Mouse Anti-IgE Recombinant Antibody (clone 47H4); Fab Fragment (CAT#: HPAB-AP102-YC-F(E))

Figure 1 M1′-specific antibody specifically binds human M1′ in the context of membrane IgE with high affinity.

(A) M1′-specific antibody 47H4 bound human membrane IgE-transfected Daudi B cells but not Daudi transfectants expressing membrane IgE that lacks the M1′ sequence. M1′-specific antibody 47H4 also bound the U266 myeloma cell line, which naturally expresses low levels of membrane IgE. Isotype control antibody staining is shown as the gray areas, and 47H4 antibody staining is shown as black lines. (B) Scatchard analysis of M1′-specific antibody 47H4 binding to membrane IgE-transfected Daudi cells. The left panel shows a competition binding curve, where each data point represents the ratio of iodinated 47H4 antibody bound to cells/total iodinated and unlabeled 47H4 antibody on the y axis vs. the total concentration of iodinated and unlabeled 47H4 antibody on the x axis. The right panel shows a Scatchard plot, where each data point represents the ratio of iodinated 47H4 antibody bound to cells/unbound iodinated 47H4 antibody on the y axis vs. the concentration of bound iodinated 47H4 antibody on the x axis The mean binding affinity ± SD from 2 separate experiments is 0.54 ± 0.02 nM.

Brightbill, H. D., Jeet, S., Lin, Z., Yan, D., Zhou, M., Tan, M.,... & Wong, T. (2010). Antibodies specific for a segment of human membrane IgE deplete IgE-producing B cells in humanized mice. The Journal of clinical investigation, 120(6), 2218-2229.

Figure 2 M1′-specific antibody prevents primary and memory IgE responses in human M1′ knockin mice.

(A) Experimental design for TNP-OVA–induced primary and memory immune responses. Human M1′ knockin mice were immunized with TNP-OVA/alum on day 0. In the primary IgE response model (n = 26 per group), mice were treated with 10 mg/kg M1′-specific antibody 47H4 or mIgG1 control antibody 3 times a week from day 0–28. Treatment with M1′-specific antibody 47H4 antibody (B) prevented the primary TNP-OVA–specific IgE response, but (C) did not affect the primary TNP-OVA–specific IgG1 response. In the memory IgE response model (n = 10 per group), mice were challenged with TNP-OVA alone on day 28 after the primary immunization, and mice were treated with 10 mg/kg M1′-specific antibody 47H4 or mIgG1 control antibody 3 times a week from day 28–49. Treatment with M1′-specific antibody 47H4 antibody (D) reduced the memory TNP-OVA–specific IgE response, but (E) did not affect the memory TNP-OVA–specific IgG1 response. Results are mean ± SD. *P < 0.05 (Bonferroni correction for pairwise comparisons).

Brightbill, H. D., Jeet, S., Lin, Z., Yan, D., Zhou, M., Tan, M.,... & Wong, T. (2010). Antibodies specific for a segment of human membrane IgE deplete IgE-producing B cells in humanized mice. The Journal of clinical investigation, 120(6), 2218-2229.

Figure 3 Therapeutic treatment with M1′-specific antibody specifically reduces IgE in N. brasiliensis infection.

(A) Experimental design for therapeutic M1′-specific antibody treatment of N. brasiliensis infection. Human M1′ knockin mice (n = 10 per group) were infected with 500 N. brasiliensis L3 larvae on day 0. Mice were treated with 10 mg/kg M1′-specific antibody 47H4 or mIgG1 control antibody 3 times per week from day 11 after infection to the end of the study at day 21 after infection. Treatment with M1′-specific antibody 47H4 antibody reduced (B) total serum IgE levels and (C) the number of IgE-producing cells in the mesenteric lymph nodes, but did not affect (D) the percentage of total syndecan+ plasma cells in the mesenteric lymph nodes. Results are mean ± SD. *P < 0.05 (Bonferroni correction for pairwise comparisons); **P < 0.0001 (Dunnett's test).

