All antibody positive samples also contained neutralizing antibodies (Fig. 1B). For this group of patients, the correlation coefficient R2 between log10 titers obtained in the non-cell-based NAb assays and those obtained in an antiviral assay varies between 0.867 and 0.910. For a selected number of patients (cohort C), sequential samples (n = 31) were tested in both cell-based
and non-cell-based Compound Library assays. Results showed that the samples identified as positive in cell-based assays were also positive in the non-cell-based NAb assay. The NAbs titers correlated highly with those obtained either in the antiviral or the reporter gene assay. The correlation factor between log10 titers obtained in the non-cell-based assay and those obtained in a reporter gene assay is R2 = 0.938; while the correlation coefficient between log10 titers obtained in the non-cell-based assay and those obtained in an antiviral assay is R2 = 0.910 (Fig. 5B & C). Using the binding and the neutralizing ECL assays to evaluate sequential serum samples from patients selected on the basis of absence of neutralizing antibodies
in the cell-based assay, we were able BIBW2992 purchase to identify a very small number of patients developing non-neutralizing antibodies. The binding activity is positive, although low, while no neutralization is observed in the time course for which we have samples (Fig. 6). It is recognized that NAb assays are an important element of the assessment of immunogenicity of a biotherapeutic. While the US draft guidance urges the use of cell-based assays for determination of NAbs, the European guideline allows the use of a non-cell-based assay if cell-based assays are not feasible or available (U.S. Department of Health and Human services, Food and Drug Administration, 2009 and European Medicines Agency (EMEA), 2007) and even recommend it as a method of choice in some instances (EMEA/CHMP/BMWP/86289/2010, 2012). Therefore,
the comparison of cell-based and non-cell-based assays for determination of NAbs during product development and clinical phases, as well as during post-marketing surveillance, is a much debated topic. The feasibility of developing non-cell-based NAb assays has been illustrated previously for detection of neutralizing auto-antibodies against the cytokine IL-17 Ergoloid in auto-immune patients (Cludts et al, 2010). A recent publication, in which different assay formats were compared, showed the potential of competitive ligand-binding assays for NAb evaluation during product development, but no clinical data were provided (Finco et al, 2011). Here, we have explored the possibility of using a non-cell-based NAb assay for the assessment of clinical samples from IFN-β treated RRMS patients. It is recognized that after treatment with IFN-β, a significant percentage of patients develop anti-IFN-β antibodies, and that these antibodies are mostly neutralizing.
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