Neutralizing AntiBodies In Multiple Sclerosis

The development of Neutralizing AntiBodies (NABs), or neutralizing activity in the absence of NABs, is a potential complication of therapy with Interferon-beta (IFN-ß) for patients with Multiple Sclerosis, limiting therapeutic efficacy.

Discontinuation of IFN-ß therapy in patients found to have sustained Titers of NABs > 1:100 over an interval of 3 - 6 months has been recently proposed as a Level A recommendation.

The extent to which NABs are causative, rather than an epiphenomenon, in determining drug failure has been a matter of numerous investigations and is still controversial.

Thus, further studies are warranted for determining the role that NABs may play in reducing the response to the drug.

In particular, the effects of NABs in reducing the efficacy of IFN-ß therapy beyond clinical relapse rate and lesion load on conventional imaging are not as yet fully understood.

Therapy-induced binding and Neutralizing AntiBodies is a major problem in Interferon-beta (IFN-ß) treatment of Multiple Sclerosis.

The objective of this study was to provide guidelines outlining the methods and clinical use of the measurements of binding and Neutralizing AntiBodies.

Systematic search of the Medline database for available publications on binding and Neutralizing AntiBodies was undertaken. Appropriate publications were reviewed by one or more of the task force members.

Grading of evidence and recommendations was based on consensus by all task force members.

Measurements of binding AntiBodies are recommended for IFN-ß AntiBody screening before performing a Neutralizing AntiBody (NAB) assay (Level A recommendation).

Measurement of NABs should be performed in specialized laboratories with a validated cytopathic effect assay or MxA production assay using serial dilution of the test Sera.

The NAB Titer should be calculated using the Kawade formula (Level A recommendation).

Tests for the presence of NABs should be performed in all patients at 12 and 24 months of therapy (Level A recommendation).

In patients who remain NAB-negative during this period measurements of NABs can be discontinued (Level B recommendation).

In patient with NABs, measurements should be repeated, and therapy with IFN-ß should be discontinued in patients with high Titers of NABs sustained at repeated measurements with 3- to 6-month intervals (Level A recommendation).

Persistent high-Titer Neutralizing AntiBodies (NAB) to therapeutic Interferon-beta(IFN-ß)in Multiple Sclerosis patients reduce therapeutic efficacy.

Difficulties in standardization of cell-based bioactivity assays have hindered interlaboratory comparison of NAB Titers and the determination of a clinically relevant definition of seropositivity.

We determined NAB status in Australaian Multiple Sclerosis patients receiving IFN-ß using both the Antiviral Cytopathic Effect (CPE) assay (n = 227) and the more specific ELISA for the type I Interferon-inducible MxA protein (n = 350).

While the log(10) Titers determined in the two assays were highly correlated (p < 0.0001; r = 0.967) with similar distributions, the MxA assay was more sensitive, detecting lower concentrations of NAB than the CPE assay.

The range of Titers determined in the CPE assay was 10 to >7290; and 9 to 53,700 in the MxA assay, with ranked Titer distribution highlighting the arbitrary nature of currently accepted definitions of NAB seropositivity.

Bioactivity of injected IFN-ß was significantly reduced in NAB-positive patients (p = 0.006; NAB MxA Titers = 184 to 5340) compared to NAB-negative patients as assessed ex vivo using real-time RT-PCR analysis of MxA gene induction.

The range of MxA mRNA levels in healthy controls was remarkably consistent with previously published results, regardless of the assay standardization method [Gilli, F., Sala, A., Marnetto, F., Lindberg, R.L., Leppert, D. and Bertolotto, A. (2003) Comparison of IFN-ß bioavailability evaluations by MxA mRNA using two independent quantification methods.

Abstract, ECTRIMS Meeting, Milan, Italy; Pachner, A., Narayan, K., Price, N., Hurd, M. and Dail, D. (2003a) MxA Gene Expression Analysis as an Interferon-beta Bioactivity Measurement in Patients with Multiple Sclerosis and the Identification of Antibody-Mediated Decreased Bioactivity. Mol. Diagn. 7, 17-25].

Assessment of IFN-ß response ex vivo accounts for both circulating factors and the cellular response to IFN-ß, and the data support the development of the MxA gene induction assay for the routine screening of patients receiving IFN-ß.

Neutralizing AntiBodies (NAB) against Interferon-ß (IFN-ß) develop in about a third of treated Multiple Sclerosis patients and are believed to reduce therapeutic efficacy of IFN-ß on clinical and MRI measures.

