James Hunt

Conformational changes in IgE contribute to its uniquely slow dissociation rate from receptor FcɛRI

Among antibody classes, IgE has a uniquely slow dissociation rate from, and high affinity for, its cell surface receptor FcɛRI. We show the structural basis for these key determinants of the ability of IgE to mediate allergic hypersensitivity through the 3.4-Å-resolution crystal structure of human IgE-Fc (consisting of the Cɛ2, Cɛ3 and Cɛ4 domains) bound to the extracellular domains of the FcɛRI α chain. Comparison with the structure of free IgE-Fc (reported here at a resolution of 1.9 Å) shows that the antibody, which has a compact, bent structure before receptor engagement, becomes even more acutely bent in the complex. Thermodynamic analysis indicates that the interaction is entropically driven, which explains how the noncontacting Cɛ2 domains, in place of the flexible hinge region of IgG antibodies, contribute together with the conformational changes to the unique binding properties of IgE.

Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells

Trastuzumab (Herceptin®), a humanized IgG1 antibody raised against the human epidermal growth factor receptor 2 (HER2/neu), is the main antibody in clinical use against breast cancer. Pre-clinical evidence and clinical studies indicate that trastuzumab employs several anti-tumour mechanisms that most likely contribute to enhanced survival of patients with HER2/neu-positive breast carcinomas. New strategies are aimed at improving antibody-based therapeutics like trastuzumab, e.g. by enhancing antibody-mediated effector function mechanisms. Based on our previous findings that a chimaeric ovarian tumour antigen-specific IgE antibody showed greater efficacy in tumour cell killing, compared to the corresponding IgG1 antibody, we have produced an IgE homologue of trastuzumab. Trastuzumab IgE was engineered with the same light- and heavy-chain variable-regions as trastuzumab, but with an epsilon in place of the gamma-1 heavy-chain constant region. We describe the physical characterisation and ligand binding properties of the trastuzumab IgE and elucidate its potential anti-tumour activities in functional assays. Both trastuzumab and trastuzumab IgE can activate monocytic cells to kill tumour cells, but they operate by different mechanisms: trastuzumab functions in antibody-dependent cell-mediated phagocytosis (ADCP), whereas trastuzumab IgE functions in antibody-dependent cell-mediated cytotoxicity (ADCC). Trastuzumab IgE, incubated with mast cells and HER2/neu-expressing tumour cells, triggers mast cell degranulation, recruiting against cancer cells a potent immune response, characteristic of allergic reactions. Finally, in viability assays both antibodies mediate comparable levels of tumour cell growth arrest. These functional characteristics of trastuzumab IgE, some distinct from those of trastuzumab, indicate its potential to complement or improve upon the existing clinical benefits of trastuzumab.

Soluble CD23 Monomers Inhibit and Oligomers Stimulate IGE Synthesis in Human B Cells

The low affinity IgE receptor, CD23, is implicated in IgE regulation and the pathogenesis of allergic disease. CD23 is a type II integral membrane protein, comprising a lectin “head,” N-terminal “stalk,” and C-terminal “tail” in the extracellular sequence. Endogenous proteases cleave CD23 in the stalk and the tail to release soluble fragments that either stimulate or inhibit IgE synthesis in human B cells. The molecular basis of these paradoxical activities is not understood. We have characterized three fragments of CD23, monomeric derCD23, monomeric exCD23, and oligomeric lzCD23. We show that the monomers inhibit and the oligomer stimulates IgE synthesis in human B cells after heavy chain switching to IgE. CD23 fragments could be targets for therapeutic intervention in allergic disease.

A Fluorescent Biosensor Reveals Conformational Changes in Human Immunoglobulin E Fc IMPLICATIONS FOR MECHANISMS OF RECEPTOR BINDING, INHIBITION, AND ALLERGEN RECOGNITION

