Research ArticleAsthma

Targeting membrane-expressed IgE B cell receptor with an antibody to the M1 prime epitope reduces IgE production

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Science Translational Medicine  02 Jul 2014:
Vol. 6, Issue 243, pp. 243ra85
DOI: 10.1126/scitranslmed.3008961

Abstract

Elevated serum levels of both total and allergen-specific immunoglobulin E (IgE) correlate with atopic diseases such as allergic rhinitis and allergic asthma. Neutralization of IgE by anti-IgE antibodies can effectively treat allergic asthma. Preclinical studies indicate that targeting membrane IgE–positive cells with antibodies against M1 prime can inhibit the production of new IgE and significantly reduce the levels of serum IgE. We report results from two trials that investigated the safety, pharmacokinetics, and activity of quilizumab, a humanized monoclonal antibody targeting specifically the M1 prime epitope of membrane IgE, in subjects with allergic rhinitis (NCT01160861) or mild allergic asthma (NCT01196039). In both studies, quilizumab treatment was well tolerated and led to reductions in total and allergen-specific serum IgE that lasted for at least 6 months after the cessation of dosing. In subjects with allergic asthma who were subjected to an allergen challenge, quilizumab treatment blocked the generation of new IgE, reduced allergen-induced early and late asthmatic airway responses by 26 and 36%, respectively, and reduced allergen-induced increases in sputum eosinophils by ~50% compared with placebo. These studies indicate that targeting of membrane IgE–expressing cells with anti-M1 prime antibodies can prevent IgE production in humans.

INTRODUCTION

Asthma is a complex inflammatory disorder of the airways characterized by marked heterogeneity in both its clinical course and response to treatment (13). Although several different asthma phenotypes have been identified, allergic asthma, mediated by immunoglobulin E (IgE) antibodies, accounts for at least half of incidence rates worldwide (3, 4). Common inhaled environmental allergens such as pollens, house dust mites, and animal dander are all well-established triggers for allergic asthma (5, 6). The discovery and characterization of the IgE antibody class were a crucial advance toward understanding the immunologic component of asthma and other allergic diseases, and subsequent research has confirmed that IgE is the key molecule involved in mediating type 1 hypersensitivity reactions (7). Allergen-specific IgE antibodies bind to high-affinity IgE receptors (FcεRI) on the surface of mast cells and basophils (8, 9). Upon repeat exposure to allergens, these IgE/FcεRI receptor complexes are cross-linked, setting up a chain of events that lead to mast cell degranulation and the subsequent release of allergic mediators that contribute to the pathogenesis of asthma (8, 9).

IgE represents an attractive therapeutic target for the treatment of asthma and other allergic diseases (911). Currently, the only licensed asthma therapy that specifically targets IgE is omalizumab, a monoclonal antibody that binds to soluble IgE antibody and prevents it from binding to IgE receptors on the surface of immune effector cells (12). Treatment with omalizumab neutralizes free IgE in the serum but does not affect IgE production (13). To maintain low levels of free soluble IgE in the serum, subjects are treated continuously every 2 or 4 weeks. Only subjects whose weight and baseline IgE levels fall within a restricted range are indicated for omalizumab treatment (14, 15).

IgE antibody is produced and secreted from IgE plasma cells, which are derived from naïve B cells that undergo isotype class switching and differentiation to become IgE-positive B cells and plasmablasts that express the membrane B cell receptor form of IgE (8, 16). It is well established that plasma cells can be short-lived (days to weeks) or long-lived (years to decades), with long-lived plasma cells typically residing in the bone marrow and short-lived plasma cells typically residing in spleen, lymph nodes, and other tissues (17, 18). Although studies in mice and humans indicate that IgE production occurs in mucosal tissues, secondary lymphoid organs, and bone marrow, the contribution of short- and long-lived plasma cells to IgE production is poorly understood. Studies in mice indicate that IgE plasma cells are predominantly short-lived, although long-lived IgE plasma cells can be found in the bone marrow (12, 1923). Studies of humans with allergic diseases have documented seasonal fluctuations in allergen-specific IgE levels that are consistent with a proportion of allergen-specific IgE being produced by short-lived plasma cells (2427); however, case reports of the transfer of allergic disease with bone marrow transplantation suggest that long-lived IgE plasma cells may reside in the bone marrow (28, 29).

Preclinically, targeting membrane IgE–expressing cells inhibits the production of new IgE plasma cells, ultimately leading to reductions in serum IgE because existing short-lived IgE plasma cells die and are not replenished with new IgE-producing plasma cells (3035). Thus, inhibiting IgE production by targeting membrane IgE–expressing cells may enable more subjects to be treated (that is, subjects with baseline IgE levels higher than that indicated for omalizumab), less frequent dosing, and/or a potential for sustained effects upon the cessation of therapy, as compared to the neutralization of IgE. Human membrane IgE contains an extracellular 52–amino acid segment that has been called M1 prime, M1′, me.1, or CemX and that is not expressed in secreted IgE antibody (3644). Antibodies targeting the M1 prime segment of membrane IgE reduce serum IgE levels and IgE-producing plasma cells in multiple preclinical models of IgE immune responses (33).

