Christian Doppler Laboratory for Innovative Immunotherapeutics, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.
Institute of Molecular Modeling and Simulation, Department of Material Sciences, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.
PLoS One. 2018 Apr 9;13(4):e0195442. doi: 10.1371/journal.pone.0195442. eCollection 2018.
We have designed a complete antibody-like construct where the CH1 and Cκ domains are exchanged for a pair of the CH3 domains and efficient pairing of the heavy and light variable domain is achieved using "Knobs-into-Holes" strategy. This construct, composed of only naturally occurring immunoglobulin sequences without artificial linkers, expressed at a high level in mammalian cells, however exhibited low solubility. Rational mutagenesis aimed at the amino acid residues located at the interface of the variable domains and the exchanged CH3 domains was applied to improve the biophysical properties of the molecule. The domain-exchanged construct, including variable domains of the HER2/neu specific antibody trastuzumab, was able to bind to the surface of the strongly HER2/neu positive cell line SK-BR3 4-fold weaker than trastuzumab, but could nevertheless incite a more potent response in an antibody-dependent cell cytotoxicity (ADCC) reporter assay with FcγRIIIa-overexpressing T-cells. This could be explained with a stronger binding to the FcγRIIIa. Importantly, the novel construct could mediate a specific ADCC effect with natural killer cells similar to the parental antibody.
我们设计了一种完整的抗体样构建体，其中 CH1 和 Cκ 结构域被一对 CH3 结构域取代，并且通过“Knobs-into-Holes”策略实现了重链和轻链可变区的有效配对。该构建体仅由天然存在的免疫球蛋白序列组成，没有人工接头，在哺乳动物细胞中高水平表达，但表现出低溶解度。为了改善分子的物理化学性质，我们对位于可变区和交换的 CH3 结构域界面的氨基酸残基进行了合理的突变。包括曲妥珠单抗（HER2/neu 特异性抗体）可变区的结构域交换构建体，与 SK-BR3 细胞系（表面高度表达 HER2/neu）的结合能力比曲妥珠单抗弱 4 倍，但在 FcγRIIIa 过表达 T 细胞的抗体依赖性细胞细胞毒性（ADCC）报告试验中，仍能引起更强的反应。这可以用与 FcγRIIIa 的更强结合来解释。重要的是，新型构建体可以与自然杀伤细胞介导特异性 ADCC 效应，类似于亲本抗体。
