Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Okayama 700-8530, Japan.
J Biosci Bioeng. 2010 Sep;110(3):351-8. doi: 10.1016/j.jbiosc.2010.03.006. Epub 2010 Apr 11.
The chicken B cell line DT40 undergoes hypermutation of immunoglobulin variable region (IgV) genes during culture, thereby constituting an antibody (Ab) library. We previously established an in vitro Ab generation system using an engineered line DT40-SW whose hypermutation machinery can be switched on and off. Abs for various antigens (Ags) can be obtained from the DT40-SW library and the specificity of the Ag-specific clones can be stabilized by stopping hypermutation. Furthermore, the affinity of obtained monoclonal Abs (mAbs) can be improved through further mutation followed by selection, a process analogous to "affinity maturation" that occurs in vivo. Although gene conversion dominantly diversifies the IgV genes in DT40 cells, point mutation is considered to be more favorable for fine-tuning Ab properties during affinity maturation. Here, we examined whether affinity maturation occurs more efficiently when the hypermutation pattern was transformed from gene conversion into point mutation in DT40-SW cells. To this end, we disrupted the XRCC3 gene that is essential for gene conversion. It was found that hemizygous disruption of the XRCC3 gene was sufficient to increase the point mutation frequency. Since hemizygous disruption is conducted more easily, we tested whether the XRCC3 (+/-) mutant generates high-affinity Abs through affinity maturation more efficiently than the wild type. Using this affinity maturation technique, we generated an improved 4-hydroxy-3-nitrophenylacetyl-specific mAb with approximately 600-fold lower K(D) than that of the original mAb. Taken together, hemizygous disruption of the XRCC3 gene is considered to be useful for obtaining high-affinity mAbs from DT40-SW cells though affinity maturation.
鸡 B 细胞系 DT40 在培养过程中会对免疫球蛋白可变区（IgV）基因发生超突变，从而构成抗体（Ab）文库。我们之前建立了一种使用工程化 DT40-SW 细胞的体外 Ab 生成系统，其超突变机制可以开启和关闭。可以从 DT40-SW 文库中获得针对各种抗原（Ags）的 Ab，并且可以通过停止超突变来稳定 Ag 特异性克隆的特异性。此外，通过进一步突变和选择，可以提高获得的单克隆 Ab（mAb）的亲和力，这一过程类似于体内发生的“亲和力成熟”。虽然基因转换主导地多样化了 DT40 细胞中的 IgV 基因，但点突变被认为更有利于在亲和力成熟过程中微调 Ab 特性。在这里，我们研究了当 DT40-SW 细胞中的超突变模式从基因转换转变为点突变时，亲和力成熟是否更有效。为此，我们破坏了 XRCC3 基因，该基因对于基因转换是必需的。结果发现，XRCC3 基因的杂合破坏足以增加点突变频率。由于杂合破坏更容易进行，我们测试了 XRCC3（+/-）突变体是否比野生型更有效地通过亲和力成熟产生高亲和力的 Ab。使用这种亲和力成熟技术，我们生成了一种改进的 4-羟基-3-硝基苯乙酰特异性 mAb，其 K(D)值比原始 mAb 低约 600 倍。总之，XRCC3 基因的杂合破坏被认为对于通过亲和力成熟从 DT40-SW 细胞中获得高亲和力的 mAb 是有用的。
