Fc-Fc interactions of human IgG4 require dissociation of heavy chains and are formed predominantly by the intra-chain hinge isomer.

Human IgG4 antibodies are remarkable not only because they can dynamically exchange half-molecules (Fab-arm exchange) but also for their ability to interact with the Fc part of IgG4 and other IgG subclasses. This rheumatoid factor-like binding of IgG4 does not appear to take place spontaneously, because it is only observed to solid-phase or antigen-bound IgG. We hypothesized that Fc-Fc interactions might involve (partial) dissociation of heavy chains. We investigated the molecular basis of these Fc-Fc interactions, and found that the structural features important for the exchange reaction also control the Fc binding activity. In particular, if arginine-409 in the CH(3)-CH(3) interface in IgG4 is mutated to lysine (the equivalent in IgG1), Fc-Fc interactions are formed 3 orders of magnitude less efficiently compared to the wild-type. This mutation was previously found to increase the CH(3)-CH(3) interaction strength in IgG4. Furthermore, of the two hinge isomers of IgG4, the intra-chain (non-covalently linked) form was found to form Fc-Fc interactions, but not the inter-chain form. Together, these results demonstrate that Fc-Fc interactions of IgG4 involve (partial or complete) dissociation of heavy chains. The promiscuity to other IgG subclasses suggests that IgG4 might act as scavenger to IgG molecules with impaired structural integrity.

Crystal structure of the human IgG4 C(H)3 dimer reveals the role of Arg409 in the mechanism of Fab-arm exchange.

Antibodies of the human IgG4 subclass uniquely undergo a process of Fab-arm exchange in which the heavy-chains of antibodies of different specificities can dissociate and then recombine. The mechanism by which the resulting functionally monovalent but bi-specific antibodies are formed is not only key to understanding their biological role, but is also important for the design of therapeutic monoclonal antibodies. Both the hinge region and the C(H)3 domain interface are known to be involved, and of the residues that differ between human IgG1 and IgG4 in C(H)3, residue 409, the only difference at the interface itself, has been implicated. We report the high resolution (1.8Å) structure of the C(H)3 domain dimer of IgG4, and find that Arg409 in IgG4, when compared with Lys409 observed in high resolution IgG1 structures, disrupts a network of water-mediated hydrogen bonding that is conserved in IgG1. Other conformational differences were detected that are a consequence of the presence of Arg409, such as a widening of the separation between residues Asn390 in one domain and Ser 400 in the other, which opens up a groove at the edge of the interface in IgG4 compared with IgG1. The effect of all these differences on the C(H)3 interface, doubled as a result of the interface's two-fold symmetry, is weakening of the inter-domain interaction in IgG4 compared with IgG1. This suggests a mechanism by which Arg409 weakens the C(H)3 interface in IgG4, predisposing this human antibody subclass to Fab-arm exchange.

Species-specific determinants in the IgG CH3 domain enable Fab-arm exchange by affecting the noncovalent CH3-CH3 interaction strength.

A distinctive feature of human IgG4 is its ability to recombine half molecules (H chain and attached L chain) through a dynamic process termed Fab-arm exchange, which results in bispecific Abs. It is becoming evident that the process of Fab-arm exchange is conserved in several mammalian species, and thereby represents a mechanism that impacts humoral immunity more generally than previously thought. In humans, Fab-arm exchange has been attributed to the IgG4 core-hinge sequence (226-CPSCP-230) in combination with unknown determinants in the third constant H chain domain (CH3). In this study, we investigated the role of the CH3 domain in the mechanism of Fab-arm exchange, and thus identified amino acid position 409 as the critical CH3 determinant in human IgG, with R409 resulting in exchange and K409 resulting in stable IgG. Interestingly, studies with IgG from various species showed that Fab-arm exchange could not be assigned to a common CH3 domain amino acid motif. Accordingly, in rhesus monkeys (Macaca mulatta), aa 405 was identified as the CH3 determinant responsible (in combination with 226-CPACP-230). Using native mass spectrometry, we demonstrated that the ability to exchange Fab-arms correlated with the CH3-CH3 dissociation constant. Species-specific adaptations in the CH3 domain thus enable Fab-arm exchange by affecting the inter-CH3 domain interaction strength. The redistribution of Ag-binding domains between molecules may constitute a general immunological and evolutionary advantage. The current insights impact our view of humoral immunity and should furthermore be considered in the design and evaluation of Ab-based studies and therapeutics.

Hybrid IgG4/IgG4 Fc antibodies form upon 'Fab-arm' exchange as demonstrated by SDS-PAGE or size-exclusion chromatography.

Human IgG4 antibodies are dynamic molecules that in vivo exchange half-molecules to become bispecific antibodies. Here we show that IgG4 antibodies and IgG4 Fc fragments similarly exchange resulting in hybrid antibodies (a single Fab+Fc) with a molecular weight of ca. 100kDa. These antibodies can be separated from IgG4 and IgG4 Fc using size-exclusion chromatography and non-reducing SDS-PAGE. This method does not rely on a cross-linking immunoassay with its potential for false-positive results due to aggregation and it unambiguously demonstrates that the 'Fab-arm' exchange process depends entirely on the Fc part (hinge+CH2+CH3).

Traces of pFc' in IVIG interact with human IgG Fc domains and counteract aggregation.

To prevent multimer formation, intravenous immunoglobulin (IVIG) is often treated with traces of pepsin. So far, the mechanism behind this treatment has been unclear. Recently, we reported that human IgG4 binds other IgG molecules via Fc-Fc interactions. Here we show that IVIG treated with traces of pepsin (Nanogam) inhibits these interactions. We found that--besides IgG4--peptides corresponding to IgG1 and IgG2 pFc' (products of limited pepsin digestion) are responsible for the inhibitory action. Using radiolabeled pFc', it was found that pFc' binds directly to IgG1. Furthermore, recombinant CH3 fragments were found to also possess binding activity, and potencies of inhibition varied over 3 orders of magnitude amongst the subclasses, IgG4 being most potent. We propose that pFc' formation explains how limited pepsin digestion diminishes adverse effects of IVIG. In particular, the presence of this fragment can enhance the stability of IgG products including IVIG and therapeutical monoclonal antibodies. Indeed, using a model system it was found that acid-induced aggregation of IgG is reduced in the presence of pFc', suggesting a 'chaperone-like' activity of this fragment. Thus, pFc' can modulate Fc interactions and may therefore reduce adverse effects of IVIG, in particular by preventing oligomerization.

Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange.

Antibodies play a central role in immunity by forming an interface with the innate immune system and, typically, mediate proinflammatory activity. We describe a novel posttranslational modification that leads to anti-inflammatory activity of antibodies of immunoglobulin G, isotype 4 (IgG4). IgG4 antibodies are dynamic molecules that exchange Fab arms by swapping a heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, which results in bispecific antibodies. Mutagenesis studies revealed that the third constant domain is critical for this activity. The impact of IgG4 Fab arm exchange was confirmed in vivo in a rhesus monkey model with experimental autoimmune myasthenia gravis. IgG4 Fab arm exchange is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 antibodies.