BJP
British Journal of Pharmacology
British Journal of Pharmacology (2016) 173 1629–1638
1629
RESEARCH PAPER Intranasal exposure to monoclonal antibody Fab fragments to Japanese cedar pollen Cry j1 suppresses Japanese cedar pollen-induced allergic rhinitis Correspondence Dr Shin Yoshino, Department of Pharmacology, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan. E-mail: [email protected] Received 1 August 2015; Revised 27 January 2016; Accepted 14 February 2016
S Yoshino and N Mizutani Department of Pharmacology, Kobe Pharmaceutical University, Kobe, Japan
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 JCPinduced 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
LIGANDS
Other proteins
Certolizumab
TNF-α
Omalizumab
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). © 2016 The British Pharmacological Society
DOI:10.1111/bph.13463
BJP
S Yoshino and N Mizutani
Introduction
Methods
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 allergenexposed 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 antiOVA 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 JCPinduced allergic rhinitis in mice.
Animals
1630
British Journal of Pharmacology (2016) 173 1629–1638
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 JCPsensitized 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 phosphataseconjugated 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 (0.5μg·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
Suppression of allergic rhinitis by antibody Fab
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.
BJP
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 oneway ANOVA followed by Bonferroni’s multiple comparison test using GRAPHPAD PRISM 6 software (GraphPad Software, San Diego, CA, USA). P-values of
British Journal of Pharmacology
British Journal of Pharmacology (2016) 173 1629–1638
1629
RESEARCH PAPER Intranasal exposure to monoclonal antibody Fab fragments to Japanese cedar pollen Cry j1 suppresses Japanese cedar pollen-induced allergic rhinitis Correspondence Dr Shin Yoshino, Department of Pharmacology, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan. E-mail: [email protected] Received 1 August 2015; Revised 27 January 2016; Accepted 14 February 2016
S Yoshino and N Mizutani Department of Pharmacology, Kobe Pharmaceutical University, Kobe, Japan
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 JCPinduced 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
LIGANDS
Other proteins
Certolizumab
TNF-α
Omalizumab
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). © 2016 The British Pharmacological Society
DOI:10.1111/bph.13463
BJP
S Yoshino and N Mizutani
Introduction
Methods
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 allergenexposed 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 antiOVA 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 JCPinduced allergic rhinitis in mice.
Animals
1630
British Journal of Pharmacology (2016) 173 1629–1638
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 JCPsensitized 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 phosphataseconjugated 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 (0.5μg·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
Suppression of allergic rhinitis by antibody Fab
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.
BJP
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 oneway ANOVA followed by Bonferroni’s multiple comparison test using GRAPHPAD PRISM 6 software (GraphPad Software, San Diego, CA, USA). P-values of
