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

在本研究中，我们尝试用编码单链抗体和T细胞受体甘氨酸链的cdna组成的融合基因转染稳定的T细胞克隆。采用RT-PCR方法从杂交瘤中提取的总RNA中扩增出抗muc1粘蛋白单克隆抗体MUSE11的VH和VL cdna，并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中，瞬时转染到包装细胞中获得病毒。然而，到目前为止，已经获得了足以建立稳定的EB病毒转化T细胞转染体的病毒滴度，目前正在评估各种病毒产生条件。另一方面，我们最近利用含有融合素基因的真核细胞表达载体，通过常规G418选择获得了稳定的转染物。一些转染克隆显示出与muc1阳性培养肿瘤细胞的结合活性。目前正在对信号链抗体的表达水平、转染物与培养细胞结合的特异性等进行详细的研究。T细胞受体γ链的信号转导和转染物在体内的结合能力也将被研究。

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.