The odd one out – Mass spectrometric detection of the ever-changing IgG4 landscape

Immunoglobulin G4 (IgG4) is the least abundant subclass of the four human IgG antibodies in plasma, but the second most used format for therapeutic antibodies. IgG4s exhibit unique structural properties that diminish a) binding to most Fcy receptors, b) binding to C1q and enable c) facile Fab-arm exchange (FAE). These properties result in low antibody dependent cellular cytotoxicity (ADCC) activation, reduced complement activation, and inability to cross-link antigens. The ability of IgG4 to bind inhibitory FcyIIB in combination with the reduced binding to activating Fcy receptors leads to a shift towards cellular inhibition. While this anti-inflammatory character has benefits in allergy and parasitic infections, studies in recent years have shed light on IgG4-mediated pathologies, such as IgG4-autoimmune diseases (IgG4-AID) and IgG4-related diseases (IgG4-RD).

Studying immunoglobulin repertoires in plasma with clonal resolution can provide novel insights into basic questions in immunology. Ig repertoire profiling allows to follow on antibody plasticity, to get novel insights into antibody maturation, and to detect mature Ig sequences directly from donors.

In recent years, our group has presented new mass spectrometric approaches to profile antibody repertoires. Therefore, we capture all IgG1 and/or IgA1 and employ specific heavy chain (HC) hinge-directed proteases to generate their Fab fragments. Subsequently, liquid chromatography coupled online to mass spectrometry (LC-MS) is applied to measure plasma Ig clones in a quantitative manner while preserving information on heavy and light chain pairing.

Here we build further on this approach by introducing plasma IgG4 clonal profiling at the level of the intact Fabs. Our initial attempts on IgG4 profiling were focused on the intact Fab2 fragments, which would allow extensive studies on FAE. In middle-up MS experiments, most of the measured signal was however dispersing in unusually high noise. We deduced, that the inflated complexity originated from FAE facilitated stochastic Fab:Fab pairings. This assumption was supported by the observation that reduction of the same Fab2 samples resulted in far less complex data. By that, the complexity originating from stochastic Fab:Fab pairings was reduced to the mere light chain and Fd diversity. Notably, this is in line with earlier claims that almost all serum IgG4 undergo FAE.

One way to reduce the spectral complexity is to measure IgG4 Fabs instead of Fab2s. For that we take advantage of the IgG4 property that a fraction of all HCs is held together exclusively by non-covalent interactions in the CH3 domain. This unique structural property gives rise to two distinct digestion products after hinge digestion; Fab2 for the molecules with covalent HC:HC interaction, and Fabs for those with only non-covalent interaction. These products can then be separated by SEC to enrich for the lower abundant Fabs prior to MS analysis.