Nagahira K, Fukuda Y, Nasu T, Kawashima H, Noguchi C, Kurihara T, Oikawa S, Nakanishi T
Suntory Institute for Biomedical Research, Osaka, Japan.
Immunol Lett. 1998 Dec;64(2-3):139-44. doi: 10.1016/s0165-2478(98)00097-2.
A mouse anti-human tumor necrosis factor-alpha (TNF-alpha) monoclonal antibody (MoAb), designated as 3B10, has previously been produced and characterized by our laboratory. We report here the construction and the expression of mouse-human chimeric antibody derived from the MoAb. cDNAs encoding variable regions of heavy and light chains were prepared from 3B10 cells by polymerase chain reaction, and introduced to mammalian expression vectors containing cDNA for human gamma1 and kappa constant regions, respectively. Cotransfection of the vectors into CHO cells resulted in production of antibody reacting with human TNF-alpha. In SDS-PAGE analysis, the chimeric antibody, c3B10, migrated at 170 kDa under a nonreducing condition, whereas two bands with 58 and 28 kDa appeared following treatment with 2-mercaptoethanol. Both c3B10 and mouse 3B10 neutralized the cytotoxic activity of human TNF-alpha to the same level, indicating that c3B10 holds the binding activity of its original MoAb. These findings suggest that the introduced genes for chimeric heavy and light chains are transcribed and translated to produce the chimeric heavy and light chain peptides, and that the peptides are assembled to form native IgG molecule. The chimeric anti-TNF-alpha antibody described in this study is expected to be less immunogenic and thus more suitable for possible clinical use.
一种小鼠抗人肿瘤坏死因子-α（TNF-α）单克隆抗体（MoAb），命名为3B10，此前已由我们实验室制备并进行了表征。我们在此报告源自该MoAb的小鼠-人嵌合抗体的构建和表达。通过聚合酶链反应从3B10细胞中制备编码重链和轻链可变区的cDNA，并分别导入含有人类γ1和κ恒定区cDNA的哺乳动物表达载体。将这些载体共转染到CHO细胞中，产生了与人TNF-α反应的抗体。在SDS-PAGE分析中，嵌合抗体c3B10在非还原条件下迁移至170 kDa，而用2-巯基乙醇处理后出现了58 kDa和28 kDa的两条带。c3B10和小鼠3B10均将人TNF-α的细胞毒性活性中和至相同水平，表明c3B10保留了其原始MoAb的结合活性。这些发现表明，引入的嵌合重链和轻链基因被转录和翻译以产生嵌合重链和轻链肽，并且这些肽组装形成天然IgG分子。本研究中描述的嵌合抗TNF-α抗体预计免疫原性较低，因此更适合可能的临床应用。
