Invited Symposium: Novel Cellular and Molecular Mechanisms in Allergic Inflammation
Since the discovery of the function of IgE over two decades ago, scientists have considered various strategies aiming at the selective inhibition of IgE antibody (1). IgE antibodies are crucial mediators of allergic reactions. In the course of an allergic immune response, allergen-specific B cells are stimulated and switched to IgE antibody production by IL-4/13 provided by TH2 cells. IgE antibodies act by arming cells bearing either high-affinity (FceRI) or low affinity (CD23) receptors such as mast cells and basophils and other inflammatory cells. Interaction of allergen with IgE : FceRI complexes on mast cells and basophils causes cross-linking of receptors which triggers the release of a variety of pharmacological mediators, cytokines and chemokines. Hence, anti-IgE antibodies that block binding of IgE to its high-affinity receptor are of potential therapeutic value in the treatment of allergy.
The engineering of a safe and effective anti-IgE molecule began with the identification of the binding site on human immunoglobulin E for its high affinity receptor (2). Based on a model of the IgE Fce3 (which is homologous to the second constant domain of IgG), homology scanning mutagenesis and replacement of individual residues were used to determine the specific amino acids of human IgE involved in binding to human FceRI. These amino acids are localized in three loops, which form a putative ridge on the most exposed side of the Fce3 domain of IgE.
To create a novel specific inhibitor of IgE which could block binding to FceRI but lacked the capacity to stimulate degranulation, the strategy was to develop a murine monoclonal antibody (MAb) directed against IgE, which would bind circulating IgE at the same site as the high-affinity receptor. This antibody should lack the harmful side effects of inducing receptor cross-linking because it would be unable to bind to IgE on cells since the FceRI would already occupy the immunoglobulin epitope. By virtue of binding to this epitope, the antibody would have the inherent ability to interfere with IgE responses by blocking binding of IgE to FceRI. Using the technique of scanning mutagenesis, a murine antibody, MAE11, was identified, which appears to recognize the identical residues in the Ce3 domain of IgE as does FceRI.
However, as a therapeutic, the murine antibody would not be the molecule of choice because experience in clinical use of non-human antibodies has identified three fundamental problems. First, non-human antibodies cause a human immune response that can reduce therapeutic value of the non-human antibody (3,4). Second, therapeutic efficacy is reduced by the relatively rapid clearance of the non-human antibody compared with human ones (5). Third, non-human antibodies generally show only weak recruitment of effector functions which may be essential for efficacy (6).
Humanization was undertaken to avoid the problems of antigenicity associated with murine MAbs (7). Version 25 (rhuMAb-E25) was selected since it possessed IgE binding affinity and biological activity comparable to the murine antibody. It was important for the anticipated clinical development that this antibody was characterized in a number of in vitro assays designed to evaluate its safety and efficacy (8).
The binding of rhuMAb-E25 to human peripheral blood basophils was assessed when basophils (CD9 and CD25, positive) from 12 normal donors sensitized with ragweed-specific IgE were challenged with antigen. Only ragweed antigen induced histamine release while rhuMAb-E25 failed to elicit histamine release from any donor. In other studies the ability of rhuMAb-E25 to block IgE binding to human lung mast cells was assessed using strips of normal human lung perfused overnight with ragweed-specific human IgE to sensitize the lung mast cells. Challenge with ragweed antigen induced mast cell degranulation as measured by histamine release and muscle contraction. In contrast, rhuMAb-E25 completely inhibited this response. These data (8) confirm that rhuMAb-E25 is effective in blocking degranulation and subsequent mediator release.
Studies were then undertaken to examine the effects of rhuMAb-E25 in vivo. The ability of rhuMAb-E25 to affect IgE responses was measured in cynomolgus monkeys since rhuMAb-E25 has nearly equivalent affinity for IgE purified from cynomolgus monkey serum (3X10-10 M) as for human IgE (1.7X10-10 M). To determine whether rhuMAb-E25 would activate cutaneous mast cells in vivo, 1 µg of the Ab was injected into the skin of normal cynomoglus monkeys. The Ab failed to elicit the wheal and flare reaction indicative of mast cell degranulation. In contract, a positive response was elicited with as little at 1 ng of a cross-linking murine MAb. Furthermore, rhuMAb-E25 failed to induce hive formation in monkey skin presensitized with 27 ng of human ragweed-specific IgE. These data are identical to the results obtained following systemic administration. Even at doses as high as 50 mg/kg, rhuMAb-E25 did not induce systemic anaphylaxis (8).
Clinical Safety and Efficacy
Based on the safety of E25 in preclinical model systems, clinical trials have been undertaken to determine the safety and efficacy of these antibodies in allergic patients. A series of single and multi-dose Phase I studies revealed that E25 was well tolerated and resulted in a dose dependent decrease in serum free IgE levels (9). Although free IgE had been suppressed, total IgE comprised of free IgE and anti-IgE:IgE complexes increased indicating a slow clearance of IgE complexes. No detectable antibody response to the therapeutic antibody was seen in any patient treated with the rhuMAb-E25.
The immune complexes found in vitro with IgE by both rhuMAb-E25 and a distinct monoclonal, chimeric murine-human antihuman IgE antibody (10) were shown to be of limited size by size-exclusion chromatography (SEC) (10, 11) and analytical ultracentrifugation (11). These studies showed that the size of rhuMAb-E25:IgE complexes formed in vitro was dependent on the molar ratio of rhuMAb-E25 and IgE. At molar equivalence, the largest complex of approximately one million molecular weight predominates. The data were best described by a cyclic hexamer structure, which consisted of three molecules of each immunoglobulin. This cyclic structure would account for the limited size of the complexes.
