Allergology International 69 (2020) 178e186

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Allergology International

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Invited Review Article The roles of IL-5 and anti-IL-5 treatment in eosinophilic diseases: , eosinophilic granulomatosis with polyangiitis, and eosinophilic chronic rhinosinusitis

* ** *** Hiroyuki Nagase a, , 1, Shigeharu Ueki b, , 1, Shigeharu Fujieda c, , 1 a Division of Respiratory Medicine and Allergology, Department of Medicine, Teikyo University School of Medicine, Tokyo, Japan b Department of General Internal Medicine and Clinical Laboratory Medicine, Akita University Graduate School of Medicine, Akita, Japan c Division of Otorhinolaryngology - Head & Neck Surgery, Department of Sensory and Locomotor Medicine, Faculty of Medical Science, University of Fukui, Fukui, Japan article info abstract

Article history: IL-5 is the most potent activator of and is produced by Th2 cells and ILC2s. A role for IL-5 in Received 8 February 2020 extracellular trap cell death, i.e., a proinflammatory cell death, has also been reported. Available online 2 March 2020 and benralizumab are humanized mAbs that target IL-5 and the IL-5 receptor a, respec- tively, and their therapeutic efficacy for severe asthma has been established. Although consistent dif- Keywords: ferences in the efficacies of those drugs have not been proven, benralizumab extensively depleted Benralizumab eosinophils via Ab-dependent cell-mediated cytotoxicity. Blood eosinophil count, but not FeNO or IgE, is Chronic rhinosinusitis with nasal polyps the best-established predictive biomarker of the efficacy of anti-IL-5 treatment. Regarding the choice of Eosinophils IL-5 biologics, the balance between blood eosinophil count and FeNO, indication of comorbidities, longitu- Mepolizumab dinal safety, and interval of injection should be considered. Mepolizumab was also effective in main- taining the remission of refractory eosinophilic granulomatous polyangiitis. Moreover, mepolizumab decreased the proportion of patients who required surgery and lowered the nasal polyp score in patients with chronic rhinosinusitis with nasal polyps; a further extensive trial is currently under way. In a phase II benralizumab study performed in Japan, no significant effect on nasal polyp score at week 12 was observed, suggesting a requirement for longer treatment. In this review, the role of IL-5 in eosinophil biology and the current status of anti-IL-5 therapy are discussed. The longitudinal safety of anti-IL-5 therapy has been increasingly established, and this strategy will be continuously indicated for eosino- philic diseases as a specific treatment for eosinophilic inflammation. Copyright © 2020, Japanese Society of Allergology. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Basic aspects of IL-5 and eosinophilic inflammation homeostatic conditions, the half-life of blood eosinophils is approximately 25 h.1 The majority of senescent (or apoptotic) eo- For more than 100 years, eosinophils have been linked to sinophils are cleared in the liver and spleen, where they are taken- allergic and parasitic diseases. Eosinophils are terminally differ- up by macrophages of the reticuloendothelial system. The þ entiated myeloid cells derived from CD34 hematopoietic stem remaining eosinophils are distributed across the gastrointestinal cells in the bone marrow. The process is regulated by transcription tract, thymus, lung, uterus, and adipose tissue, indicating physio- factors, including the GATA-binding factor 1. Once eosinophils logical function in each organ2 (reviewed in Ref. 3). In response to reach maturity, they are released into the blood stream. In normal type 2 reactions, eosinophils are markedly mobilized from the bone

