Biological activity of human IgE monoclonal antibodies targeting Der p 2, Fel d 1, Ara h 2 in basophil mediator release assays

Abstract

BackgroundHuman Immunoglobulin E monoclonal antibodies (hIgE mAb) are unique tools for investigating IgE responses. Here, the biological activity of hIgE mAb, derived from immortalized B cells harvested from the blood of allergic donors, targeting three allergens (Der p 2, Fel d 1 and Ara h 2) was investigated.MethodsThree Der p 2-, three Fel d 1- and five Ara h 2-specific hIgE mAb produced by human B cell hybridomas, were combined in pairs and used to passively sensitize humanized rat basophilic leukemia cells and compared with sensitization using serum pools. Sensitized cells were stimulated with corresponding allergens (recombinant or purified), allergen extracts or structural homologs, having 40-88% sequence similarity, and compared for mediator (β-hexosaminidase) release.ResultsOne, two and eight pairs of Der p 2-, Fel d 1- and Ara h 2-specific hIgE mAb, respectively, produced significant mediator release (>50%). A minimum hIgE mAb concentration of 15-30 kU/L and a minimum antigen concentration between 0.01-0.1 µg/mL were sufficient to induce a pronounced mediator release. Individual sensitization with one Ara h 2-specific hIgE mAb was able to induce crosslinking independently of a second specific hIgE mAb. Der p 2- and Ara h 2-specific mAb showed a high allergen specificity when compared to homologs. Mediator release from cells sensitized with hIgE mAb was comparable to serum sensitization.ConclusionThe biological activity of hIgE mAb reported here provides the foundation for novel methods of standardization and quality control of allergen products and for mechanistic studies of IgE-mediated allergic diseases, using hIgE mAb.

A Cell-Free Protein Synthesis Platform to Produce a Clinically Relevant Allergen Panel

“…After purification of the allergen (Figure S1B, lane 4), we performed a cotitration of IgE and purified CFPS-expressed Der p 2 with the AlphaLISA assay using two monoclonal IgE antibodies, 2G1 and 2F10, that each recognize a distinct epitope on opposite poles of the Der p 2 allergen. − On the final readout of an AlphaLISA assay, successful binding is indicated by an enhanced level of signal over background at some optimal concentration, with lower signal gradually fanning out as the concentration moves away from the optimum. A background signal is determined by the zero condition (“0”) where either IgE (bottom row) or Der p 2 (left column) is absent.…”

Unique allergen-specific human IgE monoclonal antibodies derived from patients with allergic disease

“…The aim was to determine (i) whether the hIgE mAb were directed against different epitopes and (ii) whether they could cross-link the high-affinity human IgE Fc ε receptor that was transfected into the RBL cell line upon allergen stimulation to induce degranulation. The mediator release data for hIgE mAb pairs to Der p 2, Fel d 1, and Ara h 2 was recently published ( 27 ). For each of the selected allergens, antibody pairs induced 40%–80% β -hexosaminidase release, providing a strong evidence for multiple non-overlapping IgE epitopes.…”

“…In the present study, β -hexosaminidase release from passively sensitized huRBL cells was used as an alternative approach to assess its biological activity. We recently published detailed comparisons of hIgE mAb potency in the huRBL system ( 27 ). In those studies, the hIgE mAb pairs to Der p 2, Fel d 1, and Ara h 2 produced high levels of mediator release and were directed against non-overlapping epitopes on each allergen.…”

“…The functional activity of the hIgE mAb was assessed by using humanized rat basophilic leukemia (huRBL) cells transfected with human FcɛRI for passive sensitization with hIgE mAb (RBL-2H3 cell line) ( 26 , 27 ). β -Hexosaminidase release was used as a marker of allergen-induced degranulation.…”