Approaches to improve drug tolerance and target tolerance in the assessment of neutralizing anti-drug antibodies.

Aim: Neutralizing anti-drug antibody (NAb) assays are inherently prone to the interference from drug and its soluble target, potentially resulting in erroneous results. An effective approach to improve drug tolerance of an NAb assay is pretreatment of samples with acid to dissociate immune complexes of NAb and drug, followed by separating NAbs from circulating drug before testing them in the assay. Methods and Results: The acid pretreatment conditions were optimized to improve drug tolerance of cell-based and non-cell-based NAb assays. NAbs were further separated from circulating drug either through direct drug removal or purification of NAb from the sample. In addition, an integrated experimental strategy was implemented to simultaneously improve drug and its soluble target tolerance for reliable NAb assessment. Conclusion: The approaches described herein would enable the development of reliable NAb assays that overcome drug and its target interference for more precise and sensitive NAb assessment.

Novel data analysis methods to overcome cut point challenges and enable comprehensive assessment of antidrug binding activity in confirmatory assays.

Immunogenicity assessments in response to drug treatment are commonly performed using a tiered approach strategy. All samples are initially tested in a screening assay followed by the evaluation of the screened positive samples in a confirmatory assay. Percent inhibition of signal intensity by the competing unlabeled drug in a confirmatory assay is typically used to measure the specificity of antidrug binding activity in samples, and has been successfully applied to most immunogenicity assays. However, the percent inhibition approach may not be suitable in cases where broadly distributed and high percent inhibition values are observed in drug-naïve subjects or when persistent operator-dependent differences in assay performance are encountered. Herein, we present the case studies faced with such challenges and provide appropriate solutions by introducing two novel data analysis methods: (1) Reference Delta, and (2) Reference Percent Inhibition, - in which relative-to-baseline signal inhibition is calculated for each sample. These novel methods significantly improve the confirmatory assay's ability to detect the samples positive for antidrug antibodies (ADA), especially when challenges are encountered using the traditional percent inhibition approach. Furthermore, both methods can be implemented in parallel with the percent inhibition method, enabling not only confirmation of ADA specificity, but also providing additional insights about the relevance of this antidrug binding activity to drug treatment.

Longitudinal assessment of the dose consistency of botulinum toxin type A (BOTOX) for cervical dystonia.

Botulinum toxin type A (BoNT/A) is the principal therapy for patients with cervical dystonia. Repeated treatments over many years are required in most cases. This retrospective review evaluates the dose of BoNT/A used to treat cervical dystonia and the interval between treatments during a 2-year observation period. Outcomes data were abstracted from the medical records of 172 patients at 3 different sites who had received BoNT/A between January and December 1998. A total of 1059 treatments were assessed. Mean per-treatment doses throughout the 2-year study ranged from 241.80 to 254.07 units. The mean interval between treatments was 108.48 days during the first year of observation and 114.14 days during the second year. These findings indicate that doses of and intervals between BoNT/A treatments for cervical dystonia were consistent throughout 2 years of observation.