The humanized monoclonal therapeutic antibody quilizumab targets M1 prime and exhibits potent in vitro and in vivo activity (33, 45). Quilizumab can be used to assess the effects of targeting membrane IgE–expressing cells in clinical studies. Here, we describe the results of two studies evaluating the activity, safety, tolerability, and pharmacokinetic profile of quilizumab in subjects with allergic rhinitis and mild allergic asthma to test the hypothesis that targeting M1 prime can reduce IgE-producing plasma cells and thereby reduce total and allergen-specific IgE.

RESULTS

Subject disposition and demographics

In an initial study, a total of 36 subjects with allergic rhinitis, but without asthma, were randomized and completed the study; 24 and 12 subjects received at least one dose of quilizumab or placebo, respectively (Fig. 1A). This sample size was considered sufficient to provide a preliminary assessment of the safety and tolerability of quilizumab, and only limited evaluations of clinical activity were assessed.

Fig. 1. Patient flow.

(A and B) Study flow chart of the phase 1b study in allergic rhinitis subjects (A) and phase 2a allergen challenge study in allergic asthma subjects (B).

In a subsequent study, 29 subjects with mild allergic asthma were randomized to receive either quilizumab (n = 15) or placebo (n = 14), and all subjects received all planned treatment interventions (Fig. 1B). In both studies, the baseline demographic characteristics were similar across active treatment and placebo groups (Table 1).

Table 1. Subject demographics and baseline characteristics.

n/a, not applicable; FEV1, forced expiratory volume in 1 s.

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Pharmacokinetics

Quilizumab exhibited the expected pharmacokinetic characteristics for an IgG1 monoclonal antibody (table S1 and fig. S1). On the basis of a noncompartmental analysis, quilizumab had a slow mean clearance in subjects with allergic rhinitis and asthma (2.2 and 1.9 ml/day per kilogram, respectively) and a long mean terminal elimination half-life (t1/2; 20 to 21 days). Across both studies, the mean relative subcutaneous bioavailability was about 55 to 66%.

Impact of quilizumab on serum IgE

In subjects with allergic rhinitis, total serum IgE levels were reduced by quilizumab treatment (administered on days 1, 29, and 57) compared with placebo. There was a mean ± SE 25 ± 4% (median, 24%) and 26 ± 3% (median, 26%) reduction from baseline in the 5.0 mg/kg intravenous (IV) and 3.0 mg/kg subcutaneous (SC) cohorts, respectively, on day 86 (Fig. 2). The effect of quilizumab on total serum IgE was less in the 1.5 mg/kg IV cohort compared to the higher-dose cohorts. At the end of the study, 6 months after the last dose was administered (day 224), total serum IgE remained 30 ± 5% and 36 ± 3% lower than baseline in the 5.0 mg/kg IV and 3.0 mg/kg SC quilizumab cohorts. Allergen-specific IgE levels were much more variable among subjects than total IgE levels, but reductions in allergen-specific IgE were also observed in the quilizumab-treated subjects. At day 224, allergen-specific IgE was reduced from baseline by a mean ± SE of 39 ± 6% (median, 40%) in the 3.0 mg/kg SC cohort compared to reductions from baseline of 9 ± 10% (median, 9%), 32 ± 14% (median, 14%), and 21 ± 13% (median, 33%) in the placebo, 1.5 mg/kg IV, and 5.0 mg/kg IV cohorts, respectively. Individual serum-specific IgE data are depicted in fig. S6.

Fig. 2. Change in serum IgE after treatment with quilizumab in subjects with allergic rhinitis (phase 1b).

Serum total IgE levels over time in allergic rhinitis subjects after administration of placebo or quilizumab (1.5 and 5 mg/kg IV and 3 mg/kg SC) on days 1, 29, and 57. Data are expressed as percentage change from baseline, where baseline is defined as the average of predose visit days −7 and −1; mean ± SE, n = 8 in each quilizumab group and n = 12 in the placebo group (IV and SC combined).