Brightbill, H. D., Jeet, S., Lin, Z., Yan, D., Zhou, M., Tan, M.,... & Wong, T. (2010). Antibodies specific for a segment of human membrane IgE deplete IgE-producing B cells in humanized mice. The Journal of clinical investigation, 120(6), 2218-2229.

Figure 4 Therapeutic treatment with M1′-specific antibody specifically reduces IgE in a mouse model of allergic asthma.

(A) Experimental design for therapeutic M1′-specific antibody treatment of mouse allergic asthma model. Human M1′ knockin mice (n = 8 per group) were immunized with TNP-OVA/alum on day 0 and challenged with 7 daily aerosol administrations of TNP-OVA, starting on day 35. Mice were treated with 100 μg of M1′-specific antibody 47H4 or control mIgG1 antibody daily from day 39 through day 45. Treatment with M1′-specific antibody 47H4 antibody reduced (B) TNP-OVA–specific IgE levels, but did not affect (C) TNP-OVA–specific IgG1 levels. Treatment with M1′-specific antibody 47H4 antibody also reduced (D) the number of IgE-producing cells, but did not affect (E) the percentage of total syndecan+ plasma cells in the spleen, as measured on day 63. Results are mean ± SD. *P < 0.05 (Bonferroni correction for pairwise comparisons); **P < 0.05 (Dunnett's test).

Brightbill, H. D., Jeet, S., Lin, Z., Yan, D., Zhou, M., Tan, M.,... & Wong, T. (2010). Antibodies specific for a segment of human membrane IgE deplete IgE-producing B cells in humanized mice. The Journal of clinical investigation, 120(6), 2218-2229.

Figure 5 Efficacy of M1′-specific antibody is mediated by apoptosis.

(A) M1′-specific antibody 47H4 antibody induced apoptosis of human membrane IgE-transfected Daudi cells. (B) Caspase inhibitor z-VAD inhibited M1′-specific antibody 47H4 antibody–induced apoptosis. Apoptosis is measured by flow cytometry using anti-Annexin V antibody. The control antibody is mIgG1. (C) M1′-specific antibody 47H4 antibody reduced the percentage of GFP+ IgE-switched B cells on day 4 in human M1′ knockin mouse splenocyte cultures stimulated with anti-CD40 antibody and recombinant IL-4. (D) M1′-specific antibody 47H4 antibody reduced the generation of soluble IgE on day 4 in human M1′ knockin mouse splenocyte cultures stimulated with anti-CD40 antibody and recombinant IL-4. (E) Experimental design for M1′-specific antibody treatment of N. brasiliensis infection. Human M1′ knockin mice (n = 9–10 per group) were infected with 500 N. brasiliensis L3 larvae on day 0. Mice were treated with 10 mg/kg M1′-specific antibody 47H4 wild-type, 47H4-DANA, or mIgG1 control antibody 3 times a week from day 0 to 21. (F) Treatment with 47H4 wild-type and 47H4-DANA antibody resulted in equivalent inhibition of N. brasiliensis–induced serum IgE. Results are mean ± SD. *P < 0.05 (Bonferroni correction for pairwise comparisons). (G) Representative flow cytometry plots of IgE-switched GFP+ B cells in the spleens of N. brasiliensis–infected mice treated with M1′-specific antibody 47H4 wild-type or mIgG1 control antibody on day 21. Numbers indicate the percentage of CD19+ GFP+ cells and are representative of at least 3 experiments. (H) M1′-specific antibody 47H4 antibody reduced the percentage of IgE-switched GFP+ B cells in the spleens of N. brasiliensis–infected mice on day 21. (A–D and H) Results are mean ± SD. (C, D, and H) *P < 0.05 (Dunnett's test).

Brightbill, H. D., Jeet, S., Lin, Z., Yan, D., Zhou, M., Tan, M.,... & Wong, T. (2010). Antibodies specific for a segment of human membrane IgE deplete IgE-producing B cells in humanized mice. The Journal of clinical investigation, 120(6), 2218-2229.

Figure 6 Characterization of a humanized M1′-specific antibody.

