Περίληψη:
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. © 2015 S. Karger AG, Basel.
Συγγραφείς:
Zuidmeer-Jongejan, L.
Huber, H.
Swoboda, I.
Rigby, N.
Versteeg, S.A.
Jensen, B.M.
Quaak, S.
Akkerdaas, J.H.
Blom, L.
Asturias, J.
Bindslev-Jensen, C.
Bernardi, M.L.
Clausen, M.
Ferrara, R.
Hauer, M.
Heyse, J.
Kopp, S.
Kowalski, M.L.
Lewandowska-Polak, A.
Linhart, B.
Maderegger, B.
Maillere, B.
Mari, A.
Martinez, A.
Mills, E.N.C.
Neubauer, A.
Nicoletti, C.
Papadopoulos, N.G.
Portoles, A.
Ranta-Panula, V.
Santos-Magadan, S.
Schnoor, H.J.
Sigurdardottir, S.T.
Stahl-Skov, P.
Stavroulakis, G.
Stegfellner, G.
Vázquez-Cortés, S.
Witten, M.
Stolz, F.
Poulsen, L.K.
Fernandez-Rivas, M.
Valenta, R.
Van Ree, R.
Λέξεις-κλειδιά:
aluminum hydroxide; guanosine phosphate; immunoglobulin G antibody; parvalbumin; placebo; allergen; calcium binding protein; Cyp c 1.01 protein, carp; fish protein; immunoglobulin E; immunoglobulin G; parvalbumin; recombinant protein, acute toxicity; adjuvant therapy; allergenicity; allergy rapid test; basophil histamine release assay; carp; circular dichroism; controlled study; Escherichia coli; experimental rabbit; female; fish allergy; fish allergy; food allergy; gel electrophoresis; gel permeation chromatography; good manufacturing practice; histamine release; human; human cell; immune response; immunization; immunogenicity; male; mass spectrometry; mouse; nonhuman; patient; peripheral blood mononuclear cell; physical chemistry; polyacrylamide gel electrophoresis; priority journal; release assay; repeated drug dose; reversed phase high performance liquid chromatography; Review; subcutaneous immunotherapy; animal; blood; chemistry; clinical trial; desensitization; double blind procedure; Food Hypersensitivity; gene expression; genetics; immunology; LD50; metabolism; mononuclear cell; multicenter study; pathology; pathophysiology; procedures; protein folding; protein stability; rabbit; randomized controlled trial; subcutaneous drug administration, Allergens; Animals; Calcium-Binding Proteins; Carps; Desensitization, Immunologic; Double-Blind Method; Escherichia coli; Female; Fish Proteins; Food Hypersensitivity; Gene Expression; Humans; Immunoglobulin E; Immunoglobulin G; Injections, Subcutaneous; Lethal Dose 50; Leukocytes, Mononuclear; Male; Mice; Parvalbumins; Protein Folding; Protein Stability; Rabbits; Recombinant Proteins