In subjects with allergic asthma, baseline serum total IgE ranged from 1 to 274.5 IU/ml. Subjects had detectable specific IgE at baseline for at least 2 and up to 10 of the allergen specificities measured (tables S3 and S4). The ratio of specific IgE to total IgE was calculated by adding all specific IgEs and dividing by the level of total IgE. Figure S3 shows the ratio of specific/total IgE of all individual subjects in both the placebo- and quilizumab-treated cohort. At baseline, the ratio of specific/total IgE ranged from 10 to 100%, indicating that, in some subjects, the 10 specific IgEs we measured constituted 10% of their serum total IgE, whereas in some subjects the 10 specific IgEs captured close to all their total IgEs. The ratio of specific to total IgE was well balanced between treatment groups.

Subjects received allergen challenge(s) during screening and at day 86. In subjects treated with placebo, total serum IgE increased by 25 ± 12% of baseline at day 86 (Fig. 3A). In contrast, in subjects treated with quilizumab, total serum IgE decreased by about 20% of baseline at day 86. The reductions in total serum IgE were observed as early as day 57, were statistically significant both before and after challenge at day 86 (P < 0.05 versus placebo), and were sustained at the end of the 4-month follow-up period, reaching a decrease of about 25% of baseline at the last available time point (day 197).

Fig. 3. Effect of quilizumab treatment on serum IgE levels in the subjects with stable mild allergic asthma (phase 2a).

(A to C) Serum total IgE (A), challenge-specific IgE (B), and non–challenge-specific IgE (C) levels over time in allergic asthma subjects after administration of placebo or quilizumab (5 mg/kg IV) on days 1, 29, and 57 and airway allergen challenge on days 1 and 86. Data are expressed as mean percentage change from baseline, where baseline is defined as the average of predose visit days −7 and −1 (mean ± SE). Individual subject data provided in figs. S4 and S5 and tables S3 to S16. Challenge-specific IgE was defined as the one allergen-specific IgE each individual subject was challenged with, whereas the non–challenge-specific IgE was defined as the average of all other measured specific IgEs that were detected. (D) Non–challenge-specific IgE levels in the quilizumab-treated patients expressed as the median percentage change from baseline IgE levels (n = 15) rather than as the mean in (C).

Allergen challenge also resulted in an approximate twofold mean increase in allergen-specific IgE in the serum at 1 month after challenge (Fig. 3B). This increase in allergen-specific IgE was confined to the IgE that was specific for the allergen used in the airway challenge of each subject, whereas IgEs that were specific for other allergens in the panel that were unrelated to the inhaled allergen were not increased (Fig. 3C).

Quilizumab treatment completely blocked allergen-induced increases in allergen-specific IgE (Fig. 3B) at day 57 and before and after challenge at day 86 (P < 0.05 versus placebo at all three time points). The individual absolute levels as well as the % baseline of total IgE, challenge-specific IgE, and non–challenge-specific IgE are depicted in figs. S4 and S5 and tables S5 to S16. After whole-lung allergen challenge in days 1 and 86, an increase was detected in challenge-specific IgE in the placebo-treated cohort, which was also reflected in a smaller increase in the serum total IgE in these subjects. In contrast, the non–challenge-specific IgE levels were not elevated after the airway challenge. There was considerable heterogeneity in the extent of challenge-specific IgE increase within the placebo group, with 4 of 14 subjects showing less than 10% increase on day 29 compared to baseline. However, it was very clear that there was a reduced increase in challenge-specific IgE and total IgE increase after the airway challenge in the quilizumab-treated subjects, with 3 of 15 subjects showing more than a 10% increase on day 29 in challenge-specific IgE. Because of the heterogeneity and variability in the response, it was difficult to detect reduction in allergen-specific IgE in the SOLARIO study. However, a trend was observed in the non–challenge-specific IgEs reducing over time when data were expressed as the median of the quilizumab subjects (fig. S6). Although the confidence intervals (CIs) were large, the trend is very consistent with the reduction in IgE observed in the phase 1 studies.

Presence of IgE plasma cells

In a subset of 10 subjects in the allergen challenge study, bone marrow aspirates were assessed on days 87 and 197 and demonstrated the ongoing presence of IgE plasma cells in subjects regardless of treatment with quilizumab or placebo (fig. S7). However, predose bone marrow aspirates were not obtained, and therefore, the quantitative effect of quilizumab on IgE plasma cells could not be as directly assessed.

Activity of quilizumab against early and late asthmatic response

Studies of omalizumab in mild allergic asthmatic subjects indicate that suppression of free serum IgE can reduce pathologic lung responses, as indicated by treatment-associated reductions in early (EAR) and late (LAR) asthmatic responses after allergen challenge (46, 47). We assessed whether blocking the production of IgE in quilizumab-treated subjects also affected EAR and LAR after allergen challenge of the allergic asthmatic subjects. At screening, both placebo and quilizumab groups had similar EAR and LAR after allergen challenge (Fig. 4A). One month after the third dose of quilizumab (day 86), both EAR and LAR were reduced in quilizumab-treated subjects compared to placebo-treated subjects (Fig. 4B). The mean reduction in the quilizumab LAR area under the concentration-time curve (AUC) was 36% (90% CI, −14 to 69%; P = 0.21), with an EAR AUC reduction of 26% (90% CI, 6 to 43%; P = 0.046) compared with the placebo group.

