A significant problem in relation to interferon-β (IFNβ) therapy is that a considerable proportion of the patients develop neutralizing anti-IFN antibodies (NAbs), which in high concentrations have been correlated to reduction of treatment response.
In a previous study, we showed that binding and neutralizing antibodies appeared faster in patients receiving IFNβ-1b compared with IFNβ-1a, and fewer became positive if 22 μg IFNβ-1a was administered once weekly compared with three times weekly. IM administration induced antibodies less frequently than subcutaneous administration. Further, we demonstrated that the frequency of NAbs against IFNβ heavily depends on the sensitivity of the neutralizing assay.1
There have been considerable controversies regarding the clinical importance of these antibodies. Initially, all phase III studies found no relation between development of NAbs and clinical efficacy.2–4⇓⇓ In some studies, however, re-evaluation of the results or prolongation of the observation period disclosed that NAbs contribute to a lack of response to INFβ therapy in some patients with multiple sclerosis (MS).5–7⇓⇓
To evaluate the effect of different serum concentrations of NAbs against IFNβ on therapeutic efficacy, we measured NAbs to IFNβ in prospectively sampled sera from patients with relapsing-remitting MS who began treatment with a commercial IFNβ preparation during the years 1996 to 1999 in Denmark.8
Patients and methods.
The study comprised 541 patients, who all were treated with the same IFNβ preparation throughout the entire observation period: Betaferon (Schering, Berlin, Germany) 8 MIU every other day in 194 patients, Rebif (Serono, Geneva, Switzerland) 22 μg weekly in 103 patients, Rebif 22 μg three times weekly in 162 patients, and Avonex (Biogen, Cambridge, MA) 30 μg once weekly in 82 patients.8
Measurements of NAbs were performed in a blinded fashion using antiviral neutralization (A549/EMC) bioassay.1 Each sample was tested with three different sensitivities of the neutralizing assay: 1) high sensitivity (addition of IFNβ, 3 LU/mL), 2) medium sensitivity (addition of IFNβ, 10 LU/mL), and 3) low sensitivity (addition of IFNβ, 100 LU/mL). The neutralizing capacity (i.e., the percentage of the added IFN, which was neutralized by the NAbs) of each serum sample was measured.
Results.
NAbs generally appeared within 12 months after start of treatment, and patients treated with IFNβ-1b developed NAbs faster than patients treated with IFNβ-1a.
After 12 months, 45.4% of the patients treated with IFNβ-1b (Betaferon) had NAbs, but, interestingly, after 36 months of therapy, a statistically significant lower proportion was NAb positive than after 12 months of treatment (p = 0.023, χ2 test). When IFNβ-1a (Rebif) was administered once weekly, significantly fewer patients (14.6%) became antibody positive at 12 months of treatment than when IFNβ-1a (Rebif) was given three times weekly (45.7%; p = 0.000, Fisher’s exact test), and when IFNβ-1a (Avonex) was administered IM, significantly fewer patients (8.5%) became antibody positive at 12 months of treatment compared with subcutaneous administration of IFNβ-1a (Rebif) once a week (p = 0.022, Fisher’s exact test).
Development of NAbs was not predicted by gender, duration of MS at start of treatment, number of relapses during the past 2 years before treatment, or progression index (Expanded Disability Status Scale [EDSS] at start of treatment divided by duration of MS).
The NAb status at 12 months was somewhat but not fully predictive of the NAb status later. If the 12-month status was negative, only 9%, 9%, 12%, and 15% became NAb positive after 2, 3, 4, and 5 years, respectively. Conversely, if the 12-month status was positive, the percentages of these patients who were still NAb positive after 2, 3, 4, and 5 years were 82%, 69%, 53%, and 46%, respectively.
The presence of NAbs had a significant effect on the relapse rate. During NAb-positive periods, the annual relapse rate was 0.64 compared with 0.43 in NAb-negative periods, yielding an odds ratio for suffering relapses of 1.55 in NAb-positive periods compared with NAb-negative periods (p < 0.01).
