Human Anti-IgE Fc Recombinant Antibody (clone MEDI4212) (CAT#: PABL-230)

Figure 1 MEDI4212 variants have increased affinity for FcγRIIIa (ELISA).

MEDI4212 variants were coated on plates and assessed for binding to: (a) FcγRIIIa-FLAG (158V) MEDI4212 afuc, 3M 2M2 and WT had EC50 values of 64.1±23.6 ng/ml, 19.0±7.3 ng/ml, 20.5±11.8 ng/ml and 272.7±78.3 ng/ml, respectively (n=3); (b) FcγRIIIa-FLAG (158F) MEDI4212 afuc, 3M 2M2, and WT had EC50 values of 72.0±25.8 ng/ml, 56.0±6.4 ng/ml, 112.0±31.2 ng/ml and 27696±4977 ng/ml, respectively (n=3). MEDI4212 variants demonstrated enhanced binding to both FcγRIIIa types compared with MEDI4212 WT.

Nyborg, A. C., Zacco, A., Ettinger, R., Borrok, M. J., Zhu, J., Martin, T.,... & Herbst, R. (2016). Development of an antibody that neutralizes soluble IgE and eliminates IgE expressing B cells. Cellular & molecular immunology, 13(3), 391.

Figure 2 MEDI4212 variants inhibit Calcium Flux and degranulation, and bind cell surface-associated IgE.

293T and NS0 cells were engineered to express IgE at the surface. (a) MEDI4212 was used to detect the IgE-expressing cells by flow cytometry. MEDI4212 variants bind to the surface of IgE-expressing cells as efficiently as MEDI4212 WT. (b) MEDI4212 variants do not bind to the surface of WT293T cells. (c) RBL-2H3 (rat basophilic cell line) cells were stably transfected with human FcεR1. Activation of FcεR1 receptor by crosslinking receptor bound IgE leads to calcium mobilization that can be detected using FLIPR (n=3, representative shown). (d) Human mast cells (LAD2 cells) naturally express human FcεR1 (n=3, representative shown). Activation of FcεR1 receptor by crosslinking receptor-bound IgE leads to activation of cells and secretion of mediators such as beta-hexosaminidase. MEDI4212 WT and ADCC-enhanced variants all inhibit calcium mobilization (c) and beta-hexosaminidase release (d) in a dose-dependent manner. ADCC, antibody-dependent cell-mediated cytotoxicity; FLIPR, Fluorometric Imaging Plate Reader; IgE, immunoglobulin E; WT, wild-type.

Nyborg, A. C., Zacco, A., Ettinger, R., Borrok, M. J., Zhu, J., Martin, T.,... & Herbst, R. (2016). Development of an antibody that neutralizes soluble IgE and eliminates IgE expressing B cells. Cellular & molecular immunology, 13(3), 391.

Figure 3 MEDI4212 variants enhance ADCC killing of IgE-expressing cells.

MEDI4212 WT and variants were evaluated for ADCC activity in four different assay systems. (a) Luciferase assay with NK/CD16-NFAT cells overexpressing FcγRIIIa (158V) and membrane IgE-transfected 293T target cells. MEDI4212 aFuc, 3M and 2M2 had average EC50 values of 28.6±11.2 ng/ml, 28.9±9.2 ng/ml and 38.9±13.5 ng/ml, respectively (n=5). (b) KC133 natural killer cells and 293T-IgE target cells at a 2.5∶1 (E/T) ratio. LDH release was quantified as a measure of cell cytotoxicity after 14 h. MEDI4212 aFuc, 3M and 2M2 had average an EC50 of 18.9±11.5 ng/ml, 13.3±9.7 ng/ml, and 21.7±6.0, respectively (n=6). (c) KC133 and NS0-IgE target cells at a 2.5∶1 (E/T) ratio. LDH release was quantified as a measure of cell cytotoxicity after 14 h. MEDI4212 aFuc, 3M and 2M2 had an EC50 of 25.6±7.2 ng/ml, 20.0±9.0 ng/ml and 41.7±12.6 ng/ml, respectively (n=6). (d) KC133 and NS0-IgE target cells at a 5∶1 (E/T) ratio. Granzyme B signaling was detected by flow cytometry after 15 min. MEDI4212 aFuc, 3M and 2M2 had an average EC50 of 160±30.2 ng/ml, 64.8.0±23.0 ng/ml and 55.4±21.6 ng/ml (n=3). MEDI4212 ADCC variants, but not MEDI4212 WT, mediated effective target cell killing in all assay formats tested. ADCC, antibody-dependent cell-mediated cytotoxicity; IgE, immunoglobulin E; LDH, lactate dehydrogenase; NK, natural killer; WT, wild-type.

Nyborg, A. C., Zacco, A., Ettinger, R., Borrok, M. J., Zhu, J., Martin, T.,... & Herbst, R. (2016). Development of an antibody that neutralizes soluble IgE and eliminates IgE expressing B cells. Cellular & molecular immunology, 13(3), 391.

Figure 4 MEDI4212 aFuc enhance killing of primary IgE class-switched human PBMCs.

Human PBMCs were collected from normal donors and class-switched by the addition of IL-4 and anti-CD40. (a) Class-switched cells were added to ELISpot wells and spots were observed in the control well. The addition of MEDI4212 aFuc and the anti-migis antibody consistently reduced the number of spots to near zero. *P<0.05 for treatment group relative to media control (anti-CD40 and IL-4). One-way ANOVA followed by Dunnetts. (b) A substantial drop in the amount of IgE expression was observed by qPCR. IgE, immunoglobulin E; PBMC, peripheral blood mononuclear cell.

Nyborg, A. C., Zacco, A., Ettinger, R., Borrok, M. J., Zhu, J., Martin, T.,... & Herbst, R. (2016). Development of an antibody that neutralizes soluble IgE and eliminates IgE expressing B cells. Cellular & molecular immunology, 13(3), 391.

Figure 5 In vitro characterization of MEDI4212. Neutralization of human IgE was assessed in FcεRI-dependent (A, B and C) or CD23-dependent (D and E) assays.

(A) Human IgE-FcεRI binding assay; (B) RBL-ER51 calcium signaling; (C) LAD2 β-hexosaminidase release; (D) IM9 binding; (E) U937 phagocytosis of IGROV1 cells. Folate receptor α (FRα). Data points represent average of duplicate determinations from a representative experiment (A) or mean ± SEM of combined data from 3–11 experiments (B–E).

Cohen, E. S., Dobson, C. L., Käck, H., Wang, B., Sims, D. A., Lloyd, C. O.,... & O’Brien, S. (2014, May). A novel IgE-neutralizing antibody for the treatment of severe uncontrolled asthma. In MAbs (Vol. 6, No. 3, pp. 755-763). Taylor & Francis.