Institute for Applied Microbiology, University of Agriculture, Nussdorfer Lände 11, 1190, Vienna, Austria.
Cytotechnology. 1996 Jan;22(1-3):129-38. doi: 10.1007/BF00353932.
The production of human monoclonal antibodies for therapeutic use is of increasing importance for treatment of viral infections such as AIDS. As human x mouse heterohybridomas rarely reach the growth rates and cell specific production rates of mouse hybridomas the transfection of standard cell lines, such as CHO or BHK, is a promising alternative. This has the additional advantage that the IgG subtype can be changed to suit the desired application. However, the use of a cell line that has not originally developed to produce antibodies, as lymphocytes and myeloma cells have, might have unrecognised drawbacks. This will be especially significant in the case of antibodies as each molecule consists of 4 chains linked by disulphide bonds which require specific intracellular factors to be properly folded and processed (Heavy chain binding protein, Protein Disulfide Isomerase a.o.). In this study we have therefore compared two cell lines: a human x mouse heterohybridoma producing IAM-2F5, a human IgG(3) antibody specific for HIV-1 with neutralising properties and a Chinese Hamster Ovary cell transfected with dihydrofolate reductase and with the heavy and light chain genes of IAM-2F5 modified to IgG(1). From each cell line three subclones were selected with low, medium and high specific production rates. Batch cultures were performed and the following cellular parameters analysed by flow cytometry; 1) total RNA content (translational activity); 2) total protein content; 3) cell cycle phase distribution; 4) concentration of light and heavy chains; 5) concentration of helper proteins such as BiP and PDI. The production rate of heterohybridoma cells was best reflected in the intracellular concentration of kappa chain, while the gamma chain concentration was comparable for all three subclones. In the CHO cells the gamma chain expression and thus gene copy number appeared to be the limiting factor. The GRP78/BiP concentration in CHO remained unchanged in spite of a 5-fold higher concentration of gamma chain in the high producing subclone. The PDI concentration in CHO cells was much lower compared to the heterohybridoma cells, irrespective of production rates.
用于治疗病毒感染（如艾滋病）的人源单克隆抗体的生产变得越来越重要。由于人源-鼠杂交瘤很少达到鼠杂交瘤的生长速度和细胞特异性产率，因此转染标准细胞系（如 CHO 或 BHK）是一种很有前途的替代方法。这具有额外的优势，即可以改变 IgG 亚型以适应所需的应用。然而，使用原本不是为产生抗体而开发的细胞系（如淋巴细胞和骨髓瘤细胞）可能存在未被认识到的缺点。在抗体的情况下尤其如此，因为每个分子由 4 条链通过二硫键连接，这些链需要特定的细胞内因子才能正确折叠和加工（重链结合蛋白、蛋白二硫键异构酶等）。在这项研究中，我们比较了两种细胞系：一种是人源-鼠杂交瘤，产生 IAM-2F5，这是一种针对 HIV-1 的人源 IgG(3)抗体，具有中和特性；另一种是转染了二氢叶酸还原酶以及 IAM-2F5 的重链和轻链基因并修饰为 IgG(1)的中国仓鼠卵巢细胞。从每种细胞系中选择了三个亚克隆，其具有低、中、高特异性产率。进行了分批培养，并通过流式细胞术分析了以下细胞参数：1）总 RNA 含量（翻译活性）；2）总蛋白含量；3）细胞周期相分布；4）轻链和重链浓度；5）辅助蛋白（如 BiP 和 PDI）的浓度。杂交瘤细胞的产率最好反映在κ链的细胞内浓度上，而三种亚克隆的γ链浓度相当。在 CHO 细胞中，γ链表达，因此基因拷贝数似乎是限制因素。尽管高产亚克隆中的γ链浓度高 5 倍，但 CHO 中的 GRP78/BiP 浓度保持不变。与杂交瘤细胞相比，CHO 细胞中的 PDI 浓度要低得多，无论产率如何。
