Naturally split intein Npu DnaE mediated rapid generation of bispecific IgG antibodies
Naturally split intein Npu DnaE mediated rapid generation of bispecific IgG antibodies
复制标题
自然分裂内含肽 Npu DnaE 介导双特异性 IgG 抗体的快速生成
DOI：
10.1016/j.ymeth.2018.10.001
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发表时间：
2019-02-01
期刊：
影响因子：
4.8
通讯作者：
Zhu, Jianwei
中科院分区：
文献类型：
--
作者：
Han, Lei;Zong, Huifang;Zhu, Jianwei
在双特异性抗体的生产中，高纯度、保留天然的免疫球蛋白结构和良好的生产效率是非常理想的。我们报道了一种名为蛋白质反式剪接双特异性抗体(BAPTS)的平台，用于合成具有天然人免疫球蛋白结构和无链错配的BsAbs。在该方法中，两个携带不同靶向性的抗体片段在哺乳动物细胞中分别表达，然后利用分裂的内含子NPU DNAE的反式剪接特性融合形成BsAbs。抗体的铰链区是一个功能影响较小的区域，用于连接两个片段。该方法包括以下步骤：(I)构建五个编码抗体组分的质粒；(Ii)分别表达和纯化两个抗体片段A和B。片段A包含一个Fab，CH3结构域和NPU DNAE(C)的“结节-入孔”突变。片段B包含另一个Fab和NPU Dna E(N)；(Iii)片段A和B在允许的还原条件下在体外混合以进行反式剪接反应；(Iv)去除不愿允许二硫键二次氧化的片段；(V)通过亲和层析从未反应的前体中分离BsAb产物。该方法允许两个重链和两个轻链的正确组装，在没有合成接头的自然结构中形成双特异性抗体。UPLC-MASS分析未发现产物中存在链错配现象。此外，观察到的动力学和较低的反应活化能证实了反式剪接是热力学有利的反应。BAPTS技术在工业应用上是可行的。
High product purity, preserving natural IgG architecture, and excellent production efficiency are highly desirable in bispecific antibody manufacturing. We have reported a platform called Bispecific Antibody by Protein Trans-Splicing (BAPTS) to synthesize BsAbs with natural human IgG structure and no chain mispairing. In the method, two antibody fragments carrying different target-specificities are separately expressed in mammalian cells and subsequently fused to form BsAbs by utilizing the trans-splicing property of the split intein Npu DnaE. The hinge region of antibody, a region with less functional impact, is selected for conjugating the two fragments. The method involves the following steps: (i) constructing five plasmids coding antibody components; (ii) separately expressing and purifying two antibody fragments A and B. Fragment A contains one Fab, "Knobs-intoHoles" mutations in the CH3 domain and NPU DnaE(c). Fragment B contains another Fab and NPU DnaE(N); (iii) mixing of fragments A and B under permissive reducing conditions in vitro to enable trans-splicing reaction; (iv) removing the reluctant to allow re-oxidation of disulfide bonds; (v) isolating BsAb product from unreacted precursors by affinity chromatography. The method allows correct assembly of two heavy and two light chains to form bispecific IgG antibodies in natural structure with no synthetic linkers. No chain mispairing was observed in the product by UPLC-MASS. In addition, the observed kinetics and low reaction activation energy confirmed that the trans-splicing is thermodynamically favored reaction. The BAPTS technology is feasible for industrial applications.