Cytolysis of tumor cells expressing the Neu/erbB-2, erbB-3, and erbB-4 receptors by genetically targeted naive T lymphocytes.
Cytolysis of tumor cells expressing the Neu/erbB-2, erbB-3, and erbB-4 receptors by genetically targeted naive T lymphocytes.
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发表时间：
1996-06
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通讯作者：
U. Altenschmidt;R. Kahl;D. Moritz;B. Schnierle;B. Gerstmayer;W. Wels;B. Groner
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作者：
U. Altenschmidt;R. Kahl;D. Moritz;B. Schnierle;B. Gerstmayer;W. Wels;B. Groner
我们正在开发将幼稚T淋巴细胞用于癌症治疗的策略。为此，我们正在获得对特定肿瘤细胞具有特异性识别的T细胞。为了指导效应淋巴细胞对肿瘤细胞，我们操纵了幼稚大鼠和小鼠T淋巴细胞以及小鼠T细胞系的识别特异性。这些细胞被嵌合t细胞受体（TCR）成分稳定地转导。TCR的zeta链由单个跨膜蛋白组成，具有短的胞外结构域和胞内结构域，用于TCR信号传导。我们为zeta链提供了细胞外肿瘤细胞识别域。使用人heregulin β 1 （erbB-3和erbB-4受体的配体）和三种不同的单链抗体特异性针对人和大鼠Neu/erbB-2受体。一个单链抗体（C11）针对大鼠Neu蛋白，一个单链抗体（FRP5）针对人erbB-2受体。针对正痘病毒Mr 14000融合蛋白的单链抗体（R-AK）作为对照。设计了一种将嵌合基因导入大鼠和小鼠原代T淋巴细胞的有效方法。将产生逆转录病毒的包装细胞系与植物血凝素和白细胞介素2激活的T细胞共培养。t细胞系通过暴露于辅助细胞系中含有逆转录病毒的上清液进行转导。采用荧光活化细胞分选法测定融合基因的表达。在感染后48小时内，80%以上的大鼠和小鼠的幼稚T细胞和85-100%的已建立的T细胞系的细胞表达融合基因。融合基因的表达在感染后至少维持10天。表达Neu/erbB-2、erbB-3或erbB-4的靶细胞在体外被表达相应识别蛋白的T细胞高特异性裂解。没有选择的标记基因是必要的，以赋予预先确定的识别特异性。所描述的实验是重要的基因治疗方法的癌症治疗与自体T细胞。
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.