Anti-IL5 therapy for asthma and beyond

Airway inflammation is considered to be the primary component contributing to the heterogeneity and severity of airway disorders. Therapeutic efficacies of diverse novel biologics targeting the inflammatory pathways are under investigation. One such target is IL-5, a type-1 cytokine that is central to the initiation and sustenance of eosinophilic airway inflammation. Over the past decade, anti-IL5 molecules have been documented to have mixed therapeutic benefits in asthmatics. Post hoc analyses of the trials reiterate the importance of identifying the IL-5-responsive patient endotypes. In fact, the currently available anti-IL5 treatments are being considered beyond asthma management; especially in clinical complications with an underlying eosinophilic pathobiology such as hypereosinophilic syndrome (HES) and eosinophilic granulomatosis and polyangitis (EGPA). In addition, closer analyses of the available data indicate alternative mechanisms of tissue eosinophilia that remain uncurbed with the current dosage and delivery platform of the anti-IL5 molecules.


Introduction
The past ten years have witnessed the development and evaluation of a number of biologics that target the Th2 cytokines involved in asthma pathophysiology, particularly those that are associated with eosinophils in the airway. Eosinophils play a key role in the pathobiology of several airway disorders presenting with chronic inflammatory pathology such as asthma [1,2], chronic obstructive pulmonary disorder (COPD) [3], eosinophilic granulamatosis and polyangitis (EGPA) [4], and hypereosinophilic syndrome (HES) [5]. Targeting Interleukin-5 (IL-5) in asthma, the central protagonist in eosinophilia (discussed in details later), was a logical derivative post promising results in animal models [6,7] and initial screenings in patients [8,9]. Deliberation arises from the mixed response of anti-IL-5 trials conducted in different asthmatic populations that document a healthy reduction in circulating eosinophils, but without much significant improvement in other clinical indices of disease severity (extensively reviewed in [10][11][12]). Again in a recent review, anti-IL-5 therapy has been conjectured to be effective in long-term management of HES patients [5]. The current review will critically evaluate the documented outcomes of the conducted clinical trials to date and subsequently assess the therapeutic implications of anti-IL-5 therapy in treating airway disorders with an aberrant eosinophilic pathobiology.

