about 20% antibodies were internalized into these cells for less than 30 minutes (Cancer lett, 1998). A cytotoxic effect against ONS-76 cells was found by the iminunotoxin which was bounded with ricin A chain and ONS-M21 antibodies, but not against antigen-negative human hepatoma HuH-7 and colon cancer SW480 cells. These results suggest that ONS-M2 I antibodies could effectively deliver toxins, chemotherapeutic agents or radionuclei to malignant glioma specifically. Now we, however, try to transduce HTK genes into the glioma cells by installments, because the molecular weight of HTK genes-binding ONS-M21 is too big to introduce HTK gene complex by the lump. Less

小鼠单克隆抗体ONS-M21（Br J Cancer，1999）识别的神经胶质瘤相关抗原是整联蛋白α 3。据报道，整合素α 3在正常脑中的表达非常低，甚至检测不到。该抗原在胶质瘤中的表达水平与胶质瘤的恶性程度和侵袭性有关。因此，研究整合素α 3在胶质瘤细胞中的表达对预测患者的预后具有重要意义。另一方面，在胶质瘤的实验性基因治疗中，利用逆转录病毒载体重构脑特异性启动子、髓鞘碱性蛋白（MBP）启动子（Gene Ther，1997年; Gene Ther，1998），证实了小鼠神经胶质瘤模型通过腹膜内施用更昔洛韦（GCV）完全恢复，如果单纯疱疹胸苷激酶（HTK）可以被引入到大约25%的神经胶质瘤细胞中。为了获得高滴度的重组逆转录病毒，我们构建了质粒pDL^#， ...更多信息 扩展了Psi区和多瘤病毒早期区，包括复制起点和早期基因。然后，我们通过稳定地引入多瘤病毒早期基因来修饰包装细胞系PA 317，并从PA 317细胞建立PAMP 51（EscherichGene Ther，1998）。通过使用这些高滴度重组逆转录病毒的体内实验，80%的小鼠神经胶质瘤模型被完全恢复（提交中的HPLCGene Ther）。这样我们就可以得到非常有效的载体，但如果肿瘤没有逆转录病毒受体，这些载体就根本不能发挥治疗作用。对于没有这些受体的肿瘤，我们研究了使用日本血凝病毒（HVJ）-脂质体将HTK基因递送到体内胶质瘤细胞中，所述脂质体由仙台病毒包膜蛋白包被。在MG小鼠的播散性神经胶质瘤细胞中观察到高效递送，并且通过施用GCV治愈了800/c表达HTK基因的这些小鼠（Gene Ther，1997）。此外，我们将通过HVJ脂质体结合人源化（Mol Immunol，1995）或单链抗体（Anticancer Res，1998）来深入研究对胶质瘤的更特异性的基因治疗。当人成神经管细胞瘤ONS-76细胞与ONS-M21抗体共培养时，约20%的抗体在不到30分钟的时间内内化到这些细胞中（Cancer lett，1998）。发现与蓖麻毒素A链和ONS-M21抗体结合的亚氨基毒素对ONS-76细胞具有细胞毒性作用，但对抗原阴性的人肝癌HuH-7和结肠癌SW 480细胞没有细胞毒性作用。这些结果表明ONS-M2 I抗体可以有效地将毒素、化疗剂或放射性核特异性地递送至恶性胶质瘤。然而，由于HTK基因结合蛋白ONS-M21的分子量太大，无法一次性将HTK基因复合物导入胶质瘤细胞，因此我们尝试将HTK基因分期导入胶质瘤细胞。少

项目成果

Moriuchi S: "Decreased N-myc expression in human medulloblastoma cell lines during differentiation." Antiancer Res. 17(1). 301-306 (1997)

Moriuchi S：“人髓母细胞瘤细胞系在分化过程中 N-myc 表达降低。”

Moriuchi S: "Decreased N-myc expression in human medulloblastoma cell lines during differentiation." Anticancer Res. 17. 301-306 (1997)

Moriuchi S：“人髓母细胞瘤细胞系在分化过程中 N-myc 表达降低。”

Yamada M: "In vitro study on intrathecal use of 5-fluoro-2'-deoxyuridine (FdUrd) for meningeal dissemination of malignant brain tumors." J Neuro-oncol. (in press). (1998)

Yamada M：“鞘内使用 5-氟-2-脱氧尿苷 (FdUrd) 治疗恶性脑肿瘤脑膜播散的体外研究。”

Kishima H: "Systemic interleukin-12 displays antitumor activity in the mouse central nervous system." Br J Cancer. (in press). (1998)

Kishima H：“全身性白细胞介素 12 在小鼠中枢神经系统中显示出抗肿瘤活性。”

Mabuchi E: "Gene delivery by HVJ-liposome in the experimental gene therapy of murine glioma." Gene Ther. (in press).

Mabuchi E：“在小鼠神经胶质瘤的实验性基因治疗中通过 HVJ 脂质体进行基因传递。”