The Significance of Clonality and Affinity of Individual Allergen-Specific IgE Antibodies for the Activation of Human Basophils

Abstract

RATIONALE: In allergic individuals symptoms arise from activation of effector cells through allergen-mediated cross-linking of FcεRI-bound IgE. The allergen-specific IgE response is polyclonal, but only little is known to what extent the clonality of the IgE-response and affinity of the individual IgE molecules affect the allergic response. Here we test a panel of well characterized recombinant IgE antibodies with varying affinities toward the major house dust mite allergen Derp2 in a human basophil degranulation assay.METHODS: Variable antibody genes from 30 different murine hybridoma cell lines expressing antibodies directed towards Derp2 were cloned into a vector system encoding the constant region of human IgE and expressed as chimeric recombinant IgE antibodies. Interactions between rIgE antibodies and rDerp2 were characterized by affinity measurements and epitope specificity using Biacore technology. Basophils from human donors were sensitized with different combinations of rIgE antibodies showing high, medium or low affinity toward rDerp2. The level of basophil degranulation was measured by FACS analysis (CD63 activation) after challenge with increasing concentrations of rDerp2.RESULTS: Antibody binding experiments identified several rIgE antibodies binding overlapping or non-overlapping epitopes on rDerp2. Binding affinities ranged from KD 200 nM to KD 0,05 nM. Different combinations of rIgE antibodies with high/high, high/medium and high/low affinities showed human basophil degranulation although CD63 activation was higher when solely combining high affinity IgE antibodies.CONCLUSIONS: Two IgE specificities are sufficient for allergen-mediated basophil activation, and the degree of activation depends on the affinity of the individual antibodies. RATIONALE: In allergic individuals symptoms arise from activation of effector cells through allergen-mediated cross-linking of FcεRI-bound IgE. The allergen-specific IgE response is polyclonal, but only little is known to what extent the clonality of the IgE-response and affinity of the individual IgE molecules affect the allergic response. Here we test a panel of well characterized recombinant IgE antibodies with varying affinities toward the major house dust mite allergen Derp2 in a human basophil degranulation assay. METHODS: Variable antibody genes from 30 different murine hybridoma cell lines expressing antibodies directed towards Derp2 were cloned into a vector system encoding the constant region of human IgE and expressed as chimeric recombinant IgE antibodies. Interactions between rIgE antibodies and rDerp2 were characterized by affinity measurements and epitope specificity using Biacore technology. Basophils from human donors were sensitized with different combinations of rIgE antibodies showing high, medium or low affinity toward rDerp2. The level of basophil degranulation was measured by FACS analysis (CD63 activation) after challenge with increasing concentrations of rDerp2. RESULTS: Antibody binding experiments identified several rIgE antibodies binding overlapping or non-overlapping epitopes on rDerp2. Binding affinities ranged from KD 200 nM to KD 0,05 nM. Different combinations of rIgE antibodies with high/high, high/medium and high/low affinities showed human basophil degranulation although CD63 activation was higher when solely combining high affinity IgE antibodies. CONCLUSIONS: Two IgE specificities are sufficient for allergen-mediated basophil activation, and the degree of activation depends on the affinity of the individual antibodies.