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.

Figure 1. Graphical presentation of secondary and tertiary structure of the CH3 domain and of an Fc fragment, and location of engineered disulfide bonds.

A: 2D-presentation (“Collier de perles” [32]) of the secondary structure of the CH3 domain. The residues that were mutated to Cysteine to form the engineered disulfide bonds are indicated in green (CysP2) and red (CysP4). Native disulfide bond residues are indicated in yellow. B: 3D cartoon presentation (created using the PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC) of the human IgG1 Fc fragment (Protein Data Bank (http://www.pdb.org/) accession 1OQO) with the positions that were mutated to create the engineered disulfide bonds indicated in green and red as in Figure 1A. The native disulfide bonds are shown in yellow.

Figure 2. Thermograms of wild-type and disulfide bond stabilized Fc mutants obtained with DSC measurements.

A: wild-type Fc, B: FcCysP2, C: FcCysP4, D: FcCysP24, E: overlay of all 5 proteins. For each protein the point of reversibility of melting of CH2 domain was determined which is shown as a dashed line in the inset graphs.

Figure 3. Thermal unfolding curves of disulfide bond stabilized Fc-mutants obtained from tCD-measurements.

Experiments were carried out at a protein concentration of 570 µg/ml in PBS at 218 nm using a 1-mm cell.

Figure 4. Spectroscopic characterization of disulfide bond stabilized Fc-mutants: (A) far-UV CD spectra and (B) near-UV CD spectra of stabilized Fc-mutants.

The measurements were carried out at a protein concentration of 570 µg/ml in PBS at 25°C.

Figure 5. Size exclusion chromatograms of wild-type Fc and disulfide bond stabilized Fc-mutants monitored at A₂₈₀.

Figure 6. BLI analyses of FcRn interaction with wild-type Fc and with disulfide bond stabilized Fc-mutants.

Biotinylated FcRn was immobilized to streptavidin tips. Association of the Fc fragment was observed at pH 6.0 and a sharp dissociation was induced by shifting the pH to 7.4.

Figure 7. Thermograms of Her2/neu binding Fcab H10-03-6 and its disulfide bond stabilized derivatives obtained with DSC measurements.