Eosinophil biology and the role of IL-5
A robust literature now support eosinophils to be pleiotropic, multifunctional leukocytes that facilitate the 'innate' response against extraneous agents in the airway, modulate the downstream 'adaptive' immunity cascade, maintain local immunity/inflammation and as the end-stage effector cells that can cause tissue damage via release of granule proteins, reactive oxygen species and cysteinyl leukotrienes (reviewed in [2,13]). In eosinophil biology (refer to Figure 1), IL-5 plays a central role in the production, mobilization, activation, recruitment, proliferation, survival and suppression of apoptosis in eosinophils at the site of inflammation (reviewed in, [2,6,12,13]), illustrated in a schematic diagram (Figure 1).
In 1996, a study reported that 'IL-5 deficient' mice failed to develop the characteristic eosinophilia and airway hyper-reactivity after ovalbumin-sensitization [7]. Soon after, 8 asthmatic patients demonstrated increase in airway eosinophil counts and methcholine PC 20 (a provocative concentration of methcholine required to induce 20% reduction in the forced expiratory volume in 1 second, FEV 1 ) when subjected to inhaled recombinant IL-5 [9]. Moreover, restraining sources of eosinophil recruitment and/or eosinophil-deficient animal models were observed to be healthy without any characteristic abnormalities [14]. As a logical derivative from the existing experimental and clinical evidences, several monoclonal antibodies (mAbs) were engineered to CD34 + myeloid progenitor cells (bone-marrow) differentiate into the IL5α + CCR3 + eosinophil-committed progenitor cells under the influence of the different transcription factors like GATA2 and C/EBPα. IL-5, IL-3 and GM-CSF stimulate their further maturation into eosinophils. Migration: release into the circulation is coordinated synergistically by IL-5 and eotaxin. Transmigration: under the influence of IL-5 and eotaxin, the eosinophils 'seep' out through the endothelium. Recruitment: Eosinophil trafficking into the site of inflammation is selectively regulated by IL-5, eotaxin and CCL5, in addition to a multitude of cytokines. Activation: IL-5 binds to IL-5Rα and activates eosinophils to release a multitude of cytokines, eosinophilic granular proteins, cysteinyl leukotrienes, that lead to tissue damage and further aggravates the inflammatory process. Survival and stabilisation: IL-5 released from different sources and products from mast cell (MC) degranulation suppresses apoptosis and allows survival of eosinophils in the submucosa. (B) Different sources of IL-5 (in red) and sustenance of eosinophilia: (i) the canonical T H 2 pathway initiated by dendritic cell (DC) activation releases IL-5. (ii) MC activation is another source of IL-5 that can be triggered by IgE binding to the FCεRI receptor or by epithelial-derived Type 2 alarmins like TSLP and IL33; or via T H 9 pathway (iii) Type-2 alarmins (IL-33, IL-25, TSLP) can activate the lineage negative ID2 + lymphoid cells resident in the tissue to differentiate into lineage negative ILC 2 s that can release IL-5 and IL-13, and drive eosinophilic inflammation (iv) IL-13 and IL-4 can recruit CD34 + progenitors cells from bone marrow into the lung tissue where it can differentiate into eosinophils in presence of IL-5. N.B. Diagram is not up to scale. Mechanisms relevant to only eosinophilic inflammation has been included.
A comparative analysis of the Anti-IL-5 trials: asthma As early as 1990, Bousquet et al., [8], correlated eosinophilia with asthma severity and demonstrated eosinophilic cation proteins (ECP) were associated with epithelial damage in 44 patients with asthma. A direct but modest correlation has been established between asthma severity, frequent exacerbations and the intensity of eosinophilia. Thereby, a sub-set of patients are being identified who suffer from 'severe refractory asthma' , consequently accounting for a high socioeconomic burden and are considered to most benefit from an eosinophil-targeted therapy [1,10].
The last 15 years have documented several clinical trials that evaluate the therapeutic relevance of anti-IL-5 biologics in asthma treatment and symptom management (refer to Table 1). As evident from the outcome summary tabulated in Table 1, Mepolizumab, a humanized mAb (IgG1) with a high affinity for binding free IL-5 (which further prevents its binding to the receptor, IL5Rα), is found to be effective in depleting eosinophil numbers in blood and the airways. On the contrary, studies in mild-moderate asthmatics documented Mepolizumab to be ineffective in improving end-point clinical symptoms (refer to Table 1, [16,19,20]), therefore raising concern over the efficacy of IL-5 as a therapeutic intervention in asthma. However, by selecting patients with persistent blood ( >0.3 × 10 9 /L) and sputum eosinophils (≥3%) coupled with frequent history of exacerbations, two independent relatively small studies in 2009 documented a significant decrease in the exacerbation frequencies (P ≤ 0.02 vs. placebo, both studies) and asthma control questionnaire (ACQ) scores (P ≤ 0.02, vs. placebo, both studies), with 750 mg infusions of Mepolizumab [21,22]; in addition to a prednisone-sparing effect [21]. Similar reduction in exacerbation frequencies with corresponding decrease in peripheral blood eosinophils was reflected in a large, double-blinded, placebo-controlled, multi-centered study conducted in 2011 [23]. Based on the dose-response observations from the DREAM study [23], 75 mg intravenous and 100 mg subcutaneous doses were investigated in a recent Phase III trial, where significant reductions in exacerbation rates by 47% and 53% respectively (P< 0.001, vs. placebo) along with depletion of blood eosinophils were recorded [28]. The 100 mg subcutaneous dose was reported in a parallel Phase III study to have a corticosteroid-sparing effect in a similar target population, with median percentage reduction of 50% in treatment group, along with 32% relative reduction in annual exacerbation rate (p= 0.04 vs placebo) [29]; a reduction though statistically significant, is less pronounced than that observed in the earlier study with higher dose and intravenous route of drug delivery [21,32]. The optimum dose, route and duration of therapy and persistence of beneficial effects for prednisone-dependent patients remain to be established.
Another anti-IL5 mAb (IgG 4/k ) Reslizumab, showed similar reduction in sputum eosinophils, significant improvement in lung function (P= 0.002, vs. placebo) and a trend towards improved asthma scores (P= 0.054, vs. placebo) in patients diagnosed with severe refractory eosinophilic asthma (see Table 1). Additionally, the authors observed the improvement in ACQ scores were most pronounced in patients with nasal polyps (P= 0.012, vs. placebo), [24] which reflected the observations of Gevaert et al., in 2003 [33]. There are recent reports of Phase III trials that demonstrate significant improvement in ACQ scores and FEV 1 (p<0.05, vs. placebo) in moderate to severe asthmatics treated with 3.0 mg/kg of intravenous Reslizumab; charting a larger improvement in asthma control for subjects with baseline eosinophils ≥400 cells/μl [30,31].
IL5Rα expressed by both mature eosinophils and eosi nophil-lineage progenitor cells [2], is targeted by Benralizumab (MEDI-563), a humanized, afucosylated mAb. Being afucosylated, this drug induces apoptosis in its target cells via enhanced antibody-mediated cellular toxicity (ADCC), and is considered to have an increased efficiency of eosinophil depletion comparative to the other anti-IL5 biologics [34]. The initial safety trial conducted by Busse et al., in 2010, documented no adverse events [25] and a further study by the same group showed 100% reduction of peripheral circulating eosinophils (Table 1) [26]. More recently, 100 mg subcutaneous Benralizumab exhibited significant improvement in annual exacerbation rates, lung function and asthma score, with greater benefits seen in patients with blood eosinophil levels ≥ 400 cells/μl [27]. Currently, there are three clinical trials registered on http://clinicaltrials.gov (NCT01914757, NCT02075255, NCT01928771, last accessed 17/09/2014), where the drug is being assessed as an adjunct therapy for 'uncontrolled' asthma.

