Duan Yajun, Zheng Junnian, Han Sufang, Wu Yi, Wang Yanming, Li Deguan, Kong Deling, Yu Yaoting
Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Science, Nankai University, Tianjin 300071, China.
J Control Release. 2008 Apr 21;127(2):173-9. doi: 10.1016/j.jconrel.2008.01.016. Epub 2008 Feb 8.
G250 is a tumor associated antigen that is found on > 90% of renal cell carcinoma (RCC). In order to develop a highly targeting gene vector for RCC gene therapy, G250 monoclonal antibody was prepared, purified and characterized. The antibody was chemically bound to Polyethylenimine (PEI) to form the IgG-PEI conjugate. The conjugate is capable of forming DNA complexes in the size of nano meters and with a narrow size distribution. The targeting effect and transfection efficiency were tested on five cell lines, ketr 3, Hela, ACHN, HepG2, and smooth muscle cells. The transfection was quantitatively determined by fluorescence activated cell sorting (FACS) and luciferase assay. The FACS results show that for G250 positive cells ketr 3 and Hela, the transfection efficiency of IgG-PEI are 2-fold higher than that of PEI. But for G250 negative cells, antibody modification has no effect on transfection. The expression of luciferase in ketr 3 cells which is expressed as enzyme activity is 15-fold and 61-fold higher than that in ACHN and SMC, respectively. In the presence of free antibody, the targeting effect of IgG-PEI is impaired and the transfection efficiency is normalized. It indicates that G250 antibody is an ideal targeting ligand for delivery of genes into RCC. Application of this IgG-PEI conjugate in RCC gene therapy will be of great interest.
G250是一种肿瘤相关抗原，在90%以上的肾细胞癌（RCC）中均可发现。为了开发用于RCC基因治疗的高靶向性基因载体，制备、纯化并鉴定了G250单克隆抗体。该抗体通过化学方法与聚乙烯亚胺（PEI）结合，形成IgG-PEI偶联物。该偶联物能够形成纳米尺寸且尺寸分布狭窄的DNA复合物。在五种细胞系ketr 3、Hela、ACHN、HepG2和平滑肌细胞上测试了靶向效果和转染效率。通过荧光激活细胞分选（FACS）和荧光素酶测定法定量测定转染情况。FACS结果表明，对于G250阳性细胞ketr 3和Hela，IgG-PEI的转染效率比PEI高2倍。但对于G250阴性细胞，抗体修饰对转染没有影响。以酶活性表示的ketr 3细胞中荧光素酶的表达分别比ACHN和SMC中的高15倍和61倍。在存在游离抗体的情况下，IgG-PEI的靶向效果受损，转染效率恢复正常。这表明G250抗体是将基因导入RCC的理想靶向配体。这种IgG-PEI偶联物在RCC基因治疗中的应用将备受关注。
