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Oktay Kirak, MD, Gert Riethmüller, MD 

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1 A novel, nonanaphylactogenic, bispecific IgE-CD3 antibody eliminates IgE+ B cells 
Oktay Kirak, MD, Gert Riethmüller, MD  Journal of Allergy and Clinical Immunology  Volume 136, Issue 3, Pages e3 (September 2015) DOI: /j.jaci Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Chromium 51 release assays were carried out to analyze cytotoxic activity mediated by bsc-IgE/CD3. A, Prestimulated CD8+ human effector T cells and J558L-IgEMS4 target cells were mixed at a 10:1 ratio in the presence of serial dilutions of bsc-IgE/CD3 antibody. B, Unstimulated PBMCs of different donors were mixed with J558L-IgEMS4 target cells at a 10:1 ratio and with different concentrations of the bsc-IgE/CD3 antibody. C, Prestimulated CD8+ human T cells and J558L-IgEMS4 target cells were mixed at a 10:1 ratio with different amounts of bsc-IgE/CD3 antibody and in the presence of serial dilutions of human IgE. Error bars indicate SEMs. Analysis was performed with GraphPad Prism software. EC50, Median effective concentration; E/T ratio, effector/target ratio. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 A and B, Chromium 51 release assays were performed with RBL-2H3 30/25 cells (Fig 2, A), which were loaded with soluble human IgE through FcεRI, or HOM2 cells (Fig 2, B), which were loaded with human IgE through the low-affinity FcεRII (CD23), to determine possible bystander cytotoxic activity against cells carrying receptor-bound human IgE. C, Release of β-hexosaminidase from RBL-2H3 30/25 cells was measured to determine potential degranulation. IgE-loaded RBL-2H3 30/25 cells were coincubated with purified T cells at an effector/target ratio (E/T ratio) of 10:1 and different concentrations of the bsc-IgE/CD3 antibody. An anti-human IgE mAb was added to the RBL-2H3 30/25 cells, either loaded or unloaded with soluble human IgE as controls. Error bars indicate SEMs. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 A, Schematic representation of the cloning strategy for the bsc-IgE/CD3 antibody. B, SDS-PAGE analysis of anti-IgE/anti-CD3. The bsc antibody has an expected relative molecular weight of approximately 55 kDa. C, Schematic picture of the mode of action of the bsc-IgE/CD3 antibody. The bsc antibody binds to IgE+ cells on one side and redirects cytotoxic activity of T cells by binding to CD3. D, Cloning strategy for generation of human transmembrane IgE in its long (top) and short (bottom) membrane isoforms. E, Fluorescence-activated cell sorting analysis of stably transfected murine J558L cells expressing the 2 isoforms MS and ML of human transmembrane IgE. Note that J558L-IgEMS4 cells have a higher expression level than J558L-IgEML7 cells. The parental cell line J558L was used as a negative control. F, Binding of the bsc-IgE/CD3 antibody to human IgE was shown by using fluorescence-activated cell sorting analysis on IgE-expressing J558L-IgEMS4 cells. For detection, we used mouse anti-His and goat anti-mouse antibodies. G, Binding to human CD3 was shown on the CD3-expressing cell line HPBALL. Negative control fluorescence-activated cell sorting analysis was performed without addition of the bsc-IgE/CD3 antibody. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 A, Activation of purified unstimulated T cells was measured based on upregulation of CD25 and CD69 after 24 hours. Upregulation of both activation markers is shown on the CD8+ T-cell subset and was only detectable in the presence of both bsc-IgE/CD3 and target cells expressing transmembrane IgE. We used the untransfected J558L cell line (left column) and the absence of bsc-IgE/CD3 (top row) as negative controls. B, Activation of T cells was also determined based on release of TNF-α, as determined by using ELISA. Unstimulated CD3+ cells were coincubated with the IgE-expressing cell lines J558L-IgEMS4 and J558L-IgEML7, respectively. The supernatant was collected after 1, 2, and 3 days, and the TNF-α concentration was measured by means of ELISA. Note that both upregulation of surface activation markers and release of TNF-α were only detectable when CD3+ effector cells, IgE-expressing target cells, and bsc-IgE/CD3 were present simultaneously. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E3 A, Loading of RBL-2H3 30/25 cells with soluble human IgE through the high-affinity FcεRI was shown by using fluorescence-activated cell sorting analysis. Unloaded RBL-2H3 30/25 cells were used as a negative control. B, Expression of the low-affinity FcεRII (CD23) on HOM2 cells was demonstrated by means of flow cytometry (left plot). An isotype control was used as a negative control. Loading of HOM2 cells with soluble human IgE was monitored by means of fluorescence-activated cell sorting analysis (right plot). Unloaded HOM2 cells were used as a negative control. C, Specificity of the IgE-CD23 interaction was confirmed by adding different concentrations of a blocking anti-CD23 antibody before loading with soluble human IgE. Note that high concentrations of anti-CD23 antibody mediate strong blocking and thus result in low binding of soluble human IgE to HOM2 cells. FITC, Fluorescein isothiocyanate; PE, phycoerythrin. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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