Further assessment of anti-IL5 Trials: clinical insights
The mixed outcomes from anti-IL-5 clinical trials highlight the need for careful endotyping of patients, since the therapy is deemed effective on those patients whose asthma is dependent on the eosinophilic inflammatory pathway [1]. The potential 'responders' to IL-5 therapy are patients who present with eosinophilia (blood >0.3 × 10 9 /L, >3% sputum), are generally steroid-responsive, and suffer from frequent exacerbations. As evident from the DREAM study [23], the atopic status is inadequate for segregating 'responders' from the 'non-responders' , since approximately 50% of the patients who responded to Mepolizumab had negative radioallergosorbent test to the four most common allergens. In addition, sub-sets of patients that are aspirin-   sensitive/induced asthma or present with sinusitis might also benefit from anti-IL5 therapy [35].
Using 'sputum eosinophils' as a biomarker to identify IL5-treatment responsive patient-groups as well as a marker for its therapeutic outcome is a topic under debate [1]. Studies that considered patients with ≥ 2.5 -3% sputum eosinophilia in their inclusion criteria, independently recorded significant improvements with asthma scores and lung function [21,22] compared to others (see Table 1). Similarly post hoc analysis showed that patients treated with 1.0 mg/kg Reslizumab, with baseline sputum levels< 3% did not show improvement in the FEV 1 , (even with depleted peripheral eosinophil levels) [18]. In fact, sputum eosinophils do not correlate with a change in circulating eosinophil numbers in the severe prednisone-dependent asthmatic patients and the former is markedly reduced before an event of exacerbations [1].
Subcutaneous doses of Mepolizumab showed neither significant reduction in sputum eosinophils (for 75 and 250 mg dosage groups) nor any relevant improvements in the symptom scores or lung function (see Table 1, [23]). The recent phase III trials documented depletion of blood eosinophils, significant improvement in asthma symptom scores and moderate reduction in exacerbation rates with lower (100 mg) subcutaneous doses (refer to Table 1), without any indication of whether the luminal eosinophilia generally exhibited in the specific patient group was resolved or not [28,29]. In contrast, 750 mg intravenous infusions in the previous two studies of similar disease profiles [21,22], were able to reduce both circulating and sputum eosinophils, allow significant improvement in ACQ, FEV 1 and quality of life score along with pronounced reduction in exacerbations. This discrepancy may reflect the therapeutic significance of the drug delivery platform and dose used, a concern addressed in a recent editorial [32].

