Department of Biotechnology, Christian Doppler Laboratory for Antibody Engineering, University of Natural Resources and Life Sciences, Vienna, Austria.
PLoS One. 2012;7(1):e30083. doi: 10.1371/journal.pone.0030083. Epub 2012 Jan 17.
We report the stabilization of the human IgG1 Fc fragment by engineered intradomain disulfide bonds. One of these bonds, which connects the N-terminus of the CH3 domain with the F-strand, led to an increase of the melting temperature of this domain by 10°C as compared to the CH3 domain in the context of the wild-type Fc region. Another engineered disulfide bond, which connects the BC loop of the CH3 domain with the D-strand, resulted in an increase of T(m) of 5°C. Combined in one molecule, both intradomain disulfide bonds led to an increase of the T(m) of about 15°C. All of these mutations had no impact on the thermal stability of the CH2 domain. Importantly, the binding of neonatal Fc receptor was also not influenced by the mutations. Overall, the stabilized CH3 domains described in this report provide an excellent basic scaffold for the engineering of Fc fragments for antigen-binding or other desired additional or improved properties. Additionally, we have introduced the intradomain disulfide bonds into an IgG Fc fragment engineered in C-terminal loops of the CH3 domain for binding to Her2/neu, and observed an increase of the T(m) of the CH3 domain for 7.5°C for CysP4, 15.5°C for CysP2 and 19°C for the CysP2 and CysP4 disulfide bonds combined in one molecule.
我们通过工程化的域内二硫键稳定了人 IgG1 Fc 片段。其中一个连接 CH3 结构域 N 端与 F 链的二硫键，使该结构域的熔点比野生型 Fc 区的 CH3 结构域提高了 10°C。另一个连接 CH3 结构域 BC 环与 D 链的工程化二硫键，使 Tm 升高了 5°C。两个域内二硫键结合在一个分子中，使 Tm 升高了约 15°C。所有这些突变都没有影响 CH2 结构域的热稳定性。重要的是，新生儿 Fc 受体的结合也不受突变的影响。总的来说，本报告中描述的稳定的 CH3 结构域为 Fc 片段的工程化提供了一个极好的基础支架，用于抗原结合或其他所需的额外或改进的特性。此外，我们还在 CH3 结构域 C 末端环中引入了域内二硫键，用于结合 Her2/neu，观察到 CysP4 的 CH3 结构域 Tm 升高了 7.5°C，CysP2 升高了 15.5°C，CysP2 和 CysP4 二硫键结合在一个分子中升高了 19°C。
