Two-Allergen Model Reveals Complex Relationship between IgE Crosslinking and Degranulation

SummaryAllergy is an immune response to complex mixtures of multiple allergens, yet current models use a single synthetic allergen. Multiple allergens were modeled using two well-defined tetravalent allergens, each specific for a distinct IgE, thus enabling a systematic approach to evaluate the effect of each allergen and percentage of allergen-specific IgE on mast cell degranulation. We found the overall degranulation response caused by two allergens is additive for low allergen concentrations or low percent specific IgE, does not change for moderate allergen concentrations with moderate to high percent specific IgE, and is reduced for high allergen concentrations with moderate to high percent specific IgE. These results provide further evidence that supraoptimal IgE crosslinking decreases the degranulation response and establishes the two-allergen model as a relevant experimental system to elucidate mast cell degranulation mechanisms

Inhibition of weak-affinity epitope-IgE interactions prevents mast cell degranulation

Development of specific inhibitors of allergy has had limited success, in part, owing to a lack of experimental models that reflect the complexity of allergen-IgE interactions. We designed a heterotetravalent allergen (HtTA) system, which reflects epitope heterogeneity, polyclonal response and number of immunodominant epitopes observed in natural allergens, thereby providing a physiologically relevant experimental model to study mast cell degranulation. The HtTA design revealed the importance of weak-affinity epitopes in allergy, particularly when presented with high-affinity epitopes. The effect of selective inhibition of weak-affinity epitope-IgE interactions was investigated with heterobivalent inhibitors (HBIs) designed to simultaneously target the antigen- and nucleotide-binding sites on the IgE Fab. HBI demonstrated enhanced avidity for the target IgE and was a potent inhibitor of degranulation in vitro and in vivo. These results demonstrate that partial inhibition of allergen-IgE interactions was sufficient to prevent mast cell degranulation, thus establishing the therapeutic potential of the HBI design

A heterobivalent ligand inhibits mast cell degranulation via selective inhibition of allergen-IgE interactions in vivo

Current treatments for allergies include epinephrine and antihistamines, which treat the symptoms after an allergic response has taken place; steroids, which result in local and systemic immune suppression; and IgE-depleting therapies, which can be used only for a narrow range of clinical IgE titers. The limitations of current treatments motivated the design of a heterobivalent inhibitor (HBI) of IgE-mediated allergic responses that selectively inhibits allergen-IgE interactions, thereby preventing IgE clustering and mast cell degranulation. The HBI was designed to simultaneously target the allergen binding site and the adjacent conserved nucleotide binding site (NBS) found on the Fab of IgE Abs. The bivalent targeting was accomplished by linking a hapten to an NBS ligand with an ethylene glycol linker. The hapten moiety of HBI enables selective targeting of a specific IgE, whereas the NBS ligand enhances avidity for the IgE. Simultaneous bivalent binding to both sites provided HBI with 120-fold enhancement in avidity for the target IgE compared with the monovalent hapten. The increased avidity for IgE made HBI a potent inhibitor of mast cell degranulation in the rat basophilic leukemia mast cell model, in the passive cutaneous anaphylaxis mouse model of allergy, and in mice sensitized to the model allergen. In addition, HBI did not have any observable systemic toxic effects even at elevated doses. Taken together, these results establish the HBI design as a broadly applicable platform with therapeutic potential for the targeted and selective inhibition of IgE-mediated allergic responses, including food, environmental, and drug allergies

Synthetic Allergen Design Reveals The Significance of Moderate Affinity Epitopes in Mast Cell Degranulation

This study describes the design of a well-defined homotetravalent synthetic allergen (HTA) system to investigate the effect of hapten–IgE interactions on mast cell degranulation. A library of DNP variants with varying affinities for IgEDNP was generated (Kd from 8.1 nM to 9.2 μM), and 8 HTAs spanning this range were synthesized via conjugation of each DNP variant to the tetravalent scaffold. HTAs with hapten Kd < 235 nM stimulated degranulation following a bell-shaped dose response curve with maximum response occurring near the hapten Kd. HTAs with hapten Kd ≥ 235 nM failed to stimulate degranulation. To mimic physiological conditions, the percent of allergen specific IgE on cell surface was varied, and maximum degranulation occurred at 25% IgEDNP. These results demonstrated that moderate hapten–IgE affinities are sufficient to trigger mast cell degranulation. Moreover, this study established the HTA design as a well-defined, controllable, and physiologically relevant experimental system to elucidate the mast cell degranulation mechanism

