Expert Commentary

Brian R. Apatoff, MD, PhD, Director, Multiple Sclerosis Clinical Care & Research Center, Clinical Attending and Associate Professor, Department of Neurology and Neuroscience, New York Presbyterian Hospital, Weill Medical College of Cornell University, New York, New York

The advent of disease-modifying therapies for treatment of multiple sclerosis (MS) has been a major advance for patients and physicians. Despite the effectiveness of these agents in slowing disease progression and preventing relapse, a number of clinical challenges remain. Patients experience breakthrough relapse of disease despite treatment, compliance with self-injectable medications can be problematic, and the immunogenicity of the medications themselves continues to be a concern. These issues, and many others, were addressed by clinical presentations at the 19th Congress of the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS) held in September 2003 in Milan, Italy.

Two large clinical trials revealed additional data on comparisons between available disease-modifying therapies. The Quality Assessment in Multiple Sclerosis Therapy (QUASIMS) Study,¹ the largest head-to-head study to date, retrospectively showed similar efficacy between intramuscular interferon beta-1a (Avonex), subcutaneous interferon beta-1b (Betaferon), and subcutaneous interferon beta-1a (Rebif 22 mcg). QUASIMS demonstrated that interferon beta efficacy was not related to dose or frequency of administration.

A 5-year extension of the Controlled High-Risk Subjects Avonex Multiple Sclerosis Prevention Study (CHAMPS), the CHAMP In Ongoing Neurological Surveillance (CHAMPIONS) study provided further evidence for the continued benefit of early treatment with Avonex after a patient's first clinical demyelinating event.² The CHAMPS study reported that initiation with Avonex once weekly during a first clinical demyelinating event significantly slowed the rate of development of clinically-definite MS and the development of new magnetic resonance imaging (MRI) abnormalities over two years.³ CHAMPIONS will be extended an additional five years, providing even longer follow-up data on the benefits of early versus delayed treatment of MS.

A number of studies focused on combination therapy as a strategy for patients with relapsing-remitting MS who experience breakthrough disease while on disease-modifying therapy. Combination therapies that were presented included Avonex with methotrexate and/or intravenous methylprednisolone—the Avonex Combination Trial (ACT);⁴ Avonex plus mycophenolate mofetil (CellCept);⁵ Avonex plus natalizumab (Antegren)—the SENTINEL study;⁶ and Avonex plus azathioprine.⁷

According to the results of several studies, the development of interferon beta-induced neutralizing antibodies compromises treatment efficacy as measured by relapses. The results of one study demonstrated that high titers of both neutralizing and binding antibodies correlate with disease progression.⁸ Another study found that the magnitude of neutralizing antibody titers predicts time to reversion to neutralizing antibody-negative status.⁹ Furthermore, an additional study demonstrated that neutralizing antibodies antagonize interferon beta-induced suppression of matrix metalloproteinase expression in patients with MS,¹⁰ confirming other reports that neutralizing antibodies to interferon are biologically relevant. The results of another study suggest that the new, liquid formulation of Avonex conveniently available in a pre-filled syringe for patients, offers the same low level of immunogenicity as the lyophilized Avonex.¹¹ Even though it has been well established that Avonex induces the lowest incidence of neutralizing antibodies compared to Betaseron and Rebif, making Avonex more convenient to administer is welcome news for both MS patients and physicians alike.

I hope you find the information contained within this Multiple Sclerosis Forum Report of benefit in the treatment of your patients with MS.

Treatment Comparisons

Quality Assessment in Multiple Sclerosis Therapy - QUASIMS—Comparative study to assess the efficacy and tolerability of interferon beta for relapsing-remitting multiple sclerosis (RRMS).

Limmroth V, Malessa R, Kalski G, for the QUASIMS Study Group.

Immunomodulatory therapy is the standard treatment for MS in the United States and Europe. Four different interferon beta products/dosing regimens are currently available: Avonex 30 mcg given intramuscularly once weekly, Betaferon 250 mcg given subcutaneously every other day and Rebif 22 mcg or 44 mcg given subcutaneously three times weekly. Open-label comparative studies of these products/dosing regimens have been performed with few contradictory results (Table 1). No randomized, double-blind prospective studies comparing all these regimens have been conducted, and will likely not be conducted in the future, due to the difficulties inherent in designing such studies.

