NAb-positive Patients Research Articles

Appearance and disappearance of neutralizing antibodies during interferon-beta therapy

Neutralizing antibodies (NABs) occur frequently in patients receiving interferon (IFN)-beta for multiple sclerosis (MS), but it is unclear whether occurrence of NABs is predictive for the persistence of NABs during continued IFN-beta therapy. The authors used an antiviral neutralization bioassay to measure NABs blindly from 6 months up to 78 months in patients with MS who were followed for at least 24 months during treatment with IFN-beta. Patients were classified into three groups: 1) persistently NAB-negative patients, defined as patients without any positive samples at any time; 2) definitely NAB-positive patients, defined as patients who had at least two consecutive positive samples; and 3) patients with fluctuating NAB-positive and NAB-negative samples. A total of 455 patients were included in the study. Overall, 52.3% of the patients were persistently NAB-negative, 40.9% became definitely NAB-positive, and the remaining 6.8% were fluctuating. More patients treated with IFN-beta-1a (Avonex) remained NAB-negative (p < 0.0001), whereas there was no difference between IFN-beta-1b (Betaferon) and IFN-beta-1a (Rebif). Patients who have remained NAB-negative during the first 24 months of therapy rarely developed NABs. On the contrary, the majority of patients, who had been NAB-positive from 12 through 30 months after start of therapy, remained NAB-positive. NABs should be measured in all patients treated with IFN-beta. If patients have been persistently NAB-negative for 24 months, measurements can be discontinued. Patients who have been NAB-positive for a period of 18 months or more usually remain NAB-positive for a long time.

Optimising MS disease-modifying therapies: antibodies in perspective

A proportion of people with multiple sclerosis (MS) treated with interferon (IFN) a develop neutralising anti-IFN beta antibodies (NABs). The immunogenicity of the available commercial compounds relates to the genetic structure of the IFN beta molecule, its mode of production, glycosylation status, aggregate formation, commercial formulation, potency, dose, frequency and, possibly, route of administration. At present, it is not possible to predict who will develop NABs usually appear within the first 2 years of starting therapy. In patients treated with IFN beta in whom NABs persist for a significant period of time, their presence is associated with a reduction in both the biological effects and clinical efficacy. Approximately one third of NAB-positive patients with a titre > 20 NU/mL will revert to NAB-negative status with long-term follow-up. The persistence of NABs appears to be linked to the type of IFN beta treatment as well as the titre of antibodies. The overall efficacy of IFN beta and, hence, of any biological disease-modifying treatment (DMT) would be substantially improved if the development of NABs could be prevented or reversed. Although the overall efficacy of IFN beta in MS is relatively modest, the efficacy in individuals who remain NAB-negative is considerably better than in those who become persistently NAB-positive. One could argue that when comparing the 'true' clinical efficacy of different IFN beta products, the comparisons should be limited to the cohorts that remain NAB-negative. As a corollary, the therapeutic efficacy of IFN beta could be maximised if patients who tolerate higher-dose preparations could be prevented from developing persistent NABs. Strategies employed to prevent or reverse the development of NABs with other biological compounds (e. g. insulin, factor VIII, IFN beta, recombinant human erythropoietin) include improvements in the manufacturing process, immunosuppression, induction of tolerance and deimmunisation, and these should be considered in relation to biological DMT therapy as part of future clinical studies.

Longitudinal analyses of the effects of neutralizing antibodies on interferon beta-1b in relapsing-remitting multiple sclerosis.

We have analysed data on exacerbation rates, Expanded Disability Status Scale (EDSS) scores, and lesion burdens using the results of two neutralizing antibody (NAB) assays (CPE and MxA) from the pivotal relapsing-remitting multiple sclerosis (MS) trial of interferon beta-1b (IFNB) with a longitudinal approach, where the influence of NABs in individual patients is assessed by comparing responses during NAB-positive and NAB-negative periods. There are apparent influences on exacerbation rate related to dose of IFNB, titer level, and duration of positivity. With the MxA assay, exacerbation rates after switching to NAB-positive status are estimated to be 28% higher [95% confidence interval (CI): (-15%, 92%)] and -2% higher [95% CI: (-21%, 21%)] on the low- and high-dose IFNB arms, respectively. When compared with all NAB-negative periods, exacerbation rates during NAB-positive periods are estimated to be 29% higher [95% CI: (0%, 67%)] and 18% higher [95% CI: (0%, 40%)] on the low- and high-dose IFNB arms, respectively. When NAB-positive patients again become NAB-negative, no evidence of increased exacerbation rates could then be demonstrated. More detailed exploratory analyses indicate that the effects are most evident in the approximately 20% of patients developing high titers. In these patients, the influence of NABs may be self-limited, as titers often diminish or NABs become undetectable with time.

