Evaluation of IgE Antibodies to Omalizumab (Xolair®) and Their Potential Correlation to Anaphylaxis.

Omalizumab (Xolair®) is a recombinant humanized monoclonal antibody that selectively binds to human immunoglobulin E (IgE). Omalizumab is used to treat IgE-mediated diseases such as chronic idiopathic urticaria (CIU) and moderate to severe allergic asthma. In pre-marketing clinical trials in patients with asthma, anaphylaxis was reported in 3 of 3,507 (0.1%) patients. In post-marketing spontaneous reports, the frequency of anaphylaxis attributed to omalizumab use was estimated to be at least 0.2% of patients based on an estimated exposure of about 57,300 patients from June 2003 through December 2006. To better understand the risk of anaphylaxis in patients with allergic asthma receiving omalizumab, a post-marketing pharmacosurveillance study was initiated in 2009. As part of this study, an assay was developed to detect antibodies of IgE isotype to omalizumab. Serum samples from patients in the study were evaluated using this assay. Our results indicated that there was no observable correlation between either anaphylaxis or skin test reactivity and the presence of antibodies of IgE isotype to omalizumab. Here, we discuss the development of this assay as well as the results of the immunogenicity assessment.

Evaluation of two commercial omalizumab/free IgE immunoassays: implications of use during therapy.

BACKGROUND: The anti-IgE monoclonal antibody, omalizumab, is approved in the US as add-on therapy for patients ≥12 years of age with moderate-to-severe persistent allergic asthma. Omalizumab is administered according to the US Food and Drug Administration approved dosing table included in the prescribing information. The dosing table was developed using Genentech's free IgE assay and is designed to achieve free serum IgE levels of <50 ng/mL, known to be associated with clinical benefit. Lack of clinical benefit in a subset of patients on omalizumab has prompted demand for commercial free IgE assays to guide omalizumab dosing. To date, two commercial free IgE assays marketed by ViraCor-IBT (no longer offered) and BioTeZ have been available to physicians. OBJECTIVE: This study compares the results generated from the two commercial free IgE assays with the free IgE levels generated by the Genentech assay. METHODS: Two serum sample sets were prepared using 20 samples from patients with a wide range of IgE and omalizumab from an omalizumab clinical trial and 36 samples from omalizumab-naïve patients. Different amounts of omalizumab were added to the 36 omalizumab naïve samples based on measured total IgE levels to ensure that a good range of IgE and omalizumab was represented in the study samples. Samples were randomized for blinded analysis of free IgE levels using the Genentech, ViraCor-IBT and BioTeZ free serum IgE assays. Analysis of samples in the ViraCor-IBT assay were conducted by ViraCor-IBT and the analysis of samples using the Genentech and BioTeZ assay methods were conducted by a third party contract research organization. RESULTS: The ViraCor-IBT and BioTeZ free IgE assays demonstrated significantly higher free IgE levels than the Genentech free IgE assay. Twenty-nine of 56 samples tested <50 ng/mL in the Genentech assay; of these, 12/29 (41%) and 20/29 (69%) tested >50 ng/mL in the BioTeZ and ViraCor-IBT assays, respectively. In the BioTeZ free IgE evaluations, 11/20 samples that were re-tested had inter-assay differences ranging from 40-190%. CONCLUSIONS: Free ligand (such as IgE) measurements are challenging and dependent on the method and reagents used. The Viracor-IBT and BioTeZ methods tend to over-estimate free serum IgE levels compared with the Genentech free IgE assay. Using these assays to monitor therapy and adjust omalizumab doses post treatment is considered off-label use and could lead to a potential risk for unnecessary treatment and/or risk to patient safety.

Proteolysis of the N-methyl-d-aspartate receptor by calpain in situ.

N-Methyl-D-aspartate (NMDA) receptors are calcium-permeable glutamate receptors that play putative roles in learning, memory, and excitotoxicity. NMDA receptor-mediated calcium entry can activate the calcium-dependent protease calpain, leading to substrate degradation. The major NMDA receptor 2 (NR2) subunits of the receptor are in vitro substrates for calpain at selected sites in the C-terminal region. In the present study, we assessed the ability of calpain-mediated proteolysis to modulate the NR1a/2A subtype in a heterologous expression system. Human embryonic kidney (HEK293t) cells, which endogenously express calpain, were cotransfected with NR1a/2A in addition to the calpain inhibitor calpastatin or empty vector as control. Receptor activation by glutamate and glycine as co-agonists led to calpain activation as measured by succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-aminomethyl coumarin (Suc-LLVY-AMC). Calpain activation also resulted in the degradation of NR2A and decreased binding of (125)I-MK-801 ((125)I-dizocilpine) to NR1a/2A receptors. No stable N-terminal fragment of the NMDA receptor was formed after calpain activation, suggesting calpain regulation of NMDA receptor levels in ways distinct from that previously observed with in vitro cleavage. NR2 subunit constructs lacking the final 420 amino acids were not degraded by calpain. Agonist-stimulated NR1a/2A-transfected cells also had decreased calcium uptake and produced lower changes in agonist-stimulated intracellular calcium compared with cells cotransfected with calpastatin. Calpastatin had no effect on either calcium uptake or intracellular calcium levels when the NR2A subunit lacked the final 420 amino acids. These studies demonstrate that NR2A is a substrate for calpain in situ and that this proteolytic event can modulate NMDA receptor levels.