AntiIgE Therapy for Asthma

Department of Medicine, Unit of Clinical Immunology and Allergy, Karolinska University Hospital Stockholm, Sweden

ROLAND BUHL

Pulmonary Department, Mainz University

Hospital Mainz, Germany

I. Introduction

Allergic diseases, such as allergic asthma, are hypersensitivity reactions initiated by immunological mechanisms (1,2). They are usually mediated by IgE antibodies, triggering an inflammation characterized by an increase in production of Th2-type cytokines at a mucosal surface, the interface between the external and the internal environments. Allergic diseases usually occur in atopic individuals who are genetically predisposed to producing IgE antibodies in response to low doses of general environmental allergens, e.g., pollens, mites, and danders. Although allergies mediated by other immuno-globulins (e.g., IgG-immune complexes that can activate complement) or lymphocytes (e.g., allergic contact dermatitis to chromium and nickel) also exist, the major part, if not all, of allergic asthma is IgE mediated. The cross-linking of mast cell/basophil membrane cell-bound IgE antibodies by allergen results in the release of inflammatory mediators that are responsible for the signs and symptoms of allergy. IgE sensitization to an allergen can develop in childhood or throughout life, and subsequent allergen contact, which may occur years later, can initiate a severe attack of allergic asthma.

An anti-IgE antibody, omalizumab (Xolair®), was approved in the United States in July 2003 for the treatment of moderate to severe allergic asthma in adults and adolescents. Omalizumab is licensed for use in allergic asthma in Australia and is under evaluation for use in patients with uncontrolled severe persistent allergic asthma in Europe. In this chapter, we will describe the role of IgE in allergic asthma and the rationale for anti-IgE therapy. We will present clinical data illustrating proof of the anti-IgE concept and results from the pivotal phase-III clinical studies showing efficacy of omalizumab in adult and pediatric asthma patients. Consideration will be given to the anti-inflammatory effects of anti-IgE treatment with omalizumab and which patients are most likely to benefit from anti-IgE therapy.

The discovery of IgE in 1968 represented a major breakthrough in our understanding of allergic disease (3). Although allergy had been recognized for centuries, and the possible existence of the "reagins" responsible had been reported in the early 20th century, allergology had been regarded with suspicion until this new immunoglobulin was declared.

IgE has a molecular weight of 190 kDa. Its structure is shown in Figure 1. The heavy chain includes four constant regions, Ce1-4, of which

Figure 1 The primary structure of IgE. Variable domains bind antigen, while constant domains determine secondary biological function (e.g., cell surface binding). Abbreviations: VL, variable domain of the light chain; VH, variable domain of the heavy chain; CL, constant domain of the light chain; Ce 1-4, constant domains of the heavy chain; Fab, antigen binding fragments; Fc, crystallizable fragments.

Figure 1 The primary structure of IgE. Variable domains bind antigen, while constant domains determine secondary biological function (e.g., cell surface binding). Abbreviations: VL, variable domain of the light chain; VH, variable domain of the heavy chain; CL, constant domain of the light chain; Ce 1-4, constant domains of the heavy chain; Fab, antigen binding fragments; Fc, crystallizable fragments.

Ce2-4 constitute the Fc fragment. As in other antibodies, the antigen-binding site is contained in the Fab fragment (at the VL/VH domains). The Ce3 domains of Fc bind either of the two IgE receptors, the high-affinity receptor FceRI [KD = (1—2) x 10—9M], or the low-affinity receptor FceRII (Kd = 1 x 10—6M). Monomeric IgE, free in circulation, has been reported to have a half-life of two to three days but recent studies of transfused IgE antibodies showed a half-life as short as 1.13 days (4). However, once IgE binds to receptors it can remain stable for weeks. Its concentration in the serum is highly dependent on age and sex (decreasing from the age of 20 years) and is very low. The range is approximately 1-100 mg/L, which corresponds to 20-40 IU/mL using the NIBSC/WHO reference 75/ 502 (5-7), which is considerably lower than that of any other immunoglobulin, e.g., 1/100,000 of IgG. Levels are typically higher in allergic populations, e.g., allergic asthma (10-1000 mg/L) (8), and highest in comorbid patients with more than one allergic disorder, e.g., in patients with asthma and "atopic dermatitis" (9,10). However, high serum IgE levels, without any related IgE antibodies, have been reported in viral infections (11), in response to air pollution like cigarette smoke (12), and also in immunological interactions like graft-versus-host disease after bone marrow transplantation (13).