* Corresponding author. Division of Respiratory Medicine and Allergology, Department of Medicine, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan. ** Corresponding author. Department of General Internal Medicine and Clinical Laboratory Medicine, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita 010- 8543, Japan. *** Corresponding author. Division of Otorhinolaryngology - Head & Neck Surgery, Department of Sensory and Locomotor Medicine, Faculty of Medical Science, University of Fukui, 23-3 Matsuokashimoaizuki, Eiheiji-cho, Yoshida-gun, Fukui 910-1193, Japan. E-mail addresses: [email protected] (H. Nagase), [email protected] (S. Ueki), [email protected] (S. Fujieda). Peer review under responsibility of Japanese Society of Allergology. 1 These authors equally contributed to this article. https://doi.org/10.1016/j.alit.2020.02.002 1323-8930/Copyright © 2020, Japanese Society of Allergology. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). H. Nagase et al. / Allergology International 69 (2020) 178e186 179 marrow and blood pool to local sites, creating a tissue . pathological conditions of asthma, such as airway hypersensitiv- The excessive accumulation and activation of eosinophils can have ity.11 In addition, nasal appreciation of recombinant IL-5 induced a deleterious effect and contribute to tissue damage. selective recruitment of eosinophils in the mucosa.12,13 Eosinophil differentiation is mediated by IL-5, IL-3, and the In a variety of allergic diseases, circulating blood eosinophils are granulocyte macrophage colony-stimulating factor (GM-CSF). in a resting state and do not degranulate until they have reached These are called b common cytokines, because of their their target tissue.14 The proinflammatory chemokines, especially shared b receptor structure. The b common receptor eotaxins (CCL11, CCL24, and CCL26), that are produced by structural comprises a cytokine-specific a chain and a common b chain that is cells (including bronchial epithelial cells and fibroblasts) primarily essential for receptor signaling and assembly.4 Among them, IL-5 regulate the accumulation of eosinophils in the airways.15 The ca- has been considered to be a therapeutic target for allergic/eosino- pacity to generate reactive oxygen species and the secretory func- philic diseases because of the presence of exclusive IL-5 receptor tion of eosinophils are important features of their role as tissue- (IL-5R) expression in eosinophils and basophils (in humans) and its damaging cells. Eosinophil pack approximately 200 granules that critical role in eosinophilopoiesis.5 contain not only cytotoxic proteins (i.e., major basic protein, In the allergen-induced adaptive immunity process, IL-5 is pri- eosinophil peroxidase, eosinophil cationic protein (ECP), and mary produced by Th2 cells. In addition, ILC2s produce abundant eosinophil-derived neurotoxin), but also over 30 cytokines.16 This IL-5 in response to IL-33 and other alarmins, thus playing a critical cargo is an efficient system for short-lived eosinophils, as de novo role in innate immunity settings.6 A previous study indicated that protein synthesis is time consuming.17,18 Eosinophils can rapidly IL-5-deficient mice exhibit a slight reduction in circulating eosin- release selected cytokines from granular storage through piecemeal ophils under baseline conditions, but completely fail to induce degranulation and possibly exocytosis. Moreover, eosinophils also parasite-induced eosinophilia.7 In contrast, IL-5 transgenic mice release their total cellular content through cytolytic degranulation showed high-level eosinophilia at the baseline.8 Familial eosino- (cytolysis). Cytolytic degranulation in inflamed tissues has been e philia is a rare autosomal dominant inherited disorder that is shown to occur in 30%e80% of eosinophils.19 21 Recent evidence characterized by prolonged peripheral eosinophilia likely second- revealed that cytolysis is an active cell death program, i.e., extra- ary to IL-5 overproduction in peripheral blood mononuclear cells.9 cellular trap cell death (ETosis).22 Unlike apoptosis, eosinophil To date, a considerable amount of evidence indicates that the sys- ETosis (EETosis) releases the total cellular content of these cells, temic increase of IL-5 is important for promoting eosinophilia. including damage-associated molecular patterns. Thus, EETosis is It is noteworthy that the systemic increase of IL-5 is not always considered to be a potentially proinflammatory type of cell death. sufficient to cause an eosinophil-mediated pathological condition. In tissues, the presence of multiple inflammatory signals (e.g., IL-5 transgenic mice remain physically normal, with the exception adhesion molecules, complements, lipid mediators, cytokines, and of the development of splenomegaly.8 In patients with familial pathogens) that work synergistically with IL-5 are required for the eosinophilia, clinical manifestations related to the eosinophilia are “full” activation of eosinophils.23 In in vitro systems, isolated human uncommon.9 At a mechanistic level, IL-5 signaling in mature eo- eosinophils spontaneously undergo apoptosis within 48 h; how- sinophils activates several signaling molecules, such as the JAK- ever, the survival of eosinophils is significantly prolonged in the STAT and MAPK pathways, inducing priming (augmentation of presence of a low concentration of IL-5.17 Of interest, the stimula- effector functions), chemokinesis/chemotaxis, the activation of tion of eosinophils with IL-5 and the platelet-activating factor in integrins, and the prolongation of survival by inhibiting apoptosis.10 low serum/albumin conditions leads to the rapid EETosis,22 sug- Tissue-specific IL-5 production might be important in some cases, gesting that activated eosinophils in tissues and/or after luminal as transgenic mice that express lung-specific IL-5 can recreate the entry can degranulate through suicidal EETosis (Fig. 1, 2). EETosis has been implicated in the pathogenesis of several eosinophilic diseases, including asthma,24,25 allergic bronchopulmonary asper- gillosis,26,27 eosinophilic otitis,28,29 eosinophilic chronic rhinosi- nusitis (ECRS) or chronic rhinosinusitis with nasal polyps (CRSwNP),29,30 hypereosinophilic syndrome,22,31 COPD,32 and Wells syndrome (eosinophilic cellulitis).33 The immunoregulatory capacities of eosinophils could be explained by their accomplishment of the highly selective process of granule-derived proteins, i.e., piecemeal degranulation. In response to exogenous stimulation, granule-stored cytokines are differentially sorted into vesicular-tubular compartments and mobilized to the outer plasma membrane.34,35 In addition to the selective release system, recent evidence suggests the existence of several specific subtypes of eosinophils.36,37 In situ differentiation of eosinophil- committed cells might be associated with eosinophil heterogeneity. þ Several studies have demonstrated that hematopoietic CD34 pro- genitor cells migrate to the sites of allergic inflammation, where they e undergo further proliferation and final maturation.38 40

