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. 2016 Apr 6;173(10):1629–1638. doi: 10.1111/bph.13463

Intranasal exposure to monoclonal antibody Fab fragments to Japanese cedar pollen Cry j1 suppresses Japanese cedar pollen‐induced allergic rhinitis

S Yoshino 1,✉, N Mizutani 1
PMCID: PMC4842921  PMID: 26895546

Abstract

Background and Purpose

Fab fragments (Fabs) of antibodies have the ability to bind to specific allergens but lack the Fc portion that exerts effector functions via binding to receptors including FcεR1 on mast cells. In the present study, we investigated whether intranasal administration of the effector function‐lacking Fabs of a monoclonal antibody IgG1 (mAb, P1‐8) to the major allergen Cry j1 of Japanese cedar pollen (JCP) suppressed JCP‐induced allergic rhinitis in mice.

Experimental Approach

Balb/c mice sensitized with JCP on days 0 and 14 were challenged intranasally with the pollen on days 28, 29, 30 and 35. Fabs prepared by the digestion of P1‐8 with papain were also administered intranasally 15 min before each JCP challenge.

Key Results

Intranasal administration of P1‐8 Fabs was followed by marked suppression of sneezing and nasal rubbing in mice with JCP‐induced allergic rhinitis. The suppression of these allergic symptoms by P1‐8 Fabs was associated with decreases in mast cells and eosinophils and decreased hyperplasia of goblet cells in the nasal mucosa.

Conclusions and Implications

These results demonstrated that intranasal exposure to P1‐8 Fabs was effective in suppressing JCP‐induced allergic rhinitis in mice, suggesting that allergen‐specific mAb Fabs might be used as a tool to regulate allergic pollinosis.

Abbreviations

Fabs

Fab fragments

i.n.

intranasally

JCP

Japanese cedar pollen

P1‐8

mAb to Cry j1

pAb

polyclonal antibody

O1‐10

mAb to OVA

OVA

ovalbumin

Tables of Links

TARGETS
Other proteins
TNF‐α
LIGANDS
Certolizumab
Omalizumab
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These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015).

Introduction

Allergic rhinitis is the most common allergic disease, and it affects 1.4 billion people worldwide according to a recent report by Settipane and Schwindt (2013). The clinical symptoms include rhinorrhoea, sneezing, nasal itching and nasal congestion (Skoner, 2001; Borish, 2003; Galli et al., 2008). Elevated levels of allergen‐specific IgE and infiltration of inflammatory cells such as mast cells and eosinophils in the nasal mucosa have been observed in patients with allergic rhinitis (Skoner, 2001; Borish, 2003; Galli et al., 2008), suggesting their role in the allergic disease. For instance, histamine released following degranulation of allergen‐exposed mast cells has been shown to clinically play a role in rhinorrhoea, sneezing and nasal itching (Cingi et al., 2009; Meltzer, 2013; Mygind, 2014).

Treatments for allergic rhinitis include anti‐histamine drugs, leukotriene receptor antagonists, a‐adrenoceptor agonists and glucocorticoids (Balle et al., 1982; Douglas, 1985; Pipkorn et al., 1987; Simons, 1989; Quraishi et al., 2004). However, these drugs do not act as allergen‐specific suppressors and thus cause various side effects. For instance, first‐generation anti‐histamines cause sedation, fatigue, cognitive decline and urinary retention (De Vos et al., 2008; Ferrer et al., 2010). While subcutaneous and sublingual immunotherapies are currently available as an allergen‐specific immunotherapy in IgE‐mediated diseases, including pollinosis allergy, (Dretzke et al., 2013; Casale and Stokes, 2014), immediate beneficial effects are not expected from these immunotherapies (Abramson et al., 1995; Pipet et al., 2009; Keles et al., 2011; Passalacqua and Canonica, 2011).

