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

摘要 肥大细胞上IgE与其同源高亲和力受体结合的变应原交联性 嗜碱性细胞释放一系列介体，导致广泛的过敏性疾病。尽管它的 作为核心角色，人们对自然产生的人类免疫球蛋白E分子知之甚少。我们的大多数人 人类对过敏原的抗体反应的知识来自于使用多克隆血清或 从鼠源性单抗中推断出来的。直到最近，自然产生的全长人类的一代 针对特定免疫原的单抗几乎是不可能的。在这里，我们使用了人类B细胞杂交瘤 方法：通过与非分泌性骨髓瘤进行电融合使记忆B细胞永生化 合作伙伴，首次产生自然产生的全长人类IgE单抗。我们的 初步数据显示，过敏性血液循环中产生IgE的记忆B细胞的频率 患者的比例非常低，平均每10万个B细胞中就有一个。尽管如此，我们还是能够 产生人类杂交瘤的面板，分泌全长自然产生的IgE抗体。这个 患者的临床信息，血清总IgE滴度和特异性IgE滴度被用来选择包含以下内容的样本 细胞针对所需的过敏原--侧重于花生和食物过敏原。一旦制造出单抗， 将与Robert合作使用Phadia进行细微变应原特异性的确定 约翰·霍普金斯大学的汉密尔顿。每个创建的过敏原特异性IgE单抗都将经历一个 希望组装mAbb面板以最终用于特定功能研究的一系列测试 过敏原蛋白。由于缺乏可用的方法和试剂，我们开发了一种特异性的IgE 免疫亲和层析法纯化人IgE单抗。纯化的过敏原特异性 单抗在过敏原竞争分析中进行测试，以将它们组装成反映抗原位置的组。 将详细测定它们与天然变应原相互作用的化学计量和结合动力学。 将评估每种过敏原特异性IgE单抗的遗传和功能方面。抗体 将获得重链和轻链序列，以确定种系的使用、 体细胞高度突变和CDR长度。此信息将用于生成等类型开关变量 免疫球蛋白单抗。最后，将对每种功能IgE的嗜碱性粒细胞脱颗粒的动力学进行全面评估。 面板内的MAB配对。重组开关变异体IGGS将被测试其拮抗能力 过敏原特异性嗜碱性粒细胞脱颗粒，以量化其治疗潜力。我们已经创建了方法 并首次开始产生和研究自然分泌的人类杂交瘤 出现过敏原特异性的IgE单抗。这项工作的目标是改进我们的分子 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.