title: Mechanism of immunoglobulin G4 Fab-arm exchange.
abstract: Immunoglobulin G (IgG) antibodies are symmetrical molecules that may be regarded as covalent dimers of 2 half-molecules, each consisting of a light chain and a heavy chain. Human IgG4 is an unusually dynamic antibody, with half-molecule exchange ("Fab-arm exchange") resulting in asymmetrical, bispecific antibodies with two different antigen binding sites, which contributes to its anti-inflammatory activity. The mechanism of this process is unknown. To elucidate the elementary steps of this intermolecular antibody rearrangement, we developed a quantitative real-time FRET assay to monitor the kinetics of this process. We found that an intrinsic barrier is the relatively slow dissociation of the CH3 domains that noncovalently connect the heavy chains, which becomes rate determining in case disulfide bonds between the heavy chains are reduced or absent. Under redox conditions that mimic the previously estimated in vivo reaction rate, i.e., 1 mM of reduced glutathione, the overall rate is ca. 20 times lower because only a fraction of noncovalent isomers is present (with intra- rather than interheavy chain disulfide bonds), formed in a relatively fast pre-equilibrium from covalent isomers. Interestingly, Fab arms stabilize the covalent isomer: the amount of noncovalent isomers is ca. 3 times higher for Fc fragments of IgG4 (lacking Fab domains) compared to intact IgG4, and the observed rate of exchange is 3 times higher accordingly. Thus, kinetic data obtained from a sensitive and quantitative real-time FRET assay as described here yield accurate data about interdomain interactions such as those between Fab and/or Fc domains. The results imply that in vivo, the reaction is under control of local redox conditions.
authors.affiliation: Sanquin Research, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands. T.Rispens@sanquin.nl
authors.forename: Theo
authors.initials: T
authors.lastname: Rispens
authors.forename: Pleuni
authors.initials: P
authors.lastname: Ooijevaar-de Heer
authors.forename: Onno
authors.initials: O
authors.lastname: Bende
authors.forename: Rob C
authors.initials: RC
authors.lastname: Aalberse
mesh_terms: D000595:Amino Acid Sequence; D018033:Antibodies, Bispecific / Q000737:chemistry* / Q000276:immunology*; D002850:Chromatography, Gel; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology; D007700:Kinetics; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary
chemical_list: D018033:Antibodies, Bispecific; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G