Accumulated immune complexes of IgE and omalizumab trap allergens in an in vitro model.

The best understood mechanisms of omalizumab are that it neutralizes free IgE and down-regulates high-affinity IgE.Fc receptors (FcepsilonRI) on basophils and mast cells. It has been proposed that since complexes of IgE and omalizumab are accumulated to 5-10 times the basal levels of IgE, they may trap incoming allergens, contributing to omalizumab's effectiveness. In order to investigate the ability of IgE:omalizumab complexes in trapping allergens and inhibiting basophil activation in an in vitro reconstitution model, the ability of IgE:omalizumab complexes to tie up antigen and hence inhibit (a) antigen binding to IgE bound by FcepsilonRI, and (b) antigen-mediated activation of basophils, was examined. The free IgE was prepared by mixing different proportions of antigen-nonspecific IgE secreted by U266 cells and antigen-specific IgE, SE44 IgE, which recognizes a synthetic 15 a.a. peptide, R15K. The antigen was (R15K)(8)-ova, i.e. ovalbumin conjugated with an average of 8 copies of R15K per molecule. The solid-phase FcepsilonRI was a recombinant protein representing the extracellular portion of the alpha chain of the FcepsilonRI receptor complex. The model FcepsilonRI(+) basophilic cell line was RBL.SX-38, a rat basophilic leukemic line transfected with the genes for alpha, beta and gamma subunits of human FcepsilonRI. The results showed that the IgE:omalizumab complexes trapped increasing amounts of antigen with increasing (a) concentration of IgE, (b) proportion of antigen-specific IgE in total IgE, and (c) concentration of total immune complexes. Such trapping decreased the antigen-induced activation of FcepsilonRI(+) cells that had been pulsed with antigen-specific IgE, resulting in decreased mediator release. These results suggest that the rapidly accumulated IgE:omalizumab complexes in omalizumab-treated patients can capture allergens and consequently contribute to the pharmacological effects of omalizumab.

Enhancing oxidative resistance of Agrobacterium radiobacter N-carbamoyl D-amino acid amidohydrolase by engineering solvent-accessible methionine residues.

N-Carbamoyl D-amino acid amidohydrolase (D-NCAase) that catalyzes the stereospecific hydrolysis of N-carbamoyl D-amino acids to their corresponding D-amino acids is valuable in pharmaceutical industry. Agrobacterium radiobacter D-NCAase is sensitive to oxidative damage by hydrogen peroxide. To investigate the role of methionine residues in oxidative inactivation, each of the nine methionine residues in A. radiobacter D-NCAase was substituted with leucine, respectively, by site-directed mutagenesis. Except for two mutants (Met5Leu and Met31Leu) with similar activities, seven mutants (Met73Leu, Met167Leu/Met169Leu, Met184Leu, Met220Leu, Met239Leu, Met244Leu, and Met239Leu/Met244Leu) were found to have reduced activities. In the presence of H(2)O(2), three mutants (Met239Leu, Met244Leu, and Met239Leu/Met244Leu) with substitution of highly solvent-accessible methionines by leucines retained their activities. The other mutants were also considerably resistant to chemical oxidation than was the wild-type enzyme. Thus, substitution of solvent-accessible methionine residues with leucine to enhance oxidative stability of D-NCAase is practical but might be with compromised activity.