Altenschmidt U, Kahl R, Moritz D, Schnierle B S, Gerstmayer B, Wels W, Groner B
Institute for Experimental Cancer Research, Tumor Biology Center, Breisacher Strasse 177, D-79106 Freiburg, Germany.
Clin Cancer Res. 1996 Jun;2(6):1001-8.
We are developing strategies to use naive T lymphocytes in cancer therapy. For this purpose, we are deriving T cells with specificity of recognition for defined tumor cells. To direct effector lymphocytes toward tumor cells, we have manipulated the recognition specificity of naive rat and mouse T lymphocytes and a mouse T-cell line. The cells were stably transduced with a chimeric T-cell receptor (TCR) component. The zeta chain of the TCR consists of a single transmembrane protein with a short extracellular domain and an intracellular domain for TCR signaling. We provided an extracellular tumor cell recognition domain to the zeta chain. Human heregulin beta1 (ligand to the erbB-3 and erbB-4 receptors) and three different single-chain antibodies specific for the human and rat Neu/erbB-2 receptors were used. One single-chain antibody (C11) is directed against the rat Neu protein, and one single-chain antibody (FRP5) is directed against the human erbB-2 receptor. The single-chain antibody (R-AK) directed against the Mr 14,000 fusion protein of orthopox viruses served as a control. An efficient procedure was devised to introduce the chimeric genes into primary rat and mouse T lymphocytes. Retrovirus-producing packaging cell lines were cocultured with the T cells activated by phytohemagglutinin and interleukin 2. T-cell lines were transduced by exposure to retrovirus-containing supernatants from helper cell lines. Expression of the fusion genes was determined by fluorescence-activated cell sorting analysis. More than 80% of the naive rat and mouse T cells and 85-100% of the cells from the established T-cell lines expressed the fusion genes within 48 h after infection. The expression of the fusion genes was maintained for at least 10 days after infection. Target cells expressing Neu/erbB-2, erbB-3, or erbB-4 were lysed in vitro with high specificity by T cells expressing the corresponding recognition proteins. No selection of a marker gene is necessary to confer a predetermined recognition specificity. The described experiments are important for a gene therapy approach to cancer treatment with autologous T cells.
我们正在研发利用天然T淋巴细胞进行癌症治疗的策略。为此，我们正在获取对特定肿瘤细胞具有识别特异性的T细胞。为了将效应淋巴细胞导向肿瘤细胞，我们对天然大鼠和小鼠T淋巴细胞以及小鼠T细胞系的识别特异性进行了操控。这些细胞用嵌合T细胞受体（TCR）组件进行了稳定转导。TCR的ζ链由单个跨膜蛋白组成，具有短的细胞外结构域和用于TCR信号传导的细胞内结构域。我们为ζ链提供了一个细胞外肿瘤细胞识别结构域。使用了人heregulin beta1（erbB - 3和erbB - 4受体的配体）以及三种针对人和大鼠Neu / erbB - 2受体的不同单链抗体。一种单链抗体（C11）针对大鼠Neu蛋白，一种单链抗体（FRP5）针对人erbB - 2受体。针对正痘病毒Mr 14,000融合蛋白的单链抗体（R - AK）用作对照。设计了一种有效的方法将嵌合基因导入原代大鼠和小鼠T淋巴细胞。产生逆转录病毒的包装细胞系与由植物血凝素和白细胞介素2激活的T细胞共培养。T细胞系通过暴露于来自辅助细胞系的含逆转录病毒的上清液进行转导。通过荧光激活细胞分选分析确定融合基因的表达。超过80%的天然大鼠和小鼠T细胞以及85 - 100%的已建立T细胞系的细胞在感染后48小时内表达融合基因。感染后融合基因的表达至少维持10天。表达相应识别蛋白的T细胞在体外以高特异性裂解表达Neu / erbB - 2、erbB - 3或erbB - 4的靶细胞。赋予预定的识别特异性无需选择标记基因。所描述的实验对于用自体T细胞进行癌症治疗的基因治疗方法很重要。
