Design of an Ara h 2 hypoallergen from conformational epitopes.

INTRODUCTION: Adverse reactions are relatively common during peanut oral immunotherapy. To reduce the risk to the patient, some researchers have proposed modifying the allergen to reduce IgE reactivity, creating a putative hypoallergen. Analysis of recently cloned human IgG from patients treated with peanut immunotherapy suggested that there are three common conformational epitopes for the major peanut allergen Ara h 2. We sought to test if structural information on these epitopes could indicate mutagenesis targets for designing a hypoallergen and evaluated the reduction in IgE binding via immunochemistry and a mouse model of passive cutaneous anaphylaxis (PCA). METHODS: X-ray crystallography characterized the conformational epitopes in detail, followed by mutational analysis of key residues to modify monoclonal antibody (mAb) and serum IgE binding, assessed by ELISA and biolayer interferometry. A designed Ara h 2 hypoallergen was tested for reduced vascularization in mouse PCA experiments using pooled peanut allergic patient serum. RESULTS: A ternary crystal structure of Ara h 2 in complex with patient antibodies 13T1 and 13T5 was determined. Site-specific mutants were designed that reduced 13T1, 13T5, and 22S1 mAbs binding by orders of magnitude. By combining designed mutations from the three major conformational bins, a hexamutant (Ara h 2 E46R, E89R, E97R, E114R, Q146A, R147E) was created that reduced IgE binding in serum from allergic patients. Further, in the PCA model where mice were primed with peanut allergic patient serum, reactivity upon allergen challenge was significantly decreased using the hexamutant. CONCLUSION: These studies demonstrate that prior knowledge of common conformational epitopes can be used to engineer reduced IgE reactivity, an important first step in hypoallergen design.

Characterization of spironolactone and metabolites derivatized using Girard's reagent P using mass spectrometry and ion mobility spectrometry.

RATIONALE: Spironolactone is a steroidal drug prescribed for a variety of medical conditions and is extensively metabolized quickly after administration. Measurement of spironolactone and its metabolites remains challenging using mass spectrometry (MS) due to in-source fragmentation and relatively poor ionization using electrospray ionization. Therefore, improved methods of measurements are needed, particularly in the case of small sample volumes. METHODS: Girard's reagent P (GP) derivatization of spironolactone was employed to improve response and provide an MS-based solution to the measurement of spironolactone and its metabolites. We performed ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) and ion mobility spectrometry (IMS)-high-resolution mass spectrometry (HRMS) to fully characterize the GP derivatization products. Analytes were studied in positive ionization mode, and MS/MS was performed using nonresonance and resonance excitation collision-induced dissociation. RESULTS: We observed the successful GP derivatization of spironolactone and its metabolites using authentic chemical standards. A signal enhancement of 1-2 orders of magnitude was observed for GP-derivatized versions of spironolactone and its metabolites. Further, GP derivatization eliminated in-source fragmentation. Finally, we performed GP derivatization and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) in a small volume of murine serum (20 µL) from spironolactone-treated and control animals and observed multiple spironolactone metabolites only in the spironolactone-treated group. CONCLUSIONS: GP derivatization was proven to have advantageous mass spectral performance (e.g., limiting in-source fragmentation, enhancing signals, and eliminating isobaric analytes) for spironolactone and its metabolites. This work and the detailed characterization using ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) and IMS serve as the foundation for future developments in reaction optimization and/or quantitative assay development.