Use of a standardized MxA protein measurement-based assay for validation of assays for the assessment of neutralizing antibodies against interferon-ß.

Effective monitoring of the development of neutralizing antibodies (NAbs) against IFN-ß in multiple sclerosis (MS) patients on IFN-ß therapy is important for clinical decision making and disease management. To date, antiviral assays have been the favored approach for NAb determination, but variations in assay conditions between laboratories and the increasing use of novel assays have contributed to the reporting of inconsistent antibody data between laboratories and between products. This study, undertaken at the request of the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA), is a joint effort by manufacturers of IFN-ß products (approved in Europe) towards harmonization of a NAb assay that facilitates generation of comparable NAb data, which, in conjunction with clinical outcomes, should prove useful for clinicians treating MS patients with IFN-ß products. This article describes the standardized cellular myxovirus resistance protein A (MxA) protein measurement-based assay for detection of IFN-ß NAbs and its use for the validation of assays used for the quantitative determination of such antibodies. Although titers varied between laboratories and the products used, utilization of IFN-ß1a rather than IFN-ß1b as the challenge antigen produced more consistent results in the NAb assay. Adoption of the standardized assay improves comparability between laboratories circumventing problems that arise when different, nonstandardized assays are employed for immunogenicity assessment. Based on the data, the EMA recommended for standardization purposes, the use of IFN-ß1a in NAb assays, independent of the therapeutic product used for therapy and validation of new NAb procedures against the standardized assay described.

Measured neutralizing titers of IFN-beta neutralizing antibodies (NAbs) can depend on the preparations of IFN-beta used in the assay.

An immune response to recombinant human protein therapeutics, including type I interferons (IFNs), has the potential to have a serious negative impact on safety and efficacy. Monitoring of patients for neutralizing antibodies (NAbs) often is advisable. In the case of IFN-beta therapy for multiple sclerosis (MS), we obtained reproducible quantitative titers of NAbs using an improved and well-characterized assay based on a 10-fold reduction of a challenge dose of IFN-beta. However, the observed titer was significantly affected by the preparation of IFN-beta used as the assay challenge. NAb titers obtained using IFN-beta1b averaged 3-5-fold lower than titers of the same sample assayed using either IFN-beta1a or human fibroblast-derived IFN-beta. This was the case whether neutralizing serum was obtained from patients on therapy with IFN-beta1a or IFN-beta1b. The reason for this apparent titer difference is not fully understood but appears to be related to protein folding or other structural properties that differentiate the IFN-beta1b both from commercial IFN-beta1a preparations and from human fibroblast-derived IFN-beta.