Fig. 1. Lifespan of eosinophils in asthma. In asthmatics, eosinophils are prominent in The role of anti-IL-5 therapy for severe asthma and the airway. Eosinophils are produced in the bone marrow from hematopoietic stem cells, similar to other granulocytes. The interaction between eosinophils and endo- eosinophilic granulomatosis with polyangiitis thelial cells involves rolling, adhesion, and transmigration. In the airway tissues and lumen, migrated eosinophils are further activated by local stimuli and release their Clinical evidence of the usefulness of mepolizumab for the pre-formed cytokines and granular content via piecemeal degranulation. The survival management of severe asthma of tissue and luminal eosinophils may be prolonged, but cytolytic degranulation (EETosis) may also be accelerated by further activation. IL-5, which is primarily pro- duced by Th2 (adaptive immunity) and ILC2 (innate immunity) cells, regulates As mentioned above, IL-5 is among the most eosinophil- eosinophil functions throughout their lifespan. selective and potent activators of various cellular functions. 180 H. Nagase et al. / Allergology International 69 (2020) 178e186

Fig. 2. Eosinophil apoptosis, prolonged survival, and activation-induced ETosis. In contrast to IL-5-stimulated cells (B), most of the non-stimulated control cells (A) showed typical apoptotic morphologies (i.e., nuclear and cytoplasmic condensation). Eosinophils that underwent ETosis (C) showed disintegration of the plasma and nuclear membrane, thus releasing free granules. Isolated human peripheral blood eosinophils were cultured in RPMI 1640 supplemented with 10% fetal bovine serum without (A) or with (B) 1 ng/ml of IL-5 for 48 h. To induce ETosis, cells were stimulated with 1 ng/ml of IL-5 and 1 mM platelet-activating factor for 3 h in RPMI 1640 with 0.3% bovine serum albumin (C). The cytospin slide preparations (A, B) and paraformaldehyde-fixed slide (C) were stained with Diff-quick. Scale bar, 50 mm. Original magnification, 400.

Mepolizumab (SB 240563), which is a humanized mAb targeting IL- Features and clinical evidence of the anti-IL-5 receptor a mAb, 5, was developed in the late 1990's41 and the results of its first benralizumab clinical trial were published in 2000.42 Although mepolizumab greatly reduced the number of eosinophils in the blood and As mentioned previously, the IL-5R is composed of an IL-5- sputum, the improvement in airway hyperresponsiveness after specific a subunit and a signal-transducing b subunit that is com- allergen challenge was not observed. This negative result was mon to IL-3 and GM-CSF receptors. Benralizumab (MEDI-563) is a confirmed by a subsequent clinical study that recruited 362 pa- humanized afucosylated mAb that was developed by Kyowa Hakko tients and showed an absence of improvement in pulmonary Kirin in the late 2000's and is specific for the human IL-5Ra.49 function or symptoms.43 Although those results were disap- Afucosylation enhances the interaction between benralizumab pointing and dampened the positioning of eosinophils in the pa- and FcgRIIIa and heightens Ab-dependent cell-mediated cytotox- thology of asthma, a major drawback of those studies was that icity (ADCC) functions.50 The incubation of NK effector cells with patients were not selectively recruited based on inflammatory benralizumab in vitro induced the apoptosis of IL-5Ra-expressing phenotypes. In addition, regarding the study endpoint, a trend for a eosinophils and basophils, suggesting the ADCC activity of benra- decrease in exacerbation was observed in the latter study.43 lizumab.50 The eosinophil-depletion efficacy of the drug was rapid In a small follow-up study that recruited only patients with a and single doses of benralizumab resulted in marked reduction of sputum eosinophil percentage of more than 3% and set the exac- blood eosinophil counts within 24 h51; moreover, benralizumab erbation as a primary endpoint, a significant decrease in exacer- decreased the airway mucosal eosinophil counts by 61.9% bation was shown.44 This positive result was further confirmed in compared with the baseline (day 28).52 large-scale trials called DREAM study45 and MENSA study,46 which The clinical efficacy of benralizumab was investigated in two recruited only patients with eosinophilic inflammation. The pro- independent phase 3 trials (SIROCCO and CALIMA).53,54 Benrali- cess used for establishing the clinical efficacy of mepolizumab zumab significantly reduced the annual exacerbation rate in pa- suggested the importance of identifying the eosinophilic asthma tients with blood eosinophil counts of at least 300 cells/ml as the phenotype when applying anti-IL-5 treatment. primary endpoint and improved the forced expiratory volume in 1 s The entry criteria for the MENSA study, which was a pivotal (FEV1) in both studies. A glucocorticoid-sparing effect was also study for this clinical indication, were set to recruit patients who reported by the ZONDA study.55 Benralizumab was approved for had a peripheral blood eosinophil count 150 cells/ml at screening severe asthma in November 2017 in United States, and January 2018 or 300 cells/ml at some point during the previous year. These in European countries and Japan. thresholds were based on the analysis of the DREAM study,45 in fi which the best responders to mepolizumab were identi ed. The Anti-IL-5 mAb, reslizumab primary endpoint in the MENSA study was the rate of exacerbation and the other outcomes included pulmonary function, health- Reslizumab is another anti-IL-5 mAb that was approved for the related quality of life (HRQOL), and asthma symptoms, all of treatment of severe asthma in the United States and European fi which were signi cantly improved by mepolizumab. In addition, a countries, but not in Japan. In contrast with other IL-5-targeted comparable effect of the intravenous and subcutaneous adminis- drugs, reslizumab is administered intravenously and the adminis- tration of this drug was shown, which led to the approval of tered dose is corrected according to body weight.56 Among patients subcutaneous injection. The eosinophil criteria in the MENSA with obesity, because the administered dose of reslizumab in- study shown above were cited in the attached document for creases as the body weight increases, a fraction of those patients mepolizumab in Japan, the perusal of which is recommended responded better to reslizumab vs. mepolizumab, which was when applying this drug to patients. In subsequent clinical studies, administered at a fixed dose.57 The efficacy of subcutaneous an improvement in HROQL as a primary endpoint was shown in administration and the improvement in efficacy observed after the phase 3b MUSCA study,47 whereas a glucocorticoid-sparing 48 switching from mepolizumab to reslizumab is under investiga- effect was demonstrated in the SIRIUS study. Mepolizumab tion.56 As studies such as those of the steroid-sparing effect of was approved for severe asthma in November 2015 in United reslizumab are lacking because of the current commercial limita- States, December 2015 in European countries, and March 2016 in tions of the drug, this review will focus of benralizumab and Japan. mepolizumab as IL-5-targeted therapies. H. Nagase et al. / Allergology International 69 (2020) 178e186 181

