Therapeutic interferon-β (IFN-β) preparations have been approved for treatment for multiple sclerosis (MS). However, the development of neutralising antibodies (NAbs) against IFN-β in MS patients reduces therapeutic efficacy. Early detection of NAbs is critical for the modification of treatment regimes. IFN-β exerts its biological effects by binding to receptors on target cells and stimulating the expression of IFN-β-inducible genes. Measurement of endogenous mRNAs for these genes can form the basis of functional bioassays. In this study we have used two approaches, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and branched DNA (bDNA) technology to develop efficient, sensitive and robust non-viral assays for the quantification of IFN-β NAbs in patients with MS. We show that the rapid (4 h) induction of the type I IFN-inducible 616 mRNA in A549 lung carcinoma cells is quantifiable by qRT-PCR and by bDNA assays. The induction of 616 mRNA is sensitively and reproducibly concentration-dependent for IFN-β stimulation and is comparable for real-time PCR and bDNA. In both assay systems, a sigmoidal dose response curve was obtained within the range 0.6625 pg/ml IFN-β with a maximal response obtained between 156 and 625 pg/ml IFN-β. This is in broad agreement with data from antiviral assays in which maximum protection was obtained between 78 and 156 pg/ml IFN-β. Measurement of 616 mRNA by real-time RT-PCR and bDNA is readily adaptable for the detection and measurement of NAbs against IFN-β. Thus, sera from patients receiving IFN-β therapy (21 months) effectively neutralised IFN-β-stimulated 616 mRNA expression (100% neutralisation at a serum titre of 1:600; 50% neutralisation at a serum titre of approximately 1:4000), whilst pre-treatment serum from the same patients had no effect at the highest concentration tested. These data suggest mRNA-based assays could be used for bioactivity measurements of therapeutic products and also in a clinical setting for the monitoring of patients receiving IFNβ treatment.
03 - 07 May 2008
European Society of Endocrinology