Fab fragments (Fabs) produced by the digestion of antibodies with papain maintain the ability to bind specific allergens but lack the Fc portion which is the binding site for receptors on immune cells, which can participate in downstream inflammatory cascades (Yoshino et al., 2010). Clinically, certolizumab, which is a PEGylated TNF‐α‐specific Fabs, has been used for the treatment of patients with rheumatoid arthritis and Crohn's disease (Da et al., 2013; Deeks, 2013; Schiff et al., 2014). We previously demonstrated that intratracheal and intranasal (i.n.) exposure to ovalbumin (OVA)‐specific IgG1 monoclonal antibody (mAb) (O1‐10) Fabs down‐regulated OVA‐induced asthmatic responses (Yoshino et al., 2014) and allergic rhinitis (Matsuoka et al., 2014), respectively, in mice. In vitro studies also showed that the capture of OVA by O1‐10 Fabs in advance prevented the subsequent binding of intact anti‐OVA polyclonal antibodies (pAbs) to the captured OVA (Yoshino et al., 2014). This suggested that the suppression of OVA‐induced asthmatic responses and allergic rhinitis by O1‐10 Fabs was due to the Fab‐mediated capture of OVA in the respiratory and nasal mucosal tissues, which reduced the binding of host anti‐OVA pAbs to the OVA, an effect responsible for the induction of the allergic disease. In the present study, we show that i.n. exposure to Fabs of an IgG1 mAb (P1‐8) to the major allergen Cry j1 of Japanese cedar pollen (JCP), a well‐known allergen, which triggers allergic pollinosis in 30 to 40 million Japanese people (Kaneko et al., 2005; Saito, 2014; Yamada et al., 2014), attenuates JCP‐induced allergic rhinitis in mice.

Methods

Animals

All animal care and experimental procedures were approved by the Experimental Animal Research Committee at Kobe Pharmaceutical University. Efforts were made to minimize animal suffering and to reduce the number of animals used. All studies involving animals are reported in accordance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). The number of animals per group was five, and the total number of animals was 93. Seven‐week‐old Balb/c male mice weighing 22–26 g were obtained from Japan SLC (Hamamatsu, Japan). These mice were housed in standard polypropylene cages (five animals per cage), under a 12/12 h light/dark cycle (lights on at 07:00 h) with food and water ad libitum.

Induction of allergic rhinitis by JCP

Mice were anaesthetized with isoflurane (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Sensitization was performed by i.p. injection of 50 μg of JCP (Biostir, Hyogo, Japan) adsorbed to alum (2.5 mg·0.5 mL−1 per animal) (Wako Pure Chemical Industries) on days 0 and 14. The sensitized mice were challenged i.n. with JCP (100 μg·in 20 μL per mouse) on days 28, 29, 30 and 35.

Production and purification of a mAb (P1‐8) to JCP and its Fabs

Mice were sensitized with JCP emulsified with complete Freund's adjuvant (Sigma‐Aldrich Fine Chemicals, MI, USA) and JCP‐sensitized spleen cells were fused with NS‐1 myeloma cells, followed by screening and cloning hybridomas that produced anti‐JCP mAbs, including P1‐8, as previously described (Terato et al., 1992). P1‐8‐producing hybridomas were grown in the CELLine CL1000 with a BD‐Cell‐MAb medium (BD Biosciences, San Diego, CA, USA) supplemented with 20% heat‐inactivated FBS, 1% l‐glutamine and 1% penicillin–streptomycin. To prepare P1‐8 Fabs, the mAb purified using a protein G column (GE Healthcare UK Ltd., Little Chalfont, UK) was digested with agarose‐linked papain (Sigma‐Aldrich, St. Louis, MO, USA) according to the methods described previously (Katpally et al., 2008). P1‐8 Fabs were separated using a protein G column and identified by Western blotting using an alkaline phosphatase‐conjugated anti‐mouse κ/λ light chain (Sigma‐Aldrich Inc.) (Towbin et al., 1979). To investigate whether the capture of JCP by P1‐8 Fabs, in advance, could prevent the interaction between JCP and intact P1‐8, P1‐8 Fabs (0.01–30 μg·mL−1) were incubated with JCP‐coated (100 μg·mL−1) 96‐well plates at 37°C for 1 h. After washing, this was followed by the addition of intact P1‐8 (0.5 μg·mL−1) to the plates, and they were incubated at 37°C for 1 h. Next, the alkaline phosphatase‐conjugated anti‐Fc of mouse IgG1 was added to measure the intact P1‐8 binding to JCP; the plate was developed with p‐nitrophenyl phosphate, and measurements were made at 405 nm using a microplate elisa reader. Additionally, P1‐8 Fabs, without adding intact P1‐8 (P1‐8 Fabs only), were prepared as a negative control. The observer was blinded in terms of the positive and negative controls.