A. The Role of IgE in Asthma

The role of IgE in the initiation of the allergic cascade is well established (14). The IgE-mediated allergic cascade involves a biphasic response with an immediate or early allergic response (EAR) and a late allergic response (LAR) (15). EAR is an acute response that occurs within one hour of exposure to allergen. It is characterized by constriction of the bronchi and bronchioles, contraction of smooth muscle and vasodilation of capillaries, and overstimulation of mucous glands and nerve endings. LAR occurs 4 to 24 hours after initial allergen challenge. It is characterized by chronic infiltration of the airways by immune cells, resulting in prolonged airflow obstruction and determining the severity of bronchial hyper-responsiveness. After 24 to 48 hours, infiltrating Th2 cells stimulate the release of proinflammatory cyto-kines. IgE plays a critical role in both the EAR and the LAR via interaction with the FceRI and FceRII receptors. In addition, IgE enhances the efficiency of antigen presentation to T cells via interaction with FceRI receptors on antigen-presenting cells (16).

The complete form of FceRI is a tetramer (apy2) and is expressed on a variety of cell types, predominantly on mast cells and basophils. FceRI is expressed as a trimer (ag2) on antigen-presenting cells (16), such as monocytes (17), epidermal Langerhans cells (18), and peripheral blood dendritic cells (19) (but is not expressed on their progenitors). It is also expressed on epithelial cells (20), platelets (21), and, at a low level, on eosinophils (22). The IgE-FceRI interaction has 1:1 stoichiometry (23).

FceRII (also called CD23) is expressed on B cells, eosinophils, platelets, natural killer cells, Th2 cells, follicular dendritic cells, Langerhans cells, and epithelial cells (24). FceRII exists in two forms (FceRIIa and FceRIIb). FceRIIa mediates endocytosis by B cells, and FceRIIb, the sequence of which differs only in a few amino acids, plays a role in IgE-mediated phagocytosis by diverse cells (25). Eosinophils express both forms (26). The IgE-FceRII interaction has 2:1 stoichiometry.

EAR results from IgE-mediated mast-cell degranulation. Mast cells are major players in the allergic response (27). When IgE antibodies on mast cells or basophils are cross-linked by allergen, the cells become activated. Interaction of receptor-bound IgE antibodies with soluble multiva-lent allergen leads to receptor aggregation. By signal transduction, a complex series of events ensues, including recruitment of intracellular protein kinases, phospholipases, influx of Ca2+ ions, and synthesis of proinflammatory mediators. This culminates in rapid (i.e., within minutes) degranulation, the release of the stored contents of cytoplasmic granules and of newly formed mediators by exocytosis (Fig. 2A). A plethora of mediators is released, including histamine, leukotrienes, the anticoagulant heparin, neutral proteases (such as tryptase and chymase, which constitute approximately 30% of the total granule protein), complex-carbohydrate-cleaving enzymes, platelet activating factor, chemokines, prostaglandins, and an array of cytokines [IL-3, IL-4, IL-5, IL-6, IL-10 and IL-13, tumor necrosis factor (TNF)-a, GM-CSF, and others] (28). Acute allergic symptoms are generated by interaction of these preformed and newly formed mediators with specific receptors on the target tissues. Unlike basophils, mast cells do not circulate, although they can migrate through the tissues in which they are localized, and are usually present in perivascular connective tissue, epithelia, and lymph nodes. In patients with allergic asthma, mast cells localize in the bronchial smooth muscle bundles. Asthma severity increases with smooth-muscle mast-cell density, because mast cell migration to airway mucus glands and degranulation increase bronchial hyper-responsiveness and mucous secretion (27).

The cause and significance of the LAR is less understood. A number of studies have shown that eosinophils play a major role. Eosinophilic inflammation has been shown to be related to asthma severity (29) and asthma exacerbations (30). However, long-term suppression of circulating eosinophils by an antibody to IL-5 did not protect against the LAR, indicating that eosinophils are not solely responsible for the effect (31). Eosino-phils are selectively recruited to the site of inflammation from the microcirculation (Fig. 2B). Their cytoplasmic granules have a crystalloid core of major basic protein and a matrix of eosinophil cationic protein, eosinophil-derived neurotoxin, and eosinophil peroxidase. These unique toxic inflammatory mediators and a variety of cytokines and lipid mediators are both synthesized and released by degranulation in response to

Early And Late Rsponse Asthma
Figure 2 Early and late asthmatic responses. (A) Sequence of events following mast-cell sensitization by IgE (activation and degranulation), the early allergenic response. (B) Role of eosinophils in the late allergenic response.

