Generation and characterization of full-length naturally occurring allergen-specific human IgE mAbs

摘要 IgE与肥大细胞上的同源高亲和力受体结合的变应原交联， 嗜碱性粒细胞释放一系列介体，导致各种过敏性疾病。尽管 由于IgE分子的重要作用，人们对天然存在的人IgE分子知之甚少。我们的大多数 对过敏原的人抗体应答的知识来自于使用多克隆血清或 从鼠单克隆抗体推断。直到最近，自然产生的全长人类 针对特定免疫原的mAb几乎是不可能的。在这里，我们使用人B细胞杂交瘤 方法，通过与非分泌性骨髓瘤的电细胞融合使记忆B细胞永生化 合作伙伴，以产生有史以来第一次，天然存在的全长人IgE单克隆抗体。我们 初步数据表明，过敏性疾病循环中产生IgE的记忆B细胞的频率 患者的比例非常低，平均每十万个B细胞中有一个。尽管如此，我们能够 产生一组分泌全长天然存在的IgE抗体的人杂交瘤。的 使用患者的临床信息、血清总IgE和特异性IgE滴度来选择包含以下的样品： 细胞直接针对所需的过敏原-集中在花生和食物过敏原。一旦单克隆抗体制成， 将使用Phadia与Robert合作进行精细过敏原特异性测定 约翰霍普金斯大学的汉密尔顿。每种过敏原特异性IgE mAb都将通过 所有的测试，希望组装的单克隆抗体的面板，最终用于功能研究与特定的 过敏原蛋白由于缺乏可用的方法和试剂，我们开发了一种IgE特异性 用于纯化人IgE mAb的免疫亲和层析方案。纯化过敏原特异性 在过敏原竞争测定中测试mAb以将它们组装成反映抗原位点的组。 将详细测定它们与天然变应原相互作用的化学计量学和结合动力学。 将评价每种过敏原特异性IgE mAb的遗传和功能方面。抗体 将获得重链和轻链序列，以确定种系使用、 体细胞超突变和CDR长度。此信息将用于生成同种型开关变体 IgG mAb。最后，将对每种功能性IgE的嗜碱性粒细胞脱粒动力学进行全面评价 面板内的mAb配对。将测试重组开关变体IgG拮抗 过敏原特异性嗜碱性粒细胞脱粒，以量化其治疗潜力。我们创造了一些方法 并开始第一次产生和研究人类杂交瘤的小组， 发生过敏原特异性IgE mAb。这项工作的目标是改善我们的分子 了解人类IgE抗体反应，这将提供设计所需的见解， 更好的免疫疗法和过敏疫苗。

ABSTRACT Allergen cross-linking of IgE bound to their cognate high affinity receptors on mast cells and basophils unleash a cascade of mediators that result in the wide array of allergic disease. Despite its central role, very little is known about the naturally occurring human IgE molecule. Most of our knowledge of the human antibody response to allergens has come from studies using polyclonal sera or inferred from murine mAbs. Until very recently, the generation of naturally occurring full-length human mAbs to a specific immunogen has been next to impossible. Here we employ a human B cell hybridoma method, immortalizing memory B cells through electrical cytofusion with a non-secreting myeloma partner, to generate for the first time ever, naturally occurring full-length human IgE mAbs. Our preliminary data shows that the frequencies of IgE producing memory B cells in the circulation of allergic patients are very low, averaging one per one hundred thousand B cells. Despite this, we are able to generate panels of human hybridomas that secrete full-length naturally occurring IgE antibodies. The patient's clinical information, serum total and specific IgE titers, is used to select samples that contain cells directed toward desired allergens - focusing on peanut and food allergens. Once a mAb is made, determination of fine allergen specificity will be carried out using Phadia in collaboration with Robert Hamilton at Johns Hopkins University. Each allergen-specific IgE mAb that is created will go through a gamut of tests in hopes of assembling panels of mAbs for ultimate use in functional studies with specific allergen proteins. Due to the paucity of available methods and reagents we developed an IgE-specific immunoaffinity chromatography protocol for purification of human IgE mAbs. Purified allergen-specific mAbs are tested in allergen competition assays to assemble them into groups reflecting antigenic sites. Stochiometry and binding kinetics of their interaction with natural allergen will be determined in detail. The genetic and functional aspects of each allergen-specific IgE mAb will be evaluated. Antibody heavy and light chain sequences will be obtained to determine the germ-line usage, the degree of somatic hypermutation, and CDR length. This information will be used to generate isotype switch variant IgG mAbs. Finally, the kinetics of basophil degranulation will be fully evaluated for each functional IgE mAb pairing within a panel. Recombinant switch variant IgGs will be tested for their ability to antagonize allergen-specific basophil degranulation to quantify their therapeutic potential. We have created methods and began to generate and study for the first time panels of human hybridomas secreting naturally occurring allergen-specific IgE mAbs. The goal of this work is to improve upon our molecular understanding of the human IgE antibody response, which will provide insights needed for the design of better immunotherapies and allergy vaccines.