Allergenic specificity of P1‐8

To investigate the allergenic specificity of P1‐8, the mAbs (0.01–10 μg·mL−1) were incubated with 2 μg·mL−1 of various allergens including haemocyanin, OVA, collagen, casein, Cry j1, Cry j2 and ragweed pollen coated on 96‐well plates at 37°C for 1 hr. After washing, this was followed by the addition of the alkaline phosphatase‐conjugated anti‐Fc of mouse IgG1. The plate was developed with p‐nitrophenyl phosphate, and measurements were made at 405 nm using a microplate elisa reader. The observer was blinded in terms of the types of allergens used for the elisa.

Administration of P1‐8 Fabs

P1‐8 Fabs (400 μg·20 μL−1 per mouse) dissolved in PBS were i.n. administered 15 min before each challenge with JCP on days 28, 29, 30 and 35. As a control, the Fabs of IgG purified from naïve mouse serum (Rockland Immunochemicals, Gilbertsville, PA, USA) were used. In some experiments, P1‐8 Fabs were i.n. administered only once 15 min before the last JCP challenge on day 35. The frequency of sneezing and nasal rubbing was counted for 30 min after the allergenic challenge, as described previously (Matsuoka et al., 2014). The operator was blinded in terms of P1‐8 Fabs and control Fabs administered. The animals were randomly allotted to P1‐8 Fab and control Fab treatment groups.

Histological analyses

Mice were killed by isoflurane inhalation on day 37, and their heads were fixed in 4% neutral‐buffered formalin. The skulls remained in EDTA for decalcification for 21 days before embedding in paraffin and sectioning at 4 μm, as described previously (Yoshino and Cleland, 1992; Guibas et al., 2014). Sections were stained with toluidine blue, haematoxylin and eosin and periodic acid–Schiff (PAS) to stain the mast cells, eosinophils and goblet cells respectively. Mast cells and eosinophils were counted in whole areas of the nasal septum and lateral process by a blinded observer (Matsuoka et al., 2014). Scoring for the nasal septum stained with PAS was also evaluated by a blinded observer on a scale of 0–4 with increments of 0.5 as follows: 0, 0% PAS+ cells; 1, 20%; 2, 50%; 3, 70%; and 4, >70% (Matsuoka et al., 2014).

Data and statistical analysis

This study complies with recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). Data are shown as the means ± SEM, with n indicating the number of animals. Statistical analyses have been performed using one‐way ANOVA followed by Bonferroni's multiple comparison test using graphpad prism 6 software (GraphPad Software, San Diego, CA, USA). P‐values of <0.05 were considered statistically significant. Post hoc tests were run only when F achieved P < 0.05 and there was no significant variance inhomogeneity.

Materials

Haemocyanin, OVA, collagen, and casein were supplied by Sigma Aldrich Inc. (St. Louis, MO, USA). Cry j1 and Cry j2 were purchased from Hayashibara Biochemical Lab. Inc. (Okayama, Japan) and ragweed pollen was obtained from Cosmo Bio Co., Ltd. (Tokyo, Japan).

Results

Allergenic specificity of P1‐8 and production of P1‐8 Fabs

To investigate its selective binding to Cry j1, P1‐8 was incubated in vitro with various allergens including haemocyanin, OVA, type II collagen, casein, Cry j1, Cry j2 and ragweed pollen. The results showed that P1‐8 bound to only Cry j1 (Figure 1A), but not to all other allergens including Cry j2 (Figure 1B) and ragweed pollen (Figure 1C). The digestion of P1‐8 with papain was followed by the production of Fabs proteins, with sizes between 37–50 kDa (Figure 2A). The in vitro pre‐incubation of P1‐8 Fabs with JCP resulted in the blockade of the binding of intact P1‐8 to JCP in a concentration‐dependent manner (Figure 2B).

