Therapeutic Efficacy of YH35324 on FcεRIα-Mediated Mast Cell/Basophil Activation

Abstract

Purpose: Immunoglobulin E (IgE) induces mast cell/basophil activation by binding with FcεRIα and contributes to the development of allergic disease, in which targeting IgE has been considered an effective therapeutic strategy. YH35324 (YH) is a new hybrid protein with an extracellular domain consisting of FcεRIα, and its pharmacodynamic effect and safety were validated. This study aimed to evaluate the therapeutic potential of YH as an anti-IgE immunomodulator compared with omalizumab (Oma).

Methods: To evaluate the in vitro efficacy of YH in human mast cells, YH was treated with various methods, and the changes were confirmed through flow cytometry, immunoblot analysis, and immunocytochemistry. To evaluate the ex vivo efficacy of YH, the expression of FcεRIα on the surface of blood basophils was measured in 64 subjects with allergic diseases by flow cytometry. Serum soluble FcεRIα, CD23, and Mas-Related G-Protein Coupled Receptor Member X2 levels were measured by enzyme-linked immunosorbent assay.

Results: The YH-administered group exhibited significantly lower expression of FcεRIα on peripheral basophils compared to the Oma-administered group up to 14 days post-administration. YH directly suppressed FcεRIα expression on the surface of LAD2 cells, as it was bound to IgE-unbound FcεRIα and migrated into the cells by actin-dependent endocytosis, then was recycled by FcRn binding in the lysosome in vitro. Serum soluble FcεRIα levels were increased in the YH-administered group compared to the other groups and showed a positive correlation with serum-free IgE.

Conclusions: YH represents a new therapeutic agent for IgE-mediated allergic disease. Further studies are needed to evaluate its additional effects on the FcεRIα-mediated autoimmune mechanism.

Keywords: Fc epsilon RI; IgE; allergy; anti-IgE antibodies; basophil; mast cell; urticaria.

Fig. 1. Time-course changes in FcεRIα expression on basophils in the study subjects and their correlation with serum-free IgE levels. (A) Time-course changes in FcεRIα expression on peripheral basophils in the YH (closed square, n = 39)-, Oma (open triangle, n = 16)- and placebo (open circles, n = 9)-administered groups. The linear chart shows the median MFI. P values were determined by the Multiple t-test plus the Bonferroni–Dunn method. (B) Time-course changes in FcεRIα expression (median MFI) based on YH concentrations (open diamond, 1 mg/kg, n = 8; closed diamond, 3 mg/kg; n = 8; open square, 6 mg/kg, n = 8; closed square, 9 mg/kg, n = 8). (C, D) Percent surface FcεRIα expression relative to the baseline value at the indicated time points (D7 and D14) compared among the YH- (n = 39), Oma- (n = 16), and placebo (n = 9)-administered groups. Data are presented as median percent with 95% CI. The Multiple t-test obtained P values plus the Bonferroni–Dunn method. (E-G) Correlation between FcεRIα expression on the surface of basophils (MFI) and serum-free IgE (ng/mL) levels in the YH-administered group at the indicated time points (D0, D14 and D21).

YH, YH35324; Oma, omalizumab; D, days; MFI, mean fluorescence intensity; CI, confidence interval. ^***P < 0.005, ^*****P < 0.0001 represents statistics between the YH- and placebo-administered groups. ^#P < 0.05 and ^##P < 0.01 represent statistics between the YH- and Oma-administered groups.

Fig. 2. Changes in expression of total/IgE-bound FcεRIα on the surface of LAD2 cells following YH or Oma treatment. (A, B) Flow cytometry histogram analysis of total/IgE-bound FcεRIα in the pre-treated group. LAD2 cells were pre-treated with YH or Oma at the indicated doses (0.1, 1, and 1,000 µg/mL), followed by IgE-sensitization (100 ng/mL). A single IgE treatment was used as a positive control, whereas a mock treatment was used as a negative control. (C) Flow cytometry dot plot analysis of total/IgE-bound FcεRIα expression in the YH/Oma (1 µg/mL) pre-treated group. (D, E) Flow cytometry histogram analysis of total/IgE-bound FcεRIα in the post-treated group. LAD2 cells were sensitized with IgE, followed by YH or Oma treatment at the indicated doses (100, 250, and 1,000 µg/mL). (F) Flow cytometry dot plot analysis of total/IgE-bound FcεRIα expression in the YH/Oma (100 µg/mL) post-treated group.

