Structural and functional analyses of antibodies specific for modified core N-glycans suggest a role in TH 2 responses.

BACKGROUND: Immune responses to N-glycan structures from allergens and parasites are often associated with pronounced, high affinity IgE reactivities. Cross-reactive carbohydrate determinants (CCDs) are constituted by modified N-glycan core structures and represent the most frequently recognized epitopes in allergic immune responses. Although recently accepted as potentially allergenic epitopes, the biological and clinical relevance as well as structural and functional characteristics of CCD-specific antibodies remain elusive. METHODS: In order to gain structural insights into the recognition of CCDs, two specific antibody fragments were isolated from a leporid immune repertoire library and converted into human/leporid IgE and IgG formats. The antibody formats were assessed by ELISA and surface plasmon resonance, structural and functional analyses were performed by X-ray crystallography, mediator release, and ELIFAB assays. RESULTS: The recombinant IgE exhibited highly specific interactions with different types of CCDs on numerous CCD-carrying glycoproteins. Crystal structures of two CCD-specific antibodies, one of which in complex with a CCD-derived disaccharide emphasize that mechanisms of core glycan epitope recognition are as specific as those governing protein epitope recognition. The rIgE triggered immediate cellular responses via FcεRI cross-linking and mediated facilitated antigen presentation by binding of IgE/antigen complexes to CD23, a process that also could be blocked by IgG of allergic patients. CONCLUSIONS: Our study provides evidence for the relevance of N-glycan recognition in TH 2 responses and corroborates that IgE and IgG antibodies to ubiquitous carbohydrate epitopes can be equivalent to those directed against proteinaceous epitopes with implications for diagnostic and immunotherapeutic concepts.

Structure of the omalizumab Fab.

Omalizumab is a humanized anti-IgE antibody that inhibits the binding of IgE to its receptors on mast cells and basophils, thus blocking the IgE-mediated release of inflammatory mediators from these cells. Omalizumab binds to the Fc domains of IgE in proximity to the binding site of the high-affinity IgE receptor FcℇRI, but the epitope and the mechanisms and conformations governing the recognition remain unknown. In order to elucidate the molecular mechanism of its anti-IgE activity, the aim was to analyse the interaction of omalizumab with human IgE. Therefore, IgE Fc Cℇ2-4 was recombinantly produced in mammalian HEK-293 cells. Functionality of the IgE Fc was proven by ELISA and mediator-release assays. Omalizumab IgG was cleaved with papain and the resulting Fab was purified by ion-exchange chromatography. The complex of IgE Fc with omalizumab was prepared by size-exclusion chromatography. However, crystals containing the complex were not obtained, suggesting that the process of crystallization favoured the dissociation of the two proteins. Instead, two structures of the omalizumab Fab with maximum resolutions of 1.9 and 3.0 Šwere obtained. The structures reveal the arrangement of the CDRs and the position of omalizumab residues known from prior functional studies to be involved in IgE binding. Thus, the structure of omalizumab provides the structural basis for understanding the function of omalizumab, allows optimization of the procedure for complex crystallization and poses questions about the conformational requirements for anti-IgE activity.