Figure 1.

Figure 1

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P1‐8 specifically binds to Cry j1. The indicated amounts of P1‐8 were added to various allergens including haemocyanin, OVA, collagen, casein, Cry j1 (A), Cry j2 (B) and ragweed pollen (C) coated on the plate. This was followed by the further addition of the alkaline phosphatase‐conjugated anti‐Fc of mouse IgG1. Then, p‐nitrophenyl phosphate was added to the plates before measuring absorbance at 405 nm using an elisa reader.

Figure 2.

Figure 2

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Production of P1‐8 Fabs by digestion with papain and their specificity. (A) Western blotting analysis. P1‐8 Fabs produced by papain digestion of the intact mAb were separated using a protein G column and identified by Western blotting using an alkaline phosphatase‐conjugated anti‐mouse κ/λ light chain. (B) Prevention of the binding of intact P1‐8 to JCP by P1‐8 Fabs. The indicated amounts of P1‐8 Fabs were added to JCP coated on the plates. This was followed by the addition of intact P1‐8 to the plates, and the alkaline phosphatase‐conjugated anti‐Fc of IgG1 was added. Then, p‐nitrophenyl phosphate was added to the plates before measuring absorbance at 405 nm using an elisa reader. As a negative control, P1‐8 Fabs without added intact P1‐8 were used.

Effect of i.n. exposure to P1‐8 Fabs on the frequency of sneezing

Allergic rhinitis was induced by sensitization (S) with JCP on days 0 and 14 followed by i.n. challenge (C) with the pollen on days 28, 29, 30 and 35. P1‐8 Fabs (Fab) were i.n. administered 15 min before each JCP challenge to the sensitized and challenged mice (S‐C‐Fab group, n = 5) (Figure 3). Normal naive IgG Fabs were used as a control (S‐C group, n = 5). JCP was also i.n. administered to non‐sensitized (NS) mice (NS‐C group, n = 5). The results showed that statistically significant difference in the number of sneezes between the NS‐C and S‐C groups was only found after the last challenge (Figure 4A–D). Treatment with i.n. P1‐8 Fabs in the S‐C‐Fab group resulted in a trend of decrease in sneezing in the first three challenges and a significant reduction in the fourth challenge, compared with that in the S‐C group. There was a statistically significant difference in sneezing between three groups when the last challenge was made (ANOVA).

Figure 3.

Figure 3

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Experimental protocols. To induce allergic rhinitis, mice were i.p. sensitized (S) with JCP plus alum on days 0 and 14 followed by i.n. challenge (C) with 100 μg of the same pollen on days 28, 29, 30 and 35. The P1‐8 Fabs (Fab; 400 μg) were administered 15 min before each JCP challenge (S‐C‐Fab group). Normal IgG Fabs were i.n. administered to mice sensitized and challenged with JCP as a positive control (S‐C group). JCP was i.n. administered to non‐sensitized mice as a negative control (NS‐C group). The frequency of sneezing and nasal rubbing observed during the 30 min after the JCP challenge was counted.

Figure 4.

Figure 4

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Repeated i.n. exposure to P1‐8 Fabs suppresses the frequency of sneezing in mice with JCP‐induced allergic rhinitis. P1‐8 Fabs (Fab) were i.n. administered 15 min before each JCP challenge (C) on days 28 (A), 29 (B), 30 (C) and 35 (D) in mice sensitized (S) with JCP on days 0 and 14 (S‐C‐Fab group). Control IgG Fabs were also i.n. administered in mice sensitized and challenged with JCP as a positive control (S‐C group). As a negative control, JCP was i.n. administered to non‐sensitized mice (NS‐C group). The number of sneezes observed during the 30 min after the JCP challenge in each group was counted. Each value is the mean ± SEM (n = 5). *P < 0.05, significantly different from NS‐C group. # P < 0.05, significantly different from S‐C group.

