Traxlmayr Michael W, Lobner Elisabeth, Hasenhindl Christoph, Stadlmayr Gerhard, Oostenbrink Chris, Rüker Florian, Obinger Christian
Christian Doppler Laboratory for Antibody Engineering at Department of Chemistry and Department of Biotechnology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria; Department of Chemistry, Vienna Institute of BioTechnology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria.
Biotechnol J. 2014 Aug;9(8):1013-22. doi: 10.1002/biot.201300483.
For most therapeutic proteins, a long serum half-life is desired. Studies have shown that decreased antigen binding at acidic pH can increase serum half-life. In this study, we aimed to investigate whether pH-dependent binding sites can be introduced into antigen binding crystallizable fragments of immunoglobulin G1 (Fcab). The C-terminal structural loops of an Fcab were engineered for reduced binding to the extracellular domain of human epidermal growth factor receptor 2 (Her2-ECD) at pH 6 compared to pH 7.4. A yeast-displayed Fcab-library was alternately selected for binding at pH 7.4 and non-binding at pH 6.0. Selected Fcab variants showed clear pH-dependent binding to soluble Her2-ECD (decrease in affinity at pH 6.0 compared to pH 7.4) when displayed on yeast. Additionally, some solubly expressed variants exhibited pH-dependent interactions with Her2-positive cells whereas their conformational and thermal stability was pH-independent. Interestingly, two of the three Fcabs did not contain a single histidine mutation but all of them contained variations next to histidines that already occurred in loops of the lead Fcab. The study demonstrates that yeast surface display is a valuable tool for directed evolution of pH-dependent binding sites in proteins.
对于大多数治疗性蛋白质而言，人们期望其具有较长的血清半衰期。研究表明，在酸性pH条件下抗原结合能力的降低可延长血清半衰期。在本研究中，我们旨在探究是否能够将pH依赖性结合位点引入免疫球蛋白G1（Fcab）的抗原结合可结晶片段中。与pH 7.4相比，对Fcab的C端结构环进行工程改造，使其在pH 6时与人类表皮生长因子受体2（Her2-ECD）细胞外结构域的结合减少。交替选择酵母展示的Fcab文库，使其在pH 7.4时具有结合能力，而在pH 6.0时不具有结合能力。当展示在酵母上时，所选的Fcab变体对可溶性Her2-ECD表现出明显的pH依赖性结合（与pH 7.4相比，在pH 6.0时亲和力降低）。此外，一些可溶性表达的变体与Her2阳性细胞表现出pH依赖性相互作用，而它们的构象和热稳定性与pH无关。有趣的是，三个Fcab中的两个没有单个组氨酸突变，但它们都在组氨酸旁边发生了变异，这些变异已经出现在先导Fcab的环中。该研究表明，酵母表面展示是蛋白质中pH依赖性结合位点定向进化的一种有价值的工具。
