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 与肥大细胞上同源高亲和力受体结合的过敏原交联 嗜碱性粒细胞释放一系列介质，导致多种过敏性疾病。尽管其 尽管人类 IgE 分子具有核心作用，但人们对天然存在的 IgE 分子知之甚少。我们的大多数 关于人类抗体对过敏原反应的知识来自使用多克隆血清或 从鼠单克隆抗体推断。直到最近，自然发生的全长人类的产生 针对特定免疫原的单克隆抗体几乎是不可能的。在这里，我们使用人类 B 细胞杂交瘤 方法，通过与非分泌性骨髓瘤进行电细胞融合使记忆 B 细胞永生化 合作伙伴，首次产生天然存在的全长人类 IgE 单克隆抗体。我们的 初步数据表明，过敏体循环中产生 IgE 的记忆 B 细胞的频率 患者的 B 细胞数量非常低，平均为十万分之一。尽管如此，我们还是能够 产生分泌全长天然 IgE 抗体的人类杂交瘤组。这 患者的临床信息、血清总 IgE 滴度和特异性 IgE 滴度用于选择含有 细胞针对所需的过敏原 - 重点关注花生和食物过敏原。一旦制备出单克隆抗体， 将与 Robert 合作使用 Phadia 确定精细过敏原特异性 约翰·霍普金斯大学的汉密尔顿。每个产生的过敏原特异性 IgE mAb 都会经过一个 进行一系列测试，希望组装单克隆抗体面板，最终用于具有特定功能的研究 过敏原蛋白质。由于缺乏可用的方法和试剂，我们开发了 IgE 特异性 用于纯化人 IgE mAb 的免疫亲和层析方案。纯化的过敏原特异性 mAb 在过敏原竞争测定中进行测试，将其组装成反映抗原位点的组。 将详细确定它们与天然过敏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 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。该信息将用于生成同种型转换变体 IgG 单克隆抗体。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估 面板内的单克隆抗体配对。将测试重组开关变体 IgG 的拮抗能力 过敏原特异性嗜碱性粒细胞脱粒以量化其治疗潜力。我们已经创建了方法 并开始首次生成和研究自然分泌的人类杂交瘤组 出现过敏原特异性 IgE mAb。这项工作的目标是改进我们的分子 了解人类 IgE 抗体反应，这将为设计提供所需的见解 更好的免疫疗法和过敏疫苗。