FUNCTIONAL ANALYSIS OF PATHOGENIC AND PROTECTIVE PEANUT ALLERGEN-SPECIFIC HUMAN ANTIBODIES

食物过敏反应是世界范围内发病率和死亡率不断增加的原因，并由 与肥大细胞和嗜碱性粒细胞效应细胞结合的过敏原特异性IgE抗体。其分子特征是 从含有以下物质的多克隆血清中很难研究某些人类IgE抗体的致病性 未知数量和比例的抗体类型的混合物。同样，IgG4和IgG4的作用机制 口服免疫疗法(OIT)诱导的IgG1抗体可保护患者免受过敏反应的影响 通过研究治疗后患者的已知序列的明确抗体澄清。 我们将研究确定的花生变应原特异性人IgE、IgG4和IgG1单抗的功能活性 从有症状的过敏患者中分离出的抗体(MAbs)与致敏但不过敏的对照组进行比较， 以及治疗后对OIT反应不同的患者。培养的原代人类肥大细胞将是 用作效应细胞，并将通过三种方法进行研究，从早期细胞激活(钙内流)开始， 通过效应器的释放，到后来的细胞因子分泌。将评估免疫球蛋白克隆的过敏原特异性， 结合亲和力和表位识别，并进行组合测试，以确定哪些物种可以致敏 肥大细胞。将测试单抗IgG4和IgG1对肥大细胞激活的阻断作用，并使用Fc突变来 确定Fcγ受体是否有助于肥大细胞效应器功能的降低。最后，IgE和其他 从患者胃肠道活检组织中分离的抗体亚型将与分离的抗体进行比较。 从血液中，确定粘膜部位是否存在潜在致病IgE的浓缩 发生过敏反应的地方。 这些研究可能会对基础和转化性人类过敏研究产生重大影响，因为 他们将使用一个完全人类的、但定义明确的实验系统来识别 过敏患者血液和胃肠道中的致病IgE克隆，并评估潜在的IgG4和IgG1- OIT后的中介保护机制。提高对IgE克隆亲和力、表位的理解 反应性和导致肥大细胞敏化的组合可以实现更好的诊断和 过敏患者的预后检测，同时更多地了解导致保护性IgG4和 IgG1可以指导治疗试验和改进免疫治疗方案的开发。

Food allergic reactions are an increasing cause of morbidity and mortality worldwide, and are triggered by allergen-specific IgE antibodies bound to mast cell and basophil effector cells. The molecular features that make some human IgE antibodies pathogenic have been difficult to study from polyclonal sera that contain mixtures of unknown numbers and proportions of antibody types. Similarly, the mechanisms by which IgG4 and IgG1 antibodies elicited by oral immunotherapy (OIT) can protect patients from allergic reactions would be clarified by studying defined antibodies of known sequence from patients following treatment. We will study the functional activity of defined peanut allergen-specific human IgE, IgG4 and IgG1 monoclonal antibodies (mAbs) isolated from symptomatic allergic patients compared to sensitized but non-allergic controls, and from patients post-treatment who vary in their response to OIT. Cultured primary human mast cells will be used as effector cells, and will be studied with three assays extending from early cell activation (Calcium influx), through effector release, to later cytokine secretion. IgE clones will be evaluated for their allergen specificity, binding affinity, and epitope recognition, and tested in combinations to determine which species can sensitize mast cells. Blocking of mast cell activation by mAb IgG4 and IgG1 will be tested, and Fc mutations used to determine whether Fcγ receptors contribute to decreases in mast cell effector functions. Finally, IgE and other antibody isotypes isolated from patient gastrointestinal tract biopsies will be compared to antibodies isolated from the blood, to determine whether there is enrichment for potentially pathogenic IgE in the mucosal sites where allergic reactions occur. These studies are likely to have a significant impact on basic and translational human allergy research, as they will use a fully human, but well-defined experimental system to identify the molecular features of pathogenic IgE clones in the blood and GI tracts of allergic patients, and evaluate potential IgG4 and IgG1- mediated mechanisms of protection following OIT. Improved understanding of IgE clone affinities, epitope reactivities, and combinations that result in mast cell sensitization could enable better diagnostic and prognostic testing in allergic patients, while more knowledge about the criteria leading to protective IgG4 and IgG1 could guide therapeutic trials and the development of improved immunotherapy regimens.