IgE binding to the FceRI receptor. However, whether this is the major pathway of the LAR is uncertain, due to the low level of FceRI expression on eosinophils (32). Major basic protein and eosinophil cationic protein have profound cytotoxic effects on the airway epithelium (33), and for this reason, eosinophils are often regarded as the primary effector cells in asthma.

Interaction of IgE with the FceRII receptor has been implicated in allergy, although its role has not yet been fully elucidated. FceRII is multifunctional and its roles include the induction of IgE synthesis (34-36) and the maintenance and modulation of the IgE response (35). IgE binding to the FceRII receptor has been shown to be responsible for rapid and specific transepithelial antigen transport in allergic rats (36). As asthmatic airway smooth muscle expresses surface FceRII, and expression is upregulated by IgE-FceRII binding (37), it is possible that FceRII is involved in a similar transepithelial migration pathway in humans, acting like an adhesion molecule to facilitate the phagocytosis of IgE-bound antigen. FceRI has also been implicated in the IgE-mediated presentation of allergen on antigen-presenting cells (38). In addition, although not as predominant as its role in binding IgE, membrane-bound FceRII (and the soluble form) has functions in the allergic response that do not involve interaction with IgE, such as in cell-cell interaction, acting as an adhesion molecule that binds p integrins, and in cytokine-like activities (39).

Allergen presentation to T cells is enhanced by IgE-FceRI complexes on antigen-presenting cells (16), including dendritic cells (40), macrophages (41), and Langerhans cells (42). Allergen presentation leads to Th2-cell-mediated allergic reactions and their associated clinical symptoms. Circulating myeloid dendritic cells are rapidly recruited to the airway epithelia following allergen inhalation (43,44), and numbers of dendritic cells are significantly higher in the airways of patients with asthma compared with control individuals (p < 0.02) (45). Dendritic cells express FceRIa, but not FceRI p (46,47), and expression of FceRIa is significantly increased in patients with asthma compared with control individuals (p < 0.003) (45). Allergens can thus be internalized and presented by dendritic cells by cross-linking of allergen-IgE antibodies bound to the a chain of FceRI

(48). However, the p chain is necessary for signal transduction (48).

These roles of T cells, B cells, mast cells, and eosinophils in the early and late asthmatic reactions are summarized in Figure 3.

III. Anti-IgE as a Therapeutic Strategy

The majority of asthma is allergic in nature and initiated by IgE antibody

(49). Targeting of factors involved in the allergic response, such as IgE, represents a novel strategy for the development of new therapeutic agents for allergic diseases. The importance of FceRI-mediated mast-cell degranulation and FceRI and FceRII-mediated enhancement of antigen presentation in the development of an allergic reaction make these two processes particularly suitable for therapeutic intervention. IgE binding to its Fc receptors mediates both processes and therefore represents an ideal target for therapeutic attenuation of the allergic cascade. This IgE-receptor-binding step might be blocked by inhibitory peptides with structures based on the receptor. However, such receptor-derived peptides may elicit an anti-peptide immune response and anaphylaxis through receptor cross-linking. A preferable strategy is to use a monoclonal anti-IgE antibody that binds

Allergen o *PC

Symptoms of allergic inflammation

8 cell

8 cell

Selection Expansion

Histamine ■*• Lipid mediators allergen

Mast I cell IL-5,IL-3

Selection Expansion

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allergen

GM-CSF

Mast I cell IL-5,IL-3

Enzymes Cytokines

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• Chest tightness

■Recruitment, activation -

Basic proteins Enzymes Lipid mediators Cytokines Chemokines

• Bronchial hyper-

responsiveness {BHR)

Chronic

GM-CSF

Eosinophil

Eosinophil

Figure 3 The interactions between mast cells, B cells, antigen-presenting cells, eosinophils, and airway tissues that are mediated by IgE during chronic asthma. Source: From Ref. 49.

free, but not receptor-bound, IgE and thereby inhibits initiation of the allergic cascade by preventing IgE binding to receptors.