Fig. 4. Effect of quilizumab treatment on the EAR and LAR in subjects with stable mild allergic asthma (phase 2a).

(A and B) Allergen-induced percent change in FEV1 after challenges at screening (A) and day 86 (B) in allergic asthma subjects after administration of placebo or quilizumab (5 mg/kg IV) on days 1, 29, and 57. FEV1 was collected every 10 min for the first 90 min and then every hour from 2 to 7 hours after allergen challenge. The FEV1 responses between 0 and 3 hours are defined as the EAR, and the FEV1 responses between 3 and 7 hours are defined as the LAR. Data expressed as means ± SE.

Effect of quilizumab on eosinophil levels in subjects with mild allergic asthma

We also assessed whether treatment with quilizumab affected inflammation in subjects with allergic asthma by measuring blood eosinophils and allergen-induced sputum eosinophils in subjects with allergic asthma. Quilizumab treatment numerically reduced allergen-induced sputum eosinophil increases, 7 hours after challenge, by 50% compared with placebo (P = 0.23) (Fig. 5A). Blood eosinophils were also numerically decreased in subjects treated with quilizumab over time, showing mean reductions in absolute counts of 20 and 28% of baseline levels at day 140 (P = 0.10 versus placebo) and day 196 (P = 0.15 versus placebo), respectively (Fig. 5B).

Fig. 5. Effect of quilizumab treatment on eosinophil counts in subjects with stable mild allergic asthma (phase 2a).

(A and B) Sputum eosinophils (A) and peripheral blood eosinophils (B) in allergic asthma subjects after administration of placebo or quilizumab (5 mg/kg IV) on days 1, 29, and 57 and airway allergen challenge on days 1 and 86. Sputum was collected before challenge and 7 and 24 hours after challenge during screening and at day 86. Sputum data are expressed as percentage eosinophils in sputum (mean ± SE). Peripheral blood eosinophil data are expressed as percentage from baseline, where baseline was the value collected at screening (mean ± SE).

Safety

Quilizumab was well tolerated at concentrations of 5.0 mg/kg administered intravenously or 3.0 mg/kg administered subcutaneously. In subjects with allergic rhinitis, treatment-emergent adverse events (TEAEs) were experienced by 83% (20 of 24) of quilizumab-treated subjects and 75% (9 of 12) of placebo-treated subjects. Most of these were mild (83% quilizumab, 67% placebo). A single severe TEAE of gastroenteritis was reported during the study by a subject receiving placebo. The most frequently reported TEAEs in subjects receiving quilizumab were upper respiratory tract infection [n = 7 (29%) quilizumab, compared with n = 2 (25%) in placebo IV].

In allergic asthmatic subjects, quilizumab was well tolerated, with no treatment-related TEAEs in the active group, and no serious or severe TEAEs, or TEAEs leading to discontinuation of study drug. The most frequent TEAE was headache, reported for five subjects (two quilizumab subjects, three placebo subjects). Other TEAEs reported in at least two subjects in either arm were nasopharyngitis (four subjects quilizumab, one subject placebo), chest discomfort (two subjects quilizumab), and dizziness and oropharyngeal pain (two placebo subjects each). Most events were mild (80% in quilizumab subjects and 64% in placebo), and the rest moderate. No anti-therapeutic antibody responses were detected in quilizumab-treated subjects in either of the two studies.

DISCUSSION

Here, we have described two early-stage clinical trials that demonstrate the clinical activity and tolerability of quilizumab, a monoclonal antibody directed against the M1 prime segment of the human membrane IgE B cell receptor. In both allergic rhinitis and mild asthmatic subjects, quilizumab treatment significantly reduced total and allergen-specific IgE in the serum, and these reductions were sustained for at least 6 months after the last dose of quilizumab. Similar reductions of total serum IgE were observed in a phase 1 trial in healthy volunteers who received a single dose of quilizumab (48). Upon allergen challenge of asthmatic subjects, quilizumab completely inhibited the production of new allergen-specific IgE. This was associated with a reduction of the allergen-induced EAR compared with placebo. In addition, allergen-induced sputum and blood eosinophils, as well as the LAR, were decreased by quilizumab treatment, although these differences were not statistically significant when compared with placebo. Quilizumab has been clinically well tolerated in the subjects exposed thus far, and most reported adverse events have been mild in severity. Overall, our findings are consistent with the proposed mechanism of action of quilizumab, indicating that targeting M1 prime–expressing cells may prevent production of new IgE and may be effective for the treatment of allergic asthma.