[Ed. Betaseron is interferon beta-1b marketed in the United States. Betaferon is interferon beta-1b marketed outside the United States.]

To gain a clearer understanding of the comparative efficacy of these therapies, a large, retrospective, open-label, four-arm, international, multicenter study was designed. According to first author Volker Limmroth, MD, PhD, of Neurologische Universitдtsklinik, Essen Germany, QUASIMS is the largest head-to-head study that has been undertaken in MS.¹ QUASIMS is based on data from 510 centers in Germany, Switzerland and Austria evaluating a total of 4,754 patients with relapsing-remitting MS over a period of two years. Patients were eligible for enrollment if they had received at least two years of uninterrupted therapy with Avonex 30 mcg once weekly, Betaferon 250 mcg every other day, or Rebif 22 mcg or 44 mcg three times weekly with continuous documentation of relapses and disability progression. Study endpoints included disability progression, percentage of relapse-free patients, annualized relapse rate, and reasons for therapy change.

Results of QUASIMS confirm the findings of the pivotal phase III studies of these agents. After two years of treatment, comparable efficacy was demonstrated between Avonex, Betaferon, and Rebif 22 mcg in relation to change in Expanded Disability Status Scale (EDSS), percentage of relapse-free patients, and relapse rate. Rebif 44 mcg was associated with a significantly lower percentage of progression-free patients compared with Avonex and Rebif 22 mcg and significantly higher relapse rates over two years. A significantly higher percentage of patients treated with Avonex were progression-free (defined as <1 point change in EDSS within two years of treatment) compared with both Betaferon and Rebif 44 mcg (P<0.008) (Figure 1). Rebif 22 mcg was significantly superior to Rebif 44 mcg as reflected by progression-free status (P<0.008). The use of Rebif 44 mcg was used significantly less often as initial therapy (P<0.008), but significantly more often as follow-up therapy.

Over two years of treatment, a significantly higher percentage of patients treated with Avonex (47.0%), Betaferon (44.7%), and Rebif 22 mcg (42.4%) were relapse-free compared with Rebif 44 mcg (32.9%)(P<0.008) (Figure 2).

There was no significant difference in annual relapse rates between Avonex (0.51), Betaferon (0.54), and Rebif 22 mcg (0.53) over the 2-year treatment period. However, all three regimens showed significantly lower annual relapse rates compared with Rebif 44 mcg (0.70) (P<0.008).

There were no significant differences between the use of Avonex, Betaferon, and Rebif 22 mcg in relation to their use as initial or follow-up therapy.

Summarizing the presentation of the results, Dr. Limmroth advised, "These data suggest that there is no correlation between dose and/or frequency of interferon beta treatment and efficacy."

Sustained Long-term Efficacy of Interferon Beta-1a

Initial results of the CHAMPIONS study, an open-label 5-year extension of the CHAMPS study.

Kinkel RP and the CHAMPIONS Study Group.

The CHAMPS study showed that initiation of treatment with Avonex after a first clinical demyelinating event in the presence of at least two T2 lesions delayed the development of clinically-definite MS as well as new MRI abnormalities over a 2-year period.³ Based on the findings of CHAMPS, Avonex was approved by the Food and Drug Administration (FDA) for use in patients with a first demyelinating event and MRI lesions consistent with MS.²⁴

Following the completion of CHAMPS, the investigators initiated an open-label extension study (CHAMPIONS) to determine if the effects of early treatment with Avonex were sustained up to five additional years. The preliminary results of CHAMPIONS, presented at a late-breaking news session at ECTRIMS 2003, demonstrated that the benefits of early initiation of Avonex persisted in slowing the progression to clinically-definite MS over five years compared to initiation of therapy at ≥2 years following the first clinical demyelinating event.² R. Peter Kinkel, MD, reported that enrollment of CHAMPIONS was closed in August 2003 and additional results will be presented at future medical meetings.

