title: Stabilisation of the Fc fragment of human IgG1 by engineered intradomain disulfide bonds.
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 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.
authors.affiliation: Department of Biotechnology, Christian Doppler Laboratory for Antibody Engineering, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Gordana
authors.initials: G
authors.lastname: Wozniak-Knopp
authors.forename: Johannes
authors.initials: J
authors.lastname: Stadlmann
authors.forename: Florian
authors.initials: F
authors.lastname: Rüker
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D044366:Transition Temperature
chemical_list: D004220:Disulfides; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D018719:Erb-b2 Receptor Tyrosine Kinases