Studies in mice indicate that most IgE is produced by short-lived plasma cells (19, 20, 23, 49). Consistent with this, anti-M1 prime antibody treatment in mouse models inhibits new IgE production and ultimately results in >90% reductions in serum IgE levels because short-lived IgE plasma cells die and are not replenished with new IgE-producing plasma cells (33). These preclinical studies also indicate that the effects of anti-M1 prime antibodies on serum IgE levels are due to the depletion of membrane IgE–positive IgE-switched B cells and plasmablasts (33).

In humans, the contributions of short- and long-lived plasma cells to IgE production are not well understood, although seasonal fluctuations in allergen-specific IgE levels in allergic subjects suggest that a proportion of allergen-specific IgE may be produced by short-lived plasma cells (2427). Our studies suggest that quilizumab treatment blocks new IgE production, as indicated by the inhibition of new allergen-specific IgE production upon allergen challenge of asthmatics. However, the 20 to 30% reductions in total and allergen-specific serum IgE levels observed in quilizumab-treated subjects in our studies are less than those observed in preclinical mouse models. These data suggest that only ~20 to 30% of serum IgE in humans are produced by short-lived plasma cells, with the remainder of the serum IgE produced by long-lived plasma cells. Consistent with this, we detected IgE plasma cells in bone marrow aspirates of a subset of 10 subjects in the allergic asthma study in both placebo- and quilizumab-treated subjects on days 87 and 197 (fig. S7). Alternatively, more prolonged treatment with quilizumab beyond the three monthly doses used in our studies may be required to decrease serum IgE levels further.

We observed similar reductions in total serum IgE in healthy volunteers (48), allergic rhinitis, and allergic asthmatics treated with quilizumab. This suggests that a similar proportion of total IgE is produced by short-lived plasma cells in all three subject populations, although it should be noted that many of the allergic rhinitis and allergic asthma subjects in the studies here did not have elevated total or allergen-specific IgE levels compared to the healthy volunteers at baseline.

M1 prime–positive cells are very rare in humans and reside mostly in tissues and lymphoid organs. We were not able to assess depletion of M1 prime–positive cells in these locations in our studies, and it was also not technically feasible to demonstrate depletion of membrane IgE–positive cells in the peripheral blood. To what extent quilizumab treatment results in direct depletion of M1 prime–expressing B cells will have to be further investigated in future studies.

Neutralization of total serum IgE with the monoclonal anti-IgE antibody omalizumab reduces asthma exacerbations and is a licensed therapy for the treatment of allergic asthma (50, 51). Omalizumab does not appear to affect IgE production in the short term (13), although long-term use over the course of several years may provide sustainable benefits beyond treatment cessation (5254). In contrast, quilizumab treatment inhibits the production of new allergen-specific IgE within 4 weeks after a single dose and leads to reductions in total and allergen-specific serum IgE within months, consistent with a rapid and direct effect on IgE production that contrasts with what is observed with omalizumab. Moreover, these reductions in IgE are sustained for at least 6 months after treatment cessation, which suggests that treatment with quilizumab may affect long-term IgE memory. The sustained reductions in serum IgE observed in studies of quilizumab in healthy volunteers, allergic rhinitics, and allergic asthmatics after the cessation of dosing, along with the long half-life of the antibody (t1/2 of 20 to 21 days), raise the potential of a convenient dosing schedule. Moreover, if quilizumab treatment not only prevents new IgE production but also affects IgE memory responses that arise from membrane IgE–expressing memory B cells, this may lead to sustained clinical benefits for IgE-mediated disease and could provide an alternative to current anti-IgE therapy paradigms.

It remains unknown how much of the reported morbidity of asthma is driven by new IgE production, although exposure to environmental allergens has been strongly linked to asthma exacerbations (55). In addition, several studies have shown a relationship between levels of allergen-specific serum IgE and asthma outcomes (5659), although some studies have not (60, 61). Ongoing and future studies will assess the clinical relevance of quilizumab on longer-term asthma endpoints including exacerbations.

Overall, our data provide proof of concept of targeting the M1 prime segment on membrane IgE in subjects with atopic diseases, such as allergic rhinitis and mild allergic asthma. These data support the continued investigation of quilizumab as a potentially clinically meaningful treatment for asthma; a phase 2b clinical trial (NCT01582503) of quilizumab in subjects with uncontrolled allergic asthma despite inhaled corticosteroid and second controller medication is currently ongoing. In addition, the clinical trials described in this article involved a limited number of subjects, such that conclusions regarding the safety of quilizumab will require data from more widespread use in future clinical studies. Furthermore, longer-term trials are also needed to investigate the potential of a sustained response to quilizumab therapy.

