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

人类抗花生 IgE 抗体反应的综合抗原图谱

基本信息

批准号：
10310446
负责人：
Scott Alan Smith
金额：
$ 89.64万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-01 至 2025-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10310446
关键词：
Adaptive Immune System Affinity Albumins Allergens Allergic Allergic Disease Allergy to peanuts Anaphylaxis Antibodies Antibody Response Antigenic Specificity B-Lymphocytes Basophils Binding Biological Assay Blood Circulation Cells Cellular Assay Clinical Clinical Data Clone Cells Complex Developed Countries Developing Countries Development Diagnostic Disease Dose Enzyme-Linked Immunosorbent Assay Epidemic Epitopes Exposure to Food Freezing Generations Goals Helminths Human Hybridomas Hypersensitivity IgE Immunoglobulin G Immunotherapy In Vitro Incidence Individual Knowledge Location Maps Measures Mediating Mediator of activation protein Methods Molecular Monoclonal Antibodies Pathogenesis Pathologic Patients Persons Play Population Process Proteins Recombinants Research Subjects Resolution Role Serum Severities Severity of illness Site Specificity Structure Therapeutic Time Tissues Transgenic Mice Vaccines Variant Work X-Ray Crystallography associated symptom clinical predictors clinically relevant cross reactivity crosslink design in vivo insight mast cell novel passive sensitization peripheral blood preservation prototype receptor response tool

项目摘要

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.

项目概要过敏性疾病发病机制的核心是 IgE 分子。在敏感个体中，重新接触有害过敏原会导致 IgE 结合，导致 Fcε 受体交联并肥大细胞和嗜碱性粒细胞的激活。这会触发介质释放到局部组织中，从而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，研究人类 IgE 分子及其针对过敏原的靶向表位的研究非常有限。我们的大多数这一过程的知识来自于使用过敏患者血清的研究，其中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，并且具有许多不同的亲和力；因此，IgE 与目标过敏原分子相互作用的研究非常深入。有缺陷的。研究这一过程的理想方法是使用天然存在的人类 IgE 单克隆抗体（单克隆抗体），从过敏受试者中分离出来。不幸的是，由于许多不可逾越的内在技术障碍，没有以前已经制备过此类抗体。我们现在已经建立了一种方法来生长、识别和通过从过敏症患者的外周血中制备人杂交瘤，使编码 IgE 的 B 细胞永生化个人。在这个提案中，我们开发了第一个天然花生的综合面板过敏原特异性人 IgE mAb，用于定义最有效的诱导剂的分子相互作用过敏反应。我们已经开始全面的绘图研究，以确定关键的免疫优势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进行绘图将通过人类 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单克隆抗体并表达为重组 IgG mAb，用作使用人类进行高级绘图研究的工具血清。 IgG mAb 与制备它们的 IgE mAb 的结合方式相同，将用于使用一组花生过敏研究对象的冷冻血清和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其中发挥的作用以及花生过敏个体之间。该信息将用于绘制疾病的临床相关性并选择拥有未被我们的面板阻断的 IgE 抗体的研究对象，从而允许在目标 1 中生成新的位点特异性 IgE mAb。最后，在目标 3 中，我们将获得原子分辨率 X-精确定义 Ara h 2 和 6 上第一个天然存在的人类 IgE 表位的结构射线晶体学。这项工作的目标是创建一个明确的、完整的和全面的分子人类 IgE 抗体对花生主要过敏原蛋白的反应图。结果将作为急需的路线图，以便设计新的免疫疗法和过敏疫苗。