The time to first relapse in NAb-negative patients was significantly increased by 270 days compared with NAb-positive patients using Kaplan–Meier analysis of the probability of remaining exacerbation free (log-rank test p = 0.028; figure 1). The percentage of NAb-negative patients who remained free of relapses throughout the study period was 39% compared with 27% in the NAb-positive group (p = 0.0064, χ2 test).
Figure 1. Kaplan–Meier analysis of the time to first relapse and proportion of relapse-free neutralizing antibody (NAb)-positive and NAb-negative patients (log-rank test p = 0.039).
NAb-positive patients showed an increase in mean EDSS after 42 months. The differences between NAb-positive and NAb-negative patients were statistically significant only at 42 months (p = 0.049) and at 48 months (p = 0.008) after treatment start (Mann–Whitney U test). Time to confirmed progression of one point or more on EDSS, sustained for at least 6 months, did not differ significantly between patients who were NAb positive or NAb negative at 12 months of treatment start (log-rank test = 2.19, p = 0.14; figure 2).
Figure 2. Time to confirmed progression in neutralizing antibody (NAb)-positive and NAb-negative patients.
Discussion and conclusions.
The Danish National IFN-beta Project is the largest independent study that has examined the impact of NAbs on the therapeutic effect of IFNβ. The results of the study show that the frequency of NAbs against IFNβ depends on the sensitivity of the assay; the occurrence of NAbs cannot be predicted from pretreatment clinical characteristics; and the occurrence of NAbs depends on the preparation, dose, frequency, and route of administration. NAbs develop faster with IFNβ-1b than with IFNβ-1a, but after 2 years a significant decrease in the proportion of NAb-positive patients is seen during continuous treatment with IFNβ-1b.8
Results from the pivotal trials of the three commercially available IFNβ preparations have been conflicting regarding the frequency of NAbs and the impact of NAbs on the therapeutic effects. In the phase III study of IFNβ-1b, 35% of the patients developed NAbs. NAb-positive patients showed higher relapse rates, number of enlarging T2 lesions, and new lesion formation compared with NAb-negative patients.5 In the phase III study of IFNβ-1a (Avonex) administered IM once weekly, 19% of the patients developed NAbs, and no influence of NAbs on efficacy was reported.3 However, when the results were reanalyzed, a trend toward a reduced effect on MRI activity was seen.6 The authors of the PRISMS study of IFNβ-1a (Rebif) administered subcutaneously three times weekly reported that NAbs was found in approximately 20% of the patients and that NAbs had no impact on the therapeutic effect of IFNβ,4 but 4-year data from the PRISMS study showed that NAb-positive patients had higher relapse rates, number of T2 active lesions, and burden of disease compared with NAb-negative patients.7 The detrimental effects of NAbs on clinical efficacy in these studies were observed after 18 months of treatment, indicating that studies shorter than 2 years cannot adequately assess the efficacy of IFNβ.
In conclusion, NAbs impact on the clinical activity of patients with MS treated with IFNβ, certainly on relapses (but not proven on disease progression) and on disease activity measured with MRI. Examination of NAbs should be performed routinely with standardized assays at least 1 and 2 years after start of treatment because few patients become NAb positive more than 2 years after initiation of IFNβ therapy. Treatment decisions should still primarily be based on clinical outcome, but for patients with high concentrations of NAbs, it would be reasonable to substitute treatment, particularly if significant clinical activity is observed.
Acknowledgments
Christian Ross, Katja Maria Clemmesen, Klaus Bendtzen, Jette Lautrup Frederiksen, Kai Jensen, Ole Kristensen, Thor Petersen, Soren Rasmussen, Mads Ravnborg, Egon Stenager, Nils Koch-Henriksen and the Danish Multiple Sclerosis Study Group are acknowledged for collaboration in the Danish Interferon-beta Project.
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