Effect of i.n. exposure to P1‐8 Fabs on the frequency of nasal rubbing

Significantly increased numbers of episodes of nasal rubbing as an indicator of itching in allergic rhinitis were observed following each JCP challenge in the S‐C group, compared with that in the NS‐C group (Figure 5A–D). When P1‐8 Fabs were administered, animals had a marked decrease in nasal rubbing in the S‐C‐Fab group compared with the S‐C group. There were statistically significant differences in nasal rubbing among the three groups when the first to fourth challenges were made (ANOVA).

Figure 5.

Figure 5

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Repeated i.n. exposure to P1‐8 Fabs suppresses the frequency of nasal rubbing in mice with JCP‐induced allergic rhinitis. P1‐8 Fabs (Fab) were administered i.n. 15 min before each JCP challenge (C) on days 28 (A), 29 (B), 30 (C) and 35 (D) in mice sensitized (S) with JCP on days 0 and 14 (S‐C‐Fab group). Control IgG Fabs were also administered i.n. to mice sensitized and challenged with JCP as a positive control (S‐C group). As a negative control, JCP was given i.n. to non‐sensitized mice (NS‐C group). The number of episodes of nasal rubbing observed during the 30 min after the JCP challenge in each group was counted. Each value is the mean ± SEM (n = 5). *P < 0.05, significantly different from NS‐C group. # P < 0.05, significantly different from S‐C group.

Effect of i.n. exposure to P1‐8 Fabs on the infiltration of mast cells and eosinophils in the nasal mucosal tissue

Because mast cells and eosinophils appear to play critical roles in allergic rhinitis (Skoner, 2001; Borish, 2003; Galli et al., 2008), these inflammatory cells present in the nasal mucosa were counted, based on positive toluidine blue and eosin staining. Significantly fewer mast cells (Figure 6A–D) and eosinophils (Figure 7A–D) were found in the nasal mucosa of the S‐C‐Fab group than in the S‐C group.

Figure 6.

Figure 6

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Repeated i.n. exposure to P1‐8 Fabs decreases mast cells in the nasal mucosa. Mast cells in the nasal septum were stained with toluidine blue on day 37 and counted in the NS‐C (A), S‐C (B) and S‐C‐Fab (C) groups. Bar = 50 μm. Each value is the mean ± SEM (n = 5) (D). *P < 0.05, significantly different from NS‐C group. # P < 0.05, significantly different from S‐C group.

Figure 7.

Figure 7

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Repeated i.n. exposure to P1‐8 Fabs decreases eosinophils in the nasal mucosa. Eosinophils in the nasal septum were stained with haematoxylin and eosin on day 37 and counted in the NS‐C (A), S‐C (B) and S‐C‐Fab (C) groups. Bar = 50 μm. Each value is the mean ± SEM (n = 5) (D). *P < 0.05, significantly different from NS‐C group. # P < 0.05, significantly different from S‐C group.

Effect of i.n. exposure to P1‐8 Fabs on the number of mucus‐producing goblet cells in the nasal mucosal tissue

The S‐C group appeared to show a number of goblet cells producing mucus in the nasal septum (Figure 8A–D) and these mucus‐producing cells were significantly reduced in the S‐C‐Fab group. There was a statistically significant difference in the number of mucus‐producing cells among the three groups (ANOVA).

Figure 8.

Figure 8

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Repeated i.n. exposure to P1‐8 Fabs decreases the number of goblet cells producing mucus in the nasal mucosa. Goblet cells in the nasal septum were stained with PAS on day 37, and the number of goblet cells producing mucus was scored in the NS‐C (A), S‐C (B) and S‐C‐Fab (C) groups. Bar = 50 μm. Each value is the mean ± SEM (n = 5) (D). *P < 0.05, significantly different from NS‐C group. # P < 0.05, significantly different from S‐C group.

Effect of single i.n. exposure to P1‐8 Fabs on the frequency of sneezing and nasal rubbing

To determine whether or not single i.n. exposure to P1‐8 Fabs was also effective in suppressing JCP‐induced allergic rhinitis, the allergen‐specific Fabs were administered only once, 15 min before the last JCP challenge, on day 35. The results showed that both sneezing and nasal rubbing in the S‐C‐Fab group were markedly decreased compared with the S‐C group (Figure 9). The frequency of sneezing and nasal rubbing in the S‐C‐Fab group was similar to that in the NS‐S group.