Predictive factors of the efficacy of anti-IL-5 treatments treatment was similar to rates in the placebo arms and rates in age- and sex-adjusted general population from Surveillance Blood eosinophil count is the best-established biomarker for the Epidemiology and End Results Registry for 2007e2011 in the prediction of the efficacy of anti-IL-5 treatments. A post-hoc anal- United States.67 ysis of phase 3 trials showed that mepolizumab58 and benralizu- For the assessment of systemic reactions after mepolizumab and mab59 reduced the rate of exacerbation as blood eosinophil count benralizumab use showed that hypersensitivity reactions were increased. Regarding mepolizumab, the exacerbation rate reduc- present in 2% and 1%e3% of patients, respectively; moreover, an tion afforded by this agent vs. the placebo increased progressively, anaphylactic reaction was observed in one patient who received from 52% in patients with a baseline blood eosinophil count benralizumab.67,68 Pyrexia was reported in less than 1% of patients 150 cells/ml to 70% in patients with a count 500 cells/ml.58 A treated with mepolizumab, and in 3%e4% of those treated with significant effect was not observed in patients from the DREAM benralizumab.53,54 Headache was observed in 20% of patients study with a baseline blood eosinophil count <150 cells/ml.46 The treated with mepolizumab (17% in the placebo group) and 7%e9% criteria “eosinophil count 150 cells/ml at treatment start” and in those who received benralizumab (5%e7% in the placebo “eosinophil count 300 cells/ml at some time during the previous group).53,54 year” were compared, and the rate of reduction in exacerbation was The analysis of anti-drug Ab (ADA) in patients treated with comparable.60 Thus, the selection of patients for mepolizumab mepolizumab and benralizumab revealed that 8%67 and 15%e17%69 therapy based on the criteria adopted in the MENSA study is rec- of the subjects had positive ADA, respectively. Neutralizing drug ommended also in clinical settings. Abs were not detected in patients who received mepolizumab,67 Regarding benralizumab, although a significant effect was whereas 9%e13% of patients treated with benralizumab were observed in patients with an eosinophil count 150 cells/ml, the positive for these Abs.69 Regarding benralizumab, although a slight effect was also dependent on blood eosinophil count59 and a sig- decrease in eosinophil-depleting activity was observed in patients nificant effect was not observed in patients with an eosinophil with high titers of ADA, positivity for ADA was not clearly associ- count <150 cells/ml.53,54 ated with drug efficacy or hypersensitivity.69 In a study that investigated the glucocorticoid-sparing effect, A review article published by Gleich commented that no specific patients were eligible for benralizumab study if they had a blood complications were observed in patients who lost eosinophils eosinophil count 150 cells/ml55 or fulfilled the criteria used in the because of autoimmune diseases.70 The safety of anti-IL-5 treat- MENSA mepolizumab study.48 Thus, the effect of anti-IL-5 therapy ment seems to be increasingly established, although a close in glucocorticoid-dependent patients with an eosinophil count monitoring of its long-term safety is desirable. <150 cells/ml has not been established. FeNO and the levels of serum IgE, which are regulated by IL-4 Comparison of the clinical efficacy of anti-IL-5 Abs and/or IL-13, were not effective predictive markers of the efficacy of mepolizumab60 or benralizumab.61 Regarding the clinical back- The efficacy of benralizumab in decreasing the number of eo- ground, the efficacy of mepolizumab was not different between sinophils in blood and lung tissues seems to be more prominent patients with or without prior treatment with omalizumab.62 Oral compared with that of mepolizumab.52,71 In addition, as basophils glucocorticoid use, comorbid nasal polyps, forced vital capacity express IL-5Ra, apoptosis of basophils was induced by the ADCC <65% of the predicted value, and more than 3 exacerbation events activity of benralizumab in an in vitro experiment,50 and the in the previous year were correlated with a higher efficacy of decrease in blood basophil