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

Handlogten, Michael W.; Kiziltepe, Tanyel; Alves, Nathan J.; Bilgiçer, Başar

doi:10.1021/cb300193f

Cited by 17 publications

(34 citation statements)

References 36 publications

(84 reference statements)

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“…Through a combination of experimental approaches and molecular modeling, it has been demonstrated that the average distance between the two Fab domains of IgE is 11–13 nm and that, owing to the differences between the extended and in-solution length of ethylene glycol, a PEG 3350 linker (extended length of 29 nm) is required to span the two antigen-binding sites on a single IgE 26–28 . Previously, we identified that ethylene glycol with an extended length of ∼6 nm is optimal for haptens to bind multiple antibodies without bridging the two antigen-binding sites on a single antibody 29–33 . Consequently, in our tetravalent allergen design, the four hapten moieties were conjugated to the core of the molecule with 8 units of ethylene glycol, which provided an extended length of 3.2 nm, yielding a maximum separation of 6.4 nm between haptens (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2013

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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.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2013

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“…Therefore, we selected DNP/IgE DNP as the high affinity pair in our design. The hapten DNP can be chemically modified to alter the affinity of this interaction to generate weaker affinity pairs [17, 24]. In order to create a second hapten/IgE pair with weaker affinity, we synthesized a DNP variant, DNP-proline (DNP-Pro).…”

Section: Resultsmentioning

confidence: 99%

Design of a heterotetravalent synthetic allergen that reflects epitope heterogeneity and IgE antibody variability to study mast cell degranulation

Handlogten

Kiziltepe

Bilgiçer

2012

Biochemical Journal

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SYNOPSIS This study describes the design of a heterotetravalent allergen (HtTA) as a multi-component experimental system that enables an integrative approach to study mast cell degranulation. The HtTA design allows presentation of two distinct haptens, each with a valency of two, thereby better reflecting the complexity of natural allergens by displaying epitope heterogeneity and IgE antibody variability. Using the HtTA design, synthetic allergens HtTA-1 and HtTA-2 were synthesized to model a combination of epitope/IgE affinities. HtTA-1 presented DNP and dansyl haptens (Kd = 22 and 54 nM for IgEDNP and IgEdansyl respectively), and HtTA-2 presented dansyl and the weak affinity DNP-Pro haptens (Kd = 550 nM for IgEDNP). Both HtTAs effectively induced degranulation when mast cells were primed with both IgEDNP and IgEdansyl antibodies. Interestingly, tetravalent DNP-Pro or bivalent dansyl were insufficient in stimulating a degranulation response, illustrating the significance of valency, affinity, and synergy in allergen-IgE interactions. Importantly, maximum degranulation with both HtTA-1 and HtTA-2 was observed when only 50% of the mast cell-bound IgEs were hapten specific (25% IgEdansyl + 25% IgEDNP). Taken together, this study establishes the HtTA system as a physiologically relevant experimental model and demonstrates its utility in elucidating critical mechanisms of mast cell degranulation.

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“…In our previous work, we describe the design, synthesis, and characterization of synthetic tetravalent allergens (Handlogten et al, 2013; Handlogten et al, 2012; Handlogten et al, 2013). These well-defined tetravalent allergens have several advantages compared to the widely used haptenated proteins, such as DNP conjugated to BSA (DNP-BSA), as model allergens.…”

Section: Resultsmentioning

confidence: 99%

“…We previously established that the relative concentration of each IgE in solution is preserved on the surface of the mast cells (Handlogten et al, 2012). The mast cells were then exposed to mixtures of HmTA [DNP 4 ] and HmTA [dansyl 4 ] with concentrations ranging from 0.5 nM to 1000 nM (Figure 3, Figure S2).…”

Section: Resultsmentioning

confidence: 99%

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

Handlogten

Deak

Bilgiçer

2014

Chemistry & Biology

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Summary Allergy 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 percent 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 supra-optimal IgE cross-linking decreases the degranulation response and establishes the two allergen model as a relevant experimental system to elucidate mast cell degranulation mechanisms.

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

Cited by 17 publications

References 36 publications

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

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

Design of a heterotetravalent synthetic allergen that reflects epitope heterogeneity and IgE antibody variability to study mast cell degranulation

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

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