All patients who had participated in CHAMPS were offered open-label treatment with intramuscular Avonex 30 mcg once weekly, although this was a not a prerequisite for participation in CHAMPIONS, Dr. Kinkel explained, some patients did not opt for any therapy. Outcome measures included the rate of development of clinically-definite MS as determined by a blinded, independent outcomes committee, disability and change in MRI parameters at five years. Alternative therapy due to side effects was offered at the discretion of individual investigators.

A total of 203 of 383 subjects in CHAMPS enrolled in CHAMPIONS and 98% (n = 198) of patients completed at least five years of the study. Of the 203 patients, 100 had originally received Avonex and 103 had received placebo. The patient characteristics of the treatment groups were well matched, with the exception of a greater T2 lesion volume and greater gadolinium-enhancing lesions in the CHAMPS Avonex-treated patients.

Dr. Kinkel advised listeners that analysis of the data suggests that patients who opted to continue in the CHAMPIONS extension had a slightly lower rate of clinically-definite MS than those from the original CHAMPS study who chose not to enter the five-year extension phase.

At baseline, 76% of CHAMPIONS patients were being treated with Avonex, 17% were receiving no therapy, and 7% were being treated with alternative disease-modifying agents. At the five-year visit, 80% of patients were being treated with Avonex, 14% were receiving no therapy, and 6% were being treated with alternative disease-modifying agents.

In the CHAMPS study, 21% of Avonex-treated patients and 38.6% of placebo-treated patients developed clinically-definite MS at 2 years, representing a 44% reduction of risk.³ In CHAMPIONS, after five years of follow-up, 21% of Avonex-treated patients (immediate treatment) and 35% of placebo-treated patients (delayed treatment) developed clinically-definite MS (rate ratio, 0.65; 95% Confidence Interval (CI), 0.43-0.97; P = 0.03). This represents a statistically significant 35% reduction in the risk of developing clinically-definite MS with immediate initiation of Avonex treatment at the onset of the first clinical demyelinating event.

Over the 5-year extension period, a 47% reduction in total relapses was observed in the Avonex-treated patients (immediate treatment) compared with placebo-treated patients (delayed treatment). Forty-three patients in the Avonex treatment group had at least one relapse over the 5-year extension period compared with 61 in the placebo treatment group, and 18 (19%) patients and 30 (32%) patients were in active relapse at the end of 5 years, respectively. A 42% reduction in the number of new T2 or enlarging lesions was observed in the Avonex-treated patients compared with placebo-treated patients. Dr. Kinkel said the five-year data on change in EDSS were not yet available.

CHAMPIONS showed that immediate treatment with Avonex at the onset of the first clinical demyelinating event had continued long-term benefit in preventing the development of clinically-definite MS compared with initiation of therapy at a later time point. CHAMPIONS has been extended for another five years to yield 10-year follow-up data.

Richard A. Rudick, MD, of the Mellen Center, Cleveland Clinic Foundation, Cleveland, Ohio, said that the advantage of early initiation of Avonex therapy versus placebo in the CHAMPS study was clearly evident in the long-term CHAMPIONS trial as shown by the significant benefits that persisted at five years—less likelihood of a relapse, of being diagnosed with clinically-definite MS as defined by having a second relapse, and of having new MRI lesions at Year 5 compared with baseline in CHAMPS.

"The clinical differences were statistically significant and the magnitude of difference looks clinically meaningful to me. There were differences in the original Avonex versus placebo groups of about 40% to 50% in favor of earlier treatment with Avonex," Dr. Rudick commented.

Dr. Rudick also observed that the outcome at five years in both treatment groups included in the CHAMPIONS study was fairly benign compared with expected outcomes in relapsing-remitting MS, suggesting long-term benefit of Avonex therapy.

"However, this is not definitive, and would become more clear with additional analyses and longer follow-up. The results are indicative of beneficial effects of starting early therapy with Avonex versus delaying therapy for a couple of years," stated Dr. Rudick.