The expression of the Interferon acute-response protein, MyxoVirus resistance protein A (MxA) is a sensitive measure of the biological activity of therapeutically applied IFN-ß and of its reduced bioavailability due to NAB.

However, MxA may not be operative in the pathogenesis of Multiple Sclerosis or the therapeutic effect of IFN-ß.

Instead, Matrix MetalloProteinases (MMPs) are increased in Brain tissue, CSF and blood circulation of Multiple Sclerosis patients and function as effector molecules in several steps of Multiple Sclerosis pathogenesis.

One of the molecular mechanisms by which IFN-ß exerts its beneficial effect in Multiple Sclerosis is reduction of MMP-9 expression and increase of its endogenous Tissue Inhibitor, TIMP-1.

Quantitative PCR measurements of MMP-2 and MMP-9, TIMP-1 and TIMP-2, and MxA were performed in peripheral MonoNuclear Cells from clinically stable Multiple Sclerosis patients with Relapsing/Remitting disease course after short-term and long-term treatment with IFN-ß.

IFN-ß therapy down-regulated the expression of MMP-9 and abolished that of MMP-2 in long-term, but not short-term treated Multiple Sclerosis, while levels of MxA were increased in both instances.

The presence of NAB reversed these effects, i.e. led to reduced MxA and increased MMP-2/MMP-9 expression levels compared with NAB- patients.

In contrast, expression of TIMPs in peripheral blood MonoNuclear Cells remained unaffected by IFN-ß therapy and the presence of NAB.

While MxA is able to detect the biological action and reduced bioavailability of IFN-ß on the basis of single injections, only MMP-9 shows quantitative correlation with the NAB Titer.

Together with evidence that an imbalance between MMP and TIMP expression is a crucial pathogenetic feature in Multiple Sclerosis, these findings support the concept of a significant role of NAB in reducing the therapeutic efficacy of IFN-ß.

Neutralizing AntiBodies (NABs) can develop in a large proportion of patients with MS who receive treatment with Interferon-beta (IFN-ß).

Data show that Interferon-ß-1b is more Immunogenic than Interferon-ß-1a and that IFN-ß-1a-Rebif((R)) is more immunogenic than Interferon-ß-1a-Avonex((R)).

This article reviews the long-term data from large phase III clinical trials showing that NABs can reduce the clinical efficacy of IFN-ß in patients with MS.

Patients who have a positive result on NAB testing have a higher relapse rate and more disease activity, as measured by Brain MRI, than do patients with a negative result.

The detrimental effects of NABs were not observed until after 18 months of treatment, suggesting that short-term clinical trials cannot adequately assess the efficacy of IFN-ß products in MS.

Clinicians should consider the possible development of NABs when starting patients on treatment and in patients with disease progression while on IFN-ß treatment.

Background
A new liquid formulation of Avonex (Interferon-beta-1a [Interferon-ß-1a]) in a prefilled syringe has been developed to make administration of the drug easier for patients with Multiple Sclerosis (MS).

This formulation does not contain Human Serum Albumin (HSA), often added to Interferon (IFN) products for stabilization.

However, formulation changes may alter the secondary, tertiary, and quaternary structures of IFN-ß products.

These kinds of structural changes could lead to the formation of AntiBodies directed against IFN-ß. Some of these Anti-IFN AntiBodies may neutralize the biologic activity of IFN-ß.

Objective
This study was designed to determine the Immunogenicity and safety of the new prefilled syringe (liquid) HSA-free formulation of Avonex in patients with Relapsing MS.

Methods
This was a multicenter, single-arm, open-label study. Patients with Relapsing MS received liquid, HSA-free Avonex 30 microg by IM injection from a prefilled syringe once weekly for up to 24 months.

Immunogenicity and safety were assessed every 3 months.

Serum levels of Neutralizing AntiBodies (NABs) were measured at baseline and every 3 months using a 2-step enzyme-linked Immunosorbent assay and AntiViral Cytopathic effect assay.

Results
A total of 153 patients (121 women, 32 men; mean [SD] age, 39.6 [9.9] years; age range, 19.0-59.0 years) were enrolled in the study.

Sera were available for analysis from 125 and 119 patients after 18 and 24 months of treatment, respectively.

By 18 months, 1 patient (1%) had > or =2 consecutive Titers of > or =20, a level at which the persistent presence of NABs has been shown in some studies to have clinical consequences.

By 24 months, 1 additional patient (total 2%) had > or =2 consecutive Titers of > or =20.

At 18 months, 5 patients (4%) had > or =1 NAB Titer of > or =5; at 24 months, 6 patients (5%) had > or =1 NAB Titer of > or =5. The safety profile of liquid Avonex was comparable to the lyophilized form containing HSA.