CONCLUSIONS

Targeting of IgE-switched B cells via the M1 prime segment of membrane IgE by the antibody quilizumab could potentially be an effective treatment for atopic diseases such as allergic asthma. An ongoing phase 2b clinical trial in subjects with moderate-to-severe asthma that remains uncontrolled despite adherence to standard-of-care treatments aims to assess the effect of quilizumab treatment on asthma symptoms and exacerbation events.

MATERIALS AND METHODS

Study design/subjects

A phase 1b, randomized, double-blind, placebo-controlled, single-center (inVentiv Health), multiple ascending dose trial (NCT01160861) evaluated the safety, tolerability, and pharmacokinetic profile of quilizumab at doses of 1.5 or 5.0 mg/kg IV or 3.0 mg/kg SC every 28 days for 3 months in subjects with seasonal or perennial allergic rhinitis (fig. S3A).

A phase 2a, randomized, double-blind, placebo-controlled, multicenter (sites in Canada and Sweden), parallel-group trial (NCT01196039) evaluated the activity, safety, and tolerability of quilizumab (5 mg/kg IV) in an allergen-challenge model in subjects with a diagnosis of stable, mild allergic asthma. The study consisted of a screening period (days −35 to 1), a 12-week treatment period, and a 16-week safety follow-up period (fig. S3B). During the screening period, subjects underwent allergen challenges to obtain a cohort with documented EAR and LAR. After screening, subjects received three intravenous doses of placebo or quilizumab administered 4 weeks apart. Posttreatment allergen challenge was conducted on day 86, 1 month after the last dose.

For both studies, all subjects provided written informed consent, and the protocol was approved by applicable independent review committees or institutional review boards.

Inclusion criteria

Subjects with allergic rhinitis enrolled in the phase 1b study were required to have total IgE serum levels of >10 IU/ml and one or more positive allergen-specific IgE >0.1 kIU/liter. Subjects with mild allergic asthma enrolled in the phase 2a study were required to have baseline pre-bronchodilator FEV1 values ≥70% predicted, with a positive skin prick test to common standard aeroallergen extracts and a positive allergen-induced EAR and LAR. Positive allergen-induced EAR was defined as a ≥20% decline from baseline FEV1 measured 0 to 3 hours after allergen challenge; the LAR was defined as a ≥15% decline from baseline FEV1 measured 3 to 7 hours after allergen challenge.

Exclusion criteria

Exclusion criteria for both studies included history or clinical manifestations of unstable medical disease, use of prohibited concomitant medications, history of or current malignancy, and pregnancy. In the phase 1b study of allergic rhinitis, subjects with a history of asthma diagnosis requiring use of a daily controller medication or rescue use of a short-acting bronchodilator within the last 3 years were excluded from study entry. Furthermore, subjects in the phase 2a study could not have received corticosteroids (oral, systemic, or inhaled), immunosuppressive agents, anticoagulants, or any medications that may interact with the study drug within 4 weeks before enrollment. Chronic use of other medication for treatment of allergic lung disease other than short-acting β2 agonists or ipratropium bromide was not permitted.

Additional exclusion criteria for the phase 1b study included FEV1 <80% of predicted at screening; atopic dermatitis requiring regular daily use of topical medications more potent than 1% hydrocortisone, neutropenia, or thrombocytopenia; receipt of blood products within 2 months before dosing; self-reported history of smoking within the 24 hours before day −1; and smoking during the confinement periods.

Further exclusion criteria for the phase 2a study included a worsening of asthma within 6 weeks preceding enrollment, preexisting lung disease other than asthma, current or former smoker with >10-pack-year history, and current or history of treatment with a monoclonal antibody or chimeric biomolecule within the past 5 months including omalizumab at the time of enrollment.

Dose selection

The doses used in these studies were selected on the basis of the available nonclinical toxicology and in vivo pharmacologic data. The highest dose of 5 mg/kg was selected to allow for safety at an exposure that is at least 20-fold below a no observed adverse effect level from the preclinical toxicology study based on the single-dose human equivalent dose, but also high enough to ensure adequate dosing for proof of activity.

Endpoints

The primary objective of the phase 1b study was to evaluate the safety and tolerability of two intravenous and one subcutaneous dose levels of quilizumab compared with placebo. In the phase 2a study, the primary efficacy endpoint was the LAR AUC (of percentage decline in FEV1 over time) 3 to 7 hours after allergen challenge at day 86. The secondary objectives in this study included the assessment of the EAR AUC 0 to 3 hours after allergen challenge at day 86. Additional, secondary objectives of both studies were to characterize the pharmacokinetic profile of quilizumab. Exploratory endpoints included changes from baseline in serum IgE levels, serum and sputum eosinophil levels, and bone marrow levels of IgE plasma cells.

