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

Abstract

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.

A Heterobivalent Ligand Inhibits Mast Cell Degranulation via Selective Inhibition of Allergen–IgE Interactions In Vivo

“…In our previous studies we have used ethylene glycol extensively as a linker in multivalent targeting due to its numerous favorable properties including hydrophilicity to improve solubility, and flexibility to allow multivalent binding without steric constraints (15–20). Moreover, ethylene glycol does not form non-specific interactions with proteins.…”

Nanoallergens: A multivalent platform for studying and evaluating potency of allergen epitopes in cellular degranulation

“…21 Briefly, BSA at 10 mg mL −1 in 1 mL of bicarbonate buffer (0.1 M, pH 9.0) and 100 μl of 10 mg/mL of dansyl chloride DMF were combined and incubated at room temperature for 2 hours. The conjugated BSA was purified using a 0.5 ml 10 kDa molecular mass cut-off spin concentrator (Millipore).…”

“…21 The synthetic scheme is described in Figure S-1. Briefly, protected molecules with terminal acid groups were activated with HBTU and a four-fold molar excess of DIEA for 5 minutes and then conjugated to the resin over 30 minutes.…”

“…Importantly, this system also does not reflect the epitope heterogeneity or the polyclonal nature of clinical IgE’s hence is not an appropriate model to simulate and study a natural response. 21,24 Likewise, hapten-BSA conjugates have a limited valency (approximately 20, given the number of lysines for binding) which restricts their ability to stimulate degranulation with low affinity peptide mimetics. Given the limitations of the BSA system, a model system for accurate and reliable allergen epitope presentation is urgently needed for successful adaption of the in vitro allergy research towards clinically relevant allergen proteins.…”

“…17,21,25–27 This design allowed control over the avidity between the allergen molecule to receptor bound IgE’s. This system has been exceptionally valuable in studies of IgE-FcεRI clustering and enabled us to demonstrate the significance of weak affinity epitopes in triggering cellular degranulation.…”