Synthetic Allergen Design Reveals the Significance of Moderate Affinity Epitopes in Mast Cell Degranulation

This study describes the design of a well-defined homotetravalent synthetic allergen (HTA) system to investigate the effect of hapten–IgE interactions on mast cell degranulation. A library of DNP variants with varying affinities for IgE^DNP was generated (<i>K</i>_d from 8.1 nM to 9.2 μM), and 8 HTAs spanning this range were synthesized via conjugation of each DNP variant to the tetravalent scaffold. HTAs with hapten <i>K</i>_d < 235 nM stimulated degranulation following a bell-shaped dose response curve with maximum response occurring near the hapten <i>K</i>_d. HTAs with hapten <i>K</i>_d ≥ 235 nM failed to stimulate degranulation. To mimic physiological conditions, the percent of allergen specific IgE on cell surface was varied, and maximum degranulation occurred at 25% IgE^DNP. These results demonstrated that moderate hapten–IgE affinities are sufficient to trigger mast cell degranulation. Moreover, this study established the HTA design as a well-defined, controllable, and physiologically relevant experimental system to elucidate the mast cell degranulation mechanism

Nonchromatographic Affinity Precipitation Method for the Purification of Bivalently Active Pharmaceutical Antibodies from Biological Fluids

This Article describes an affinity-based precipitation method for the rapid and nonchromatographic purification of bivalently active monoclonal antibodies by combining the selectivity of affinity chromatography with the simplicity of salt-induced precipitation. This procedure involves (i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; (ii) formation and selective precipitation of cyclic antibody complexes created by binding to trivalent haptens specific for the antibody; and (iii) membrane filtration of the solubilized antibody pellet to remove the trivalent hapten from the purified antibody. We applied this technique to the purification of two pharmaceutical antibodies, trastuzumab and rituximab, by synthesizing trivalent haptens specific for each antibody. Using this method, we were able to purify both antibodies from typical contaminants including CHO cell conditioned media, ascites fluid, DNA, and other antibodies with yields >85% and with >95% purity. The purified antibodies displayed native binding levels to cell lines expressing the target proteins demonstrating that the affinity-based precipitation method did not adversely affect the antibodies. The selectivity of the affinity-based precipitation method for bivalently active antibodies was established by purifying trastuzumab from a solution containing both active and chemically denatured trastuzumab. Prior to purification, the solutions displayed 20–76% reduction in binding activity, and after purification, native binding activity was restored, indicating that the purified product contained only bivalently active antibody. Taken together, the affinity-based precipitation method provides a rapid and straightforward process for the purification of antibodies with the potential to improve product quality while decreasing the purification costs at both the lab and the industrial scale

Enhancement of Antibody Selectivity via Bicyclic Complex Formation

This study describes a strategy where antibody selectivity for high antigen-density surfaces is enhanced by forming a thermodynamically stable bicyclic complex. The bicyclic complex was formed via multivalent interactions of the antibody with a synthetic trivalent mimotope at a 3:2 molar ratio. Complex formation was analyzed using dynamic light scattering and analytical ultracentrifugation, showing a hydrodynamic radius of ∼22 nm and a calculated molecular weight of 397 kDa, depicting a trimeric complex formation. The complex has high thermodynamic stability and results in a 10-fold higher binding affinity for the trivalent mimotope (<i>K</i>_d = 0.14 μM) compared to the monovalent mimotope (<i>K</i>_d = 1.4 μM). As bicyclic complexes, the antibodies showed ∼18% binding of the monomeric form to low antigen-density surfaces. At high antigen-density, antibody binding was equal whether delivered as a complex or a monomer. These results establish bicyclic complex selectivity for high antigen-density surfaces and suggest a potential method to enhance therapeutic antibody selectivity for diseased cells