Antibody conjugates bispecific for intercellular adhesion molecule 1 and allergen prevent migration of allergens through respiratory epithelial cell layers

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Presentation transcript:

Antibody conjugates bispecific for intercellular adhesion molecule 1 and allergen prevent migration of allergens through respiratory epithelial cell layers Christoph Madritsch, PhD, Julia Eckl-Dorna, MD, PhD, Katharina Blatt, PhD, Isabella Ellinger, PhD, Michael Kundi, MD, PhD, Verena Niederberger, MD, Peter Valent, MD, Rudolf Valenta, MD, Sabine Flicker, PhD Journal of Allergy and Clinical Immunology Volume 136, Issue 2, Pages 490-493.e11 (August 2015) DOI: 10.1016/j.jaci.2015.01.006 Copyright © 2015 The Authors Terms and Conditions

Fig 1 Visualization of P2/ICAM1 and Phl p 2 on 16HBE14o- cells by means of immunofluorescence microscopy. Images (A-D) show cells incubated with different combinations of reactants (left margin and bottom), which were stained with Alexa Fluor 488–labeled anti-mouse antibodies (green, left column) and Alexa Fluor 568–labeled anti-rabbit antibodies (red, middle column) to visualize αICAM1-mouse IgG and Phl p 2, respectively. Nuclei were stained with 4′, 6-Diamidino-2-Phenylinodole, Dihydrochloride (blue), and merged images are shown in the right column. White bars = 20 μm. Journal of Allergy and Clinical Immunology 2015 136, 490-493.e11DOI: (10.1016/j.jaci.2015.01.006) Copyright © 2015 The Authors Terms and Conditions

Fig 2 Inhibition of transepithelial migration of Phl p 2 by P2/ICAM1 conjugates leads to decreased basophil activation with basolateral samples. Samples obtained from apical and basolateral compartments of Transwell cultures were incubated with blood samples from 3 patients allergic to Phl p 2 (A-C), and basophil activation was measured by determining upregulation of the surface marker CD203c on basophils by using flow cytometry. CD203c upregulation expressed as the stimulation index is displayed on the y-axis. Results are means of triplicates, and error bars indicate SDs. *P < .05, **P < .01, and ***P < .001, ANOVA and linear contrasts. Journal of Allergy and Clinical Immunology 2015 136, 490-493.e11DOI: (10.1016/j.jaci.2015.01.006) Copyright © 2015 The Authors Terms and Conditions

Fig E1 Reactivity of P2/ICAM1 with Phl p 2 and ICAM1 in ELISAs. Conjugates formed with different ratios of anti–Phl p 2 and anti-ICAM1 (x-axes: P2/ICAM1, 1:1, 1:0.5, and 1:0.25), anti–Phl p 2 (αP2-IgG), or anti–ICAM1 (αICAM1-IgG) alone were tested for reactivity with Phl p 2 (A) or ICAM1 (B) and detected with anti-human F(ab′)2 antibodies (black bars) or anti-mouse IgG (gray bars). OD values (y-axes) corresponding to bound P2/ICAM1 conjugates are shown as means of triplicates ± SDs. Journal of Allergy and Clinical Immunology 2015 136, 490-493.e11DOI: (10.1016/j.jaci.2015.01.006) Copyright © 2015 The Authors Terms and Conditions

Fig E2 Binding of the bispecific antibody conjugates P2/ICAM1 and P5/ICAM1 to the immobilized allergens Phl p 2 and Phl p 5 and recombinant ICAM1 in ELISAs. Bound P2/ICAM1 and P5/ICAM1 were detected with alkaline phosphatase–conjugated anti-human F(ab′)2 antibodies. Buffer containing 0.5% BSA was applied as a negative control (buffer control). One representative experiment of 3 is shown. Journal of Allergy and Clinical Immunology 2015 136, 490-493.e11DOI: (10.1016/j.jaci.2015.01.006) Copyright © 2015 The Authors Terms and Conditions

Fig E3 Detection of ICAM1 or P2/ICAM1–captured Phl p 2 on respiratory epithelial cells by using flow cytometry. A, ICAM1 was detected on the surfaces of 16HBE14o- cells by using ICAM1–specific antibodies (white graph) or an isotype control (gray graph). FITC, Fluorescein isothiocyanate. B, Cells preincubated with different ratios of P2/ICAM1 (1:1; 1:0.5; and 1:0.25) and Phl p 2 were probed for cell-bound Phl p 2 with specific rabbit antibodies (white graphs) or the isotype control (gray graphs). C, Scatter plots showing percentages of Phl p 2–reactive cells with 3 different ratios of P2/ICAM1. D, Cells incubated with Phl p 2–specific human IgG1 antibodies, followed by Phl p 2 and Phl p 2–specific rabbit antibodies. One representative experiment of 3 independent experiments is shown. Journal of Allergy and Clinical Immunology 2015 136, 490-493.e11DOI: (10.1016/j.jaci.2015.01.006) Copyright © 2015 The Authors Terms and Conditions