Dr. Rudick praised CHAMPIONS for having more than 90% of patients who entered the study evaluable at five years. "This is an excellent rate of ascertainment and provides confidence that the results are correct. CHAMPIONS should continue as long as it is possible to maintain such a high ascertainment rate. Good data has been published on 15-year outcome in untreated patients following a clinically-isolated syndrome. Ideally, the CHAMPIONS patients should be followed this long, so we could compare outcome with that untreated group. This might tell us more about the long-term benefits of Avonex therapy," concluded Dr. Rudick.

Combination Therapy in Treating Multiple Sclerosis

Rationale and design of the Avonex Combination Trial.

Cohen J, Antel J, Calabresi P, et al for the ACT investigators.

Despite the proven efficacy of disease-modifying therapies in delaying the progression of relapsing-remitting MS, breakthrough relapses occur in a proportion of all patients. Optimal treatment of patients with breakthrough disease has yet to be clearly identified. Combination therapy with pharmacologic agents that have been shown to be effective and tolerable in treating MS represent a potentially promising approach.

ACT is designed to compare use of Avonex in combination with methotrexate with or without intravenous methylprednisolone. The rationale and design of ACT were presented by Jeffrey Cohen, MD of the Mellen Center, Cleveland Clinic Foundation, Cleveland, Ohio.⁴

Enrollment in ACT began in May 2003. The study investigators plan to enroll 900 patients with relapsing-remitting MS, EDSS between 0.0-5.0, and experiencing breakthrough disease (defined as having at least one relapse or gadolinium-enhancing MRI lesion in the previous year) while on Avonex monotherapy. Patients will be continued on intramuscular Avonex 30 mcg once weekly and will be randomized to add-on therapy in equal allocation to four cells of a 2x2 factorial design. Treatment group 1 is weekly oral placebo and no intravenous methylprednisolone; Treatment group 2 is weekly oral methotrexate 20 mg and no intravenous methylprednisolone; Treatment group 3 is oral placebo and intravenous methylprednisolone 1 gm/day each given for 3 days every two months; and Treatment group 4 is methotrexate and intravenous methylprednisolone. Patients are to be followed for a period of 24 months.

Dr. Cohen stated that the 2x2 factorial design allows for testing of two interventions using a total sample size comparable to that required for a 2-arm comparison. "In effect, you get two studies in one," noted Dr. Cohen. The study is powered to show that the methotrexate and intravenous methylprednisolone interventions have independent, additive effects.

Clinical efficacy parameters include relapse rate (primary methotrexate outcome), MS Functional Composite, and EDSS. MRI measures include brain parenchymal fraction (a measure of brain atrophy, the intravenous methylprednisolone primary outcome), gadolinium-enhancing lesions, T2-hyperintense lesions, and T1-hypointense lesions (black holes).

ACT will determine the safety and efficacy of adding methotrexate, intravenous methylprednisolone or a combination of both to ongoing Avonex therapy in relapsing-remitting MS patients with breakthrough disease on Avonex monotherapy. According to Dr. Cohen, noteworthy aspects of ACT are:

• Whole brain atrophy (brain parenchymal fraction) is included as a primary outcome measure.
• The MS Functional Composite (a scoring system that was designed to improve upon the EDSS in quantifying functional disability) is included prospectively as a secondary outcome measure.
• Investigators are drawn from both specialized MS clinics and community neurology practices.
• Although the study is sponsored by a pharmaceutical company, the governance and management of the study are the sole responsibility of the investigators.

"This trial will create an infrastructure and a new model for future academic-industry collaborative studies of therapies for treating multiple sclerosis," stated Dr. Cohen.

Combination of interferon beta-1a (Avonex) and mycophenolate mofetil (CellCept) phase II trial in relapsing-remitting multiple sclerosis.

Vermersch P, Waucquier N, Bourteel H, et al on Behalf of the G-SEP.

