Comprehensive antigenic mapping of the human anti-peanut IgE antibody response

项目摘要 过敏性疾病发病机制的中心是IgE分子。在敏感个体中， 暴露于有害的过敏原导致IgE接合，引起Fcε受体交联， 肥大细胞和嗜碱性粒细胞的活化。这会触发介质释放到局部组织中，导致 与过敏性疾病相关的大量症状，包括过敏性休克。迄今为止， 人类IgE分子的活性及其过敏原的靶向表位非常有限。我们的大多数 该过程的知识来自于使用过敏患者血清的研究，所述血清含有以下物质的混合物： 许多抗体，具有许多特异性，针对许多不同的表位，并具有许多 不同的亲和力;因此，IgE与靶变应原的分子相互作用的研究大大 有缺陷研究这一过程的理想方法是使用天然存在的人IgE单克隆抗体 （mAb），分离自过敏性受试者。不幸的是，由于许多不可逾越的内在技术障碍， 这种抗体以前曾被制造过。我们现在已经建立了一种方法来种植，识别和 通过从过敏性疾病患者的外周血制备人杂交瘤使编码IgE的B细胞永生化， 个体在这项提案中，我们开发了第一个全面的天然花生面板， 过敏原特异性人IgE单克隆抗体，以确定最有效的诱导剂的分子相互作用 过敏反应我们已经开始了全面的绘图研究，以确定关键的免疫显性 主要花生过敏原蛋白Ara h 1、2、3和6上的抗原位点。在目标1中，功能性抗原位点 通过花生诱导的过敏反应的映射将通过人FcεRI的被动致敏来完成 使用IgE mAb的转基因小鼠。在目标2中，将选择结合独特抗原位点的原型mAb 并表达为重组IgG mAb，用作使用人抗体进行高级作图研究的工具。 血清的IgG mAb与制备它们的IgE mAb相同地结合，将用于 使用一组花生过敏研究受试者的冷冻血清和ImmunoCAP进行阻断研究 诊断这将定义IgE抗体的每个抗原位点特异性群体在细胞内发挥的作用。 以及花生过敏个体之间的关系。这些信息将用于绘制疾病的临床相关性 并选择具有未被我们的面板阻断的IgE抗体的研究受试者， 在Aim 1中产生新的位点特异性IgE mAb。最后，在目标3中，我们将获得原子分辨率 结构，以精确地定义有史以来第一个天然存在的人IgE表位的Ara h 2和6的X- 射线晶体学这项工作的目标是建立一个明确的，完整的和全面的分子 人IgE抗体对花生主要过敏原蛋白的反应图谱。结果将作为 一个急需的路线图，以允许设计新的免疫疗法和过敏疫苗。

PROJECT SUMMARY At the center of the pathogenesis of allergic diseases is the IgE molecule. In sensitized individuals, re- exposure to the offending allergen results in IgE engagement, causing Fcε receptor cross-linking and activation of mast cells and basophils. This triggers the release of mediators into the local tissue, resulting in the vast array of symptoms associated with allergic diseases, including anaphylactic shock. To date, studies of the human IgE molecule, and its targeted epitopes on allergens, have been very limited. Most of our knowledge of this process has come from studies using allergic patient serum, which contains a mixture of many antibodies, with many specificities, directed toward many different epitopes, and having many different affinities; thus the studies of the molecular interactions of IgE with target allergens are greatly flawed. The ideal way to study this process is to use naturally-occurring human IgE monoclonal antibodies (mAbs), isolated from allergic subjects. Unfortunately, due to many impassible intrinsic technical hurdles no such antibodies have previously ever been made. We have now established a method to grow, identify and immortalize IgE encoding B cells by making human hybridomas from the peripheral blood of allergic individuals. In this proposal, we develop the first comprehensive panels of naturally-occurring peanut allergen-specific human IgE mAbs to define the molecular interactions of the most potent inducers of anaphylaxis. We have already begun comprehensive mapping studies to identify key immunodominant antigenic sites on the major peanut allergen proteins Ara h 1, 2, 3, and 6. In Aim 1, functional antigenic site mapping via peanut induced anaphylaxis will be accomplished by passive sensitization of human FcεRI transgenic mice using IgE mAbs. In Aim 2, prototype mAbs that bind unique antigenic sites will be selected and expressed as recombinant IgG mAbs to use as tools for advanced mapping studies using human serum. IgG mAbs, which bind identically as the IgE mAbs from which they were made, will be employed in blocking studies using a panel of peanut allergic research subjects’ frozen serum and ImmunoCAP diagnostics. This will define the role that each antigenic site-specific population of IgE antibodies play within and between peanut allergic individuals. This information will be used to draw clinical correlates of disease and to select research subjects which possess IgE antibodies not blocked by our panels, allowing for the generation of new site-specific IgE mAbs in Aim 1. Finally, in Aim 3, we will obtain atomic resolution structures to precisely define the first ever naturally-occurring human IgE epitopes on Ara h 2 and 6 by X- ray crystallography. The goal of this work is to create a definitive, complete and comprehensive molecular map of the human IgE antibody response to the major allergen proteins of peanut. The results will serve as a much-needed road map to allow for the design of new immunotherapies and allergy vaccines.