Further assessment of Anti-IL5 trials: molecular insights
The eosinophil biology is complex and outcomes from the anti-IL-5 clinical trials reiterate this. Many of the anti-IL-5 clinical trials (Table 1) document the presence of tissue eosinophilia in spite of nil/low circulating levels, post-treatment. Especially, both studies with Benralizumab showed 100% reduction of eosinophils in bone-marrow and peripheral blood, but presence of airway mucosal/sub-mucosal eosinophils [26] and detectable levels of ECP in the sputum [25], indicating an alternative mechanism to IL-5 for eosinophil initiation, recruitment, activation and survival in the tissues. Delving further, Haldar et al., [22] showed significant decrease (P< 0.002) for both circulating blood and sputum eosinophils (see Table 1) in the Mepolizumab study group, which was not reflected in the paired bronchialbiopsy specimens (obtained before and after the study).
In context, antisense oligonucleotide therapy (TPI ASM8), developed to suppress the expression of surface receptors CCR3 (C-C chemokine receptor type 3, binds eotaxin) and β chain (shared receptor for IL-5, IL-3 and granulocyte macrophage-colony stimulating factor, GM-CSF) [36], reduced sputum eosinophil counts by 46%; while a CCR3 antagonist was recently documented to show no effect of blood or sputum eosinophilia or to have any clinically improvement in moderate to severe asthmatics [37]. Scattered evidences [2,11,38] instrument the presence of alternative pathways in situ, that can trigger, activate and maintain eosinophils in the sub-mucosal and mucosal surfaces, independent of the classical T H 2 pathway activation triggers (refer to Figure 1).
Extraneous environment-derived factors including nonallergic sources like pathogens and epithelial damage can trigger the release of epithelium-derived 'Type-2 alarmins' -IL-25, IL-33, and thymic stromal lymphopoeitin (TSLP, see Figure 1) [15,38]. IL-25 and IL-33 can initiate mast cell (MC) response (mostly sub-mucosal localization) that leads to the release of IL-5 and CCL5. In addition, the type-2 alarmins activate the resident lineage negative, type 2, innate lymphoid cells (ILC 2 s) to release the classical T H 2 cytokines IL-5, IL-13, IL-9, that directly or indirectly support eosinophil recruitment and survival in the tissues (refer to Figure 1) [15,[38][39][40][41]. Again, in vitro experiments demonstrated TSLP in presence of pro-inflammatory stimuli IL1-beta/tumor necrosis factor alpha (mimicking an ongoing inflammatory state) activated MCs to release IL-5 and IL-13; thereby, suggesting subsidiary mechanisms that produce IL-5 and can promote eosinophil numbers in the inflamed parenchyma/airway lumen [42]. IL-13 (and IL-4 in airway smooth muscle, ASM) trigger the release of eotaxins from the ASM [43] and the bronchial epithelium [44] that promote recruitment of eosinophils and eosinophil progenitors (refer to Figure 1). Increased IL-13 and IL-4 can promote the homing of CD34 + haemopoietic progenitor cells into the airway parenchyma [45].
Evidence suggest that this mechanism maybe upstream of activation by epithelial cell-derived cytokines [46,47]. Understanding physiological processes that promote airway eosinophilia in severe asthma may be critical to the development of novel treatment modalities for optimal asthma control. Luminal eosinophilia in asthma arise as a result of (i) the recruitment of mature eosinophils from the periphery in response to locally elaborated chemoattractants such as eotaxin and/or (ii) the localized maturation of eosinophil lineage-committed progenitors, termed "in situ differentiation" in the presence of locally elaborated cytokines such as IL-5 [48,49]. That haemopoietic progenitors differentiate within the tissue is inferred from findings that there is increased recruitment of eosinophil progenitor cells into the airways in asthmatics [50]. In addition CD34 + cells extracted from human nasal polyp tissue and nasal explant tissue undergo IL-5 driven differentiation to form mature eosinophils [51,52]. In context, anti-IL5 mAbs reduce tissue and luminal eosinophils (see Table 1), and are most effective in severe prednisonedependent asthmatics with eosinophilic bronchitis [21]. These findings suggest that local eosinophilopoiesis may be a more dominant mechanism for the persistence of eosinophils in the airways of patients with moderate-to-severe asthma than chemokine-dependent (for e.g. eotaxin) recruitment of mature eosinophils. Whilst this remains to be determined, it may explain our recent findings where treatment with anti-CCR3 failed to clear luminal eosinophils likely because the treatment did not attenuate local differentiative processes [37].
Controlling the development of airway eosinophilia may involve targeting multiple factors that stimulate eosinophils recruitment and modulate local differentiative processes or prolonged tissue survival. A few conducted clinical trials with mAbs targeting IL-4 and IL-13 biology, similar to anti-IL-5, has met with a mixed response in improving clinical symptoms [10]. However, a combination therapy with drugs like Dupulimab (targets the receptor complex common both IL-4 and IL-13) [53] and an anti-IL-5 mAb could synergistically curb the mechanisms of in situ eosinophilia plausibly altered in severe asthmatics, that render the airways susceptible to maintain the clinical symptoms.