Results of a single-center, open-label phase II study presented by Patrick Vermersch, MD, Clinique Neurologique, CHU de Lille, France, demonstrated that the combination of Avonex and CellCept is safe and tolerable in patients with relapsing-remitting MS.⁵ The combination of Avonex and CellCept appears to have clinical promise in treating relapsing-remitting MS due to a low incidence of relapse, no significant changes on neurological exam, and the absence of new gadolinium-enhancing lesions observed over six months of treatment with the combination.

Dr. Vermersch advised that despite the availability of disease-modifying therapies for MS, treatment failures commonly occur, some of which may be due to poor patient adherence. There is an ongoing need to further understand the effectiveness that concomitant therapies will have in treating breakthrough disease in these patients and combination therapy is an approach worthy of investigation. The rationale for combining Avonex with CellCept rests on the demonstrated clinical efficacy of Avonex and unique pharmacologic properties of CellCept. CellCept represents a new generation of immunosuppressive drugs designed to prevent allograft rejection in organ transplantation whose use now includes treatment in autoimmune diseases, including rheumatoid arthritis and Crohn's disease.

CellCept suppresses glycosylation and expression of specific adhesion molecules important in MS, thereby decreasing the recruitment of lymphocytes and monocytes. CellCept has the potential to be neuroprotective, Dr. Vermersch indicated.

Twenty-eight of 30 enrolled patients completed the trial. At baseline, the mean age of patients was 35.3 years (range, 18 to 55 years), with a mean duration of disease of 8.3 years and mean duration of Avonex therapy of 2.8 years. The pre-study annual relapse rate was 2 and the mean EDSS score was 2.9 (range, 0.0 to 5.5). Inclusion criteria included patients with relapsing-remitting MS, EDSS score at baseline <6.0 and treatment with intramuscular Avonex 30 mcg once weekly for at least six months. Study participants had to have at least two relapses over the past two years and at least one relapse over the past six months. Eight patients had from one to eight gadolinium-enhancing lesions at baseline.

Oral CellCept was administered in increasing doses in combination with Avonex at 500 mg/day for the first 3 weeks, followed by 1 gm twice a day from Month 1 to Month 6.

Eight relapses occurred over the 6-month study period—five in the first 3 months and three in the last 3 months. At the end of the study, the annualized relapse rate went from 1.98 to 0.7 as a result of treatment with the combination of Avonex and CellCept. EDSS scores remained stable in 14 patients, went up slightly in 3 patients, and went down in 12 patients. There was no evidence of new gadolinium-enhancing lesions at study end.

Two patients discontinued the combination therapy, one due to gastrointestinal effects and personal reasons and the other for diarrhea probably related to the combination. Very few patients reported headaches, insomnia, nausea, dizziness, or benign infectious diseases and were considered non-related to therapy by the investigators. Hematologic effects and liver enzyme elevations were transient and not judged as clinically meaningful.

Based on these encouraging results, Dr. Vermersch indicated a phase III study is warranted. The study could compare Avonex against the combination of Avonex and CellCept in treating relapsing-remitting MS.

Baseline patient characteristics of the SENTINEL study: a study designed to determine the efficacy and safety of natalizumab (Antegren) in combination with interferon beta-1a (Avonex) for the treatment of relapsing-remitting multiple sclerosis (RRMS).

Rudick R, Calabresi P, Confavreux C, Galetta S, Hartung H-P, Radue EW, Stuart W, Willmer-Hulme AJ, Panzara M, Sandrock A.

Although current disease-modifying drugs represent a major advance in the treatment of MS, many patients have breakthrough disease despite treatment and the search continues for improved therapies.

The SENTINEL study will evaluate the use of Avonex in combination with Antegren, the first alpha-4 integrin antagonist in the new class of selective adhesion molecule (SAM) inhibitors.⁶ Alpha-4 integrin is expressed in a variety of immune cells that have been implicated in a number of cellular processes involved in the pathogenesis of MS and represents a potential target for therapeutic intervention.

