Hypoallergenic mutants of the Timothy grass pollen allergen Phl p 5 generated by proline mutations.
BACKGROUND
Phl p 5 is a major allergen of Timothy grass (Phleum pratense). A recombinant native Phl p 5 has already been used in clinical trials of allergen-specific immunotherapy as a component of a cocktail of allergens. Recombinant hypoallergenic allergens should further improve the treatment by reducing the risk of anaphylactic reactions at an increased therapeutic dosage. Native Phl p 5 is formed by α-helical regions separated by regions containing prolines. In order to generate hypoallergenic mutants, we studied the effect of proline mutations in single and multiple regions.


METHODS
All mutants were analyzed by IgE inhibition assays and size exclusion chromatography with on-line mass determination. Selected mutants were additionally analyzed by field-flow fractionation, dynamic light scattering, circular dichroism spectroscopy, basophil activation and T-cell proliferation assays.


RESULTS
Variants lacking prolines in a single region were obtained as soluble monomers. Six of eight molecules showed a slightly reduced IgE-binding capacity. Mutants carrying proline deletions in multiple regions formed monomers, dimers or insoluble aggregates. The mutant MPV.7 with five proline deletions and a substitution of proline 211 to leucine is monomeric, shows a strongly diminished IgE binding and maintains T-cell reactivity. The hydrodynamic radius and the content of the α-helical structure of MPV.7 are well comparable with the wild-type allergen.


CONCLUSIONS
The hypoallergenic Phl p 5 variant MPV.7 combines multiple proline deletions with a substitution of proline 211 to leucine and meets basic demands for a pharmaceutical application. MPV.7 is a promising candidate for grass pollen immunotherapy with a cocktail of recombinant hypoallergens.
Research and Development Division, Allergopharma Joachim Ganzer KG, Reinbek, Germany. martin.wald@allergopharma.de
Martin
M
Wald
Helga
H
Kahlert
Gerald
G
Reese
Nina
N
Krontal
Domen
D
Zafred
Walter
W
Keller
Oliver
O
Cromwell
Helmut
H
Fiebig
Andreas
A
Nandy
D000328:Adult; D000368:Aged; D000485:Allergens / Q000235:genetics* / Q000276:immunology*; D019943:Amino Acid Substitution; D001491:Basophils / Q000276:immunology; D003888:Desensitization, Immunologic / Q000379:methods; D005260:Female; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D008875:Middle Aged; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D031784:Phleum / Q000235:genetics / Q000276:immunology; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D011058:Pollen / Q000235:genetics* / Q000276:immunology*; D011392:Proline / Q000235:genetics; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D006255:Rhinitis, Allergic, Seasonal / Q000276:immunology / Q000628:therapy; D017384:Sequence Deletion; D012995:Solubility; D013601:T-Lymphocytes / Q000276:immunology
D000485:Allergens; D050505:Mutant Proteins; C076049:Phl p V protein, Phleum pratense; D010940:Plant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D011392:Proline