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

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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.