Curr Med Res Opin. 2014 May;30(5):913-22.
Evaluation of two commercial omalizumab/free IgE immunoassays: implications of use during therapy.
- 1Genentech , South San Francisco, CA , USA.
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.
The role of IgE antibodies in allergic asthma is well established, and the FDA-approved anti-IgE antibody omalizumab (Xolair®, Genentech) is used as an add-on therapy for patients whose symptoms are inadequately controlled with inhaled corticosteroids.1 Following omalizumab administration, free serum IgE levels fall rapidly, while total IgE levels increase due to formation of omalizumab-IgE complexes, which can remain elevated for up to 1 year after therapy is discontinued.1,2
To ensure that free serum IgE is reduced to a clinically efficacious level of <50 ng/mL2, the dose of omalizumab is determined before therapy starts using an approved dosing table that was derived using a free IgE assay developed by Genentech, Inc. Because not all patients respond to therapy, a demand for an omalizumab-specific free-IgE assay has risen to identify patients who may have been underdosed by examining whether free IgE serum falls within the therapeutic range. Adjustment of omalizumab dosing based on free IgE measurements has not been approved and is considered off-label use. However, two commercial free serum IgE assays, one from Viracor-IBT (no longer available), and the second from BioTeZ have been marketed to allow physicians to monitor free IgE in patients treated with omalizumab.
Detecting free ligands (such as IgE) is technically very challenging and results can vary between assays. Given that the commercial free IgE assays may potentially be used to make treatment decisions, we sought to compare IgE data obtained from these assays with the Genentech assay. To ensure a representative range of IgE and omalizumab levels were used for this evaluation, we prepared two sets of samples (in vivo and in vitro) using sera from allergic asthma patients. The 20 in vivo samples were prepared by pooling sera from 32 patients who received omalizumab and had similar free IgE and omalizumab profiles. The 36 in vitro samples were prepared from sera of 15 omalizumab-naïve patients by spiking with various levels of omalizumab to obtain a wide range of omalizumab/total IgE molar ratios.
The Viracor-IBT free-IgE assay uses the same capture reagent as the Genentech free IgE assay, but utilizes high binding ImmunoCAP instead of ELISA plates (Figure 1) and has a different detection reagent, sample incubation times, and sample dilutions. The BioTeZ recovery ELISA assay claims to simultaneously quantify free IgE and total omalizumab (Figure 2). Samples were randomized and blindly evaluated to determine free IgE (using Genetech, Viracor-IBT, and BioTeZ assays), total IgE (using Phadia’s ImmunoCap® assay) and omalizumab (using BioTeZ and Genentech assays) levels. The ViraCor-IBT and the ImmunoCap assays were conducted by ViraCor-IBT; the Genentech and BioTeZ assays were conducted by a third party contract research organization. The Phadia ImmunoCap assay was verified by Genentech to have no interference from omalizumab. The molar ratio of omalizumab/total IgE was calculated for each sample using the results from Genentech’s total omalizumab assay and Phadia’s total IgE assay.
As expected, all free-IgE assays showed a trend of decreasing free IgE with increasing omalizumab/total IgE molar ratios (Figure 3). However, both the Viracor-IBT and BioTeZ assays tended to measure significantly higher levels of free IgE compared with the Genentech assay in the 29 omalizumab-positive samples. Notably, 12 (41%) and 20 (69%) of the omalizumab-positive samples with free IgE levels <50 ng/mL in the Genentech assay had corresponding free IgE levels >50 ng/mL in the BioTeZ and Viracor-IBT assays, respectively. In addition, the BioTeZ assay reported extreme values (<30% or >300% of the corresponding Genentech data) for approximately one third of the total 56 samples.
The BioTeZ omalizumab assay was also highly variable, detecting approximately 20% to 3000% of omalizumab reported by the Genentech assay (Figure 4). Moreover, the BioTeZ assay generated false-positive results in 3 of the 12 omalizumab-negative samples and false-negative results in 8 of the 44 omalizumab-positive samples. Repeat evaluation of these samples demonstrated poor reproducibility of the BioTeZ assay for both free IgE and omalizumab measurements. In the free IgE evaluations, 11/20 samples that were re-tested had inter-assay differences ranging from 40–190%.
Regardless of the inconsistencies observed with the BioTeZ assay, both commercial assays tended to have over-recovery compared with the Genentech assay, which may arise from the use of assay methods and conditions that promote disruption of omalizumab-IgE complexes. The stability of the IgE/omalizumab complex can be affected by multiple factors, including the assay format, assay reagents, sample dilution, and incubation times. The Viracor-IBT free IgE method uses the Phadia technology, combination of the high binding capacity of the ImmunoCap® cellulose sponge and the high binding affinity of the capture reagents (IgE receptor) compared with omalizumab can promote disruption of the omalizumab-IgE complex. The unusually long sample incubation times and high sample dilution can be responsible for over recovery of free IgE in the BioTeZ method.
These results demonstrate that the commercial assays evaluated are not reliable for monitoring free IgE and, if used during omalizumab treatment, may overestimate free IgE levels. Overestimation of free IgE levels could lead physicians to unnecessarily increase the omalizumab dose to achieve further reductions in free IgE, or prematurely discontinue treatment on the mistaken assumption that the treatment is not effectively reducing free IgE.
We do not intend to claim that the Genentech free IgE assay is superior to that of the commercial assays evaluated in this study. Rather, we wish to re-emphasize that the Genentech free IgE assay is the clinically relevant assay because it was used to generate the FDA-approved dosing table for omalizumab. The omalizumab product label clearly states that on-treatment re-testing of serum total IgE levels cannot be used to guide dosing, and re-initiation of treatment after a period of treatment of less than 1 year should be based on the original pre-treatment serum levels of IgE. Furthermore, the product label does not state that free IgE levels should be monitored during therapy.1
Multiple factors can impact the efficacy of omalizumab in non-responders and these should be considered in addition to absolute free IgE levels3. As specified by the prescribing information, the most effective method for evaluating continued need for omalizumab is still periodic assessment of the patient’s disease severity and level of asthma control.1
- Genentech, Novartis US. XOLAIR FDA Prescribing Information. Available at: http://www.gene.com/gene/products/information/pdf/xolair-prescribing.pdf [Last accessed November 25, 2013].
- Hochhaus G, Brookman L, Fox H, et al. Pharmacodynamics of omalizumab: implications for optimised dosing strategies and clinical efficacy in the treatment of allergic asthma. Curr Med Res Opin 2003;19: 491–8
- Heaney LG, Robinson DS. Severe asthma treatment: need for characterising patients. Lancet 2005;365: 974–6.