Yonsei Med J.  2016 Nov;57(6):1412-1419. 10.3349/ymj.2016.57.6.1412.

Antibody to FcεRIα Suppresses Immunoglobulin E Binding to High-Affinity Receptor I in Allergic Inflammation

• Hong JY
• Bae JH
• Lee KE
• Kim M
• Kim MH
• Kang HJ
• Park EH
• Yoo KS
• Jeong SK
• Kim KW
• Kim KE
• Sohn MH

• KMID: 2427159
• DOI: http://doi.org/10.3349/ymj.2016.57.6.1412

Abstract

PURPOSE
High-affinity receptor I (FcεRI) on mast cells and basophils plays a key role in the immunoglobulin E (IgE)-mediated type I hypersensitivity mediated by allergen cross-linking of the specific IgE-FcεRI complex. Thus, prevention of IgE binding to FcεRI on these cells is an effective therapy for allergic disease. We have developed a strategy to disrupt IgE binding to FcεRI using an antibody targeting FcεRIα.
MATERIALS AND METHODS
Fab fragment antibodies, which lack the Fc domain, with high affinity and specificity for FcεRIα and effective inhibitory activity against IgE-FcεRI binding were screened. IgE-induced histamine, β-hexosaminidase and Ca2+ release in basophils were determined by ELISA. A B6.Cg-Fcer1a(tm1Knt) Tg(FCER1A)1Bhk/J mouse model of passive cutaneous anaphylaxis (PCA) was used to examine the inhibitory effect of NPB311 on allergic skin inflammation.
RESULTS
NPB311 exhibited high affinity to human FcεRIα (KD=4 nM) and inhibited histamine, β-hexosaminidase and Ca2+ release in a concentration-dependent manner in hFcεRI-expressing cells. In hFcεRIα-expressing mice, dye leakage was higher in the PCA group than in controls, but decreased after NPB311 treatment. NPB311 could form a complex with FcεRIα and inhibit the release of inflammation mediators.
CONCLUSION
Our approach for producing anti-FcεRIα Fab fragment antibody NPB311 may enable clinical application to a therapeutic pathway in IgE/FcεRI-mediated diseases.

Keyword

Immunoglobulin E (IgE); high-affinity IgE receptor I (FcεRI); Fab fragment; antibody affinity; passive cutaneous anaphylaxis

MeSH Terms

Allergens
Animals
Antibodies, Monoclonal/metabolism/*pharmacology
Basophils/*immunology/metabolism
Humans
Hypersensitivity/immunology
Immunoglobulin E/immunology/*metabolism/physiology
Immunoglobulin Fab Fragments/*metabolism
Inflammation/metabolism
Mast Cells
Mice
Receptors, IgE/*immunology/metabolism/physiology
Allergens
Antibodies, Monoclonal
Immunoglobulin Fab Fragments
Receptors, IgE
Immunoglobulin E

Figure

• 

  Fig. 1 Development of an anti-FcεRI Fab fragment antibody NPB311. (A) Human FcεRIα was coated on the plate, followed by incubation with the indicated antibodies. Specific antibody binding was detected by further incubation with hIgE, followed by incubation with horseradish peroxidase (HRP)-conjugated mouse-anti-hIgE and development with Immune Glo substrate. (B) hFcεRIα-expressing RBL-SX38 cells were seeded in a 96-well plate followed by incubation with the indicated antibodies. Specific binding was detected by further incubation with HRP-conjugated anti-hIgG and development by Immune Glo substrate. FcεRI, high-affinity IgE receptor I; hIgE, human immunoglobulin E; RBL, rat basophilic leukemia.

• 

  Fig. 2 Quality and purity of purified anti-FcεRIα Fab fragment antibody NPB311. (A) Size-exclusion chromatography analysis of NPB311. After papain digestion, the purity of NPB311 was checked by HPLC (TSKgel column). 1:IgG control, 2:NPB311. (B) After freeze-drying, NPB311 was incubated at 4℃ for the indicated times. Protein samples in sodium dodecyl sulfate (SDS) buffer were subjected to electrophoresis under non-reducing and reducing conditions through a SDS-PAGE gel. The gel was stained with Coomassie brilliant blue G-250. FcεRI, high-affinity immunoglobulin E receptor I.

• 

  Fig. 3 Effect of NPB311 on [Ca2+] in RBL-SX38 cells. (A) RBL-SX38 cells (5×104/well) were incubated at 37℃ for 24 h. NP-IgE were treated with/without NPB311 (0, 0.16, 0.8, 4, 20, 100, and 500 nM) and incubated at 37℃ for 2 h, then FLIPR calcium 4 dye were added. After incubation for 1 h, NP-BSA was added and then FLUO-4AM (relative fluorescence units, RFU) was detected every 2 sec and 160 times. (B) RBL-SX38 cells (5×104/well) were incubated at 37℃ for 24 h. NPB311 (0 and 500 nM) were incubated 37℃ for 2 h, then FLIPR calcium 4 dye were added. After incubation, NP-BSA was added and then FLUO-4AM was detected every 2 sec and 90 times. Results are presented as mean±SD from four independent experiments. FLIPR, Fluorometric Imaging Plate Reader.

• 

  Fig. 4 Effect of NPB311 on IgE-mediated degranulation in RBL-SX38 cells. (A) Surface plasmon resonance (SPR) traces of the NPB311 binding to FcεRIα. Binding curves for FcεRIα were performed at 0, 6.25, 12.5, 25, 50, and 100 nM. (B and C) NP-hIgE was treated with various concentrations of antibodies (NPB311 or omalizumab or hIgG), and then 0.5 µg of NP-BSA was added. Supernatants were analyzed for β-hexosaminidase activity and histamine release. Results are given as mean±SD, from three separate experiments. Compared with hIgG, *p<0.05; compared with omalizumab, †p<0.05. IgE, immunoglobulin E; hIgE, human IgE; RBL, rat basophilic leukemia; FcεRI, high-affinity IgE receptor I; NP, nitrophenylacetyl.

• 

  Fig. 5 Effect of NPB311 on dye leakage in a passive cutaneous anaphylaxis model. Human FcεRIα-expressing transgenic mice received an injection of NP-hIgE with/without NPB311 (0.05 mg/kg) intradermally into the ear. After (A and B) NPB311 or (C) IgG injection, mice were intravenously administrated with 1 mg/mL of NP-BSA in Evans blue dye and then the color intensity was evaluated. Left ear: NP-IgE; Right ear: NP-IgE/NPB311. FcεRI, high-affinity IgE receptor I; NP, nitrophenylacetyl; hIgE, human IgE; IgE, immunoglobulin E.

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