Fig E4 Visualization of P2/ICAM1 and Phl p 2 on 16HBE14o- cells by using immunofluorescence microscopy over a time course of 24 (A), 48 (B), and 72 (C) hours. Images showing cells incubated with different combinations of reactants (left margin and bottom), which were stained with Alexa Fluor 488–labeled anti-mouse antibodies (green, left column) and Alexa Fluor 568–labeled anti-rabbit antibodies (red, middle column) to visualize αICAM1–mouse IgG and Phl p 2, respectively. Nuclei were stained with 4′, 6-Diamidino-2-Phenylinodole, Dihydrochloride (blue), and merged images are shown in the right column. White bars = 20 μm. Journal of Allergy and Clinical Immunology 2015 136, 490-493.e11DOI: (10.1016/j.jaci.2015.01.006) Copyright © 2015 The Authors Terms and Conditions

Fig E4 Visualization of P2/ICAM1 and Phl p 2 on 16HBE14o- cells by using immunofluorescence microscopy over a time course of 24 (A), 48 (B), and 72 (C) hours. Images showing cells incubated with different combinations of reactants (left margin and bottom), which were stained with Alexa Fluor 488–labeled anti-mouse antibodies (green, left column) and Alexa Fluor 568–labeled anti-rabbit antibodies (red, middle column) to visualize αICAM1–mouse IgG and Phl p 2, respectively. Nuclei were stained with 4′, 6-Diamidino-2-Phenylinodole, Dihydrochloride (blue), and merged images are shown in the right column. White bars = 20 μm. Journal of Allergy and Clinical Immunology 2015 136, 490-493.e11DOI: (10.1016/j.jaci.2015.01.006) Copyright © 2015 The Authors Terms and Conditions

Fig E4 Visualization of P2/ICAM1 and Phl p 2 on 16HBE14o- cells by using immunofluorescence microscopy over a time course of 24 (A), 48 (B), and 72 (C) hours. Images showing cells incubated with different combinations of reactants (left margin and bottom), which were stained with Alexa Fluor 488–labeled anti-mouse antibodies (green, left column) and Alexa Fluor 568–labeled anti-rabbit antibodies (red, middle column) to visualize αICAM1–mouse IgG and Phl p 2, respectively. Nuclei were stained with 4′, 6-Diamidino-2-Phenylinodole, Dihydrochloride (blue), and merged images are shown in the right column. White bars = 20 μm. Journal of Allergy and Clinical Immunology 2015 136, 490-493.e11DOI: (10.1016/j.jaci.2015.01.006) Copyright © 2015 The Authors Terms and Conditions

Fig E5 Cell-bound P2/ICAM1 conjugates inhibit transepithelial migration of Phl p 2. A, ELISA detection of free Phl p 2 in apical (black bars) and basolateral (gray bars) compartments of cultured 16HBE14o- cell monolayers that had been preincubated with (+) or without (−) P2/ICAM1 conjugates. Concentrations of free Phl p 2 corresponding to OD values (y-axis) were measured at different time points (x-axis: 24, 48, and 72 hours) in wells with or without cells (no cells). In the negative control Phl p 2 was omitted (no Phl p 2). B, Simultaneous detection of P2/ICAM1–Phl p 2 complexes in the same samples as in Fig E5, A. Shown is one of 3 independent experiments producing comparable results. Journal of Allergy and Clinical Immunology 2015 136, 490-493.e11DOI: (10.1016/j.jaci.2015.01.006) Copyright © 2015 The Authors Terms and Conditions

Fig E6 Cell-bound P2/ICAM1 conjugates inhibit transepithelial migration of Phl p 2 (A) but not of the unrelated allergen Bet v 1 (B). ELISA detection of free Phl p 2 (Fig E6, A) or free Bet v 1 (Fig E6, B) in apical (black bars) and basolateral (gray bars) compartments of cultured 16HBE14o- cell monolayers that had been preincubated with (+) or without (−) P2/ICAM1 conjugates. Concentrations of free Phl p 2 or Bet v 1 corresponding to OD values (y-axis) were measured at 72 hours in wells with or without cells (no cells). In the negative control Phl p 2 was omitted (no Phl p 2). ***P < .001, significant difference. Journal of Allergy and Clinical Immunology 2015 136, 490-493.e11DOI: (10.1016/j.jaci.2015.01.006) Copyright © 2015 The Authors Terms and Conditions

Fig E7 Basophils were detected on the basis of side-scatter characteristics and expression of CD203c and analyzed with Paint-a-gate in the FlowJo program. Basophils are defined as CD203c+ cells in all samples. PE, Phycoerythrin; SSC, side scatter. Journal of Allergy and Clinical Immunology 2015 136, 490-493.e11DOI: (10.1016/j.jaci.2015.01.006) Copyright © 2015 The Authors Terms and Conditions