UCB Pharma, Slough, Berkshire SL1 3WE, United Kingdom.
Protein Sci. 2012 Sep;21(9):1315-22. doi: 10.1002/pro.2118. Epub 2012 Aug 9.
The stability of therapeutic antibodies is a prime pharmaceutical concern. In this work we examined thermal stability differences between human IgG1 and IgG4 Fab domains containing the same variable regions using the thermofluor assay. It was found that the IgG1 Fab domain is up to 11°C more stable than the IgG4 Fab domain containing the same variable region. We investigated the cause of this difference with the aim of developing a molecule with the enhanced stability of the IgG1 Fab and the biological properties of an IgG4 Fc. We found that replacing the seven residues, which differ between IgG1 C(H) 1 and IgG4 C(H) 1 domains, while retaining the native IgG1 light-heavy interchain disulfide (L-H) bond, did not affect thermal stability. Introducing the IgG1 type L-H interchain disulfide bond (DSB) into the IgG4 Fab resulted in an increase in thermal stability to levels observed in the IgG1 Fab with the same variable region. Conversely, replacement of the IgG1 L-H interchain DSB with the IgG4 type L-H interchain DSB reduced the thermal stability. We utilized the increased stability of the IgG1 Fab and designed a hybrid antibody with an IgG1 C(H) 1 linked to an IgG4 Fc via an IgG1 hinge. This construct has the expected biophysical properties of both the IgG4 Fc and IgG1 Fab domains and may therefore be a pharmaceutically relevant format.
治疗性抗体的稳定性是制药行业的首要关注点。在这项工作中，我们使用热荧光法检查了含有相同可变区的人 IgG1 和 IgG4 Fab 结构域之间的热稳定性差异。结果发现，IgG1 Fab 结构域比含有相同可变区的 IgG4 Fab 结构域稳定 11°C。我们研究了这种差异的原因，旨在开发一种具有 IgG1 Fab 增强稳定性和 IgG4 Fc 生物学特性的分子。我们发现，在保留天然 IgG1 轻链-重链间二硫键（L-H）的情况下，替换 IgG1 C(H)1 和 IgG4 C(H)1 结构域之间的七个残基不会影响热稳定性。将 IgG1 型 L-H 链间二硫键（DSB）引入 IgG4 Fab 中，可使热稳定性增加至与具有相同可变区的 IgG1 Fab 相同的水平。相反，用 IgG4 型 L-H 链间 DSB 替换 IgG1 L-H 链间 DSB 会降低热稳定性。我们利用 IgG1 Fab 的稳定性增加，设计了一种通过 IgG1 铰链将 IgG1 C(H)1 连接到 IgG4 Fc 的杂交抗体。该构建体具有 IgG4 Fc 和 IgG1 Fab 结构域的预期物理化学性质，因此可能是一种具有药物相关性的形式。
