Te Huataki Waiora School of Health, University of Waikato, Hamilton, New Zealand.
Te Aka Mātuatua School of Science, University of Waikato, Hamilton, New Zealand.
Protein Sci. 2023 Mar;32(3):e4589. doi: 10.1002/pro.4589.
The constant regions of clinical monoclonal antibodies are derived from a select number of allotypes found in IgG subclasses. Despite a long-term acknowledgment that this diversity may impact both antibody function and developability, there is a lack of data on the stability of variants carrying these mutations. Here, we generated a panel of IgG1, IgG2, and IgG3 antibodies with 32 unique constant region alleles and performed a systematic comparison of stability using red edge excitation shift (REES). This technique exploits the fluorescent properties of tryptophan residues to measure antibody structural dynamics which predict flexibility and the propensity to unfold. Our REES measurements revealed broad stability differences between subclasses with IgG3 possessing the poorest overall stability. Further interrogation of differences between variants within each subclass enabled the high-resolution profiling of individual allotype stabilities. Crucially, these observed differences were not found to be linked to N297-linked glycan heterogeneity. Our work demonstrates diverse stabilities (and dynamics) for a range of naturally occurring constant domain alleles and the utility of REES as a method for rapid and sensitive antibody stability profiling, requiring only laboratory spectrophotometry equipment.
临床单克隆抗体的恒定区来源于 IgG 亚类中发现的少数特定同种型。尽管长期以来人们已经认识到这种多样性可能会影响抗体的功能和可开发性，但对于携带这些突变的变体的稳定性却缺乏数据。在这里，我们生成了一组具有 32 个独特恒定区等位基因的 IgG1、IgG2 和 IgG3 抗体，并使用红色边缘激发位移 (REES) 进行了稳定性的系统比较。该技术利用色氨酸残基的荧光特性来测量抗体结构动力学，从而预测其灵活性和展开倾向。我们的 REES 测量结果显示，亚类之间存在广泛的稳定性差异，其中 IgG3 的整体稳定性最差。进一步研究每个亚类内变体之间的差异，能够对个体同种型稳定性进行高分辨率分析。至关重要的是，这些观察到的差异与 N297 连接的聚糖异质性无关。我们的工作表明，一系列天然存在的恒定结构域等位基因具有不同的稳定性（和动力学），并且 REES 作为一种快速、灵敏的抗体稳定性分析方法具有实用性，仅需要实验室分光光度设备。
