Immunochemistry

Project leaders: Theo Rispens PhD, Prof Rob Aalberse PhD and Prof Lucien Aarden PhD

Structural and functional properties of human IgG4

IgG4 is a unique antibody with characteristic structural properties that contribute to its status as ‘blocking’ antibody. We found that human IgG4 can exchange half-molecules with other IgG4 molecules in the blood. This usually results in antibodies with two different antigen-combining sites. The reaction is not observed upon mixing IgG4 antibodies in buffer, but is observed both in vivo (in a mouse model) and in vitro (in the presence of glutathione as catalyst). 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 found to be able to bind to other IgG molecules, in particular, IgG4. This binding is related to the exchange process and could resemble an intermediate step. IgG4 binds to all human IgG subclasses if directly immobilized, but only to IgG4, 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.

IgG4 also stands out in its appearance during a Th2-driven immune response. Whereas IgG1 antibodies are readily formed upon antigenic challenge, IgG4 antibody titers rise only slowly upon persistent antigenic stimulation. However, the IgG4 response dominates in the end. The underlying mechanisms that control switch to and proliferation of IgG4-producing B cells are only partially understood. We initiated a study 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 positive B cells is correspondingly low. Tools are currently being developed to investigate features that might explain why the immune regulation of IgG4 differs from other isotypes, 1) developmental stages of the IgG4 B cell; 2) how and when the isotype switch is induced; and 3) at which stage the cell becomes an IgG-secreting cell.

Pro- and anti-inflammatory fractions in intravenous immunoglobulin (IVIG)

Besides being used for replacement therapy in patients with antibody deficiency, intravenous immunoglobulin (IVIG) is used in high doses in conditions such as idiopathic thrombocytopenia purpura (ITP), Kawasaki syndrome and Guillain-Barré. In applications other than replacement therapy, the mechanisms of action are largely uncertain, and proposed mechanisms include effects due to scavenging of complement activation products, blockade of Fc receptors, effects of IgG dimers, and effects of specific antibodies (for example: cytokine neutralization).

We investigated properties such as stability of the IgG dimers present in IVIG under different physical conditions including size exclusion chromatography and sodium dodecyl sulfate (SDS) electrophoresis. A substantial fraction of dimers dissociate rapidly under conditions mimicking those in patients after administering IVIG, but part of the dimers remain stable. Formation of dimers and larger aggregates may result in part from slightly denatured IgG. We found that aggregation of IgG may be counteracted by addition of fragments of IgG.

Treatments of conditions such as ITP require high doses of IVIG. It is reported that only the fraction of IgG molecules containing sialic acid is responsible for its anti-inflammatory action. These findings are based mainly on an arthritis mouse model. In cooperation with Sanquin Plasma Products, IVIG was enriched for sialic acid (SA). The SA-enriched and -depleted IVIG is currently being tested in a mouse model for ITP.

Furthermore, monomeric precursors for aggregation of IgG are difficult to detect. Usually, hydrophobic fluorescent probes such as 1-anilino-8-naphthalenesulfonate are used that may detect exposed hydrophobic surfaces as a result of partial unfolding. We extended this approach by detecting binding of such probes using isothermal titration calorimetry. In addition to fluorescent probes, non-fluorescent probes, including peptides, can be used to probe native or non-native configurations. Furthermore, IgG-derived peptides can be used to detect epitopes that are normally shielded.

pFc modulates Fc-Fc interactions of IgG and prevents aggregation
pFc’ modulates Fc-Fc interactions of IgG and prevents aggregation. Pepsin digests IgG into F(ab)2, pFc’ (black) and a number of smaller fragments. B) Two CH3 or pCH3 domains form a non-covalent dimer in solution. C) Papain digestion results in formation of an Fc fragment. D) HP-SEC analysis of pepsin digests of IgG1 at 37oC after 90 minutes (solid line) and 1000 minutes (dotted line). Open circles indicate inhibition of Fc-Fc interactions by fractions of the pepsin digest after 90 minutes (right axis). E) Aggregation of IgG induced by acid shock in absence or presence of pFc’.

Key publications