Shimizu N, Chen J, Gamou S, Takayanagi A
Department of Molecular Biology, Keio University School of Medicine, Tokyo, Japan.
Cancer Gene Ther. 1996 Mar-Apr;3(2):113-20.
In this article we describe an improved method to produce a conjugate of anti-erythrocyte growth factor (EGF) receptor monoclonal antibody with polylysine via thio-ether bonds. The resulting antibody/polylysine conjugate was found to be a much more stable DNA (gene) carrier than the previous conjugate formed via disulfide bonds. We designated the conjugate as an "immunoporter" and the immunoporter/DNA (gene) complex as an "immunogene." The fluorescent microscopic observation showed that the immunoporter as well as immunogene bound specifically to the EGF receptors on the cell surface, and the loaded reporter gene, such as beta-galactosidase (beta-GAL), was detected in the cell nucleus at 2 hours after transfection. The enzyme activity from the beta-GAL gene was detected at 12 hours and increased for 3 to 5 days. Similar kinetics were obtained for another reporter gene, chloramphenicol acetyltransferase. Furthermore, the immunoporter delivered the herpes simplex virus thymidine kinase gene and induced substantial suicide effects on tumor cells when gancyclovir or acyclovir was added. Thus, the immunogene approach was successful in delivering therapeutic genes to EGF receptor overexpressing tumor cells. Further technical refinement may prove useful as a supplementary treatment of patients with squamous cell carcinomas.
在本文中，我们描述了一种改进的方法，通过硫醚键制备抗红细胞生长因子（EGF）受体单克隆抗体与聚赖氨酸的缀合物。结果发现，所得的抗体/聚赖氨酸缀合物是一种比先前通过二硫键形成的缀合物更稳定的DNA（基因）载体。我们将该缀合物命名为“免疫转运体”，将免疫转运体/DNA（基因）复合物命名为“免疫基因”。荧光显微镜观察表明，免疫转运体以及免疫基因能特异性结合细胞表面的EGF受体，并且在转染后2小时，在细胞核中检测到了加载的报告基因，如β-半乳糖苷酶（β-GAL）。在12小时时检测到了β-GAL基因的酶活性，并且在3至5天内活性增加。对于另一个报告基因氯霉素乙酰转移酶，也获得了类似的动力学结果。此外，当加入更昔洛韦或阿昔洛韦时，免疫转运体递送单纯疱疹病毒胸苷激酶基因并对肿瘤细胞产生显著的自杀效应。因此，免疫基因方法成功地将治疗性基因递送至EGF受体过表达的肿瘤细胞。进一步的技术改进可能作为鳞状细胞癌患者的辅助治疗方法而被证明是有用的。
