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

全长天然过敏原特异性人 IgE mAb 的生成和表征

• 批准号: 9245291
• 负责人: Scott Alan Smith
• 金额: $ 21万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-19 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9245291
• 关键词: Adaptive Immune System; Affinity; Allergens; Allergic; Allergic Disease; Allergy to peanuts; Antibodies; Antibody Response; Antigens; B-Lymphocytes; Basophilic Cell; Basophils; Binding; Biochemical; Biological Assay; Biosensor; Blood; Blood Circulation; Cells; Characteristics; Clinical; Clone Cells; Collaborations; Column Chromatography; Data; Databases; Developed Countries; Developing Countries; Diagnostic; Disease; Enzyme-Linked Immunosorbent Assay; Epitopes; Foundations; Frequencies; Future; Generations; Genes; Genetic; Germ Lines; Goals; Helminths; Human; Human Activities; Hybridomas; Hypersensitivity; IgE; Immunogenetics; Immunoglobulin Class Switching; Immunoglobulin G; Immunoglobulin Somatic Hypermutation; Immunoglobulins; Immunotherapy; In Vitro; Individual; Kinetics; Knowledge; Length; Light; Maps; Mediator of activation protein; Memory B-Lymphocyte; Methods; Molecular; Molecular Cloning; Monoclonal Antibodies; Multiple Myeloma; Mutation; Pathogenicity; Pathologic; Patients; Population; Proteins; Protocols documentation; Reagent; Recombinants; Research; Role; Sampling; Serum; Site; Specificity; Structure; Techniques; Technology; Testing; Therapeutic; Therapeutic Monoclonal Antibodies; Time; Universities; Vaccines; Variant; Work; allergic response; base; crosslink; design; expression vector; falls; food allergen; group competition; human monoclonal antibodies; immunoaffinity chromatography; improved; insight; instrument; mast cell; murine monoclonal antibody; peripheral blood; programs; receptor

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.

抽象的 IgE的过敏原交联与肥大细胞上的认知高亲和力受体结合 嗜碱性粒子释放了一系列导致多种过敏性疾病的介体。尽管有它 中心作用，对天然存在的人类IgE分子知之甚少。我们的大多数 对过敏原反应的知识来自使用多克隆血清或 从鼠mabs推断。直到最近，自然发生的全长人 对特定免疫原的mAb几乎是不可能的。在这里，我们采用人类B细胞杂交瘤 方法，通过非分泌骨髓瘤的电胞质渗透使记忆B细胞永生化的记忆B细胞 合作伙伴，是有史以来第一次生成的，自然发生了全长的人Ige mab。我们的 初步数据表明，在过敏性循环中，IgE产生记忆B细胞的频率 患者非常低，平均每十万个B细胞。尽管如此，我们能够 生成人类杂交瘤的面板，这些杂交瘤分泌全长发生的IgE抗体。这 患者的临床信息，血清总和特定的IgE滴度用于选择包含的样品 针对所需过敏原的细胞 - 专注于花生和食物过敏原。一旦制造了mab， 通过Phadia与Robert合作，将对精细过敏原特异性进行确定 约翰·霍普金斯大学的汉密尔顿。创建的每个过敏原特异性IgE mab都会通过 希望组装mAB的测试范围，以在功能研究中最终使用，并具有特定的特定 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 纯化人IgE mAb的免疫亲和力色谱方案。纯化过敏原特异性 在过敏原竞争测定中测试了mAB，以将它们组装成反映抗原位点的组。 将详细确定其与天然过敏原相互作用的静态计量和结合动力学。 将评估每个过敏原特异性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量 躯体超成名和CDR长度。此信息将用于生成同型开关变体 IgG mabs。最后，将为每个功能IgE全面评估嗜碱性脱粒的动力学 在面板内进行单支配对。重组开关变体IgG将测试其对抗的能力 过敏原特异性嗜碱性脱粒，以量化其治疗潜力。我们创建了方法 并开始生成和研究人类杂种自然分泌的人类杂交瘤 发生过敏原特异性IgE mAb。这项工作的目的是改善我们的分子 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。