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

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<p><b><i>Background:</i></b> 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. <b><i>Objectives: </i></b>Preclinical characterization and good manufacturing practice (GMP) production of mutant Cyp (mCyp) c 1. <b><i>Methods:</i></b><i>Escherichia coli</i>-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. <b><i>Results:</i></b> 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)<sub>3</sub>-adsorbed drug product demonstrated at least 20 months of stability. <b><i>Conclusion:</i></b>
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.</p>
Date made available12 Jan 2017
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