Protein Interactions Group, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD USA.
MAbs. 2013 Jul-Aug;5(4):533-9. doi: 10.4161/mabs.25211. Epub 2013 May 29.
Combinatory antibody library display technologies have been invented and successfully implemented for the selection and engineering of therapeutic antibodies. Precise targeting of important epitopes on the protein of interest is essential for such isolated antibodies to serve as effective modulators of molecular interactions. We developed a strategy to efficiently isolate antibodies against a specific epitope on a target protein from a yeast display antibody library using dengue virus envelope protein domain III as a model target. A domain III mutant protein with a key mutation inside a cross-reactive neutralizing epitope was designed, expressed, and used in the competitive panning of a yeast display naïve antibody library. All the yeast display antibodies that bound to the wild type domain III but not to the mutant were selectively sorted and characterized. Two unique clones were identified and showed cross-reactive binding to envelope protein domain IIIs from different serotypes. Epitope mapping of one of the antibodies confirmed that its epitope overlapped with the intended neutralizing epitope. This novel approach has implications for many areas of research where the isolation of epitope-specific antibodies is desired, such as selecting antibodies against conserved epitope(s) of viral envelope proteins from a library containing high titer, high affinity non-neutralizing antibodies, and targeting unique epitopes on cancer-related proteins.
组合抗体文库展示技术已经被发明并成功应用于治疗性抗体的筛选和工程改造。为了使分离得到的抗体成为有效的分子相互作用调节剂，精确靶向目标蛋白上的重要表位至关重要。我们开发了一种策略，使用登革热病毒包膜蛋白结构域 III 作为模型靶标，从酵母展示抗体文库中高效分离针对靶蛋白特定表位的抗体。设计了一个关键突变位于交叉反应性中和表位内的结构域 III 突变蛋白，并在酵母展示原始抗体文库的竞争淘选中使用该蛋白。与野生型结构域 III 结合但不与突变型结合的所有酵母展示抗体都被选择性地分拣和表征。鉴定出两个独特的克隆，并显示与不同血清型的包膜蛋白结构域 III 发生交叉反应性结合。其中一种抗体的表位映射证实其表位与预期的中和表位重叠。这种新方法适用于许多需要分离表位特异性抗体的研究领域，例如从含有高滴度、高亲和力非中和抗体的文库中选择针对病毒包膜蛋白保守表位的抗体，以及针对癌症相关蛋白上的独特表位。
