Neutralizing AntiBodies In Multiple Sclerosis

Interferon-beta (IFN-ß) is generally considered an effective treatment for Multiple Sclerosis (MS).

However, some patients do not respond to this therapy, possibly due to the production of Neutrailzing AntiBodies (NAB) which can prevent the biological effect of IFN-ß.

We compared the two types of IFN-ß, the GlycoSylated IFN-ß-1a and the Non-GlycoSylated IFN-ß-1b, as their chemical differences may entail differing Immunogenic capacities.

We studied 22 Relapsing/Remitting MS patients treated with IFN-ß-1a and 31 treated with IFN-ß-1b for 1 year, using the same assay and criteria.

To compare the two types of IFN-ß in their ability to induce Binding and Neutrailzing AntiBodies and examined the correlation of the findings with the clinical data.

Binding AntiBodies to IFN-ß-1a and IFN-ß-1b were determined by Enzyme-Linked Immunosorbent Assay.

A bioassay was used to detect and quantify the NABs to IFN-ß, measuring the capacity of NABs to block the AntiViral resistance induced by IFNs.

Binding AntiBodies were found in 32 % of those treated with IFN-ß-1a and in 52 % of those treated with IFN-ß-1b; NABs were found in 14% and 24 %, respectively.

Both groups showed a significant decrease in relapse rate during the first year of treatment.

These results demonstrate that the IFN-ß-1b molecule is more Immunogenic than the IFN-ß-1a molecule.

This may be due to the Non-GlycoSylated, chemical structure of the former, which can produce aggregates and enhance AntiBody production.

No association was found between the presence of NABs and the clinical status of the patients.

Background
Neutralizing AntiBodies (NABs) against Interferon-beta (IFN-ß) have been described in one third of patients with Multiple Sclerosis treated with INF-ß. We have analyzed the frequency of NABs and their clinical consequences.

Patients And Methods
We have studied 68 patients and NABs were determined by protein A MyxoVirus assay.

Results
Positive NABs were detected in 13% of the patients after 2 years of treatment.

Conclusions
A relationship does not seem to exist between presence of NABs and a poor evolution of the disease in our patients with Multiple Sclerosis treated with Interferon-ß.

Interferon-beta (IFN-ß) is currently the most commonly used treatment of Relapsing/Remitting Multiple Sclerosis (MS).

At the time of this study, two preparations of IFN-ß were available, IFN-ß-1a (Avonex^® and IFN-ß-1b (Betaseron^®), which both can elicit an Immune Response with the development of anti-IFN-ß AntiBodies.

Direct comparisons between these two preparations regarding AntiBody frequencies have, however, been difficult to perform, because two different analysis methods measuring partly different biological effects of IFN-ß have been employed.

In the present study, binding and Neutralizing Anti-IFN-ß-1a and -1b AntiBodies were detected in parallel by an independent, well-acknowledged, Interferon research laboratory using an Immunoassay and a cytopathic virus inhibition assay.

Five per cent of patients treated with IFN-ß-1a intramuscularly (n = 20) had Neutralizing AntiBodies (NABs) compared with 44% of patients treated with IFN-ß-1b subcutaneously (n = 48).

A high degree of cross-reactivity between Neutralizing anti-IFN-ß-1a and -1b AntiBodies was observed. No effect of NABs on clinical outcome could be detected in this limited material.

Binding Anti-IFN-ß AntiBodies were observed in 20% of IFN-ß-1a treated patients compared with 81% of patients treated with IFN-ß-1b.

Only one of 17 patients examined (6%) had detectable Titres of binding Anti-IFN-ß-1b AntiBodies in the CerebroSpinal Fluid (CSF).

These data are the first using identical methodology to show that IFN-ß-1a gives rise to fewer NABs than IFN-ß-1b at recommended treatment schedules.

A proportion of these AntiBodies can reduce the biologic activity of IFN-ß, and they are, therefore, referred to as Neutralizing AntiBodies (NAB).

The remaining AntiBodies do not interfere with the biologic activity of IFN-ß and are referred to as NonNeutralizing AntiBodies (NNAB) in this paper.

ImmunoGlobulin (Ig) subtyping of these AntiBodies was carried out, and Ig subclass patterns in 20 patients with NAB were compared with those of NNAB in 39 patients.

In patients with NAB, IgG2 and IgG4 were found to occur more frequently than in patients with NNAB (30% vs. 3%, p = 0.05, and 55% vs. 18%, p = 0.003, respectively).

The NAB titer correlated strongly with the IgG4 titer (r = 0.53, p = 0.02).

Median total IgG, IgG1, and IgG4 titers were significantly higher in NAB than in NNAB patients (respectively, 8000 vs. 3200, p = 0.01; 1600 vs. 400, p = 0.0004; 200 vs. 0, p = 0.004).

It is concluded that the development of NAB is related to both the quantity and the quality of the AntiBodies against IFN-ß-1b.

The fate of the Neutralizing AntiBody (NAB) in MS patients treated with Interferons remains unclear.