IgE, immunoglobulin E; YH, YH35324; Oma, omalizumab.

Fig. 3. Intracellular migration of YH in LAD2 cells. (A) Degradation of endogenous (endo) FcεRIα in LAD2 cells in the YH/Oma pre- or post-treated group. Protein expression was normalized to the expression of GAPDH. (B) Translocation of YH in human LUVA cells. Protein expression was normalized to the expression of β-actin. (C) Changes in FcεRIα localization in LUVA cells following YH treatment. Cells were co-immunolabeled for FcεRIα (purple) and ER marker (calnexin; green) or (D) lysosome marker (LAMP1; green) and imaged by confocal microscopy. White scale bars represent 5 μm. (E) FcεRIα is co-localized at clathrin-Rab11-FcRn following YH treatment. Cells were co-immunolabeled for FcεRIα (red), clathrin (green), Rab11 (green), or FcRn (green). White scale bars represent 5 µm.

YH, YH35324; Oma, omalizumab; endo, endogenous; ER, endoplasmic reticulum; LAMP1, lysosomal-associated membrane protein 1; DAPI, 4′,6-diamidino-2-phenylindole; Rab11, Ras-related protein Rab-11; FcRn, neonatal Fc receptor.

Fig. 4. Actin-dependent endocytosis, exocytosis, and recycling of YH in human mast cells. (A) Reduced endocytosis of YH after actin polymerization inhibitor (Cyt. D) treatment. DMSO was used as a vehicle for Cyt. D. (B, C) Immunoprecipitation of YH complexes composed of FcεRIα and β-actin. (D) Exocytosis of YH from LUVA cells after replacing the medium. Cells and medium were harvested after 24 and 48 hours. Exosomes were separated from the harvested medium. (E) Efficacy of YH that can be recycled. LAD2 cells were treated with YH and replaced with the new medium. The released medium was harvested at the last exchange time (control) and 24 hours after incubation (24 hours). YH was precipitated with protein A/G agarose to detect YH in the medium. (F) The released medium was cultured with new LAD2 cells, and cell lysates were extracted 48 hours later.

YH, YH35324; Cyt. D, cytochalasin D; endo, endogenous; IP, immunoprecipitation; C, control.

Fig. 5. Time-course changes in sFcεRIα, sCD23, and sMRGPRX2 levels and their correlations with serum-free IgE levels in the serum of the study subjects. (A-C) Soluble form of FcεRIα, CD23, and MRGPRX2 was measured by ELISA at the indicated time points (D0 and D112) in the YH (closed square, n = 22)-, Oma (open triangles, n = 5)- and placebo (open circles, n = 6)-administered groups. Data are presented as the median percent (% of D0). P values were obtained by the Mann-Whitney test. (D, E) Correlations between sFcεRIα (ng/mL) and free IgE levels in the YH-administered group at D0 and D112.

YH, YH35324; Oma, omalizumab; D, days; sFcεRIα, soluble form of FcεRIα; sCD23, soluble form of CD23; sMRGPRX2, soluble form of mas-related G-protein coupled receptor family member X2; MRGPRX2, mas-related G-protein coupled receptor family member X2; ELISA, enzyme-linked immunosorbent assay. ^**P < 0.01 represent statistics between the YH- and placebo-administered groups. ^#P < 0.05 represents statistics between the YH- and Oma-administered groups.

Fig. 6. A schematic overview of a novel mechanism of YH35324 in depleting FcεRIα on the surface of mast cells/basophils. (1) High concentrations of YH35324 outside the cells are induced by actin-dependent endocytosis. In the early stage of endocytosis, IgE-unbound YH35324 could thought to selectively bind to FcεRIα, which is also unbound to IgE on the cell surface, thereby inducing the dissociation of the other subunits, β and γ. (2) In the endo-lysosomal state, YH35324 can bind to FcRn under reduced pH conditions. YH35324 bound to FcRn were not degraded by lysosomal proteases, whereas other proteins that do not bind to FcRn were degraded. FcεRIα is cleaved into the sFcεRIα form by a protease that has not yet been identified. (3) YH35324 bound to FcRn is retained in recycling endosomes (where Rab11 is located) and (4) released together sFcεRIα via exocytosis.

IgE, immunoglobulin E; FcεRIα, α subunit of FcεRI; FcRn, neonatal Fc receptor; sFcεRIα, soluble FcεRIα.