Antegren, a humanized antibody to alpha-4 integrin, inhibits the migration of leukocytes from the blood to the central nervous system and has shown promise in the treatment of MS. In a recent study, Antegren reduced the number of new gadolinium-enhancing lesions and clinical relapses in patients with relapsing-remitting and relapsing secondary-progressive MS.²⁵ Antegren has also been shown to be safe and well tolerated when used in combination with Avonex in treating patients with relapsing-remitting MS.²⁶

SENTINEL is an ongoing, multicenter, randomized, double-blind, placebo-controlled, parallel-group, phase III study designed to determine the efficacy and safety of Antegren in combination with Avonex in patients with relapsing-remitting MS. Inclusion criteria stipulated that patients had to have had at least one relapse consistent with MS while on treatment with Avonex within 12 months of randomization. Patients had to have received Avonex for two or more months prior to relapse.

The primary endpoints of SENTINEL are reduction in clinical relapse rate and slowing of disability progression. Additional endpoints are increase in the proportion of relapse-free patients, slowing of disability progression as measured by the reduction in the number and volume of new or newly enlarging T2-hyperintense lesions on brain MRI, reduction in the number and volume of T1-hypointense lesions ("black holes"), and reduction in number and volume of gadolinium-enhancing lesions on brain MRI.

A total of 1,196 patients with relapsing-remitting MS (age range, 30 to 49 years) have been randomized to treatment with Antegren 300 mg by intravenous infusion every 4 weeks or placebo intravenous infusion every 4 weeks in combination to weekly intramuscular Avonex 30 mcg for a total of 116 weeks. At baseline, 76% of patients had an EDSS score between 1.5 and 3.5 (median 2; range, 1 to 6). This level of disability is similar to that of other populations in previous MS treatment trials (MSCRG and PRISMS), indicated Dr. Richard A. Rudick, who presented the poster.

Dr. Rudick stated, "The patient population is similar to EDSS scores in AFFIRM,^[27] an ongoing study of natalizumab monotherapy in untreated patients with relapsing-remitting multiple sclerosis experiencing their first neurological episode of multiple sclerosis or with at least one relapse in the previous 12 months. Patients in SENTINEL had a longer disease duration and were older than those in AFFIRM."

Compared with the untreated patients in AFFIRM, SENTINEL patients had a longer median time from onset of first MS symptoms to breakthrough relapse (seven years compared with five years in AFFIRM), and a longer median time from diagnosis to relapse (four years for patients with breakthrough relapses and two years for untreated patients in AFFIRM). Over the 3 years prior to entry in SENTINEL, 27% of patients had two relapses, 27% had three relapses, and 34% had four or more relapses.

"The population identified by the SENTINEL study should prove valuable in defining the characteristics of patients with relapsing-remitting multiple sclerosis experiencing breakthrough relapses on interferon beta-1a treatment," advised Dr. Rudick.

"The aim of both SENTINEL and AFFIRM is to establish a treatment paradigm for natalizumab. These studies should tell us about the effectiveness of this new agent as monotherapy in patients with their first neurological episode or active untreated multiple sclerosis and its role as add-on therapy in patients who relapse on Avonex. At the end of these trials, we hope we will have a new agent with complementary indications," stated co-investigator Christian Confavreux, MD, Head of Neurology Service A, Hфpital of Neurologique, Lyon, France.

A safety study of combination treatment with interferon beta-1a (Avonex) and azathioprine in breakthrough multiple sclerosis.

Greenstein J, Rae-Grant AD, Eckert N, Multiple Sclerosis Institute, Lehigh Valley Hospital, Philadelphia, Pennsylvania.

Combination therapy is an attractive approach for treatment of relapsing MS patients with breakthrough disease. This study evaluated the safety and efficacy of combining Avonex (30 mcg weekly) with azathioprine (150 mg daily) for a 12-month period in patients with relapsing-remitting MS who had experienced at least one relapse while on Avonex therapy over the preceding 12 months.⁷ According to Jeffrey Greenstein, MD, of the Multiple Sclerosis Institute, Lehigh Valley Hospital, Philadelphia, Pennsylvania, the combination of Avonex and azathioprine was safe and generally well tolerated.