Pharmacodynamic and pharmacokinetic analyses

Quilizumab concentrations in serum were measured by enzyme-linked immunosorbent assay. The serum pharmacokinetic profile of quilizumab was summarized by estimating total exposure (AUC), Cmax, time to Cmax, total serum clearance [or CL/F (apparent clearance)], volume of distribution, and elimination t1/2. In the phase 2a study, observed maximum serum concentration after each dose (Cmax,1, Cmax,29, and Cmax,57) and trough quilizumab concentrations before the second and third doses (Ctrough,29 and Ctrough,57) were also calculated, as well as the serum concentration at 28 days after the last dose.

Serum total and allergen-specific IgE were measured by ImmunoCap (ViraCor-IBT Laboratories). Specific IgE was measured for the following allergens: cat dander, cat hair, horse, house dust mite Dermatophagoides pteronyssinus, house dust mite Dermatophagoides farinae, ragweed, June grass, redtop grass, sweet vernal, and Timothy grass. Peripheral blood eosinophil counts were obtained from standard complete blood count. Percentage of sputum eosinophils was calculated by enumerating the differential cells present in the sputum samples.

Allergen challenges

Allergen inhalation was performed as described by O’Byrne and colleagues (62).The concentration of allergen extract for inhalation was determined from a formula described by Cockcroft and co-workers (63). During a screening period, doubling concentrations of allergen were inhaled over 2 min by tidal breathing from a nebulizer until a 20% fall in FEV1 at 10 min after allergen was reached. For allergen inhalation challenge, FEV1 was measured 10, 20, 30, 45, 60, 90, and 120 min after allergen inhalation, then each hour until 7 hours after allergen inhalation. The EAR was the largest decline in FEV1 up to 3 hours after allergen inhalation, and the LAR was the largest decline in FEV1 between 3 and 7 hours after allergen inhalation. The same dose of allergen was to be administered for the day 86 allergen challenge.

Sputum eosinophils

Sputum was induced and processed using the method described by Pizzichini et al. (64). Cells were prepared on glass slides for differential counts using a cytospin and stained with Diff-Quik (American Scientific Products).

Bone marrow

Bone marrow aspirates were obtained from the posterior iliac crest using a bone marrow aspiration needle (16 × 2 inches; Sherwood Medical). Ten milliliters of bone marrow was aspirated into a 10-ml syringe containing 1 ml of sterile heparin (1000 U/ml) (Leo Laboratories). CD138 cells were isolated by positive selection using microbeads, and memory B cells were identified by flow cytometry as CD19+CD27+CD38+ and stained intracellular for IgE or IgG (65).

Serum IgE measurements in SOLARIO phase 2

Serum total IgE and allergen-specific IgE were measured by ImmunoCap (ViraCor-IBT Laboratories). The allergen-specific IgEs that were measured were selected on the basis of the airway challenge subjects received. The following 10 allergens were measured in all subjects: cat dander, cat hair, horse, house dust mite D. pteronyssinus, house dust mite D. farinae, ragweed, June grass, redtop grass, sweet vernal, and Timothy grass. In the case of the grasses, subjects were challenged with an allergen mixture of June grass, redtop grass, sweet vernal, and Timothy grass. To evaluate the effect of quilizumab on serum IgE, we analyzed the data using total IgE, challenge-specific IgE, and non–challenge-specific IgE. Challenge-specific IgE was defined as the one allergen-specific IgE each individual subject was challenged with, whereas the non–challenge-specific IgE was defined as the average of all other measured specific IgEs that were detected at predose time points (days −7 and −1). In subjects challenged with the grass allergen mixture, the challenge-specific IgE was the average of the four individual grasses. Total IgE levels, challenge-specific IgE, and non–challenge-specific IgEs were expressed as absolute values and % baseline, where baseline is defined as the average of the two predose time points (days −7 and −1). Table S2 provides an illustrative example (one subject) to clarify the specifics of the data analysis performed.

Statistical analysis

The sample size for the phase 1b study was considered sufficient to provide a preliminary assessment of the safety and tolerability of quilizumab in subjects with allergic rhinitis. Inclusion of eight subjects treated with quilizumab in each dose level provided reasonable probability to reject the dose (>70% probability) if the underlying rate of dose-limiting toxicity was 30% or higher. No hypothesis testing was performed in the phase 1b study.

The sample size for the phase 2a study was considered sufficient to account for variability in the analysis and interpretation of the primary endpoint. The main analysis goal in the phase 2a study was to obtain a point estimate and a 90% CI on the population mean of the primary and secondary efficacy outcomes. Hypothesis testing (performed at a two-sided 5% significance level) was exploratory, and P values (corresponding to t tests for the efficacy endpoints and Wilcoxon rank sum tests for the pharmacodynamic/biomarker data) were not adjusted for multiple comparisons.

