Abstract
The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest in understanding and modulating the IgG-FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG(1) and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation to map sites perturbed by binding on both partners of the IgG-FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found that several regions in the IgG Fab region also showed reduced deuterium uptake. Our findings indicate the presence of hitherto unknown FcRn interaction sites in the Fab region or a possible conformational link between the IgG Fc and Fab regions upon FcRn binding. Further, we investigated the role of IgG glycosylation in the conformational response of the IgG-FcRn interaction. Removal of antibody glycans increased the flexibility of the FcRn binding site in the Fc region. Consequently, FcRn binding did not induce a similar conformational stabilization of deglycosylated IgG as observed for the wild-type glycosylated IgG. Our results provide new molecular insight into the IgG-FcRn interaction and illustrate the capability of hydrogen/deuterium exchange mass spectrometry to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in solution.
Highlights
From the ‡Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark; §Roche Pharmaceutical Research and Early Development, Large Molecule Research, Roche Innovation Center Penzberg, Nonnenwald 2, 82377 Penzberg, Germany
An Ig is a “dimer of a dimer” consisting of light chains and heavy chains in which each light chain is linked to a heavy chain and the light– heavy dimers are connected by disulfide bridges to form the intact antibody
In this study we investigated the interaction between human FcRn and two variants of a full-length IgG1 by means of hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS)
Summary
Proteins and Variants—Recombinant human monoclonal IgG1 antibody was expressed in CHO cells and buffered in 50 mM Na phosphate, 50 mM NaCl, pH 6.5 (Roche Diagnostics, Penzberg, Germany). Hydrogen/Deuterium Exchange Mass Spectrometry—HDX-MS experiments were performed on glycosylated human IgG1 (Abwt), deglycosylated human IgG1 (Abdegly), human FcRn, and deglycosylated FcRn (FcRndegly) using the following samples. Abwt Ϯ FcRn—Abwt (73 pmol/l) and FcRn (112 pmol/l) were mixed and diluted in 99.9% D2O 50 mM Na phosphate, 50 mM NaCl, pH 6.5, to a final D2O content of 90% and Ab and FcRn concentrations of 1.2 pmol/l and 8.96 pmol/l, respectively. Hydrogen/Deuterium Exchange Mass Spectrometry with ETD— Deuterated samples were prepared using the same procedure as in the HDX-MS experiments, except the injection amount was adjusted to 100 pmol Abwt and a 5-fold dilution into D2O buffer (80% D2O) was employed, resulting in Ab and FcRn concentrations of 4 pmol/l and 14 pmol/l, respectively.
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