Figure 9.

Figure 9

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A single i.n. exposure to P1‐8 Fabs decreases the frequency of sneezing and nasal rubbing in mice with JCP‐induced allergic rhinitis. (A) P1‐8 Fabs (Fab) were administered i.n. only once, 15 min before the last JCP challenge (C) on day 35, in mice sensitized (S) with JCP on days 0 and 14 (S‐C‐Fab group). Control IgG Fabs were also given i.n. to mice sensitized and challenged with JCP as a positive control (S‐C group). As a negative control, JCP was i.n. administered in non‐sensitized mice (NS‐C group). The frequency of sneezing (B) and nasal rubbing (C) observed during the 30 min after the JCP challenge in each group was counted. Each value is the mean ± SEM (n = 5). *P < 0.05, significantly different from NS‐C group. # P < 0.05, significantly different from S‐C group.

Discussion and conclusions

The present study suggested that i.n. exposure to Fabs of an allergen‐specific mAb was effective in down‐regulating allergic rhinitis because i.n. administration of P1‐8 Fabs specific for the major allergen Cry j1 of JCP (Kaneko et al., 2005; Saito, 2014; Yamada et al., 2014) resulted in marked suppression of sneezing and nasal rubbing in mice with JCP‐induced allergic rhinitis. In addition, the suppression of the allergic symptoms by P1‐8 Fabs was associated with decreases in mast cells and eosinophils, as well as hyperplasia of goblet cells in the nasal mucosa, which play an important role in allergic diseases including pollinosis allergies (Skoner, 2001; Borish, 2003; Galli et al., 2008), further suggesting the efficacy of Cry j1‐specific mAb Fabs for JCP‐induced allergic rhinitis. We previously showed that i.n. exposure to Fabs of mAb (O1‐10) to OVA, often used as an allergen to create experimental animal models of allergic diseases (Yamaki and Yoshino, 2012a, 2012b; Nabe et al., 2015), attenuated OVA‐specific allergic rhinitis in mice (Matsuoka et al., 2014). However, OVA does not appear to be an allergen causing allergic rhinitis in humans. The present study is the first report demonstrating that the i.n. exposure to P1‐8 Fabs specific for JCP, well known as an allergen causing cedar pollinosis in 30 to 40 million Japanese people (Kaneko et al., 2005; Saito, 2014; Yamada et al., 2014), down‐regulates the pollen allergy.

The suppression of JCP‐induced allergic rhinitis by P1‐8 Fabs appears to be due to the unique structure and functions of Fabs, in that they maintain allergen‐binding sites but lack an Fc portion for binding receptors on immune and inflammatory cells, such as mast cells and eosinophils, which play a critical role in allergic diseases including pollinosis (MacKenzie et al., 2001; Shiraishi et al., 2013). Our previous studies showed that intratracheal exposure to anti‐OVA IgG1 mAb (O1‐10) Fabs, but not intact O1‐10, suppressed asthmatic responses in mice (Yoshino et al., 2014). Furthermore, the suppression of asthmatic responses by O1‐10 Fabs, but not intact O1‐10, was associated with decreased production of inflammatory cytokines, including IL‐1β and IL‐6 in lung tissue, suggesting a role of the Fc portion of the intact mAb in allergic airway inflammation. In order to avoid Fc‐mediated effector functions including mast cell degranulation and inflammatory cell activation, Fabs, but not intact antibodies, have been used to treat murine or human diseases. For instance, Hacha et al. (2012) demonstrated that nebulized anti‐IL‐13 mAbs Fabs reduced allergen‐induced asthma in mice. Digoxin‐specific Fabs have been used to treat patients with potentially life‐threatening digitalis toxicity (Antman et al., 1990; Woolf et al., 1992; Flanagan and Jones, 2004).