Conclusions
The prefilled syringe (liquid) HSA-free formulation of Avonex was well tolerated and showed a low level of Immunogenicity.

Over 24 months, 2% of patients developed persistent NABs (> or = 2 consecutive Titers of > or =20).

We have analyzed 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 (IFN-B) 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 IFN-B, 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 IFN-B 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 IFN-B 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.

We analyzed the kinetics and clinical impact of Binding AntiBodies (BABs) and Neutralizing AntiBodies (NABs) to three Interferon-beta (IFN-ß) products in patients with Relapsing/Remitting MS (RRMS).

Patients with RRMS received Interferon-ß-1b 8 MIU subcutaneously (SC) every other day, IntraMuscular (IM) Interferon-ß-1a 30 mcg once weekly, or SC IFN-ß-1a 22 mcg three times weekly for up to 4 years.

The changes of BABs and IFN-ß were measured using Enzyme-Linked Immunosorbent Assay (ELISA), and positive BAB samples were then analysed for neutralizing activity using an antiviral cytopathic effect assay.

Patients were considered BAB⁺ if they had a positive sample with an optical density (OD)>mean + 3SD of the OD of the control sample; high BAB Titers were defined as >1:500.

Patients were considered if they had Titers > or =20 LU/mL, with high NAB Titers defined as >1:100.

The impact of BABs and NABs on relapses and Expanded Disability Status Scale (EDSS) score also was evaluated. Thirty patients were enrolled in each treatment group.

Over the course of the study, 83% of patients developed BABs to Interferon-ß-1b, 13% to IM Interferon-ß-1a, and 47 % to SC IFN-ß-1a.

Forty percent of patients developed NABs to IFN-ß-1b, 6.7% to IM IFN-ß-1a, and 26.7% to SC IFN-ß-1a.

Of 22 patients, 10 patients (45.5%) demonstrated high Titers of both NABs and BABs (20% IFN-ß-1b, 3.3% IM IFN-ß-1a, 10% SC Interferon-ß-1a).

The relapse rate significantly increased after the appearance of high NAB titers (p=0.03); however, an even higher significance level (p<0.001) was observed in patients with high Titers of both NABs and BABs.

In 10 patients with high Titers of both NABs and BABs, an increase in mean EDSS score from 2.2 +/- 0.8 at baseline to 3.6 +/- 1.2 at year 2 (p<0.01) was observed.

NAB^- patients showed no significant change in EDSS score at year 2.

These findings demonstrate that high Titers of both BABs and NABs reduce the clinical efficacy of IFN-ß in patients with RRMS, which is important for the long-term efficacy of these drugs.

Background
Interferon-ß is the first-line treatment for Relapsing/Remitting Multiple Sclerosis, but the drug can induce Neutralizing AntiBodies against itself, which might reduce effectiveness.

We aimed to assess the clinical effect of Neutralizing AntiBodies.

Methods
We measured Neutralizing AntiBodies every 12 months for up to 60 months in 541 patients with Multiple Sclerosis, randomly selected from all patients who started treatment with Interferon-ß between 1996 and 1999.

Patients left the study if they changed or discontinued therapy.

AntiBodies were measured blindly, using antiviral neutralization bioassays with high, medium, and low sensitivity, and with different neutralizing capacities as cutoff value for definition of a Neutralizing-AntiBody-positive result.

Findings
Patients developed Neutralizing AntiBodies independent of age, sex, disease duration, and progression index at start of treatment.

Relapse rates were significantly higher during AntiBody-positive periods (0.64-0.70) than they were during AntiBody-negative periods (0.43-0.46; p<0.03).

When comparing the number of relapses in the Neutralizing-AntiBody-positive and Neutralizing-AntiBody-negative periods we found odds ratios in the range 1.51 to 1.58 (p<0.03).

Time to first relapse was significantly increased by 244 days in patients who were AntiBody-negative at 12 months (log rank test 6.83, p=0.009).

During this short-term study, presence of Neutralizing AntiBodies did not affect disease progression measured with the Expanded Disability Status Scale.

Interpretation
Our findings suggest that the presence of Neutralizing AntiBodies against Interferon-ß reduces the clinical effect of the drug.

In patients who are not doing well on Interferon-ß, the presence of such AntiBbodies should prompt consideration about change of treatment.

Background
MxA is an AntiViral protein exclusively induced by Type 1 Interferons (IFN) and some Viruses, and MxA gene expression is one of the most appropriate markers for measuring the biologic activity of exogenous IFN-ß.