counts was also suggested.72 Although, benralizumab.63 no head-to-head comparison of clinical efficacy is available, and it is Regarding the continuation rules at 16 weeks after the onset of impossible to compare the results of different trials, because the mepolizumab therapy, there was no evidence of a reliable marker baseline number of eosinophils and exacerbation events differ that could predict the long-term effects of the drug,64 suggesting among studies. the existence of late responders. As an approach to this type of comparison, the efficacy of mepolizumab, benralizumab, and reslizumab was compared via an Safety of the anti-IL-5 treatment indirect treatment comparison by analyzing the data from clinical studies using the drugs at licensed dose and the route of admin- As eosinophils have been reported to have potential anti- istration. The analysis matched the number of eosinophils among parasitic and anti-viral effects,65 there is a concern about the studies and showed that mepolizumab was associated with decrease in host defense during anti-IL-5 therapy. However, the significantly greater improvements in exacerbations and asthma frequency of upper-airway infection tended to be lower than that of control compared with other anti-IL-5 drugs in patients with a the placebo arm in clinical studies of the 1-year use of mepolizu- blood eosinophil count 300 cells/ml.73 In contrast, another mab46 and benralizumab.53,54 The longitudinal safety of mepoli- matching-adjusted indirect comparison analysis that matched the zumab up to 52 weeks (COSMOS study, n ¼ 558)66 or 3.5 years various parameters including number of eosinophils, IgE levels, and (COLUMBA study, n ¼ 347),67 and of benralizumab for 1 year (BORA previous exacerbations by estimating a propensity score did not study, n ¼ 1576)68 or 2 years69 was assessed, but no serious concern show significant differences between mepolizumab and benrali- about an increase in infections was raised. No parasitic infections zumab.74 These contradictory results might stem from the different were reported for both drugs.67,68 analytical method used in the various studies. Although the better Regarding malignancies, six out of 347 patients (1.7%) and 12 of efficacy of benralizumab after mepolizumab treatment was re- 1576 patients (0.8%) reported malignancies while receiving ported in case reports,75,76 a clear difference in efficacy between mepolizumab67 and benralizumab,68 respectively. The types of these drugs has not been established. malignancies included basal cell carcinoma (5.8 events per 1000 Although the specific reason for the discrepancy observed be- patient-years in mepolizumab); cancers of the nasal cavity, tween the efficacy of the elimination of eosinophils and clinical prostate (1.7 events per 1000 patient-years in mepolizumab), improvement is unclear, the following are potential explanations. breast (0.83 events per 1000 patient-years in mepolizumab), co- In animal experiments, the existence of a subset of “good eosino- lon, and pancreas; chronic myeloid leukemia; and B-cell lym- phils”, which are termed resident eosinophils (rEOS), has been phoma. The rate of incidence of malignancy during mepolizumab suggested.36 As rEOS reportedly suppress airway inflammation and 182 H. Nagase et al. / Allergology International 69 (2020) 178e186 their survival is IL-5-independent,36 mepolizumab might not eliminate those cells and the number of residual eosinophils might not be related to asthma exacerbation. However, as the existence of rEOS has not been established in human tissues, further human studies should clarify the potential existence of this subset of good eosinophils and the nature of the residual eosinophils after mepolizumab treatment. From another perspective, compared with the circulating eosinophils, the expression of IL-5Ra appears to be functionally downregulated in lung tissues.77 Another study also reported that expression of the IL-3 receptor and GM-CSF receptor, but not IL-5Ra, was observed in tissue eosinophils after an allergen challenge.78 Those observations suggest that the additional efficacy of benralizumab afforded by ADCC activity might be limited to complete elimination of tissue eosinophils.

