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Int Arch Allergy Immunol ; 166(1): 41-51, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25765512

RESUMO

BACKGROUND: The FAST (food allergy-specific immunotherapy) project aims at developing safe and effective subcutaneous immunotherapy for fish allergy, using recombinant hypoallergenic carp parvalbumin, Cyp c 1. OBJECTIVES: Preclinical characterization and good manufacturing practice (GMP) production of mutant Cyp (mCyp) c 1. METHODS: Escherichia coli-produced mCyp c 1 was purified using standard chromatographic techniques. Physicochemical properties were investigated by gel electrophoresis, size exclusion chromatography, circular dichroism spectroscopy, reverse-phase high-performance liquid chromatography and mass spectrometry. Allergenicity was assessed by ImmunoCAP inhibition and basophil histamine release assay, immunogenicity by immunization of laboratory animals and stimulation of patients' peripheral blood mononuclear cells (PBMCs). Reference molecules were purified wild-type Cyp c 1 (natural and/or recombinant). GMP-compliant alum-adsorbed mCyp c 1 was tested for acute toxicity in mice and rabbits and for repeated-dose toxicity in mice. Accelerated and real-time protocols were used to evaluate stability of mCyp c 1 as drug substance and drug product. RESULTS: Purified mCyp c 1 behaves as a folded and stable molecule. Using sera of 26 double-blind placebo-controlled food-challenge-proven fish-allergic patients, reduction in allergenic activity ranged from 10- to 5,000-fold (1,000-fold on average), but with retained immunogenicity (immunization in mice/rabbits) and potency to stimulate human PBMCs. Toxicity studies revealed no toxic effects and real-time stability studies on the Al(OH)3-adsorbed drug product demonstrated at least 20 months of stability. CONCLUSION: The GMP drug product developed for treatment of fish allergy has the characteristics targeted for in FAST: i.e. hypoallergenicity with retained immunogenicity. These results have warranted first-in-man immunotherapy studies to evaluate the safety of this innovative vaccine.


Assuntos
Alérgenos/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Dessensibilização Imunológica/métodos , Proteínas de Peixes/imunologia , Hipersensibilidade Alimentar/prevenção & controle , Parvalbuminas/imunologia , Alérgenos/administração & dosagem , Alérgenos/química , Alérgenos/genética , Animais , Proteínas de Ligação ao Cálcio/administração & dosagem , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Carpas/imunologia , Método Duplo-Cego , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Proteínas de Peixes/administração & dosagem , Proteínas de Peixes/química , Proteínas de Peixes/genética , Hipersensibilidade Alimentar/imunologia , Hipersensibilidade Alimentar/fisiopatologia , Expressão Gênica , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Injeções Subcutâneas , Dose Letal Mediana , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Masculino , Camundongos , Parvalbuminas/administração & dosagem , Parvalbuminas/química , Parvalbuminas/genética , Dobramento de Proteína , Estabilidade Proteica , Coelhos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
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