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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

基本信息

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
项目类别：
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

项目摘要

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.

摘要