Positioning of anti-IL-5 treatment for severe asthma (Table 1) Fig. 3. Distribution of severe asthma phenotypes and potential indication for biologics. Four phenotypes were defined based on a threshold value of blood eosinophil count of 150 cells/ml and a FeNO level of 25 ppb. Patients with severe asthma under inhaled In our analysis, approximately 25% of patients with severe corticosteroid treatment and not using biologics were recruited from an outpatient asthma were indicated for all biologics, including omalizumab, clinic at the Teikyo University Hospital (n ¼ 102). Patients were categorized into four mepolizumab, benralizumab, and dupilumab, if the threshold phenotypes (AeD); the proportion of patients belonging to each phenotype is shown. The potential indication for biologics is also shown for each phenotype. The percent- values of blood eosinophils and FeNO were set at 150 cells/ml and ages of patients in each category who fulfilled the indication for omalizumab are 79 25 ppb, respectively. Although a definitive biomarker for the shown in parentheses. Fifteen patients (14.7% of total patients) were treated with choice of biologics has not been established, the following infor- regular oral glucocorticoids (A, 6.7%; B, 53.3%; C, 6.7%; D, 33.3%). The diagnosis of mation should be considered in this context. severe asthma was based on the Japanese guidelines for adult asthma95 and the fi e The efficacy of anti-IL-5 therapy and dupilumab strengthens as indication for omalizumab was de ned as a total serum IgE level of 30 1500 IU/ml and positive specific IgE Ab for perennial aeroallergen. the blood eosinophil count increases (Table 1).58,59,80 As the level of FeNO is regulated by IL-4/IL-13, this parameter is a good marker for predicting the efficacy of dupilumab.80 However, the utility of FeNO Other points that should be taken into consideration regarding was not shown for mepolizumab.60 the choice of biologics are comorbidities, longitudinal safety, and We classified the patients with severe asthma (n ¼ 102) into interval of injection (Table 1). Mepolizumab is indicated for four phenotypes based on eosinophil count and FeNO (Fig. 3). The eosinophilic granulomatosis with polyangiitis (EGPA), and dupilu- FeNO single-high phenotype (C) was the smallest group in our mab is indicated for CRSwNP (in western countries) and for atopic analysis (4.9%) and in a recent phase 3 QUEST study of dupilumab dermatitis, whereas omalizumab is indicated for seasonal rhinitis (8.5%)80 and this phenotype will be indicated for dupilumab or in Japan, as well as for urticaria. Safety data covering 2 years for omalizumab therapy. The eosinophil single-high phenotype (B) and benralizumab, 4 years for mepolizumab, and 10 years for omali- the dual-high phenotype (D) encompassed the largest proportion zumab have been generated. The interval of injection in the of patients, both in our analysis (B, 39.2%; D, 31.4%) and in the maintenance phase is 8 weeks for benralizumab, 4 weeks for e QUEST study (B, 29.9%; D, 41.7%)80; and the all biologics may be mepolizumab, 2 4 weeks for omalizumab, and 2 weeks for indicated for those phenotypes. In the QUEST study, the exacerba- dupilumab. e tion rate reduction afforded by dupilumab vs. the placebo was As shown in Figure 3, omalizumab is indicated for 40% 60% of fi significant in both phenotypes, however, numerically lower in the patients with all phenotypes strati ed according to blood eosino- eosinophil single-high phenotype (B) compared with the dual-high phil counts and FeNO level. As the global evaluation of treatment phenotype (D) (B, 47% vs. D, 65%).80 Thus, anti-IL-5 therapies effectiveness at 4 months predicted the longitudinal effect as a might be relatively more effective than is dupilumab for the continuation rule, trial of omalizumab treatment for 4 months was recommended in the “care pathway” when multiple biologics are eosinophil single-high phenotype (B), although further investiga- 81 tion is required to confirm this notion. indicated.

