Preparation of HLA-unrestricted anti-tumor cytotoxic T cells by gene transfer.

通过基因转移制备 HLA 非限制性抗肿瘤细胞毒性 T 细胞。

• 批准号: 08670524
• 负责人: HINODA Yuji
• 金额: $ 1.22万
• 依托单位: Sapporo Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08670524/
• 关键词: MUC1; cytotoxic T cells; HLA-unrestricted; Single chain antibody; T cell receptor gamma chain; レトロウイルスベクター

In this study, we attempted the establishment of the stable T cell clones transfected with a fusion gene consisting of cDNAs coding for single chain antibody and T cell receptor gannma chain. The VH and VL cDNAs of an anti-MUC1 mucin monoclonal antibody MUSE11 were amplified from total RNA extracted from hybridoma by RT-PCR method, then ligated them and used as the single chain antibody gene. The fusion gene was ligated into a retroviral vector PG1EN and transient transfection into packaging cells was performed to obtein viruses. However, the virus titer sufficient for establishing stable transfectans of EB virus-transformed T cells has thus far been obtained, being now evaluating various conditions of virus production. On the other hand, we have recently obtained thr stable transfectants using eukaryocytic expression vectoe haboring the fusin gene by conventional G418 selection. Some transfectant clones were shown to express the binding activity to MUC1-positive cultured tumor cells. Detailed studies on the expression level of signal chain antibody, the specificity of the binding of transfectants to cultured cells and so on are now under ongoing. Signal transduction of T cell receptor gamma chain and the binding ability of transfectants in vivo will be aiso examined.

在本研究中，我们尝试建立转染融合基因的稳定T细胞克隆，该融合基因由编码单链抗体和T细胞受体gannma链的cDNA组成。通过RT-PCR方法从杂交瘤中提取的总RNA中扩增抗MUC1粘蛋白单克隆抗体MUSE11的VH和VL cDNA，然后将其连接作为单链抗体基因。将融合基因连接到逆转录病毒载体PG1EN上，瞬时转染包装细胞，获得病毒。然而，迄今为止已经获得了足以建立EB病毒转化的T细胞的稳定转染子的病毒滴度，目前正在评估病毒生产的各种条件。另一方面，我们最近通过常规G418选择，利用带有融合蛋白基因的真核细胞表达载体获得了稳定的转染子。一些转染子克隆表现出与 MUC1 阳性培养肿瘤细胞的结合活性。目前，信号链抗体的表达水平、转染子与培养细胞结合的特异性等详细研究正在进行中。还将检查T细胞受体γ链的信号转导和转染子的体内结合能力。

项目成果

Hinoda Y et al.: "Enhancement of reactivity of anti-MUC1 core protein antibody and killing activity of anti-MUC1 cytotoxic T cells by deglycosylation of target tissues or cells." J.Gastroenterol.33. 164-171 (1998)

Hinoda Y 等人：“通过靶组织或细胞的去糖基化增强抗 MUC1 核心蛋白抗体的反应性和抗 MUC1 细胞毒性 T 细胞的杀伤活性。”

Noto H.et al.: "Cytotoxic T lymphocytes derived from bone marrow mononuclear cells recognize an underglycosylated form MUC1 mucin." Int.Immunol.9. 791-798 (1997)

Noto H.等人：“源自骨髓单核细胞的细胞毒性 T 淋巴细胞识别糖基化不足的形式 MUC1 粘蛋白。”

Hinoda Y et al.: "Enhancement of reactivity of anti-MUC1 core protein antibody and killing activity of anti-MUC1 cytotoxic T cells by deglycosylation of target tissues or cells." J.Gastroenterol.33(in press). (1998)

Hinoda Y 等人：“通过靶组织或细胞的去糖基化增强抗 MUC1 核心蛋白抗体的反应性和抗 MUC1 细胞毒性 T 细胞的杀伤活性。”

日野田 裕治 他: "抗体分子工学と癌の遺伝子治療" 日臨免誌. 19. 453-459 (1996)

Yuji Hinoda 等：“抗体分子工程和癌症基因治疗”Nichinrin Menshi 19. 453-459 (1996)