Research and Development Division, Allergopharma Joachim Ganzer KG, Reinbek, Germany.
Int Arch Allergy Immunol. 2012;159(2):130-42. doi: 10.1159/000336651. Epub 2012 May 30.
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.
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.
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.
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.
Phl p 5 是梯牧草（Phleum pratense）的主要过敏原。重组天然 Phl p 5 已在过敏原特异性免疫治疗的临床试验中作为过敏原混合物的成分使用。重组低变应原过敏原应通过降低在增加的治疗剂量下发生过敏反应的风险来进一步改善治疗。天然 Phl p 5 由 α-螺旋区域与含有脯氨酸的区域隔开形成。为了产生低变应原突变体，我们研究了单个和多个区域脯氨酸突变的影响。
所有突变体均通过 IgE 抑制测定法和在线质量测定的排阻色谱法进行分析。选择的突变体还通过场流分级分离、动态光散射、圆二色性光谱法、嗜碱性粒细胞激活和 T 细胞增殖测定法进行分析。
在单个区域中缺乏脯氨酸的变体作为可溶性单体获得。八个分子中的六个显示出略微降低的 IgE 结合能力。在多个区域携带脯氨酸缺失的突变体形成单体、二聚体或不溶性聚集体。具有五个脯氨酸缺失和脯氨酸 211 取代为亮氨酸的突变体 MPV.7 是单体，表现出强烈降低的 IgE 结合并且保持 T 细胞反应性。MPV.7 的流体力学半径和α-螺旋结构含量与野生型过敏原非常可比。
低变应原 Phl p 5 变体 MPV.7 将多个脯氨酸缺失与脯氨酸 211 取代为亮氨酸结合在一起，并满足药物应用的基本要求。MPV.7 是使用重组低变应原混合物进行草花粉免疫治疗的有前途的候选物。