Table 1 Different characteristics of the biologics indicated for severe asthma.

Omalizumab Mepolizumab Benralizumab Dupilumab

Anti-IgE Ab Anti-IL-5 Ab Anti-IL-5Ra Ab Anti-IL-4Ra Ab

Decrease in blood eosinophils Decrease in lung tissue eosinophils ~ ND Continuation rule established ND ND Prognosis after discontinuation Study ongoing ND ND analyzed Biomarker for prediction Controversial Blood eosinophils Blood eosinophils FeNO of efficacy Blood eosinophils Serum IgE Approved comorbidity Seasonal rhinitis (Japan) EGPA None CRSwNP Urticaria (EU, USA) Atopic dermatitis Interval of injection 2 or 4 weeks 4 weeks 8 weeks 2 weeks (maintenance) Longitudinal safety data ~10 years ~4 years ~2 years ~1 year

EU, European Union; ND, Not determined; USA, United States of America. H. Nagase et al. / Allergology International 69 (2020) 178e186 183

Effect of mepolizumab on eosinophilic granulomatosis with The first randomized, double-blinded, placebo-controlled study polyangiitis of mepolizumab in patients with CRSwNP was reported in 2011.93 Objective patients exhibited failure of standard therapy for EGPA, formerly known as ChurgeStrauss syndrome, is an CRSwNP before the study. Twenty patients with severe CRSwNP eosinophilic vasculitis characterized by asthma, peripheral neu- were administrated mepolizumab (750 mg) intravenously twice ropathy, and eosinophilic vasculitis of various organs.82 Most of with an interval of 4 weeks, while 10 patients with CRSwNP were these patients are dependent on systemic glucocorticoid therapy, treated with placebo using the same protocol. The nasal polyp and relapses are common. The efficacy of mepolizumab was scores in the mepolizumab group were significantly reduced from investigated as an add-on therapy in patients with refractory the baseline (1.30) compared with the placebo group (0.00). Blood EGPA.83 Mepolizumab treatment led to a significant increase in eosinophil counts and serum ECP and IL-5Ra levels were also remission time, and remission occurred in 53% of participants in the significantly reduced in the mepolizumab group. However, re- mepolizumab group vs. 19% in the placebo group. Forty-four ductions in the LundeMackay CT score, SNOT-22, loss-of-smell percent of the members of the mepolizumab group, compared symptom score, and rhinorrhea scores were not significantly with 7% of those of the placebo group, received an average daily different between the groups. To obtain a significant reduction of dose of prednisolone of 4 mg at the end of the study. This study those scores, a greater number of patients and more frequent times showed for the first time the efficacy of mepolizumab for main- of mepolizumab administration might be required. taining remission in EGPA. Mepolizumab was approved for EGPA in The next randomized, double-blinded, placebo-controlled study December 2017 in United States and May 2018 in Japan. Further of mepolizumab in patients with CRSwNP was reported in 2017.94 A studies of the efficacy of the drug at the acute phase, the positioning total of 107 patients were randomized to receive mepolizumab in the initial treatment, and treatment sequence should be per- (n ¼ 54) or placebo (n ¼ 53) in three countries (Belgium, formed in the future. Netherlands, and the United Kingdom). Patients had a nasal polyp score of 3 or more in one nostril (and a minimum score of 2 on the Roles of anti-IL-5 treatment in chronic rhinosinusitis with other side) and a visual analog scale (VAS) nasal symptom score > 7. nasal polyps Patients were also required to be eligible for surgery as a result of being refractory to standard steroid therapy (typical corticosteroid CRSwNP is a severe persistent airway disease. In Europe and the and short course of systemic corticosteroids). Patients with severe United states, CRSwNP has been thought to be mainly characterized CRSwNP received intravenous injection of mepolizumab (750 mg) by a type-2-mediated inflammatory reaction with marked infil- every 4 weeks (Weeks 1, 5, 9, 13, 17, and 21), for a total of 6 doses. A tration of eosinophils and mast cells in the nasal mucosa and NP.84 significantly greater proportion of patients in the mepolizumab Japanese CRSwNP was recognized as a neutrophilic inflammatory group (10%) compared with the placebo group (30%) no longer disease 30 years ago. However, the infiltrated cell type in recent required surgery at Week 25 (P ¼ 0.006). The nasal polyp score reports of Japanese CRSwNP cases was changed from neutrophils to decreased significantly at Week 25 in the mepolizumab group (1.8 eosinophils.85 Patients with this type of CRSwNP complain mainly from the baseline, Fig. 5) compared with the placebo group (0.6, of loss of smell, and strong eosinophil infiltration is observed in P ¼ 0.001). The nasal polyp score was improved by 1 or more points nasal polyp tissues. This CRSwNP type is resistant to macrolide in 27 (50%) patients in the mepolizumab group and in 14 (27%) therapy and easily recurs after endoscopic sinus surgery (ESS), patients in the placebo group. The symptom VAS scores, and SNOT- suggesting the presence of intractable/refractory CRSwNP. There- 22 questionnaire score were significantly decreased in the mepo- fore, this intractable type of CRSwNP is termed eosinophilic chronic lizumab group compared with the placebo group. Headache, e rhinosinusitis (ECRS).86 89 nasopharyngitis, oropharyngeal pain, and back pain were major IL-5 might be a major target in the therapeutic strategy for adverse events in both groups. Mepolizumab was tolerated well. CRSwNP. Bachert's group from Ghent University presented 10 Blood eosinophil counts decreased from a geometric mean of clusters of CRSwNP, six of which had high concentrations of IL-5, 500 cells/ml at the baseline to 50 cells/ml at Week 25 in the mepo- ECP, and IgE, and four of which had low or undetectable levels of lizumab group; in contrast, such a decrease was not observed in the these markers.90 IL-4-stimulated Th2 cells and TSLP/IL-33- placebo group. These results suggest that mepolizumab treatment stimulated ILC2s are major IL-5-producing cells in nasal mucosa has the potential to improve QOL and reduce the surgery- and nasal polyps (Fig. 4). Mast cells in ECRS also produce IL-5 by associated burden in patients with severe CRSwNP. An extensive stimulation of TSLP and IL-1b.91,92 randomized, double-blinded, placebo-controlled international