Anti-IL-5 therapy for other lung eosinophilic disorders
As evident from the on-going discussion, IL-5 and IL-5 receptor alpha (IL5Rα) exhibit an undeniable eosinophil lineage-specificity. Indisputably, they have been considered as a potential therapeutic target in eosinophilic airway disorders.

Hypereosinophilic syndrome (HES)
HES is a heterogeneous rare disorder defined by the presence of >1500 eosinophils per μL of blood, persistent for ≥ 6 months, with eosinophil-related organ involvement or dysfunction and no identifiable secondary cause of eosinophilia [5]. Early case studies with HES patients show considerable improvement in disease symptoms, patient relief, and decrease in eosinophilia with 750 mg intravenous Mepolizumab [5]. Table 2 summarizes an open-label study with 4 patients in 2004 [54] followed by a double-blinded, multi-centered clinical trial with Mepolizumab in 2008, where 41 out of 43 patients (on the experimental drug and tapering strength of prednisone), managed to maintain a circulating blood eosinophil count< 600/μL, for ≥8 weeks (p< 0.0001, vs. placebo) [55]. Additionally, it could be an alternative to using high-dose OCS, otherwise prescribed to the patients diagnosed with T-lymphocyte variant of HES [56]. A number of studies are currently underway to evaluate other anti-IL5 molecules in addition to Mepolizumab as potential therapeutic interventions in HES. A study with HES patients is projected to end in early 2017, which evaluates the safety and efficacy of Benralizumab (NCT02130 882) in these subjects (http://clinicaltrials.gov).

Eosinophilic Granulomatosis and Polyangitis (EGPA)
EGPA, earlier known as Churg-Strauss syndrome, is a rare form of vasculitis characterized by asthma and eosinophilia, with multi-organ involvement (lung, peripheral nerves, heart, gastrointestinal tract, skin), where systemic corticosteroid is the cornerstone of therapy [4,60]. Histology shows classic evidence of an eosinophilic inflammatory response in the airway tissue of these patients and hence Mepolizumab has been reasoned to be a potential corticosteroid-sparing therapy. A recent case study reported complete regression of asthma (discontinuation of inhaled therapy) and depletion of blood and airway eosinophils in a patient with refractory EGPA with monthly infusions of 750 mg Mepolizumab [60]. Kim et al., 2010, reported a significant (75%) reduction in circulating eosinophils with 4 monthly doses of 750 mg (i.v) Mepolizumab, in a small open label trial with 7 patients, that allowed safe reduction of OCS from a mean dose of 18.8 mg to 4.6 mg [57]. In addition, Mepolizumab at the same dosage allowed complete remission in 8 out of 9 EGPA patients in a Phase II uncontrolled trial (detailed in Table 1) [58]. Though promising, further clinical investigations are necessary to ascertain the therapeutic benefit of Mepolizumab in EGPA and a large multicenter clinical trial is ongoing.