As IgE-receptor binding directs immune responses through the multiple cell types on which Fc receptors are expressed, the effects of blocking it could be expected to be manifold (Fig. 3). Blocking IgE binding to FceRI receptors on dendritic cells could reduce the efficiency of antigen presentation to T cells (16), while blocking binding to FceRI receptors on mast cells and basophils could prevent allergen-induced degranulation and avoid the effects following the release of inflammatory mediators (27). In addition, blocking IgE binding to FceRII receptors on monocytes and eosinophils could prevent IgE-mediated phagocytosis (25).

For reasons of tolerability, a therapeutic anti-IgE antibody must be non-immunogenic and non-anaphylactogenic. In addition, the binding affinity between IgE and the antibody should favor the formation of immune complexes small enough to result in a reasonable rate of clearance without immune-complex-mediated adverse reactions. To achieve therapeutic efficacy, a dose of anti-IgE capable of nearly completely removing free IgE might be necessary, as FceRI receptor density on effector cells is high (104-106 per cell) and only 2000 IgE molecules are required for half-maximal histamine release from basophils exposed to allergen (50).

IV. Anti-IgE Therapy with Omalizumab—Proof of Concept

A monoclonal humanized recombinant anti-IgE antibody (omalizumab) has been generated from a human IgG1 framework onto which is grafted the complementarity-determining region from a murine anti-IgE antibody (51). This was designed for optimal safety. As the entire molecule contains fewer than 5% murine residues, it has a low potential for immunogenicity (51). Omalizumab recognizes the Ce3 domain of free human IgE (Figs. 1 and 4). As this is the same site that binds the FceRIa and FceRII receptors, omalizumab cannot bind receptor-bound IgE and is thereby prevented from inducing mast-cell or basophil degranulation and anaphylaxis (51). This has been demonstrated by in vitro and in vivo studies (52) and clinical studies in 2845 patients. Analytical ultracentrifugation and size-exclusion chromato-graphy revealed that omalizumab-IgE complexes are generally small, the largest consisting of a cyclic or near-cyclic heterohexamer of three IgE and three anti-IgE molecules (<103kDa) (53). While this species formed at a molar ratio of 1:1, a heterotrimer of two IgE molecules and one anti-IgE was the dominant species formed at the more physiological molar ratio of 10:1 (IgE to anti-IgE), and a heterotrimer of one IgE and two anti-IgE molecules was dominant at a 1:10 molar ratio.

The therapeutic potential of omalizumab was confirmed in a multicenter, double-blind, placebo-controlled trial enrolling 240 patients, which found omalizumab to considerably reduce serum free IgE (54). In some patients, concentrations of serum free IgE decreased by >90% over 12 weeks of omalizumab administration (from 160 IU/mL to below the detection limit of 10 IU/mL, 24ng/mL), and a dose of 0.005 mg/kg/week omalizumab for each IU/mL of free IgE in serum at baseline was effective in reducing serum levels of free IgE to the lowest detectable level at steady state. Another study, which found omalizumab to reduce serum levels of free IgE to 1% of pretreatment levels, also reported a marked reduction of FceRI on basophils: the pretreatment median receptor density was 220,000 per basophil, reducing to a median of 8300 after three months of omali-zumab therapy (55). This reduction in receptor density was accompanied

Figure 4 Structures of the complexes formed by interaction of the antigen-recognition site of omalizumab with the Ce3 site of IgE. The heterotrimer is formed at molar ratios of 1:10 and 10:1, and the heterohexamer at a molar ratio of 1:1.

Figure 4 Structures of the complexes formed by interaction of the antigen-recognition site of omalizumab with the Ce3 site of IgE. The heterotrimer is formed at molar ratios of 1:10 and 10:1, and the heterohexamer at a molar ratio of 1:1.

Trimers (-490-530 kD)

Hexamer

Fceri Binding

Figure 5 The reduction in serum free IgE by omalizumab binding is associated with downregulation of the high affinity FceRI receptor. (A) Schematic showing receptor downregulation by IgE. Likewise, an increase in serum free IgE is associated with an increase in FceRI-receptor expression. This process is believed to occur in both basophils and mast cells. (B) Correlation between basophil FceRI expression and serum levels of free IgE in patients receiving omalizumab. Source: From Ref. 56.

