Designing Selective Inhibitors of IgE-mediated Mast Cell Degranulation

描述（由申请方提供）：过敏是由过敏原介导的免疫球蛋白E（IgE）分子在肥大细胞表面聚集引起的。过敏性疾病在发达国家日益引起人们的健康关注。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不等，在美国每年导致1,500人死亡。过敏症没有治愈方法，目前的治疗方法主要集中在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，意外接触食物过敏也非常频繁，使患者面临危及生命的过敏反应的风险。结合起来，需要更有效的替代治疗IgE介导的过敏反应。本申请的目的是建立一个 详细了解表位-IgE相互作用对肥大细胞脱粒的影响，并设计IgE介导的过敏反应的选择性抑制剂。由于天然存在的过敏原通常是具有多个过敏诱导表位的复杂的、结构异质的蛋白质，因此开发模拟天然过敏反应的细胞实验模型系统一直是一个挑战。首先，我们将通过开发一个多组分实验模型系统来解决这个问题，该系统将通过将表位异质性和IgE变异性结合在一起来实现综合方法，以更好地反映天然过敏原的复杂性。我们将使用这个生理上更相关的模型，以建立一个详细的了解表位IgE相互作用在肥大细胞的刺激和抑制。接下来，我们将设计高亲合力异二价抑制剂（HBI）的表位IgE相互作用的IgE介导的过敏反应的选择性抑制。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;和不太为人所知的“核苷酸结合位点”（NBS）。HBI与两个位点的同时二价结合将提供比过敏原表位更强的亲合力，从而竞争性地抑制过敏原-IgE相互作用和肥大细胞脱粒。这项建议的具体目标是： 开发反映表位异质性和IgE抗体可变性的多组分实验模型系统，以确定表位-IgE相互作用如何在生理学相关条件下影响肥大细胞脱粒; ii）工程化异二价抑制剂（HBI），其利用不那么熟知的NBS通过部分表位抑制选择性抑制肥大细胞上的IgE簇集; iii）在小鼠变态反应模型中验证多组分实验模型系统和HBI设计。所提出的工作是创新性的，因为它（i）开发了生理学相关的实验模型系统来研究肥大细胞刺激及其抑制，（ii）表征了在所有抗体上发现的不那么知名的NBS，从而揭示了抗体结构中存在的独特且未充分利用的特征;（iii）研究了保守的NBS的实用性，以及（iv）提出了选择性抑制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.