Measured Neutralizing Titers of IFN-β Neutralizing Antibodies (NAbs) Can Depend on the Preparations of IFN-β Used in the Assay

Abstract

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.

Important sources of variability in clinical studies of neutralizing antibodies against interferon beta

“…In the presence of a sufficient concentration of NAbs, IFN beta does not up-regulate MxA protein levels, which are measured by an enzyme-linked immunosorbant assay (ELISA). This assay is a highly sensitive, specific, and simple method for measuring anti-IFN beta NAbs [11,12]. While the results of these two assays were generally concordant, significant scatter was observed when the same serum samples were analyzed using both assays (Fig.…”

“…Choice of the appropriate sensitivity and cutoff value, which may differ for different IFN betas, is an unfortunately circular process. Clinical observations of worsened outcome may assist in determining the appropriate NAb assay sensitivity and cutoff values, but improper initial choice of these values may prevent observation of NAbrelated clinical changes [12,14]. Fig.…”

The implications of immunogenicity for protein-based multiple sclerosis therapies

“…Most investigators have found it simpler to use IFNβ-1a antigen as the target in antibody assays, even in patients who are receiving IFNβ-1b. This leads to overestimation of antibody levels in patients treated with IFNβ-1b compared with what would be determined using IFNβ-1b as the antigen [69]. Generally, however, antibody titers are higher in patients treated with IFNβ-1a, regardless of whether titers are assessed against IFNβ-1a or IFNβ-1b.…”

Quantification of the Neutralization of Cytokine Biological Activity by Antibody: The Ten-Fold Reduction Bioassay of Interleukin-6 as Growth Factor

“…The ten-fold reduction approach has been successfully operative with very different methodologies to measure NAbs to the IFNs, involving different biological effects, whether they be, for example, antiviral (Grossberg and others 1986), MxA protein induction (Pungor and others 1998;Files 2007), cell-growth inhibition (Redlich and Grossberg 1989), or luciferase reporter gene induction (Lallemand and others 2008;Lam and others 2008). Further, NAb titers calculated as TRU/mL determined on the same serum samples by the luciferase gene induction and by antiviral assays were comparable (Lallemand and others 2008).…”