We conducted a follow-up survey of NAB titers in 59 long-term treated patients from the London and Vancouver cohorts of the pivotal trial of Interferon-ß-1b.

NAB were measured with the MyxoVirus protein A assay and an ELISA, at a mean follow-up that exceeded 8 years. NABs disappeared in the majority of patients.

Background
Neutralizing AntiBodies (NAB) to Interferon-beta (IFN-ß) occur in about one-third of MS patients treated with IFN-ß-1b and there is an association with a loss of clinical and MRI efficacy.

However, there are no data regarding the bioavailability of IFN-ß-1b in patients with and without NAB.

Methods
The authors measured MxA in whole blood by ELISA, and Serum-Binding AntiBodies (SBA) by Western blot and ELISA in 134 samples of MS patients on IFN-ß-1b and 54 control subjects, and correlated the MxA levels and SBA titers with the NAB titer.

Results
In the IFN-ß-1b group 84 samples were NAB negative, 21 were NAB positive (i.e., titer of > or =20), and 29 had detectable NAB (i.e., Titer between 10 and 20).

The median MxA concentration in NAB-negative patients was 4.09 ng/10(5) Peripheral Blood Leukocytes (PBL), 2.37 ng/10(5) PBL in samples with detectable NAB, 0.36 ng/10(5) PBL in NAB-positive samples, and 0.27 ng/10(5) PBL in control subjects.

There was no significant difference between NAB-positive samples and control subjects, otherwise the groups differed significantly from each other.

SBA occurred in 49% of NAB-negative samples, in 79% of samples with detectable NAB, and in all NAB-positive samples. With regard to the SBA Titer, all groups differed significantly from each other. In none of the control samples were SBA detected.

Conclusion
The conversion of SBA into NAB depends to some degree on the SBA titer, but other mechanisms may be involved.

Once NAB have developed, the bioavailability of IFN-ß-1b as measured by MxA is completely inhibited.

The long-term treatment with Interferon-ß-1b of a 7-year-old male with Relapsing/Remitting Multiple Sclerosis is documented.

Thirty-two months after initiating treatment, he demonstrates dramatic clinical improvement, without relapse, despite high titers of Neutralizing AntiBodies to Interferon-ß-1b. It appears reasonable to attribute a role in his improvement to Interferon-ß-1b.

Objective
To determine whether Neutralizing AntiBodies (NABs) to Interferon-beta-1a (IFN-ß-1a) (Avonex) and IFN-ß-1b (Betaseron) cross-react.

Background
A total of 38% of MS patients treated with IFN-ß-1b and 22% of those treated with IFN-ß-1a were reported to develop NABs, which could reduce the clinical efficacy of the drug.

Methods
Blood from 10 MS patients was collected before and at 3 and 6 months after initiating treatment with IFN-ß-1a .

ELISA was performed to detect binding AntiBodies to IFN-ß-1a. Sera from patients who tested positive for binding AntiBodies to IFN-ß-1a were then screened for NABs to IFN-ß-1a in a biologic assay based on neutralization of AntiViral activity.

These Serum samples were subsequently tested for cross-reactivity with IFN-ß-1b both in the ELISA and the biologic assay.

In the second part of the study, Sera from patients who participated in the Phase III IFN-ß-1b trial at the Univ of Maryland were examined for cross-reactivity with IFN-ß-1a in the ELISA and the biologic assay.

Results
Of the 10 patients treated with IFN-ß-1a, three developed binding as well as NABs to IFN-ß-1a 6 months after treatment.

And these AntiBodies cross-reacted with IFN-ß-1b both in the binding and the biologic assay.

Similarly, Sera from six patients with NABs to IFN-ß-1b showed cross-reactivity with IFN-ß-1a in the binding assay.

Three of these six Serum samples tested for Neutralizing activity against IFN-ß-1a demonstrated the presence of NABs to IFN-ß-1a.

Conclusions
NABs to IFN-ß-1a (Avonex) and IFN-ß-1b (Betaseron) cross-react, both in the binding and the biologic assays.

This suggests that switching to alternate IFN-ß preparation in patients who develop NABs may not be clinically beneficial.

Studies examining cross-reactivity between NABs to IFN-ß-1a and IFN-ß-1b in a large number of patients are indicated.

Evidence of diminution of therapeutic efficacy in 35% of Interferon-beta-1b (IFN-ß-1b -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 IFN-ß-1b 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 (EDSS 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 IFN-ß-1b; 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. IFN-ß-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.

Objective
To evaluate the incidence and the prevalence of Neutralizing AntiBodies (NABs) to three Interferon-ß (IFN-ß) products in patients with Multiple Sclerosis (MS).

Methods
Sera were tested from 125 patients with Relapsing/Remitting MS. Patients were treated with IFN-ß-1b (Betaferon, n = 29) 8 MIU subcutaneously every other day, IFN-ß-1a (Avonex, n = 44) 30 microg intramuscularly once weekly, or IFN-ß-1a (Rebif, n = 36) 22 microg subcutaneously three times weekly for 6 to 18 months.