All efficacy analyses were based on a modified intent-to-treat population, including all randomized subjects who received at least one dose of study drug and who completed the baseline and week 12 allergen challenge tests. All safety analyses included all randomized subjects who received any amount of the study drug.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/6/243/243ra85/DC1

Fig. S1. Mean serum concentration of quilizumab versus time in subjects with allergic rhinitis (phase 1b).

Fig. S2. Study design schema.

Fig. S3. Ratio of serum-specific IgE/total IgE in individual placebo- and quilizumab-treated (5.0 mg/kg, IV) allergic asthma subjects (phase 2a).

Fig. S4. Absolute levels of serum allergen-specific and total IgE over time in all individual subjects of placebo- and quilizumab-treated cohorts (phase 2a).

Fig. S5. Percentage of change of serum allergen-specific and total IgE levels over time in all individual subjects of placebo- and quilizumab-treated cohorts (phase 2a).

Fig. S6. Effect of quilizumab on serum allergen-specific IgE in allergic rhinitis subjects (phase 1b).

Fig. S7. IgE plasma cells in bone marrow aspirates of allergic asthma subjects (phase 2a).

Table S1. Pharmacokinetic parameters of quilizumab.

Table S2. Illustrative example of specific IgE data analysis.

Table S3. Predose baseline levels of all specific IgE (kU/liter) in the placebo-treated allergic asthma subjects.

Table S4. Predose baseline levels of all specific IgE (kU/liter) in the quilizumab-treated allergic asthma subjects.

Table S5. Individual subject serum total IgE (IU/ml) levels and summary statistics in placebo-treated allergic asthma subjects.

Table S6. Individual subject serum total IgE levels (IU/ml) and summary statistics in quilizumab-treated allergic asthma subjects.

Table S7. Individual subject serum total IgE (% baseline) levels and summary statistics in placebo-treated allergic asthma subjects.

Table S8. Individual subject serum total IgE levels (% baseline) and summary statistics in quilizumab-treated allergic asthma subjects.

Table S9. Individual subject serum challenge-specific IgE (kU/liter) levels and summary statistics in placebo-treated allergic asthma subjects.

Table S10. Individual subject serum challenge-specific IgE levels (kU/liter) and summary statistics of levels in quilizumab-treated allergic asthma subjects.

Table S11. Individual subject serum challenge-specific IgE (% baseline) levels and summary statistics in placebo-treated allergic asthma subjects.

Table S12. Individual subject serum challenge-specific IgE (% baseline) levels and summary statistics in quilizumab-treated allergic asthma subjects.

Table S13. Individual subject serum levels and summary statistics of non–challenge-specific IgE (kU/liter) levels in placebo-treated allergic asthma subjects.

Table S14. Individual subject serum levels and summary statistics of non–challenge-specific IgE (kU/liter) levels in quilizumab-treated allergic asthma subjects.

Table S15. Individual subject serum levels and summary statistics of non–challenge-specific IgE (% baseline) levels in placebo-treated allergic asthma subjects.

Table S16. Individual subject serum levels and summary statistics of non–challenge-specific IgE (% baseline) levels in quilizumab-treated allergic asthma subjects.

REFERENCES AND NOTES

  1. Acknowledgments: We thank S. Kotwal for developing the IgE and IgG plasma cell assay in bone marrow aspirates. Funding: This study was supported by Genentech Inc. Support for third-party writing assistance for this manuscript, furnished by J. Brennan of MediTech Media, UK, was provided by Genentech Inc. Author contributions: G.M.G., J.M.H., H.S., W.S.P., Y.Z., X.C.L., L.C.W., J.G.M., and P.M.O. contributed to study design. G.M.G., J.M.H., L.-P.B., H.S., J.M.F., W.S.P., D.W.C., B.E.D., R.L., Y.Z., B.D., Y.W., R.M., I.M., X.C.L., L.C.W., J.G.M., and P.M.O. contributed to acquisition and interpretation of data. All authors helped to write the manuscript and approved the final version. Competing interests: G.M.G., L.-P.B., J.M.F., D.W.C., B.E.D., R.L., B.D., I.M., and P.M.O. are members of AllerGen NCE Inc., the Allergy, Genes and Environment Network, National Research Network funded by Industry Canada through the Networks of Centres of Excellence (NCE) Program, and received contractual funding support from Genentech Inc. for the conduct of the phase 2a SOLARIO Allergen Challenge Study. J.M.H., H.S., W.S.P., Y.Z., Y.W., R.M., X.C.L., L.C.W., and J.G.M. are employees of Genentech Inc., a member of the Roche Group, and may have an equity interest in Roche. A patent application relating to the subject matter of this manuscript has been filed.
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