Structural and functional properties of human IgG4
Immunoglobulin (Ig) G4 is a unique antibody with characteristic structural properties that contribute to its status as a ‘blocking’ antibody. We found that human IgG4 exchanges half-molecules with other IgG4 molecules in blood, yielding antibodies with two different antigen-combining sites. The reaction was not observed upon mixing IgG4 antibodies in buffer, but was seen in both in vivo (in a mouse model) and in vitro (in the presence of glutathione as catalyst) models. We identified the key structural features that are responsible for this phenomenon using a panel of IgG4/IgG1 mutants in collaboration with Genmab. Also, IgG4 was able to bind to other IgG molecules, in particular, IgG4. This binding is related to the exchange process and may resemble an intermediate step. IgG4 binds to all human IgG subclasses if directly immobilized, but only to IgG4 when bound to antigen. Mechanistic studies are undertaken to establish the individual steps of this process. A FRET assay has been developed to monitor the exchange reaction in real time (Figure 1).
IgG4 stands out also during a T helper cell (Th) 2-driven immune response. Whereas IgG1 antibodies are readily formed, IgG4 antibody titers rise only slowly upon persistent antigenic stimulation. However, the IgG4 response dominates in the end. The underlying mechanisms that control the switch to, and proliferation of, IgG4-producing B cells are only partially understood. We initiated research aimed at unraveling these mechanisms. We quantified the number of IgG4-positive B cells in blood with FACS analysis and in vitro culture experiments. In line with the relatively low serum levels of IgG4 (3-4% of total IgG), the number of IgG4+ B cells is correspondingly low. Tools are currently being developed to investigate features that might explain why immune regulation of IgG4 differs from other isotypes.
- Labrijn AF, Rispens T, Meesters J, Rose RJ, den Bleker TH, Loverix S, van den Bremer ET, Neijssen J, Vink T, Lasters I, Aalberse RC, Heck AJ, van de Winkel JG, Schuurman J, Parren PW. Species-Specific Determinants in the IgG CH3 Domain Enable Fab-Arm Exchange by Affecting the Noncovalent CH3-CH3 Interaction Strength. J Immunol 2011; 187, 3238-46.
- Rispens T, Ooijevaar-de Heer P, Bende O, Aalberse RC. Mechanism of Immunoglobulin G4 Fab-arm Exchange. J Am Chem Soc 2011; 133:10302.
Figure 1: Real-time monitoring of the exchange process using a fluid-phase FRET assay. A) IgG4 is labeled with either Dy488 or Dy594 fluorochrome, and the reaction is monitored by measuring the FRET signal that arises from the mixed exchange product. B) Anti-Bet v 1 IgG4 was labeled with Dy488 and anti-Fel d 1 with Dy594 and incubated (37°C/1 mM GSH), and in a cross-linking radioimmunoassay using Bet v 1 Sepharose and 125I-labeled Fel d 1 it was demonstrated that bi-specific antibodies are formed similar to non-labeled antibodies. C) Fluorescence overlay spectra of IgG1-Dy488 and IgG1-Dy594 (adalimumab, left panel) or IgG4-Dy488 and IgG4-Dy594 (natalizumab, right panel) mixed and incubated at 37°C for 60 minutes after reduction with 1 mM DTT. D) Rate profile obtained by monitoring the exchange of DTT-reduced IgG4 by measuring the appearance of a FRET signal at 620 nm. Black curve: experiment; red curve: fit of a first-order exponential. Lower panel shows residuals of fit. (J Am Chem Soc 2011; 133:10302).