We previously demonstrated that Arthus‐type reaction‐mediated antigen‐induced arthritis in mice (Yoshino, 1995; Yoshino et al., 1995) induced by passive sensitization with anti‐OVA pAbs, followed by intra‐articular injection of the antigen, was suppressed by Fabs of the anti‐OVA pAb as we expected (Yoshino et al., 2010). However, a question arises as to why JCP‐induced rhinitis was suppressed by mAb P1‐8 Fabs that were expected to recognize only one epitope of Cry j1. A similar question arose when we previously observed the marked suppression of OVA‐induced asthmatic responses by the intratracheal administration of O1‐10 Fabs 30 min before the OVA challenge in mice (Yoshino et al., 2014), because at least five different immunological epitopes of OVA have been reported (Elsayed et al., 1988; Johnsen and Elsayed, 1990; Renz et al., 1993). Therefore, in that study, we attempted to solve this question by carrying out in vitro studies, in which anti‐OVA pAbs were added to preformed complexes of OVA and O1‐10 Fabs. The results showed that larger, but not smaller, amounts of complexes consisting of O1‐10 Fabs and OVA formed, in advance, were able to block the subsequent binding of smaller doses of anti‐OVA pAbs to OVA in the complexes. Therefore, the in vitro studies also suggested in vivo that large amounts of O1‐10 Fabs (120 μg) and OVA (40 μg) complexes, formed on the surface of airway mucosal tissues, appeared to efficiently block the binding of small amounts of blood‐derived anti‐OVA pAbs to O1‐10 Fab‐captured OVA, followed by the suppression of asthmatic responses by the OVA‐specific mAb Fabs. Similarly, in the present study, 100 μg of JCP was administered as an i.n. challenge dose, while the fourfold‐higher dose of 400 μg of P1‐8 Fabs was i.n. administered to suppress JCP‐induced allergic rhinitis, suggesting that the suppression of this experimental rhinitis by i.n. exposure to P1‐8 Fabs is due to the complexes of P1‐8 Fabs and Cry j1 that were formed in advance on the surface of the nasal mucosa. These complexes were able to block the subsequent binding of anti‐Cry j1 pAbs to Cry j1 present in the complexes.

It is noteworthy that the marked suppression of JCP‐induced allergic rhinitis by P1‐8 Fabs was allergen Cry j1 specific. It is also noteworthy that the suppression of this allergic rhinitis was achieved by i.n. exposure to P1‐8 Fabs. These results suggest that the frequency of side effects of the allergen‐specific mAb Fabs exposed locally would be lower compared with the currently available therapeutic drugs for allergic pollinosis, including anti‐histamine drugs and anti‐allergic drugs, because the therapeutic drugs are not allergen‐specific and are orally administered, causing a range of systemic side effects during treatment (Balle et al., 1982; Douglas, 1985; Pipkorn et al., 1987; Simons, 1989; Quraishi et al., 2004). Although the anti‐IgE mAb omalizumab, which neutralizes free IgE and inhibits the IgE‐mediated reactions, is currently available for allergic diseases including severe asthma (Domingo, 2014; Sòler, 2014; Logsdon and Oettgen, 2015), this therapy targeting IgE is still not allergen‐specific. Subcutaneous and sublingual immunotherapies in allergic rhinitis are currently available as allergen‐specific therapies. However, a longer period of treatment with allergens is often required before patients benefit from this approach (Abramson et al., 1995; Pipet et al., 2009; Keles et al., 2011; Passalacqua and Canonica, 2011).

In conclusion, P1‐8 Fabs specific for Cry j1 were effective in suppressing JCP‐induced allergic rhinitis in mice, most likely through mAb Fab‐dependent Cry j1 allergen capture in nasal mucosal tissues. Targeting this interaction may prevent pathological interactions between anti‐Cry j1 pAbs and Cry j1, which are important for the induction of allergic rhinitis.

Author contributions

S.Y. designed the research. S.Y. and N.M. performed the research. N.M. contributed essential reagents or tools. S.Y. and N.M. analysed the data. S.Y. wrote the paper.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organizations engaged with supporting research.

Acknowledgements

These studies were supported by a Grant‐in‐Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.

Yoshino, S. , and Mizutani, N. (2016) Intranasal exposure to monoclonal antibody Fab fragments to Japanese cedar pollen Cry j1 suppresses Japanese cedar pollen‐induced allergic rhinitis. British Journal of Pharmacology, 173: 1629–1638. doi: 10.1111/bph.13463.

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