Patients with multiple sclerosis (MS) treated with interferon β (IFNβ) preparations develop varying levels of antibodies that neutralize the biological effects of IFNβ, reduce its in vivo bioavailability, and diminish its therapeutic efficacy. The aim was to determine as distinct measures of immunogenicity the occurrence (frequency) and the magnitude (level) of IFNβ neutralizing antibody (NAb) formation in a large Canadian population as a cross-sectional study of patients with MS treated in a clinical practice setting with different, equally available IFNβ products: Avonex(®) (intramuscular IFNβ-1a), Rebif(®) (subcutaneous (SC) IFNβ-1a) at 22 and 44 μg, and Betaseron(®) (SC IFNβ-1b). Over a 3-year period 3,124 serum samples from 2,711 patients with MS were submitted by neurologists in MS clinics distributed across Canada and tested for NAbs in a single independent laboratory, utilizing a quantitative, standardized NAb bioassay. NAb frequency was greatest (35%) with Rebif (SC IFNβ-1a) 44 μg and least (7.5%) with Avonex (intramuscular IFNβ-1a), whereas Betaseron (IFNβ-1b) and Rebif 22 μg were in between (22%). NAb serum levels at magnitudes considered high, ≥100 tenfold reduction units (TRU)/mL, were found in 65%-83% of patients with detectable NAbs. Nearly half (42%-47%) of NAb-positive patients given IFNβ-1a preparations had very high titers (≥ 1,000 TRU/mL), whereas only 22% of NAb-positive patients on Betaseron had titers >1,000 TRU/mL. Differences in patterns of NAb formation among the four IFNβ product-dose combinations became more evident in patients with MS when both NAb frequency and the full range of NAb titer magnitude were measured.

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The neutralizing antibodies (NAbs) that develop in patients during interferon (IFN) therapy can reduce its benefi cial effects. The universally employed method of NAb measurement currently is the constant IFN method, in which antigen at a single given concentration is mixed with serial dilutions of serum, the lowest fi nal dilution of which (usually 1:20) is constrained by the potential adverse effect of human serum on human cells in culture. The constant antibody (Ab) method described herein uses serum at a certain set dilution (usually 1:20) mixed with a series of IFN concentrations. Theoretical neutralization curves based on the previously presented model of the Ab-IFN reaction are depicted herein in terms of experimentally observable quantities. As predicted by the theoretical studies, the constant Ab method was demonstrated experimentally to extend the lower limits of detection of Ab by a factor of 10-20. The excellent agreement observed between the theoretical prediction and experimental fi ndings reinforces the validity of using as NAb unitage the titer based on 10-fold reduction of IFN activity, reportable as Tenfold Reduction Units (TRU)/mL, as previously recommended. Testing by the constant Ab method of sera previously considered negative (<20 TRU/mL by the constant IFN method) from patients treated with Rebif or Betaseron showed that ~50% had detectable NAbs; such sera from Avonex-treated patients had titers of <1 TRU/mL. The constant Ab method can be used as a quantitative, sensitive IFN NAb screening bioassay of any nature, and should be able to detect low levels of NAbs early in the course of IFN therapy. The method may be useful to test monoclonal antibodies for otherwise undetectable NAbs. In principle, the constant Ab method should be applicable to the measurement of NAbs against any cytokine or other protein-effector molecule.

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