Methods
A new quantitative-competitive PCR method was used to quantify MxA mRNA in Peripheral Blood Mononuclear Cells of 99 treatment-naive and 92 IFN-ß-treated patients with MS (22 Avonex, 17 Betaferon, and 53 Rebif-22).

Every 3 months, IFN-ß-induced Neutralizing AntiBodies (NAB) were evaluated in Sera using a cytopathic effect assay.

Three categories of patients were identified: NAB negative (NAB^-), persistent NAB positive (NAB⁺, >or=2 consecutive positive samples), and isolated NAB⁺ (one positive sample).

Results
Treatment-naive patients expressed detectable MxA mRNA levels (mean = 36 +/- 32 fg MxA/pg GlycerAldehyde-3-Phosphate DeHydrogenase (GAPDH); range 1 to 160) and an upper normal threshold was established (mean + 3 SD = 132 fg MxA/pg GAPDH).

IFN-ßtreated patients exhibited more than 11-fold higher levels (mean = 412 +/- 282 fg MxA/pg GAPDH; range 16 to 1,172). However, 17 patients did not exhibit an increase in MxA mRNA level; 15 of these 17 patients showed a concurrent NAB⁺ Titer.

Moreover, 13 were persistent NAB⁺. Isolated NAB⁺ patients did not show a decrease in bioavailability of IFN-ß (n = 9; mean = 567 +/- 366 fg MxA/pg GAPDH; range 83 to 1,120).

In NAB^- patients, bioavailability was comparable among the three different IFN-ß preparations 12 hours after injection.

Conclusion
During IFN-ß therapy, the presence of NAB reduced or abolished bioavailability in a relevant percentage of patients.

These data could be important for the early detection of patients with MS who are not responsive to IFN-ß therapy.

Neutralizing AntiBodies (NAB) to Interferon-beta (IFN-ß) occur in some Multiple Sclerosis (MS) patients, particularly during the first year of treatment.

The presence of NAB may be associated with an attenuation of the therapeutic effect.

The aim of this study was to compare the frequency of NAB occurrence in patients treated with Interferon-ß-1b with that in patients treated with IFN-ß-1b combined with monthly pulses of IntraVenous MethylPrednisolone (MP).

One hundred and sixty-one patients with Relapsing/Remitting MS were randomized in two treatment arms: 8 MIU of Interferon-ß-1b subcutaneously injected every other day either alone or in combination with 1000 mg of monthly intravenous MP.

NAB were evaluated at baseline and at months 3,6,9,12 and 15 by the MxA assay in a specialized laboratory.

Positivity was defined as a Titer of > or = 20 neutralizing units according to two different definitions:
1. I) one or more non-consecutive positive samples
2. II) at least two consecutive positive samples

NAB (definition I) were observed in 26.8% of patients in the Interferon-ß-1b alone arm and in 12.1% of patients in the combination therapy arm (p = 0.05 by the chi-square test), which corresponds to a relative reduction of 54.9%.

Whereas according to definition II, these figures dropped to 22.5 % for the IFN-ß-1b alone arm, and 10.6% for the combination therapy arm (relative reduction 52.9%, p = 0.10, NS).

The presence and titer of Neutralizing AntiBodies (NABs) was evaluated by an AntiViral biological assay in 387 samples of 111 Multiple Sclerosis (MS) patients treated with one of the three commercial preparations of Interferon-beta (IFN-ß).

Fifty NAB positive samples were found in 19 patients: 11 treated with Interferon-ß-1b (Betaferon(R)) and eight with IFN-ß-1a (five with Avonex(R) and three with Rebif(R)).

All the 38 NABs⁺ samples of patients treated with IFN-ß-1b cross-reacted with IFN-ß-1a of both commercial types.

The median level of Neutralizing Units (NUs) of the sera was higher when tested against Interferon-ß-1a than against IFN-ß-1b (p=0.000 vs. Avonexr(R) and p=0.003 vs. Rebif(R)).

In line with these data, the NABs⁺ Sera of patients treated with Interferon-ß-1a cross-reacted with Interferon-ß-1b and the level of NUs were lower when tested against IFN-ß-1b than against IFN-ß-1a (p=0.003).

The different amount of NUs against IFN-ß types 1a and 1b could be due to the presence of aggregates in the Interferon-ß-1b preparation.

The different levels of cross-reactivity of NABs could reduce the bioavailability and therapeutic efficacy of IFN-ß in NABs⁺ patients switching from Interferon-ß-1b to Interferon-ß-1a.