Key determinants in the occurrence of clonal variation in humanized antibody expression of CHO cells during dihydrofolate reductase mediated gene amplification
Key determinants in the occurrence of clonal variation in humanized antibody expression of CHO cells during dihydrofolate reductase mediated gene amplification
复制标题
DOI：
10.1021/bp000144h
复制
发表时间：
2001-01-01
影响因子：
2.9
通讯作者：
Lee, GM
中科院分区：
文献类型：
--
作者：
Kim, NS;Byun, TH;Lee, GM
用重链（HC）和轻链（LC） cDNA表达载体共转染缺乏二氢叶酸还原酶（DHFR）的CHO细胞，获得了表达乙型肝炎病毒S表面抗原人源化抗体的重组中国仓鼠卵巢（CHO）亲本克隆。对23个具有代表性的亲本无性系，如0.02、0.08、0.32和1.0 muM，进行逐步筛选，增加对甲氨蝶呤（MTX）的抗性，发现它们在抗体表达方面的克隆变异显著。在23个亲本无性系中，只有1个无性系（hu17）的特异性抗体产率（q(Ab)）随着MTX浓度的增加而显著增加，最高可达0.32 muM。与亲本克隆（hu17）相比，在0.32 muM MTX （hu17-0.32）下，hu17的q（Ab）提高了约12.5倍。为了阐明克隆变异发生的原因，对每个扩增克隆在0.32 muM MTX下获得的染色体dna进行了Southern blot分析。只有hu17-0.32克隆在基因扩增过程中没有发生严重的基因重排，并且只有一个49 kb的扩增单元，包括LC和HC cdna。荧光MTX竞争实验表明，在0.32 muM MTX下，q（Ab）没有增强的其他克隆对MTX毒性的抗性是通过MTX转运系统受损等机制获得的。综上所述，本文获得的数据表明，尽管逐步选择，克隆可以通过dhfr介导的基因扩增以外的机制获得MTX抗性，因此发现抗体表达方面的克隆变异是显著的。
Recombinant Chinese hamster ovary (CHO) parental clones expressing a humanized antibody against S surface antigen of hepatitis B virus were obtained by cotransfection of heavy chain (HC) and light chain (LC) cDNA expression vectors into dihydrofolate reductase (DHFR)-deficient CHO cells. When 23 representative parental clones were subjected to stepwise selection for increasing methotrexate (MTX) resistance, such as 0.02, 0.08, 0.32, and 1.0 muM, their clonal variations in regard to antibody expression were found to be significant. Among 23 parental clones, only one clone (hu17) showed the significant increment of specific antibody productivity (q(Ab)) With increasing MTX concentration up to 0.32 muM. Compared with the parental clone (hu17), the q(Ab) Of hu17 resistant at 0.32 muM MTX (hu17-0.32) was enhanced approximately 12.5-fold. To clarify the reason for the occurrence of clonal variations, Southern blot analyses of chromosomal DNAs derived from each amplified clone at 0.32 muM MTX were performed. Only the hu17-0.32 clone did not experience severe genetic rearrangement during gene amplification, and it had only one 49-kb amplification unit including the LC and HC cDNAs. A fluorescent MTX competition assay showed that the resistance against MTX toxicity of the other clones without enhanced q(Ab) at 0.32 muM MTX was obtained by mechanisms such as an impaired MTX transport system. Taken together, the data obtained here show that clonal variations in regard to antibody expression are found to be significant because clones can acquire MTX resistance by mechanisms other than DHFR-mediated gene amplification despite the stepwise selection.