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

本研究试图建立稳定的T细胞克隆，并将编码单链抗体和T细胞受体gannma链的cDNA融合基因转染T细胞。用RT-PCR方法从杂交瘤细胞总RNA中扩增出抗MUC 1粘蛋白单克隆抗体MUSE 11的重链和轻链cDNA，并将其连接起来作为单链抗体基因。将融合基因连接到逆转录病毒载体PG 1 EN中，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，现在正在评估病毒生产的各种条件。另一方面，我们最近利用含有融合蛋白基因的真核表达载体，通过常规的G418筛选获得了稳定的转染子。一些转染子克隆显示表达对MUC 1阳性培养的肿瘤细胞的结合活性。关于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方面的详细研究正在进行中。还将检测T细胞受体γ链的信号转导和转染子的体内结合能力。

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.