title: 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.
  
  
  OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.affiliation: Department of Experimental Immunology, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Laurian
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authors.lastname: Zuidmeer-Jongejan
authors.forename: Hans
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authors.lastname: Huber
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authors.lastname: Swoboda
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authors.lastname: Lewandowska-Polak
authors.forename: Birgit
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authors.lastname: Linhart
authors.forename: Bernhard
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authors.lastname: Maderegger
authors.forename: Bernard
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authors.lastname: Maillere
authors.forename: Adriano
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authors.lastname: Mari
authors.forename: Alberto
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authors.lastname: Martinez
authors.forename: E N Clare
authors.initials: EN
authors.lastname: Mills
authors.forename: Angela
authors.initials: A
authors.lastname: Neubauer
authors.forename: Claudio
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authors.lastname: Nicoletti
authors.forename: Nikolaos G
authors.initials: NG
authors.lastname: Papadopoulos
authors.forename: Antonio
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authors.lastname: Portoles
authors.forename: Ville
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authors.lastname: Ranta-Panula
authors.forename: Sara
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authors.lastname: Santos-Magadan
authors.forename: Heidi J
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authors.lastname: Schnoor
authors.forename: Sigurveig T
authors.initials: ST
authors.lastname: Sigurdardottir
authors.forename: Per
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authors.lastname: Stahl-Skov
authors.forename: George
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authors.lastname: Stavroulakis
authors.forename: Georg
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authors.lastname: Stegfellner
authors.forename: Sonia
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authors.lastname: Vázquez-Cortés
authors.forename: Marianne
authors.initials: M
authors.lastname: Witten
authors.forename: Frank
authors.initials: F
authors.lastname: Stolz
authors.forename: Lars K
authors.initials: LK
authors.lastname: Poulsen
authors.forename: Montserrat
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authors.lastname: Fernandez-Rivas
authors.forename: Rudolf
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authors.lastname: Valenta
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authors.lastname: van Ree
mesh_terms: D000485:Allergens / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000818:Animals; D002135:Calcium-Binding Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D002347:Carps / Q000276:immunology; D003888:Desensitization, Immunologic / Q000379:methods*; D004311:Double-Blind Method; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D029941:Fish Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D005512:Food Hypersensitivity / Q000276:immunology / Q000503:physiopathology / Q000517:prevention & control*; D015870:Gene Expression; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood; D007074:Immunoglobulin G / Q000097:blood; D007279:Injections, Subcutaneous; D007928:Lethal Dose 50; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000473:pathology; D008297:Male; D051379:Mice; D010320:Parvalbumins / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D017510:Protein Folding; D055550:Protein Stability; D011817:Rabbits; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D000485:Allergens; D002135:Calcium-Binding Proteins; C454159:Cyp c 1.01 protein, carp; D029941:Fish Proteins; D007074:Immunoglobulin G; D010320:Parvalbumins; D011994:Recombinant Proteins; D007073:Immunoglobulin E