Anti-interferon antibodies in multiple sclerosis. Molecular basis and their impact on clinical efficacy.

Low levels of naturally-occurring, high-affinity antibodies directed against cytokines can be found in the circulation of individuals who have never been exposed to exogenously-supplied cytokines. These antibodies are thought to play a regulatory role in the intensity and duration of immune response. Interferon (IFN) beta has been shown to attenuate both relapsing-remitting multiple sclerosis (MS) and secondary progressive MS in several well-powered, randomized, controlled clinical trials. IFN therapy can induce the production of anti-IFN neutralizing antibodies (NAb), usually in the second 6 months of treatment, in 3 to 45% of treated patients. This variation in the proportion of NAb-positive patients is probably due to the immunogenicity of different formulations of IFN beta, as well as the assay used, which are not currently standardized. The occurrence of NAb appears to be directly correlated with the dose of therapeutic IFN administered, up to a certain dose threshold. If the dose is increased beyond this threshold, the levels of NAb decrease. The biological significance of anti-IFN NAb is not yet known, nor has it been proven conclusively that they affect the clinical response to IFN beta therapy. The presence of NAb is therefore not an indication that treatment should be changed. Indeed, any treatment decision should be based only on the clinical response to therapy.

A sensitive radioimmunoprecipitation assay for assessing the clinical relevance of antibodies to IFN β

Background: Some multiple sclerosis (MS) patients treated with interferon beta (IFN β) develop antibodies to the drug. Neutralising antibody (NAB) assays for IFN β are expensive and the clinical relevance...

A novel and rapid assay for the detection of neutralizing antibodies against interferon-beta.

There is evidence that neutralizing antibodies (NAB) have a negative influence on the clinical and magnetic resonance imaging effects of interferon-beta (IFNbeta) in multiple sclerosis (MS) patients. The current methods for NAB detection are restricted to specialized laboratories because they require a cell culture and sometimes a viral culture. Results are typically obtained after several weeks. Therefore, the development of a simple and rapid assay for the detection of NAB was sought. Whole blood samples from 28 NAB-positive patients and 110 NAB-negative patients (52 with IFNbeta and 58 without IFNbeta therapy) were incubated with IFNbeta 976 IU/mL for 24 hours. MxA protein levels--a specific marker of class I IFN bioactivity--were measured in paired samples with and without IFNbeta incubation and the difference in MxA levels was calculated. The mean increase of MxA levels after stimulation with IFNbeta in the NAB-positive group was 8 ng/mL (range 0-44 ng/mL) and in the NAB-negative group was 84 ng/mL (range 0-302 ng/mL). Using an increase of 22.5 ng/mL as cut-off) the specificity of the MxA stimulation assay was 81.2% and the sensitivity was 96.4%. The whole blood MxA stimulation assay is virtually as sensitive as the conventional NAB assay but somewhat less specific. However, this is outweighed by the procedural advantage of the assay, which is simpler, quicker and much less expensive.

The significance of neutralizing antibodies in patients with multiple sclerosis treated with interferon beta.

