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Judit Fazekas-Singer, MSc, Josef Singer, MD, PhD, Kristina M

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Presentation on theme: "Judit Fazekas-Singer, MSc, Josef Singer, MD, PhD, Kristina M"— Presentation transcript:

1 AllergoOncology: Generating a canine anticancer IgE against the epidermal growth factor receptor 
Judit Fazekas-Singer, MSc, Josef Singer, MD, PhD, Kristina M. Ilieva, MSc, Miroslawa Matz, MSc, Ina Herrmann, DVM, Edzard Spillner, PhD, Sophia N. Karagiannis, PhD, Erika Jensen-Jarolim, MD  Journal of Allergy and Clinical Immunology  Volume 142, Issue 3, Pages e11 (September 2018) DOI: /j.jaci Copyright © Terms and Conditions

2 Fig 1 Expression and characterization of can225IgE-λ, and IgE-receptor expression on effector cell lines. Expression vector construct (A), can225IgE-λ on a silver-stained protein gel (B), and western blot developed with antidog IgE (Fc) (C) and with antidog light chain (D). Binding of can225IgE-λ to human EGFR by Western blot (E) and to EGFR on human and canine target cell lines using EGFR-negative cell line CHO K1 for control (F-I). FcεRI expression on human and canine effector cell lines (J-M) and relative CD23 expression of MPT-1 canine mast cells compared with DH82 monocytic/macrophage-like cells, normalized to beta-actin expression (N). CMV, Cytomegalovirus; HER-2, epidermal growth factor receptor 2; PE, phycoerythrin; SV40, simian vacuolating virus 40. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © Terms and Conditions

3 Fig 2 Functional characterization of can225IgE-λ. Dose-dependent binding of can225IgE-λ to Fcε-receptors on U937 human promonocytic cells (A), DH82 canine monocytic/macrophage-like cells (B), and MPT-1 canine mast cells (C), with left panels depicting fluorescence histograms and right panels quantified delta mean fluorescence intensity values (n = 3). ADCC- and ADCP-triggering potency (D) and total tumor cell killing (E) induced by can225IgE-λ compared with other antibodies with human U937 cells and human A431 tumor cells as targets. ADCC- and ADCP-triggering potency (F) and total tumor cell killing (G) of can225IgE-λ compared head-to-head to its IgG counterpart can225IgG and to a combinatory can225IgG + IgE-λ treatment, using canine DH82 cells as effectors and A431 tumor cells as targets. ADCC-triggering potency of can225IgE-λ using canine MPT-1 mast cells as effector cells and human A431 as target cells (H). Microscopic image of the ADCC assay depicted in panel H: can225IgE-λ–mediated cytotoxic killing of A431 cells (green) by MPT-1 mast cells (red) using DAPI (blue) as an indicator of dead cells (white arrowheads) (I). Receptor-specific binding of can225IgE-λ to FcεRI expressed on RBL-SX38 cells (J) and to canine CD23 on transfected CHO K1 cells (K). DAPI, 4′-6-Diamidino-2-phenylindole, dihydrochloride. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © Terms and Conditions

4 Fig E1 Gating strategies, ADCC and ADCP control assays, and phenotypical characterization of effector cells. Gating strategy in flow-cytometric ADCC and ADCP assays (A) and DH82 cell characterization (B). ADCC- and ADCP-triggering potency (C) and total tumor cell killing (D) induced by can225IgE-λ compared with other antibodies in a negative control assay using U937 as effector and EGFR-negative CHO K1 as target cells. ADCC- and ADCP-triggering potency (E) and total tumor cell killing (F) induced by can225IgE-λ compared with other antibodies in a negative control assay using DH82 as effector and EGFR-negative CHO K1 as target cells. DH82 cell characterization in regard of CD16 (G), CD14 (H), and CD80 (I) expression, pointing toward a monocyte-like phenotype. Negative control experiment to Fig 2, K: nontransfected (parental) CHO K1 did not bind can225IgE-λ (J). Evaluating IgE-mediated tumor cell killing over time confirmed previous literature reporting 2.5 hours as the optimal incubation time (K). CFSE, Carboxyfluoresceinsuccinimidyl ester; FSC-A, forward scatter-area; ns, nonsignificant; SSC-A, side scatter-area. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © Terms and Conditions

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