(A) Humanized M1′-specific 47H4 antibody (h47H4) bound human membrane IgE-transfected Daudi B cells but not Daudi transfectants expressing membrane IgE that lacks the M1′ sequence. Humanized M1′-specific 47H4 antibody also bound the U266 myeloma cell line, which naturally expresses low levels of membrane IgE. (B) Scatchard analysis of humanized M1′-specific 47H4 antibody binding to membrane IgE-transfected Daudi cells. The left panel shows a competition binding curve, where each data point represents the ratio of iodinated h47H4 antibody bound to cells/total iodinated and unlabeled h47H4 antibody on the y axis vs. the total concentration of iodinated and unlabeled h47H4 antibody on the x axis. The right panel shows a Scatchard plot, where each data point represents the ratio of iodinated h47H4 antibody bound to cells/unbound iodinated h47H4 antibody on the y axis vs. the concentration of bound iodinated h47H4 antibody on the x axis. The mean binding affinity ± SD from 2 separate experiments is 1.50 ± 0.14 nM.

Brightbill, H. D., Jeet, S., Lin, Z., Yan, D., Zhou, M., Tan, M.,... & Wong, T. (2010). Antibodies specific for a segment of human membrane IgE deplete IgE-producing B cells in humanized mice. The Journal of clinical investigation, 120(6), 2218-2229.

Figure 7 Humanized M1′-specific antibody induces apoptosis of membrane IgE-transfected Daudi cells and specifically reduces IgE in an atopic human PBMC-SCID model.

(A) Humanized M1′-specific 47H4 antibody (h47H4) induced apoptosis of human membrane IgE-transfected Daudi cells. (B) Experimental design for atopic human PBMC-SCID model. Sublethally irradiated SCID-beige mice (n = 11–12 per group) were injected with 108 PBMCs from an atopic human donor. Mice were treated on days 2, 3, and 4 with 100 ng recombinant human IL-4 and on days 0 and 3 with 100 μg each of anti–human IFN-γ and anti–human IL-12 neutralizing antibodies. Then, 300 μg humanized M1′-specific 47H4 antibody or control hIgG1 antibody was delivered 3 times a week, starting on day 0. Treatment with humanized M1′-specific 47H4 antibody reduced (C) total serum human IgE levels and (D) the number of splenic human IgE-producing plasma cells, but did not affect (E) total human IgM levels or (F) the frequency of total CD38+ PC+ splenic plasma cells. Results are mean ± SD. *P < 0.0001 (Dunnett's test).

Brightbill, H. D., Jeet, S., Lin, Z., Yan, D., Zhou, M., Tan, M.,... & Wong, T. (2010). Antibodies specific for a segment of human membrane IgE deplete IgE-producing B cells in humanized mice. The Journal of clinical investigation, 120(6), 2218-2229.

Figure 8 IgE suppression by passive immunization in the new IgE-huEMPD mouse line.

(A) Schematic representation of the human EMPD knock-in (grey box) replacing the shorter murine EMPD region drawn in scale. The adjacent regions encoding Ce3, Ce4 as well as transmembrane and cytoplasmic regions remained untouched. Boxes separated by connecting lines represent exons spaced by introns. (B) The treatment schedule combines passive immunization (AB) at weekly intervals with IgE induction by three ovalbumin injections (ova). Numbers represent days after start of treatment. (C) Total IgE levels were measured by ELISA before the first mAB application and after the treatment as shown in B. Each animal is represented by one dot. Samples were measured once (before) or twice (after) in 1–3 dilutions, the mean is shown. Significance levels were calculated in each group between pre and post treatment levels and between the groups based on the delta (post-pre levels) as described in the Materials and methods section, ns non-significant p> 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 19 (IgG2a), 16 (47H4) or 19 (15cl12) mice. (D) Ovalbumin-specific IgE was measured by ELISA before the first mAB application and after the treatment as in C. Samples were measured once (before), twice or three times (after), the mean is shown. Before treatment, all animals but two were below the limit of quantification indicated by the asterisk (*) and the grey area (< 40 ng/ml). Groups sizes and statistical significance as in C.

Vigl, B., Salhat, N., Parth, M., Pankevych, H., Mairhofer, A., Bartl, S., & Smrzka, O. W. (2017). 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, 449, 28-36.