Chronic eosinophilic pneumonia (CEP)
Chronic eosinophilic pneumonia (CEP) is an idiopathic condition that presents with peripheral eosinophilia, eosinophilic infiltrates in the lung parenchyma and may occasionally be associated with asthma. Increased levels of IL-5 and release of cytotoxic granular proteins from eosinophils constitutes an important pathomechanism in pulmonary tissue damage observed in CEP [61]. Conventionally OCS therapy is used for treatment; however, an eosinophil-targeted therapy with Mepolizumab might result in steroid-sparing therapeutic benefit in CEP patients.

Chronic bronchitis (in COPD)
Chronic bronchitis is a primary component of COPD that encompasses a high level of heterogeneity. A sputum database analysis of 2443 patients with airway diseases, revealed one-fifth of the COPD patients experience eosinophilic bronchitis (EB). Additionally, EB was also associated with the severity of airflow obstruction in non-asthmatic COPD. 18% COPD patients with frequent exacerbations were documented to have EB and associated eosinophilia. Targeting eosinophils and IL-5 levels with Mepolizumab may decrease exacerbation rates and improve lung functions for this sub-set of COPD patients [62], as documented for other eosinophil-driven airway  disorders (discussed previously). However, as per the recent reports of Brightling and co-workers, no reduction in annual exacerbation rates was observed in moderate to severe COPD patients with eosinophilia (>3% sputum eosinophils) when treated with a monthly/bimonthly subcutaneous dose of 100 mg Benralizumab, even though the treatment effectively depleted both airway and circulating eosinophils (refer to Table 2) [59]. Nevertheless, online database (http://clinicaltrials.gov, last accessed 16/09/2014) enlists ongoing independent studies investigating efficacy and safety of Mepolizumab as an adjunct treatment in COPD management (registration number: NCT02105961); in COPD with EB (NC T01463644); reducing exacerbations in severe COPD (NCT02105948); and Phase III trials for Benralizumab in moderate to very severe COPD (NCT02155660, NC T02138916), addressing safety and efficacy of the drug.

Summary
The ongoing anti-IL5 clinical trials, show promise of a safe, effective treatment option for the severe 'eosinophilic' asthma endotype independent of their atopic status [63]. Beyond asthma, small pilot studies have documented their potential in treating HES and EGPA patients. IL-5 may not be the sole determinants of persistent airway eosinophilia. Recently described immune cells such as the ILC 2 s and epithelium-derived type-2 alarmins that release T H 2 cytokines like IL-13 may also play important rolesHence, targeting IL-5 and IL-13 to curb the eosinophil-derived clinical symptoms needs to be investigated in select patient-subtypes. Finally, while blood eosinophil count or other indirect assessments such as the eosinophil/lymphocyte ratios may help to identify an "eosinophilic phenotype" to initiate therapy with an anti-eosinophil biologic drug in patients with moderate asthma, this strategy may not be as effective as measuring sputum eosinophils to monitor response to therapy particularly in more severe systemic corticosteroiddependent asthmatic patients. The key to successful therapy would be to select the appropriate patient population.
The mere presence of eosinophils in blood or sputum may not be sufficient. The patients who are likely to respond are those whose disease is truly largely dependent on eosinophil biology. Identification of these patients require clinical acumen, clinical criteria and demonstration of persistent (not transient) systemic and airway eosinophilia.