Hashimoto Koji, Kurosawa Kohei, Seo Hidetaka, Ohta Kunihiro
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan.
Methods Mol Biol. 2019;1904:307-317. doi: 10.1007/978-1-4939-8958-4_14.
We previously developed the in vitro method to generate monoclonal antibodies (mAbs) from libraries constructed with chicken B-cell line DT40 (referred to as the "ADLib system"). As the wild-type DT40 cells express immunoglobulin M (IgM), the original ADLib system provides monoclonal antibodies in chicken IgM format. For the therapeutic, diagnostic, and research purposes, the Fc regions of IgMs should be exchanged to other classes and species, for example human or murine IgG. However, the Fc engineering by conventional bioengineering process is laborious and takes plenty of time. Here, we developed a method to enable the seamless replacement of the Fc regions of antibodies generated by the ADLib system, using recombination-mediated cassette exchange (RMCE). In this system, two Cre recombinase recognition sites were inserted into the IgM's Fc region of the DT40 genome, allowing the exchange of the Fc region to the sequences of interest by co-transfection of a donor sequence and a Cre recombinase expression vector. We describe the detailed protocol of the technology: how to construct the RMCE host strains, select mAbs by the ADLib system, and exchange their Fc regions to generate chimeric mAbs.
我们之前开发了一种体外方法，用于从用鸡B细胞系DT40构建的文库中产生单克隆抗体（mAb，称为“ADLib系统”）。由于野生型DT40细胞表达免疫球蛋白M（IgM），原始的ADLib系统以鸡IgM形式提供单克隆抗体。出于治疗、诊断和研究目的，IgM的Fc区域应替换为其他类别和物种，例如人或鼠IgG。然而，通过传统生物工程方法进行Fc工程既费力又耗时。在此，我们开发了一种方法，利用重组介导的盒式交换（RMCE）实现对ADLib系统产生的抗体的Fc区域进行无缝替换。在该系统中，两个Cre重组酶识别位点被插入到DT40基因组的IgM的Fc区域，通过共转染供体序列和Cre重组酶表达载体，可将Fc区域替换为感兴趣的序列。我们描述了该技术的详细方案：如何构建RMCE宿主菌株，通过ADLib系统选择单克隆抗体，以及交换它们的Fc区域以产生嵌合单克隆抗体。
