Designing Selective Inhibitors of IgE-mediated Mast Cell Degranulation

描述(申请人提供)：过敏是由过敏原介导的肥大细胞表面免疫球蛋白E(IgE)分子的聚集引起的。在发达国家，过敏性疾病正在增加人们对健康的担忧。根据过敏反应的严重程度，结果可能从简单的瘙痒到过敏性休克，后者在美国每年导致1500人死亡。过敏没有治愈的方法，目前的治疗方法侧重于治疗急性症状或慢性局部免疫抑制。即使在最严格的饮食限制下，意外接触食物过敏的情况也很常见，这会使患者面临危及生命的过敏反应。总而言之，有必要对IgE介导的过敏反应进行更有效的替代治疗。此应用程序的目标是建立一个 详细了解表位-IgE在肥大细胞脱颗粒中的相互作用，并设计IgE介导的过敏反应的选择性抑制剂。由于自然产生的过敏原通常是具有多个过敏诱导表位的复杂、结构不同的蛋白质，因此开发模拟自然过敏反应的细胞实验模型系统一直是一个挑战。首先，我们将通过开发多组分实验模型系统来解决这个问题，该系统将通过结合表位异质性和IgE变异性来实现综合方法，以更好地反映天然过敏原的复杂性。我们将利用这个生理上更相关的模型来建立一个详细的了解表位-IgE在肥大细胞刺激及其抑制中的相互作用。下一步，我们将设计高亲和力的表位-IgE相互作用的异二价抑制剂(HBI)，以选择性地抑制IgE介导的过敏反应。HBI将被设计为同时靶向位于IgE Fab结构域上的两个附近的结合位点：抗原结合位点和不太知名的“核苷酸结合位点”(NBS)。HBI与这两个位点的同时二价结合将提供比变应原表位更强的亲和力，从而竞争性地抑制变应原-IgE相互作用和肥大细胞脱颗粒。这项建议的具体目的是：i) 开发一个反映表位异质性和IgE抗体变异性的多组分实验模型系统，以确定在生理相关条件下表位-IgE相互作用如何影响肥大细胞脱颗粒；ii)设计异二价抑制剂(HBI)，利用不为人所知的NBS通过部分表位抑制选择性抑制肥大细胞上的IgE聚集；iii)在小鼠过敏模型中验证多组分实验模型系统和HBI设计。这项拟议的工作具有创新性，因为它(I)开发了一个与生理相关的实验模型系统来研究肥大细胞的刺激及其抑制，(Ii)表征了在所有抗体上发现的不太为人所知的NBS，从而揭示了抗体结构中存在的一个独特且未被充分利用的特征；(Iii)研究了保守的NBS的用途，以及(Iv)带来了选择性抑制IgE介导的过敏反应的新的分子设计方法。

DESCRIPTION (provided by applicant): Allergies are elicited by the allergen-mediated-clustering of the immunoglobulin E (IgE) molecules on the surface of mast cells. Allergic diseases are increasing health concerns in developed nations. Depending on the severity of the allergic reaction, the results can vary from a simple itch to anaphylactic shock, which results in 1,500 deaths each year in the US. There are no cures for allergies, and current therapies focus on treatment of acute symptoms or chronic localized immune suppression. Even with the most restricted diets, accidental exposure is very frequent for food allergies, putting patients at riskfor life threatening anaphylaxis. Combined, there is a need for more effective, alternative treatments for IgE-mediated allergic responses. The objective of this application is to establish a detailed understanding of epitope-IgE interactions on mast cell degranulation, and to design selective inhibitors of IgE-mediated allergic responses. Because naturally occurring allergens are typically complex, structurally heterogeneous proteins with multiple allergy-inducing epitopes, it has been a challenge to develop cellular experimental model systems that mimic natural allergic responses. First, we will address this problem by developing a multicomponent experimental model system, which will enable an integrative approach by incorporating epitope heterogeneity and IgE variability to better reflect the complexity of natural allergens. We will us this physiologically more relevant model to establish a detailed understanding of epitope-IgE interactions in mast cell stimulation and its inhibition. Next, we will engineer high avidity heterobivalent inhibitors (HBI) of epitope-IgE interactions for selective inhibition of IgE-mediate allergic reactions. The HBI will be designed to simultaneously target two nearby binding sites located on the Fab domain of an IgE: the antigen binding site; and the not-so-well-known "nucleotide binding site" (NBS). The simultaneous bivalent binding of the HBI to both sites will provide enhanced avidity over that of the allergen epitope, thereby competitively inhibiting allergen-IgE interaction and mast cell degranulation. The specific aims of this proposal are to: i) develop a multicomponent experimental model system that reflects epitope heterogeneity and IgE antibody variability to establish how epitope-IgE interactions affect mast cell degranulation under physiologically relevant conditions; ii) to engineer heterobivalent inhibitors (HBI) that utilize the not-so-well-known NBS for selective inhibition of IgE clustering on mast cells via partial epitope inhibition; iii) to validate of the multicomponent experimental model system and HBI design in mouse allergy models. The proposed work is innovative because it (i) develops a physiologically relevant experimental model system to study mast cell stimulation and its inhibition, (ii) characterizes the not-so-well-known NBS found on all antibodies, and thereby reveals a unique and underutilized feature present in the antibody structure; (iii) investigates a utility for the conserved NBS, and (iv) brings a novel molecular design approach for selective inhibition of IgE-mediated allergic responses.