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Development of a Hypoallergenic Recombinant Parvalbumin for First-in-Man Subcutaneous Immunotherapy of Fish Allergy

2015; Karger Publishers; Volume: 166; Issue: 1 Linguagem: Inglês

10.1159/000371657

1423-0097

Laurian Zuidmeer, H. Huber, Ines Swoboda, Neil M. Rigby, Serge A. Versteeg, Bettina M. Jensen, Suzanne Quaak, Jaap H. Akkerdaas, Lars Blom, Juan A. Asturias, Carsten Bindslev‐Jensen, Maria Livia Bernardi, Michael Clausen, Rosa Ferrara, Martina Hauer, Jet Heyse, Stephan Kopp, Marek L. Kowalski, Anna Lewandowska‐Polak, Birgit Linhart, Bernhard Maderegger, Bernard Maillère, Adriano Mari, Alberto Martı́nez, E. N. Clare Mills, Angela Neubauer, Claudio Nicoletti, Nikolaos G. Papadopoulos, Antonio Portolés, Ville Ranta‐Panula, S. Santos‐Magadán, Heidi Julius Schnoor, Sigurveig Sigurdardottir, Per Stahl Skov, George Stavroulakis, Georg Stegfellner, Sonia Vázquez‐Cortés, Marianne Witten, Frank Stolz, Lars K. Poulsen, Montserrat Fernández‐Rivas, Rudolf Valenta, Ronald van Ree,

Asthma and respiratory diseases

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.