Blocking Allergic Reaction through Targeting Surface-Bound IgE with Low-Affinity Anti-IgE Antibodies

Abstract

Allergic disorders have now become a major worldwide public health issue, but the effective treatment options remain limited. We report a novel approach to block allergic reactivity by targeting the surface-bound IgE of the allergic effector cells via low-affinity anti-human IgE Abs with dissociation constants in the 10^-6 to 10^-8 M range. We demonstrated that these low-affinity anti-IgE mAbs bind to the cell surface-bound IgE without triggering anaphylactic degranulation even at high concentration, albeit they would weakly upregulate CD203c expression on basophils. This is in contrast to the high-affinity anti-IgE mAbs that trigger anaphylactic degranulation at low concentration. Instead, the low-affinity anti-IgE mAbs profoundly block human peanut- and cat-allergic IgE-mediated basophil CD63 induction indicative of anaphylactic degranulation; suppress peanut-, cat-, and dansyl-specific IgE-mediated passive cutaneous anaphylaxis; and attenuate dansyl IgE-mediated systemic anaphylaxis in human FcεRIα transgenic mouse model. Mechanistic studies reveal that the ability of allergic reaction blockade by the low-affinity anti-IgE mAbs was correlated with their capacity to downregulate the surface IgE and FcεRI level on human basophils and the human FcεRIα transgenic mouse bone marrow-derived mast cells via driving internalization of the IgE/FcεRI complex. Our studies demonstrate that targeting surface-bound IgE with low-affinity anti-IgE Abs is capable of suppressing allergic reactivity while displaying an excellent safety profile, indicating that use of low-affinity anti-IgE mAbs holds promise as a novel therapeutic approach for IgE-mediated allergic diseases.

Figure 1

Basophil CD63 expression profiles of the allergic subjects induced by allergens and anti-IgE Abs. Three-color FACS analysis was employed, and the CD123⁺/HLA-Dr⁻ population was gated as basophils for measurement of CD63 expression. (A–C) The dose-response basophil CD63 expression of three peanut allergic subjects induced by Ara h2, E4.15, and LAIGE p6.2. (D and E) The dose-response basophil CD63 expression profiles of two cat allergic subjects. (F) Summary of the kinetic dose-response curves of CD63 expression to allergens (Ara h2+Fel d1), E4.15 and p6.2 (n=5).

Figure 2

Basophil CD63 and CD203c expression profiles of the cat allergic and normal healthy subjects induced by Fel d1 and anti-IgE Abs. Four-color FACS analysis was employed to simultaneously determine the basophil CD63 and CD203c expression. The CD123⁺/HLA-Dr⁻ population was gated as basophils. (A) The dose-response basophil CD63/CD203c expression of the cat allergic subjects (n=3) induced by Fel d1, E4.15, and LAIGE p6.2. (B) The dose-response basophil CD63/CD203c expression of the normal healthy subjects (n=5) induced by PAE, E4.15, and LAIGE p6.2. (C and D) Dose-dependent CD203c upregulation promoted by LAIGE p6.2 in both cat allergic and normal healthy subjects, respectively (solid lines). The dot lines represent the maximal CD203c upregulation driven by Fel d 1, PAE and E4.15 for comparison. (E and F) Summary of the kinetic dose-response curves of CD63 (open symbols) and CD203c (filled symbols) expression driven by Fel d1, E4.15 and LAIGE p6.2. (G and H) Histamine release levels from cat allergic (n=3) and normal subjects (n=5).

Figure 3

Basophil degranulation-triggering capacity of anti-IgE mAbs. (A) Dose-response BAT of anti-IgE mAbs with differential affinities to IgE (n=3). (B) Basophil IL-4 production induced by LAIGE p6.2, with PAE as a positive control (n=3).

Figure 4

Safety profiles of LAIGE p6.2 tested with hFcεRIα Tg mouse model. (A) PCA. The various concentrations of p6.2 as labelled were used to locally challenge IgE systemically sensitized mice. PAE was included as a positive, and PBS as negative, control. (B) Quantitative assessment of p6.2 induced PCA using EBD extraction (n=4). (C) Systemic anaphylaxis clinical scores from the p6.2 and E4.15 challenged mice (n=3). (D) Systemic anaphylaxis core body temperature changes from p6.2 (250 μg) and E4.15 (50 μg) challenged mice (n=3). (E–J) Comparison of allergic mediator, cytokine and chemokine release triggered by E4.15 and p6.2 challenged mice (n=4).

Figure 5

LAIGE p6.2 blocks allergen-induced basophil CD63 expression. (A) FACS profile of the peanut allergen induced CD63^high expression inhibited by p6.2. (B) Summary of the inhibitory effects of p6.2 on peanut allergen induced CD63^high expression (n=5). (C) FACS profile of the cat allergen Fel d1 induced CD63^high expression inhibited by p6.2. (D) Summary of the inhibitory effects of p6.2 on cat allergen Fel d1 induced CD63^high expression (n=4).

Figure 6

The therapeutic index of high and low affinity anti-IgE mAbs. The therapeutic index of the peanut BAT based approach was calculated as “degranulation triggering dose” dividing by the “therapeutic dose”. The BAT triggering dose was defined as the lowest anti-IgE mAb concentration capable of inducing >5% basophil CD63 expression, whereas the effective therapeutic dose was arbitrarily defined as the lowest anti-IgE mAb dose sufficient to inhibit >50% of peanut allergen triggered CD63 expression (n=3).

Figure 7

LAIGE p6.2 blocks IgE-mediated PCA in hFcεRIα Tg mice. (A) p6.2 inhibited peanut allergic IgE-mediated PCA. (B) Quantitative EBD assessment from the peanut allergic IgE-mediated PCA spots. (C & D) p6.2 inhibited cat allergic IgE-mediated PCA. (E & F) p6.2 inhibited dansyl specific IgE-mediated PCA. The plus (+) was the PAE positive control, whereas the minus (-) was PBS negative control. N=4 mice for each group.

Figure 8

P6.2 attenuates dansyl IgE-mediated systemic anaphylaxis. (A). The rectal temperature changes of the dansyl IgE sensitized hFcεRIα Tg mice in control (mIgG₁) and p6.2 treated mice (n=4). (B) The anaphylactic clinical scores in above two groups of mice.

Figure 9

Down-regulation of surface IgE and FcεRI on basophils and BMMCs by p6.2. (A) Effects of p6.2 on expression of the basophil surface FcεRI and IgE levels determined by flow cytometry. CD123 staining was included as an internal staining control. The data shown is the representative of three experiments. (B) Effects of p6.2 on the BMMC surface FcεRI and IgE expression. The c-kit staining was included as an internal staining control. Shown is the representative data from two experiments.

Figure 10

p6.2 triggers the surface IgE internalization and co-localization with FcεRI. (A) Effect of p6.2 on promoting surface IgE internalization. (B) Internalized FITC-IgE in the confocal sections. The highlighted area of the middle panel of Figure 10A was subjected to confocal section analysis. The data are the representative of two experiments with similar results. (C) Co-localization of IgE with FcεRI in the cytoplasmic compartment of BMMCs. The FcεRI was stained as red, IgE as green and the co-localization of red and green merge as yellow, whereas the nucleus was stained as blue. The BMMCs were treated for 24 (panel 1–6) and 48 (panel 7–12) hours respectively. The data are the representative of >20 cells from each condition analyzed.