T HE DEVELOPMENT of neutralizing antibodies(NABs)against interferons is well described in patients with viral hepatitis and with some cancers that are treated with interferon. In patients with hepatitis or cancer, thedevelopmentofNABshas been associated with reduction of interferon-relatedadverseeffects (eg, fever, chills, and myalgia) and with the recurrence of disease activity. TreatmentfailureduetoNABsinsuch patientshasbeenmucheasier to identify, by measuring the more sensitive diseaseactivitymarkers(eg,transaminase levels in patients with hepatitis ortumorortumormarkers inpatients with cancer). The development of NABs in patients with multiple sclerosis (MS) treated with interferons is a more recent observation, which has generated substantial controversy. Approximately one third of patients treated with interferon beta-1b developed NABs in the pivotal trial of that agent. The NABs were identified by neutralization of a standard dose of interferon in a virus plaque assay and by the reduction of interferon induction of the MxA protein in cell culture. Disease activity markers, barring the use of magnetic resonance imaging (MRI), are not yet available in MS. Measurement of the clinical effect of NABs has generally depended on measuring attacks or disability in periods subsequent to the development of a positive titer. Interferon-treated patients with MS who develop NABs generally do so in the second half of the first year of treatment. Many clinical trials have not been powered to show an effect of the NAB after this period. Furthermore, pursuing the identification of a deleterious effect of such antibodies has not been a leading corporate interest. It appears that all interferons are capable of inducing the formation of NABs and that interferon NABs are generally cross-reactive. It is difficult to compare the data from the pivotal trials of the 3 major interferons: interferon beta-1b (Betaseron) and 2 types of interferon beta-1a (Avonex and Rebif). The antibody titers were derived from different assays, and the definitions of positive titers were different. From studies of the development of NABs outside clinical trials, it has been noted that interferon beta-1b is more immunogenic than interferon beta-1a and that the subcutaneous route of administration might increase this risk, compared with intramuscular administration. In the study of interferon beta1b, the development of NABs appeared to be associated with loss of efficacy, especially in the observation period between 18 and 24 months. Patients who were positive for NABs experienced an attack rate in this time frame approximating that of placebo-treated patients. This clinical observation was also supported by parallel changes in the MRI studies and by reduction of interferon adverse effects. For example, skin necrosis occurred only in patients without NABs. The NABs that developed in 22% of the longest-treated (104week subset) patients in the trial of interferon beta-1a (Avonex) were not shown to significantly alter attack rates, although an effect was seen on MRI activity and on the induction of neopterin. In the most recent study of interferon beta-1a (Avonex) in which the drug was used after the first attack, NABs developed in fewer than 1% of patients. Why the rates of NAB positivity keep decreasing with each successive observation from this group remains enigmatic. An effect of NABs was not identified in the PRISMS Study of interferon beta-1a (Rebif) at 2 years, but extension of this study for another 2 years revealed definite differences between attack, disability, and MRI outcome measures in favor of the patients who remained NAB negative. According to the findings of studies of adequate duration, there appears to be a convincing effect of NABs on primary outcome measures, by which the efficacy of these drugs has been shown. Is all interferon efficacy lost after NAB development? Earlier studies of interferon treatment in hepatitis and cancer suggested a reduction but not total abrogation of efficacy. There is also some suggestion that disease progression might be less likely in NABpositive patients with MS, but this observation requires corroboration. Observations over the shortterm had suggested that NABs could disappear in a substantial proportion of patients. Of interest, in the pivotal trial with interferon beta1b, 60% of the patients who had a NAB titer were subsequently shown to have a negative NAB titer. In a cohort of patients studied with frequent MRI analyses, approximately 50% of the patients who met the definitions for NABs ultimately From the Multiple Sclerosis Clinic, University of Western Ontario, London. CONTROVERSIES IN NEUROLOGY

Neutralizing antibodies during treatment of multiple sclerosis with interferon beta-1b: experience during the first three years. The IFNB Multiple Sclerosis Study Group and the University of British Columbia MS/MRI Analysis Group.

Evidence of diminution of therapeutic efficacy in 35% of interferon beta-1b (IFNB)-treated multiple sclerosis (MS) patients who developed neutralizing antibodies (NABs) led to extensive study of the effects of NABs on therapeutic benefits, side effects, and magnetic resonance imaging (MRI) data. First, we validated the IFNB NAB assay used in the multicenter trial by having representative stored serum samples reanalyzed by an independent laboratory. When NABs developed (as defined), usually in the first year, the exacerbation rates after 18 months resembled placebo rates, the numbers of enlarging MRI lesions significantly increased compared with those in NAB-negative patients, and there was increased new lesion formation in the MRI (p = 0.067). However, worsening of the mean Expanded Disability Status Scale score in the 8-MIU treatment arm was higher in patients who remained NAB-negative in the third year (p = 0.083). NAB-positive patients were not overrepresented among the noncompleters, or in five patients having at least one episode of skin-site necrosis. After 18 months, flu-like symptoms were about twice as common in NAB-negative as in NAB-positive patients, although the frequency did not exceed 21% in any semester. Decisions to discontinue IFNB therapy should be made individually based on clinical response and a positive titer of NABs in the serum with the use of a reliable assay. ELISA and Western blot techniques measure binding antibodies, not NABs specifically, and are unsuitable for use. Possible, but as yet unproven, means of dealing with NAB positivity should be studied in properly designed trials. IFNB-1b remains an effective therapy for a majority (65%) of MS patients having relapses. The annual exacerbation rates in NAB-negative patients receiving the 8-MIU dosage regimen are about 50% of those seen in untreated patients, a greater reduction than the one-third reduction earlier reported for the entire high-dose arm, and a meaningful treatment benefit.