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

Laurian Zuidmeer-Jongejan; Hans Huber; Ines Swoboda; Neil Rigby; Serge A Versteeg; Bettina Margrethe Jensen; Suzanne Quaak; Jaap H Akkerdaas; L. 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; H J Schnoor; Sigurveig T Sigurdardottir; P Stahl Skov; George Stavroulakis; Georg Stegfellner; Sonia Vázquez-Cortés; M Witten; Frank Stolz; Lars K. Poulsen; Montserrat Fernandez-Rivas; Rudolf Valenta; Ronald van Ree

doi:10.1159/000371657

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

Laurian Zuidmeer-Jongejan, Hans Huber, Ines Swoboda, Neil Rigby, Serge A Versteeg, Bettina Margrethe Jensen, Suzanne Quaak, Jaap H Akkerdaas, L. 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 LinhartBernhard 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, H J Schnoor, Sigurveig T Sigurdardottir, P Stahl Skov, George Stavroulakis, Georg Stegfellner, Sonia Vázquez-Cortés, M Witten, Frank Stolz, Lars K. Poulsen, Montserrat Fernandez-Rivas, Rudolf Valenta, Ronald van Ree

Department of Clinical Medicine

65 Citations (Scopus)

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.

Original language	English
Journal	International Archives of Allergy and Immunology
Volume	166
Issue number	1
Pages (from-to)	41-51
Number of pages	11
ISSN	1018-2438
DOIs	https://doi.org/10.1159/000371657
Publication status	Published - 6 Apr 2015

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10.1159/000371657

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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., ... van Ree, R. (2015). Development of a Hypoallergenic Recombinant Parvalbumin for First-in-Man Subcutaneous Immunotherapy of Fish Allergy. International Archives of Allergy and Immunology, 166(1), 41-51. https://doi.org/10.1159/000371657

Zuidmeer-Jongejan, L, Huber, H, Swoboda, I, Rigby, N, Versteeg, SA, Jensen, BM, Quaak, S, Akkerdaas, JH, Blom, L, Asturias, J, Bindslev-Jensen, C, Bernardi, ML, Clausen, M, Ferrara, R, Hauer, M, Heyse, J, Kopp, S, Kowalski, ML, Lewandowska-Polak, A, Linhart, B, Maderegger, B, Maillere, B, Mari, A, Martinez, A, Mills, ENC, Neubauer, A, Nicoletti, C, Papadopoulos, NG, Portoles, A, Ranta-Panula, V, Santos-Magadan, S, Schnoor, HJ, Sigurdardottir, ST, Stahl Skov, P, Stavroulakis, G, Stegfellner, G, Vázquez-Cortés, S, Witten, M, Stolz, F, Poulsen, LK, Fernandez-Rivas, M, Valenta, R & van Ree, R 2015, 'Development of a Hypoallergenic Recombinant Parvalbumin for First-in-Man Subcutaneous Immunotherapy of Fish Allergy', International Archives of Allergy and Immunology, vol. 166, no. 1, pp. 41-51. https://doi.org/10.1159/000371657

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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. 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.",

author = "Laurian Zuidmeer-Jongejan and Hans Huber and Ines Swoboda and Neil Rigby and Versteeg, {Serge A} and Jensen, {Bettina Margrethe} and Suzanne Quaak and Akkerdaas, {Jaap H} and L. Blom and Juan Asturias and Carsten Bindslev-Jensen and Bernardi, {Maria L} and Michael Clausen and Rosa Ferrara and Martina Hauer and Jet Heyse and Stephan Kopp and Kowalski, {Marek L} and Anna Lewandowska-Polak and Birgit Linhart and Bernhard Maderegger and Bernard Maillere and Adriano Mari and Alberto Martinez and Mills, {E N Clare} and Angela Neubauer and Claudio Nicoletti and Papadopoulos, {Nikolaos G} and Antonio Portoles and Ville Ranta-Panula and Sara Santos-Magadan and Schnoor, {H J} and Sigurdardottir, {Sigurveig T} and {Stahl Skov}, P and George Stavroulakis and Georg Stegfellner and Sonia V{\'a}zquez-Cort{\'e}s and M Witten and Frank Stolz and Poulsen, {Lars K.} and Montserrat Fernandez-Rivas and Rudolf Valenta and {van Ree}, Ronald",

year = "2015",

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doi = "10.1159/000371657",

language = "English",

volume = "166",

pages = "41--51",

journal = "International Archives of Allergy and Immunology",

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T1 - Development of a Hypoallergenic Recombinant Parvalbumin for First-in-Man Subcutaneous Immunotherapy of Fish Allergy

AU - Zuidmeer-Jongejan, Laurian

AU - Huber, Hans

AU - Swoboda, Ines

AU - Rigby, Neil

AU - Versteeg, Serge A

AU - Jensen, Bettina Margrethe

AU - Quaak, Suzanne

AU - Akkerdaas, Jaap H

AU - Blom, L.

AU - Asturias, Juan

AU - Bindslev-Jensen, Carsten

AU - Bernardi, Maria L

AU - Clausen, Michael

AU - Ferrara, Rosa

AU - Hauer, Martina

AU - Heyse, Jet

AU - Kopp, Stephan

AU - Kowalski, Marek L

AU - Lewandowska-Polak, Anna

AU - Linhart, Birgit

AU - Maderegger, Bernhard

AU - Maillere, Bernard

AU - Mari, Adriano

AU - Martinez, Alberto

AU - Mills, E N Clare

AU - Neubauer, Angela

AU - Nicoletti, Claudio

AU - Papadopoulos, Nikolaos G

AU - Portoles, Antonio

AU - Ranta-Panula, Ville

AU - Santos-Magadan, Sara

AU - Schnoor, H J

AU - Sigurdardottir, Sigurveig T

AU - Stahl Skov, P

AU - Stavroulakis, George

AU - Stegfellner, Georg

AU - Vázquez-Cortés, Sonia

AU - Witten, M

AU - Stolz, Frank

AU - Poulsen, Lars K.

AU - Fernandez-Rivas, Montserrat

AU - Valenta, Rudolf

AU - van Ree, Ronald

PY - 2015/4/6

Y1 - 2015/4/6

N2 - 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.

AB - 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.

U2 - 10.1159/000371657

DO - 10.1159/000371657

M3 - Journal article

C2 - 25765512

SN - 1018-2438

VL - 166

SP - 41

EP - 51

JO - International Archives of Allergy and Immunology

JF - International Archives of Allergy and Immunology

IS - 1

ER -

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

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