Stabilisation of the Fc Fragment of Human IgG1 by Engineered Intradomain Disulfide Bonds

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 Tm of 5°C. Combined in one molecule, both intradomain disulfide bonds led to an increase of the Tm 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 Tm 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.本研究报道了通过工程化域内二硫键（engineered intradomain disulfide bonds）稳定人类IgG1 Fc片段（human IgG1 Fc fragment）的工作。其中一种连接CH3结构域（CH3 domain）N端与F链（F-strand）的二硫键，相较于野生型Fc区域（wild-type Fc region）中的CH3结构域，可使该结构域的解链温度（melting temperature, Tm）提升10℃。另一种工程化二硫键连接CH3结构域的BC环（BC loop）与D链（D-strand），可使Tm升高5℃。将这两种域内二硫键整合至同一分子中时，可使Tm提升约15℃。所有上述突变均未对CH2结构域（CH2 domain）的热稳定性产生影响。值得注意的是，新生儿Fc受体（neonatal Fc receptor）的结合活性也未受这些突变影响。总体而言，本研究中报道的稳定化CH3结构域可为用于抗原结合或其他期望新增或优化特性的Fc片段工程化改造提供优异的基础骨架。此外，我们已将上述域内二硫键引入至经工程化改造的、可结合Her2/neu的CH3结构域C端环（C-terminal loops）的IgG Fc片段中，并观察到：当引入CysP4突变时，CH3结构域的Tm升高7.5℃；引入CysP2突变时，Tm升高15.5℃；而当CysP2与CysP4二硫键同时整合至同一分子中时，Tm升高19℃。