Abstract
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.
Highlights
Monoclonal antibodies with high affinity and specificity are well established therapeutics and invaluable tools for biological research
We aimed at increasing the stability of the human CH3 domain in the context of an Fc fragment, in order to increase its tolerance to mutations in loop regions, which may be introduced at a later stage with the purpose of creating antigen binding sites in this unconventional region of the antibody molecule
Out of 29 disulfide bonds that were predicted by DSDBASE to be possible in the CH3 domain, we selected 5 that seemed to be the ones with the highest likelihood of success as judged by visual examination of the crystal structure of an Fc fragment: Thr350 and Leu441 (CysP1), Pro343 and Ala431 (CysP2), Ser375 and Phe404 (CysP3), Ser375 and Pro396 (CysP4) and Val348 and Lys439 (CysP5)
Summary
Monoclonal antibodies with high affinity and specificity are well established therapeutics and invaluable tools for biological research. The Fc part itself is composed of two CH2 domains and two CH3 domains that form a homodimeric region at the Cterminal end of the antibody molecule. These regions associate with each other with two disulfide bridges in the hinge region which is located between the CH1 and CH2 domains and by strong non-covalent interactions between the two CH3 domains [1]. The CH2 domains harbour the binding sites for all effector molecules such as the Fcc receptors as well as complement factor C1q. At the interface between CH2 and CH3 there is the binding site for neonatal Fc receptor (FcRn), which mediates the long half-life of antibodies
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