Background: Immunoglobulin E (IgE) antibodies play a role in allergic disease. Results: IgE has a bent conformation in solution that becomes more bent upon binding to the FcϵRI receptor but less bent upon binding the anti-IgE omalizumab. Conclusion: Conformational change is critical for FcϵRI-mediated IgE activity. Significance: The bent structure provides a molecular rationale for the susceptibility of IgE-FcϵRI complexes to allergenic stimulation. IgE binding to its high affinity receptor FcϵRI on mast cells and basophils is a key step in the mechanism of allergic disease and a target for therapeutic intervention. Early indications that IgE adopts a bent structure in solution have been confirmed by recent x-ray crystallographic studies of IgEFc, which further showed that the bend, contrary to expectation, is enhanced in the crystal structure of the complex with receptor. To investigate the structure of IgEFc and its conformational changes that accompany receptor binding in solution, we created a Förster resonance energy transfer (FRET) biosensor using biologically encoded fluorescent proteins fused to the N- and C-terminal IgEFc domains (Cϵ2 and Cϵ4, respectively) together with the theoretical basis for quantitating its behavior. This revealed not only that the IgEFc exists in a bent conformation in solution but also that the bend is indeed enhanced upon FcϵRI binding. No change in the degree of bending was seen upon binding to the B cell receptor for IgE, CD23 (FcϵRII), but in contrast, binding of the anti-IgE therapeutic antibody omalizumab decreases the extent of the bend, implying a conformational change that opposes FcϵRI engagement. HomoFRET measurements further revealed that the (Cϵ2)2 and (Cϵ4)2 domain pairs behave as rigid units flanking the conformational change in the Cϵ3 domains. Finally, modeling of the accessible conformations of the two Fab arms in FcϵRI-bound IgE revealed a mutual exclusion not seen in IgG and Fab orientations relative to the membrane that may predispose receptor-bound IgE to cross-linking by allergens.

Disulfide linkage controls the affinity and stoichiometry of IgE Fcepsilon3-4 binding to FcepsilonRI

IgE antibodies cause long-term sensitization of tissue mast cells and blood basophils toward allergen-induced cross-linking and triggering of allergic inflammation. This persistence of IgE binding is due to its uniquely high affinity for the receptor FcϵRI and in particular its slow rate of dissociation once bound. The binding interface consists of two subsites, one contributed by each Cϵ3 domain of IgE Fc in a 1:1 complex. We have investigated the contributions of Cϵ3 disulfide linkage and glycosylation to the kinetics and affinity of binding of an Fc subfragment (Fcϵ3–4) to a soluble receptor fragment (sFcϵRIα). In contrast to IgG Fc where deglycosylation abrogates receptor binding activity, the removal of the N-linked carbohydrate at Asn-394 in Fcϵ3–4 only reduces binding affinity by a factor of 4, principally because of a faster off-rate. Removal of the inter-heavy chain disulfide bond unexpectedly resulted in a fragment with a much faster off-rate and the potential to form a complex with a 2:1 stoichiometry (sFcϵRIα:Fcϵ3–4). This permitted the determination of the affinity of a single, natively folded Cϵ3 domain for the first time. The low affinity Ka ≈ 105-106 m–1, similar to that determined previously for an isolated and partially folded Cϵ3 domain, demonstrates that substantial reduction in affinity can be achieved by preventing the engagement of one of the two Cϵ3 domains. Recent structural data indicate that conformational change in IgE is required to allow both Cϵ3 domains to bind, and thus an allosteric inhibitor that prevents access to the second Cϵ3 has the potential to reduce the ability of IgE to sensitize allergic effector cells.

Attenuation of IgE Affinity for FcϵRI Radically Reduces the Allergic Response in Vitro and in Vivo

The high affinity of IgE for its receptor, FcϵRI (Ka ∼ 1010 m–1), is responsible for the persistence of mast cell sensitization. Cross-linking of FcϵRI-bound IgE by multivalent allergen leads to cellular activation and release of pro-inflammatory mediators responsible for the symptoms of allergic disease. We previously demonstrated that limiting the IgE-FcϵRI interaction to just one of the two Cϵ3 domains in IgE-Fc, which together constitute the high affinity binding site, results in 1000-fold reduced affinity. Such attenuation, effected by a small molecule binding to part of the IgE:FcϵRI interface or a distant allosteric site, rather than complete blocking of the interaction, may represent a viable approach to the treatment of allergic disease. However, the degree to which the interaction would need to be disrupted is unclear, because the importance of high affinity for immediate hypersensitivity has never been investigated. We have incorporated into human IgE a mutation, R334S, previously characterized in IgE-Fc, which reduces its affinity for FcϵRI ∼50-fold. We have compared the ability of wild type and R334S IgE to stimulate allergen-induced mast cell activation in vitro and in vivo. We confirmed the expected difference in affinity between wild type and mutant IgE for FcϵRI (∼50-fold) and found that, in vitro, mast cell degranulation was reduced proportionately. The effect in vivo was also marked, with a 75% reduction in the passive cutaneous anaphylaxis response. We have therefore demonstrated that the high affinity of IgE for FcϵRI is critical to the allergic response, and that even moderate attenuation of this affinity has a substantial effect in vivo.