An additional 16 patients were treated with Rebif 22 microg intramuscularly once or twice weekly. NABs were assessed using the cytopathic effect assay before treatment and every three months during treatment.

Patients with two or more consecutive positive samples were considered to be persistent NAB positive (NAB⁺).

Results
At baseline, no patients were NAB⁺. NABs developed during the first three months of treatment and continued to develop until month 18.

Over 18 months of treatment, the risk of being persistent NAB⁺ was 31% for Betaferon, 15% for Rebif, and 2% for Avonex (Betaferon versus Avonex, p = 0.001; Betaferon versus Rebif, p = 0.19; Rebif versus Avonex, p = 0.04).

In all patients with one or more NAB⁺ samples, the risk of becoming NAB⁺ was 38% for Betaferon, 18% for Rebif, and 7% for Avonex (Betaferon versus Avonex, p = 0.0007; Betaferon versus Rebif, p = 0.10; Rebif versus Avonex, p = 0.07).

At month 18, the prevalence of persistent NAB⁺ patients was 31.6% for Betaferon, 18.7% for Rebif, and 4% for Avonex. Numbers of NAB⁺ patients observed were similar with intramuscular Rebif and with subcutaneous Rebif.

Conclusion
The three IFN-ß preparations have different degrees of immunogenicity, with Betaferon producing the highest incidence of NABs and Avonex the lowest.

These differences should be considered by Neurologists when selecting treatment for their patients with MS because NABs can reduce both BioAvailability and clinical efficacy of IFN-ß.

Background
MxA gene expression is one of the most appropriate markers of biological activity of exogenous Interferon-beta (IFN-ß).

Methods
We quantified MxA mRNA for five consecutive days in 62 patients treated with IFN-ß (16, Avonex; 10, Betaferon; 24, Rebif 22; 12, Rebif 44), by quantitative-competitive polymerase chain reaction.

Every three months, IFN-ß induced Neutralizing AntiBodies (NABs) were evaluated in Sera using a cytopathic effect assay.

Results
Two categories of patients were identified:

1. One group (49/62) had a sharp post-injection increase in MxA expression
   • Defined as "IFN-ß biological responder"
2. The other group (13/62) had no MxA induction after IFN-ß administrations
   • Defined as "IFN-ß biological non-responder"

In 11/13 biological non-responders, the persistent presence of NABs correlated with abolished biological activity, independently of treatment regimen. The two remaining IFN-ß biological non-responders were NAb^-.

Among the 49 IFN-ß biological responders, biological activity was comparable between the four preparations on day 2 and 3 (+12 and +36 hours post-injection), but it was greater in Betaferon and both Rebif preparations on day 1, 4, and 5.

In biological responders treated three times a week, only 82% (59/72) of injections were considered effective, compared with 100% (13/13) of Avonex injections.

Conclusion
Our results suggest that an optimal IFN-ß regimen is not yet available: Avonex, given once a week, shows lower cumulative biological activity.

On the other hand, both Betaferon and Rebif, given three times a week, show 18% biologically ineffective injections and higher risk of developing NABs, which abolish biological activity.

Objective
To analyze the impact of Neutralizing AntiBodies (NABs) on the clinical efficacy of IFN-ß.

Methods
This was an open-label study involving 78 patients with Multiple Sclerosis treated with Betaferon 8 million IU (MIU) subcutaneously (SC) every other day (n = 20), Rebif 22 micro g SC 3 times weekly (n = 25), or Avonex 30 micro g IM once weekly (n = 33).

Every 3 months, blood samples were collected and sera were analyzed for NABs using an AntiViral cytopathic effect assay.

Patients were categorized according to their NAB status: NAB negative (NAB^-); isolated NAB positive (NAB⁺), patients with > or =1 positive sample (titer > or = 20); and persistent NAB⁺, patients with > or =2 consecutive positive samples (Titer > or = 20).

Patients who were NAB^- and persistent NAB⁺ were compared for relapse rate, time between first and second relapse, percentage of relapse-free patients, and percentage of patients who had a sustained progression of > or =1 point on the Expanded Disability Status Scale (EDSS).

Results
The incidence of persistent NAB⁺ patients was:
• 35% for Betaferon
• 20% for Rebif
• 3% for Avonex

During IFN-ß treatment, both NAB⁺ and NAB^- patients showed a reduction in relapse rate; this reduction (25%) was not significant in NAB⁺ patients but was significant (67%; p < 0.0001) in NAB- patients.

In addition, the mean relapse rate was higher (p = 0.039), mean time between first and second relapse was shorter (13 vs 21 months; p = 0.0064), and there was a trend suggesting that NABs affected the probability of remaining relapse free (p = 0.08).

A higher percentage of NAB⁺ patients versus NAB^- patients had worsening of EDSS scores during follow-up (p = 0.013).

Conclusion
Persistent NABs significantly reduce the clinical efficacy of IFN-ß.