Development of a hypoallergenic recombinant parvalbumin for first-in-man subcutaneous immunotherapy of fish allergy.

Abstract

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.

• Authors: Laurian Zuidmeer-Jongejan, Hans Huber, Ines Swoboda, Neil Rigby, Serge A Versteeg, Bettina M Jensen, Suzanne Quaak, Jaap H Akkerdaas, Lars Blom, Juan Asturias, Carsten Bindslev-Jensen, Maria L Bernardi, Michael Clausen, Rosa Ferrara, Martina Hauer, Jet Heyse, Stephan Kopp, Marek L Kowalski, Anna Lewandowska-Polak, Birgit Linhart, Bernhard Maderegger, Bernard Maillere, Adriano Mari, Alberto Martinez, E N Clare Mills, Angela Neubauer, Claudio Nicoletti, Nikolaos G Papadopoulos, Antonio Portoles, Ville Ranta-Panula, Sara Santos-Magadan, Heidi J Schnoor, Sigurveig T Sigurdardottir, Per Stahl-Skov, George Stavroulakis, Georg Stegfellner, Sonia Vázquez-Cortés, Marianne Witten, Frank Stolz, Lars K Poulsen, Montserrat Fernandez-Rivas, Rudolf Valenta, Ronald van Ree
• Journal: International archives of allergy and immunology (Int Arch Allergy Immunol) Vol. 166 Issue 1 Pg. 41-51 ( 2015) ISSN: 1423-0097 [Electronic] Switzerland
• PMID: 25765512 (Publication Type: Journal Article, Multicenter Study, Randomized Controlled Trial)
• Copyright: © 2015 S. Karger AG, Basel.

Chemical References

• Allergens
• Calcium-Binding Proteins
• Cyp c 1.01 protein, carp
• Fish Proteins
• Immunoglobulin G
• Parvalbumins
• Recombinant Proteins
• Immunoglobulin E

Topics

• Allergens (administration & dosage, chemistry, genetics, immunology)
• Animals
• Calcium-Binding Proteins (administration & dosage, chemistry, genetics, immunology)
• Carps (immunology)
• Desensitization, Immunologic (methods)
• Double-Blind Method
• Escherichia coli (genetics, metabolism)
• Female
• Fish Proteins (administration & dosage, chemistry, genetics, immunology)
• Food Hypersensitivity (immunology, physiopathology, prevention & control)
• Gene Expression
• Humans
• Immunoglobulin E (blood)
• Immunoglobulin G (blood)
• Injections, Subcutaneous
• Lethal Dose 50
• Leukocytes, Mononuclear (immunology, pathology)
• Male
• Mice
• Parvalbumins (administration & dosage, chemistry, genetics, immunology)
• Protein Folding
• Protein Stability
• Rabbits
• Recombinant Proteins (administration & dosage, chemistry, genetics, immunology)