Fig. 4. IL-5 plays an important role in the pathogenesis of nasal polyps. TSLP, IL-33, and IL-1b are released from nasal epithelial cells after infection with microorganism (vi- Fig. 5. Changes in the nasal polyp score from the baseline. Mepolizumab significantly ruses and bacteria). TSLP induces IL-5 production by Th2 cells and ILC2s. TSLP and IL- reduced the nasal polyp score at Week 25 compared with the baseline. p*** <0.001. BL, 1b induce IL-5 release from mast cells. baseline; LS, least squares; SE, standard error. 184 H. Nagase et al. / Allergology International 69 (2020) 178e186 clinical trial is currently under way, to assess the effect of mepoli- AstraZeneca and research grants from Maruho. SF received speaker fees from Kyorin, zumab on severe CRSwNP in with a combined cohort stemming Taiho and Mitsubishi Tanabe Pharma and has been an advisory board member for GlaxoSmithKline, Kyowa Kirin and Sanofi, and received research grants from Mar- from many countries of Europe and the United states. uho, Tsumura, Mitsubishi Tanabe Pharma and Sanofi. We conducted a phase II, multicenter, randomized, double- blinded, placebo-controlled study of ECRS for benralizumab in Japan. Fifty-six patients were enrolled (placebo, n ¼ 11; benrali- References zumab single shot, n ¼ 22; benralizumab three times every 4 weeks, n ¼ 23). Blood eosinophil count reached zero by benrali- 1. Farahi N, Singh NR, Heard S, Loutsios C, Summers C, Solanki CK, et al. Use of zumab. Several cases in the active group exhibited a decrease in the 111-Indium-labeled autologous eosinophils to establish the in vivo kinetics of human eosinophils in healthy subjects. 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The effect of recombinant human interleukin-5 on eosinophil accumulation and degranu- Conclusion lation in human nasal mucosa. J Allergy Clin Immunol 1992;90:160e8. 13. Terada N, Konno A, Natori T, Tada H, Togawa K. Interleukin-5 preferentially In this review, the role of IL-5 and the current status of anti-IL-5 recruits eosinophils from vessels in nasal mucosa. Acta Otolaryngol Suppl 1993;506:57e60. therapy in eosinophilic diseases, including severe asthma, EGPA, 14. Malm-Erjefalt M, Greiff L, Ankerst J, Andersson M, Wallengren J, Cardell LO, and ECRS or CRSwNP, was discussed. In the near future, biologics et al. Circulating eosinophils in asthma, allergic rhinitis, and atopic dermatitis targeting alarmins, including IL-33 or TSLP, which are more up- lack morphological signs of degranulation. Clin Exp Allergy 2005;35:1334e40. stream molecules that broadly suppress the downstream pathways, 15. Yamada T, Miyabe Y, Ueki S, Fujieda S, Tokunaga T, Sakashita M, et al. Eotaxin-3 as a plasma biomarker for mucosal eosinophil infiltration in chronic rhinosi- may be available. As anti-IL-5 therapy is specific to eosinophilic nusitis. Front Immunol 2019;10:74. inflammation, this strategy will be continuously indicated for 16. Neves JS, Perez SA, Spencer LA, Melo RC, Reynolds L, Ghiran I, et al. Eosinophil eosinophilic diseases once its longitudinal safety and efficacy is granules function extracellularly as receptor-mediated secretory organelles. Proc Natl Acad Sci U S A 2008;105:18478e83. better established. 17. Ueki S, Mahemuti G, Oyamada H, Kato H, Kihara J, Tanabe M, et al. Retinoic acids are potent inhibitors of spontaneous human eosinophil apoptosis. J Immunol 2008;181:7689e98. Acknowledgments 18. Ueki S, Nishikawa J, Fukuchi M, Konno Y, Takeda M, Moritoki Y, et al. ICAM-1 upregulation is not required for retinoic acid-induced human eosinophil sur- This review was supported in part by a research grant from vival. Immunol Lett 2018;196:68e73. 19. Saffari H, Hoffman LH, Peterson KA, Fang JC, Leiferman KM, Pease 3rd LF, et al. Environmental Restoration and Conservation Agency (HN), the Electron microscopy elucidates eosinophil degranulation patterns in patients Japan Agency for Medical Research and Development (AMED; SU: with eosinophilic esophagitis. J Allergy Clin Immunol 2014;133:1728e34.e1. JP19ek0410055, SF: JP17ek0410040, JP16ek0109062), the Mochida 20. Erjefalt JS, Greiff L, Andersson M, Adelroth E, Jeffery PK, Persson CG. Degran- ulation patterns of eosinophil granulocytes as determinants of eosinophil Memorial Foundation for Medical and Pharmaceutical Research driven disease. Thorax 2001;56:341e4. (SU), Japanese Society of Laboratory Medicine Fund for Promotion 21. Persson C, Uller L. Theirs but to die and do: primary lysis of eosinophils and of Scientific Research (SU), Japan Society for the Promotion of Sci- free eosinophil granules in asthma. Am J Respir Crit Care Med 2014;189: e ence KAKENHI (SU: 15KK0329, 16K08926, 17K09993, FS: 628 33. 22. Ueki S, Melo RC, Ghiran I, Spencer LA, Dvorak AM, Weller PF. Eosinophil 17H04344), and a Health Labour Sciences Research Grant (H30- extracellular DNA trap cell death mediates lytic release of free secretion- Nantitou(nan)-Ippan-016, SF). competent eosinophil granules in humans. Blood 2013;121:2074e83. The authors are grateful to Ms. Satomi Misawa for outstanding 23. Kita H. Eosinophils: multifaceted biological properties and roles in health and disease. Immunol Rev 2011;242:161e77. assistance in drawing the figure and Ms. Noriko Tan and Dr. Yui 24. Mukherjee M, Bulir DC, Radford K, Kjarsgaard M, Huang CM, Jacobsen EA, et al. Miyabe for excellent technical assistance. Thanks are also extended Sputum autoantibodies in patients with severe eosinophilic asthma. J Allergy e to Ms. Asako Tsukamoto and Ms. Eriko Endo for their outstanding Clin Immunol 2018;141:1269 79. 25. Choi Y, Le Pham D, Lee DH, Lee SH, Kim SH, Park HS. Biological function of secretarial assistance. The author would like to thank Enago (www. eosinophil extracellular traps in patients with severe eosinophilic asthma. Exp enago.jp) for the English language review. Mol Med 2018;50:104. 26. Muniz VS, Silva JC, Braga YAV, Melo RCN, Ueki S, Takeda M, et al. Eosinophils release extracellular DNA traps in response to Aspergillus fumigatus. J Allergy Conflict of interest Clin Immunol 2018;141:571e85. e7. HN received speaker fees from AstraZeneca, GlaxoSmithKline, Novartis and 27. Omokawa A, Ueki S, Kikuchi Y, Takeda M, Asano M, Sato K, et al. Mucus Sanofi, and has been an advisory board member for GlaxoSmithKline, and received plugging in allergic bronchopulmonary aspergillosis: implication of the research grants from Boehringer Ingelheim. SU received speaker fees from eosinophil DNA traps. Allergol Int 2018;67:280e2. H. Nagase et al. / Allergology International 69 (2020) 178e186 185

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