Fab-arm exchange affects each and all endogenous serum IgG4 evenly

Antibodies are key molecular elements of the human immune system and account for an increasingly large proportion of therapeutics. Next to the well-studied and explored IgG1, several other classes (e.g. IgA, IgM) and sub-classes (e.g., IgG2, IgG3 and IgG4) exist in humans. In particular IgG4 is worth a closer examination as it has unique natural properties and is regularly used as a scaffold in biologicals. IgG4 stands out from the other IgG by its ability to dissociate and form two half-molecules which can interchange with those of other clones to form novel bivalent antibodies. Detailed analysis of endogenous IgG4 so far has been hampered by a lack of analytical methods to dissect and analyze IgG4 molecules with clonal resolution. Here, we present an LC-MS-based approach enabling the analysis of IgG4 repertoires with clonal resolution, which we used to monitor endogenous serum IgG4 repertoires from seven healthy donors. Most strikingly, our data reveal the combinatorial explosion in diversity of the serum IgG4 clonal repertoire. This phenomenon is explained by the stochastic behavior of Fab-arm exchange, making virtually each IgG4 molecule in serum bispecific. Although the endogenous IgG4 clonal repertoire is therefore extremely diverse, we demonstrate that this IgG4 repertoire persists over time within an individual healthy donor for more than one year. This newly established method now enables repertoire analysis for IgG4, which plays a critical role in a plethora of disease settings including allergy, autoimmunity and vaccination.