Fox et al (12) characterized the rhuMAb-E25: IgE complexes formed in vivo and examined the disposition of the antibody in a relevant animal model. 125I-rhuMAb-E25 was administered as an intravenous bolus dose to wild caught cynomolgus monkeys that have high levels of IgE. SEC of serum samples showed that the rhuMAb-E25:IgE complexes were of limited size and were similar to the small complexes formed in vitro with human IgE at antigen excess. No specific up-take of radioactivity was seen in any of the tissues collected from the cynomolgus monkeys at 1 hour and 96 hour postadministration.
In studies designed to expand the safety database and explore efficacy, rhuMAb-E25 was administered to mild allergic asthmatics in two studies using the technique of aeroallegen bronchoprovocation. Inhaled allergens, acting through IgE-dependent mechanisms, are important triggers of asthma symptoms, inducers of airway hyperresponsiveness and airway inflammation. The effect of rhuMAb-E25 on the provocation concentration of allergen causing a 15% fall in FEV1 (allergen PC15) during the allergen-induced early asthmatic response (EAR) was assessed in a multicenter, randomized, double-blind, parallel group study (13). rhuMAb-E25 was well tolerated and only one patient in the active group was withdrawn because of a generalized urticarial rash after the first dose. Compared with baseline values, the median allergen PC15 on Days 27, 55, and 77 were increased by 2.3, 2.2, and 2.7 doubling doses (Delta log PC15/0.3) respectively with rhuMAb-E25 and –0.3, +0.1, and –0.8 doubling does with placebo (p (less than/equal to)0.002). Methacholine PC20 improved slightly after E25, this change becoming statistically significant on Day 76 (p <0.05).
In a complementary study (14), the effects of 9 weeks of treatment with rhuMAb-E25 was examined in a parallel group, randomized, double-blind, placebo-controlled study of 19 mild allergic asthmatic subjects. Treatment with rhuMAb-E25 reduced serum IgE, increased the dose of allergen needed to provoke an early asthmatic response, reduced the mean maximal fall in FEV1 during the early response (30 ± 10% at baseline to 18.8 ± 8%, versus placebo 33 ± 8% at baseline to 34 ± 4%; p = 0.01), and reduced the mean maximal fall in FEV1 during the late response (24 ± 20% at baseline to 9 ± 10% versus placebo 20 ± 17% at baseline to 18 ± 17%; p = 0.047). Maximal bronchoconstriction during the late phase was reduced by 60%, eosinophilia in induced sputum 24 hours after allergen challenge was reduced 11-fold, and the PC20 for methacholine 24 hours after allergen challenge was increased by 1.6 doubling doses. Together these findings suggest a major role for IgE in orchestrating the airway narrowing, airway tissue eosinophilia, and airway hyperractivity of the late-phase response (14, 15).
More recently, moderate to severe allergic asthmatics have been enrolled in double-blind, placebo controlled trials (16, 17). Following a 4-week run-in in which corticosteroid (CS) and beta-agonist dosages were adjusted, 317 subjects were randomized to IV biweekly rhuMAb-E25 [low dose (Low) = 0.006 or high dose (High) = 0.014 mg/kg/IU/ml] or placebo for a period of 12 weeks (16). Subjects in all groups were demographically alike at baseline with a FEV1 percent predicted (mean = 71%), PEFR L/min (mean = 380), symptom score (median = 4 on a 7-point scale), inhaled CS (median = 800 ug/day), oral CS (median = 10 mg/day) and inhaled beta-agonist (median = 7 puffs daily); baseline serum IgE (ng/ml): Low 619, High 564 and placebo 480. Each active treatment was associated with a 99% mean fall in serum free IgE. Mean symptom score improvement of 42% in both High (p=0.003) and Low (p=0.004) E25 groups compared to 23% in Placebo. An arbitrary (greater than/equal to) 50% improvement in symptoms occurred in 49% of High (p=0.01) and 46% of Low (p=0.02) compared to only 25% in placebo. Significant increases in PEFR occurred in High (AM PEFR = + 31 L/min; p+0.007 and PM PEFR = + 20 L/min; p=0.003). These findings (16) demonstrated that 12 weeks of rhuMAb-E25 administration leads to a significant improvement in symptoms and lung function in moderate to severe allergic asthma.
Continuing treatment for 20 weeks permitted an assessment of the corticosteroid-sparing effect of E25 (17). Corticosteroid (CS) use decreased from baseline: placebo 24%; rhuMAb-E25 Low dose 41% decrease (p=0.02); rhuMAb-E25 High dose 50% decrease (p=0.04). Mean decrease in oral steroid use also favored rhuMAb-E25 treatment groups: 65% decrease in Low rhuMAb-E25, 50% in High rhuMAb-E25 versus 0% placebo. During the course of the study, rhuMAb-E25 treatment decreased asthma exacerbations: 45% of placebo patients exacerbated versus 28% of Low dose rhuMAb-E25 (p=0.01) and 30% o High dose E25 (p=0.03). Daily total symptom scores and beta-agonist rescue were significantly decreased in the rhuMAb-E25 treatment groups. Therefore, rhuMAb-E25 can be safely administered while decreasing exacerbations and use of CS and rescue medications.
This outstanding safety and efficacy record of rhuMAb-E25 in early clinical studies is the result of meticulous, rigorous preclinical experiments (vide supra). Late stage clinical trials confirming and extending these results are underway. Years before rhuMAb-E25 becomes an accepted therapy for allergic asthma, the MAb will have taught us much about the contribution of IgE to the immunopathobiology of asthma.
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|Fick, R.B.; Smigelski, S.S; Anchick, M.J; (1998). Anti-IgE as a Probe to Understand Allergic Inflammantion.. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/denburg/fick0528/index.html|
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