Figure 5 The reduction in serum free IgE by omalizumab binding is associated with downregulation of the high affinity FceRI receptor. (A) Schematic showing receptor downregulation by IgE. Likewise, an increase in serum free IgE is associated with an increase in FceRI-receptor expression. This process is believed to occur in both basophils and mast cells. (B) Correlation between basophil FceRI expression and serum levels of free IgE in patients receiving omalizumab. Source: From Ref. 56.

by a reduction in responsiveness of basophils to stimulation by allergen of approximately 90%, suggesting that FceRI density on basophils is regulated by serum levels of free IgE (Fig. 5A,B) (56). The mast-cell response, as measured by skin tests, was also markedly reduced (55), and it is likely that similar FceRI down-regulation occurs in mast cells, which are morphologically very similar to basophils. This suggests that FceRI-receptor density is regulated by circulating levels of free IgE, and that moderately reducing free IgE with omalizumab is very effective in reducing FceRI expression.

Two preliminary studies further support the therapeutic use of omalizumab in patients. In patients with allergic asthma, nine weeks' omalizumab therapy (57) reduced serum free IgE to levels below or approaching the detection limit and increased the dose of allergen required to provoke an allergic response (for bronchoconstriction, increased from 1:870 to 1:459; for cutaneous reaction, increased from 1:10,000 to 1:2000). In addition, it attenuated both the EAR [mean maximum fall in forced expiratory volume in one second (FEV1) during which EAR decreased from 30% to 18.8%, p = 0.01 vs. placebo], and the LAR (mean maximum fall in FEV1 during which EAR decreased from 24% to 9%, p = 0.047; induced sputum eosinophil count reduced 11-fold; methacholine responsiveness PC20 improved). Similarly, 11 weeks' omalizumab therapy (58) reduced serum free IgE by 89%, and attenuated the EAR [scored as improvements in methacholine responsiveness (PC20, p < 0.05, final measurement) and allergen responsiveness (PC15, p < 0.002, throughout)].

Clinical benefit with omalizumab is observed when free IgE levels in serum are reduced to 50ng/mL (20.8IU/mL) or less [target 25ng/mL (10.4 IU/mL)]. The ability of omalizumab to reduce free IgE levels to this extent depends on dose and the patient's weight and baseline IgE level. To simplify dosing and ensure that free IgE reduction is achieved, an individualized tiered dosing table was developed. According to this table, patients receive omalizumab, 150-375 mg, by subcutaneous injection for every two or four weeks, depending on weight and starting IgE level (Fig. 6) (59).

Body weight (kg)

30-60

>60-70

>70-80

>80-90

>90-150

Baseline IgE (IU/mL)

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>30-100

150

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375

Not dosed

Figure 6 Omalizumab subcutaneous doses for adolescents and adults with allergic asthma. Source: From Ref. 59.

V. Pivotal Studies in Asthma

Pivotal in the clinical evaluation of omalizumab were three large, multicenter, randomized, double-blind, placebo-controlled, phase III studies conducted in a total of 1405 children, adolescents, and adults (aged 6 to 76 years) with moderate to severe allergic asthma. Patients had a positive skin prick test to one or more common allergens to which they were exposed, and serum total IgE levels 30 to 700 (or an upper limit of 1200 in children) IU/mL (60-62). These three studies had a similar design (Fig. 7): a four- to six-week run-in phase prior to randomization; a 16-week "steroid-stable" phase, where placebo or active treatment was given in addition to stable inhaled corticosteroid (ICS) treatment [beclomethasone dipropionate (BDP)]; and a 12-week "steroid-reduction" phase, in which ICS therapy was gradually reduced to the optimal lowest dose required for an acceptable level of asthma control, ending with four weeks at a constant, minimal ICS dose. Subcutaneous injections of 150-750 mg omalizumab were given every four or two weeks (doses above 225 mg were divided into two and given every two weeks). The dose was calculated from patient baseline IgE and body weight to provide at least 0.016 mg/kg per IU/mL of IgE per four weeks. Baseline characteristics of the patients enrolled are shown in Table 1. The primary endpoint for the studies in adults was reduction in asthma exacerbations during the steroid-stable or steroid-reduction phases. Exacerbations were defined as a worsening of asthma requiring treatment with oral or intravenous corticosteroids or doubling of baseline ICS dose.

1 year

Screening tests

Core study Double-blind extension* Qff

Omalizumab or placebo Omalizumab or placebo study

I drug

Coping with Asthma

Coping with Asthma

If you suffer with asthma, you will no doubt be familiar with the uncomfortable sensations as your bronchial tubes begin to narrow and your muscles around them start to tighten. A sticky mucus known as phlegm begins to produce and increase within your bronchial tubes and you begin to wheeze, cough and struggle to breathe.

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