Anti-Human IGHE Recombinant Antibody (Omalizumab) (CAT#: TAB-007)

Figure 1 Comparison of effect of treatment with omalizumab and placebo on changes in absolute and % predicted forced expiratory volume in 1 s (FEV1) between baseline and times A and B.

Bars represent the median and interquartile range. Mann–Whitney U-Test used for comparison.

Pillai, P., Chan, Y. C., Wu, S. Y., Ohm-Laursen, L., Thomas, C., Durham, S. R., ... & Corrigan, C. J. (2016). Omalizumab reduces bronchial mucosal IgE and improves lung function in non-atopic asthma. European Respiratory Journal, 48(6), 1593-1601.

Figure 2 A single IgE mutation prevents omalizumab binding.

SPR-binding assays with immobilized omalizumab.

Pennington, L. F., Tarchevskaya, S., Brigger, D., Sathiyamoorthy, K., Graham, M. T., Nadeau, K. C., ... & Jardetzky, T. S. (2016). Structural basis of omalizumab therapy and omalizumab-mediated IgE exchange. Nature communications, 7, 11610.

Figure 3 E2_79 and omalizumab bind to both WT and Cys-335 IgE-Fc proteins.

A , E2_79 binds to both WT IgE-Fc ( open circles , solid line ) and Cys-335 IgE-Fc ( filled circles , dashed line ) in an ELISA assay. IgE-Fc proteins were plated and incubated with E2_79 at the indicated concentrations, and E2_79 binding was detected with an anti-His tag antibody. B , omalizumab binds to both WT IgE-Fc ( open circles , solid line ) and Cys-335 IgE-Fc ( filled circles , dashed line ) in an ELISA assay. IgE-Fc proteins were plated and incubated with omalizumab, and omalizumab binding was detected with a polyclonal anti-IgG antibody. C and D , side ( C ) and top ( D ) views of the omalizumab-binding region on the IgE-Fc C ⑀ 3 domain. The main chain conformation of the IgE-Fc Cys-335 structure and the disulfide bond at residue 335 are shown in magenta , whereas residues mapped to the omalizumab binding region are shown in stick representation . Omali- zumab binding interactions have been mapped to IgE residues 421– 432 including the FG loop residues 424 – 426 (HLP) that lie within the Fc ⑀ RI ␣ binding interface (33). Despite the conformational restriction imposed by the Cys-335 disulfide, the omalizumab epitope remains accessible to binding.

Wurzburg, B. A., Kim, B., Tarchevskaya, S. S., Eggel, A., Vogel, M., & Jardetzky, T. S. (2012). An engineered disulfide bond reversibly traps the IgE-Fc3-4 in a closed, non-receptor binding conformation. Journal of biological chemistry, jbc-M112.

Figure 4 Omalizumab has no inhibitory effect on anti-IgE-induced degranulation.

(a) Freshly isolated human skin mast cells were coincubated with or without omalizumab (10 ug/mL) for 24 h or with DARPin Fc (100 pmol/mL) for 1 h as positive control for FceRI-FccRIIb cross linking. Mast cells were then activated with anti-IgE (1 ug/mL) for 1 h and histamine release determined. Results are expressed as mean SEM histamine release (% total) of three independent experiments. Panel (b) shows peripheral blood-derived monocytes from healthy donors that were coincubated with or without omalizumab (10 ug/mL) for 1 h and then activated using anti-IgE (5 ug/mL) for another hour to induce degranulation. Results are expressed as mean SEM histamine release (% total) of five independent experiments.

Gericke, J., Ohanyan, T., Church, M. K., Maurer, M., & Metz, M. (2015). Omalizumab may not inhibit mast cell and basophil activation in vitro. Journal of the European Academy of Dermatology and Venereology, 29(9), 1832-1836.

Figure 5 Inducible expression of IgE BCR on IMIGEC cells - a cell reporter for membrane IgE EMPD crosslinking.

Upper panel: Surface expression of membrane IgE BCR on IMIGEC cells was measured by flow cytometry at different time points of dox incubation (0-48 h, x-axis) with mABs recognizing various regions of the IgE BCR (MFI, y-axis) or respective isotype controls. The right part of the plot shows expression kinetics after 24 h dox treatment followed by 24 h dox withdrawal. Pooled data of 3 biological replicates are shown as mean ± SD. Lower panels: Exemplary histograms and MFI values (FL-2/PE channel) after 18 h are shown.

Oskar, W. (2016). Quantitative in vitro and in vivo models to assess human IgE B cell receptor crosslinking by IgE and EMPD IgE targeting antibodies. Journal of Immunological Methods.