May 6, 2019April 9, 2019Free AccessEstablishment of Neurofilament Light Chain (NfL) SIMOA assay as well as a conversion factor to enable comparison to historical results using Bovine calibrators. (P2.2-074)Robert Hendricks, teresa davancaze, Christopher Harp, Ann Herman, Dana Baker, Jochen Brumm, H. Von Budingen, Mike Townsend, Erica Eggers, and Sally FischerAuthors Info & AffiliationsApril 9, 2019 issue92 (15_supplement)https://doi.org/10.1212/WNL.92.15_supplement.P2.2-074 Letters to the Editor

Background: Neurofilament light (NfL) chain is an established cerebrospinal fluid (CSF) biomarker for neuroaxonal injury. The highly sensitive Quanterix Simoa™ platform is evaluated for NfL measurement in both CSF and blood. There is a need to link historical ELISA data that use bovine NfL to that of Simoa using a recombinant human (rhuman) NfL standard. Results/Methodology: The Simoa NF-light ® Advantage Kit was validated for CSF and qualified for serum and plasma, using both rhuman and bovine NfL calibrators. Matched CSF, serum and plasma samples from 112 multiple sclerosis patients were analyzed using both calibrators. Conclusion: In multiple sclerosis, there is a good correlation between blood and CSF NfL levels. A conversion factor of approximately 5:1 was established between bovine and rhuman NfL calibrators.

An in-depth evaluation of the Quanterix© Simoa™ platform was undertaken by scientists from the AAPS Emerging Technologies Focus Group to determine the overall performance of the technology as well as provide guidance to future users. In order to test the platform in a non-GLP bioanalytical setting, a cross-site evaluation of the Quanterix IL-6 biomarker kit was performed. Parameters tested during this evaluation included sensitivity, accuracy and precision, and parallelism in human serum from normal individuals. The results demonstrated improved sensitivity compared to the claimed sensitivity of other commercially available IL-6 kits and showed excellent site-to-site reproducibility. Observed issues included difficulties with system reliability and a lack of parallelism and specificity in a subset of samples. Overall, these results demonstrate that while there are challenges to the Simoa platform this technology offers automation capabilities and excellent sensitivity that enhance bioanalysis especially of low-abundance analytes.

Post-translational modifications of Tau protein are of central importance in Alzheimer's disease (AD) pathogenesis. In addition to the well-established role of hyperphosphorylation in AD, emerging evidence indicates the involvement of proteolytic cleavages in Tau pathology development. In this study we explored the disease-related changes in the composition of Tau protein species, both full length and fragments, in cortical tissue from AD subjects. Cryosections of fresh-frozen fusiform gyrus cortex from AD subjects and cognitively normal, age matched control subjects were characterized using immunoprecipitation, ELISA, immunoblotting and transcriptomic analyses. Additionally, fresh-frozen frontal cortical tissues from control subjects were used to identify proteolytic cleavage sites using proteomic methods. Full-length Tau isoforms showed changes in protein expression pattern with high correlation to disease severity. Specifically, the longest Tau isoform, Tau441 (2N4R), is selectively enriched in AD patients versus controls, with the highest enrichment in Braak stages V and VI. No difference in Tau spliced forms were observed at the RNA level, indicating the enrichment of specific isoforms occurs post-transcriptionally. One possible mechanism is differential fragmentation of Tau isoforms via proteolytic cleavage. We observed that the Tau fragmentation pattern is common and stereotypical across all patient samples, regardless of disease status, with fragments representing ∼25% of total Tau signal. The extent of Tau fragmentation, however, is inversely correlated with the extent of tauopathy and Tau441 levels. Further analyses of the fragments suggest at least one common cleavage site for all isoforms, generating a N terminal-mid domain fragment and a C-terminal fragment. Our data suggest a proteolytic mechanism that shifts the Tau protein landscape toward the enrichment of longer isoforms during Alzheimer's disease progression. The fragments we identified are likely precursors of CSF Tau fragments, and therefore have implications for interpretation of current mid-domain Tau biomarkers for AD diagnosis and progression. Further understanding of the mechanisms of Tau fragmentation would provide insight into the biological significance of CSF Tau measurements beyond the most commonly used mid-domain assays.

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.

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.

Data generated using various immunoassay methods are an integral part of the development of protein therapeutics. These assays are used in clinical and preclinical studies to establish the pharmacokinetic (PK) and pharmacodynamic (PD) characteristics as well as to assess the immunogenicity properties of a therapeutic. PK assays measure therapeutic levels post-administration which is essential for understanding the effective dose and dose regimen for a therapeutic. Anti-OX40L is a fully humanized monoclonal antibody designed for the potential treatment of an autoimmune disease. The anti-OX40L human PK assay is required to be sensitive, robust, and precise. To address challenges due to assay sensitivity and reproducibility, as well as assay technology limitations, during development of the anti-OX40L human PK assay, three different assays, including an MSD-based electrochemiluminescence assay (ECLA), a fluorometric enzyme-linked immunosorbent assay (ELISA), and a colorimetric ELISA, were evaluated. The MSD-based assay was the most sensitive but posed risk of inter-well signal crosstalk. The fluorescence ELISA fell short on reproducibility. The colorimetric ELISA was ultimately chosen for supporting sample analysis. This paper presents characterization data obtained from each of these assay formats, challenges that were encountered in the development of the assay, and the rationale for selecting the ultimate assay format.

Modulating the binding affinities to IgE or changing the FcγR binding properties of anti-IgE antibodies offers an opportunity to enhance the therapeutic potential of anti-IgE antibodies, but the influence of increased affinity to IgE or reduced Fc effector function on the pharmacological properties of anti-IgE therapies remains unclear. Our studies were designed to characterize the pharmacokinetics, pharmacodynamics and immune-complex distribution of two high-affinity anti-IgE monoclonal antibodies, high-affinity anti-IgE antibody (HAE) 1 and 2, in mice and monkeys. HAE1, also known as PRO98498, is structurally similar to omalizumab (Xolair®), a humanized anti-IgE IgG1 marketed for the treatment of asthma, but differs by 9 amino acid changes in the complementarity-determining region resulting in a 23-fold improvement in affinity. HAE2 is similar to HAE1, but its Fc region was altered to reduce binding to Fcγ receptors. As expected given the decreased binding to Fcγ receptors, systemic exposure to pre-formed HAE2:IgE complexes in mice was greater (six-fold) and distribution to the liver lower (four-fold) compared with HAE1:IgE complexes. In monkeys, systemic exposure to HAE1 was similar to that previously observed for omalizumab in this species, but required comparatively lower serum drug concentrations to suppress free IgE levels. HAE2 treatment resulted in greater exposure and greater increase of total IgE, relative to HAE1, because of decreased clearance of HAE2:IgE complexes. Overall, these data suggest that increased binding affinity to IgE may provide a more effective therapeutic for asthma patients, and that retaining FcγR binding of the anti-IgE antibody is important for elimination of anti-IgE:IgE complexes.