The study included 18 patients with a mean age of 46.2 years—nine with relapsing-remitting MS and nine with relapsing MS with progression. Mean age at disease onset was 43.1 years (range, 17 to 53 years), mean duration of MS was 10.2 years (range, 4 to 29 years), and mean EDSS at study entry was 4.63 (range, 1.0 to 6.5). No prior immunosuppressive therapy or corticosteroid use was allowed within the previous three months of study entry. At baseline, no patient tested positive for neutralizing antibodies.

Mean EDSS at study exit was 4.07 (baseline 4.63). The 25-foot walk test improved from a mean of 12.94 seconds at baseline to 7.34 seconds at study exit. Similarly, the dominant 9-hole peg test improved from a baseline of 29.87 seconds to 26.26 seconds at study exit. The Paced Auditory Serial Addition Test (PASAT) results were 37.17 at baseline and 36.57 at study exit.

Dr. Greenstein indicated intolerability to azathioprine developed in four patients. One patient developed a white blood cell count <3000/mm³ and three patients had serum alanine aminotransferase (ALT) elevations greater than twice the upper limit of normal. Dr. Greenstein suggested that the results of this study suggest that a large-scale, double-blind study is warranted to compare the combination of Avonex and azathioprine to Avonex monotherapy.

Neutralizing Antibodies

Interferon beta antibodies and their clinical impact: a titer-related effect.

Perini P, Calabrese M, Biasi G, Gallo P, Department of Experimental Pathology, University of Padua, Padua, Italy.

The clinical relevance of interferon-induced neutralizing antibodies has been widely debated. Neutralizing antibodies are a subset of antibodies that bind to interferon (binding antibodies). Typically, testing for binding antibodies occurs first, with further testing of binding antibody-positive sera tested for the presence of neutralizing antibodies. Laboratory and clinical studies have suggested that the presence of neutralizing antibodies is associated with compromised interferon efficacy in treating MS. A recent study showed that the presence of binding antibodies was correlated with treatment failure, while neutralizing antibody-negative status was associated with treatment success.²⁸

A prospective study presented at ECTRIMS 2003 showed that high titers of both binding and neutralizing antibodies had a significant impact on reducing the clinical effectiveness of interferon beta therapy.⁸ As has been demonstrated in previously published studies, this study also showed that of three available interferon beta products, Betaseron was the most immunogenic, followed by Rebif and with Avonex being the least immunogenic.

First author Paola Perini, MD, Department of Experimental Pathology, University of Padua, Padua, Italy suggested that the titer-related effect on therapeutic efficacy observed in the study may explain discrepancies in reported studies of neutralizing antibody-positive MS patients. The effects of both binding and neutralizing antibodies are important for long-term efficacy of interferon beta-based therapies, Dr. Perini added.

Thirty patients were included in one of three treatment arms: Betaseron 8 MIU subcutaneously every other day, Avonex 30 mcg intramuscularly once weekly or Rebif 22 mcg subcutaneously three times weekly. Treatment duration was four years. Binding antibodies were measured using enzyme-linked immunosorbent assay (ELISA) with positive samples further analyzed for neutralizing antibodies using an antiviral cytopathic effect assay (CPE). High binding antibodies titers were defined as >1:500 and high neutralizing antibodies titers were defined as >1:100.

Over the course of the study, binding antibodies developed in 83% of Betaseron-treated patients, 47% of Rebif-treated patients, and 13% of Avonex-treated patients. Neutralizing antibodies were detected in 40% of Betaseron-treated patients, 26.7% of Rebif-treated patients, and 6.7% of Avonex-treated patients.

Of 22 neutralizing antibody-positive patients, 10 (45.5%) demonstrated high titers of both binding and neutralizing antibodies—6 (20%) patients had been treated with Betaseron, 3 (10%) patients had been treated with Rebif and one (0.3%) patient had been treated with Avonex. However, not all titers of neutralizing antibodies correlated with clinical activity. With the emergence of high neutralizing antibody titers, relapse rate significantly increased (P = 0.03); however, an even higher significance level (P<0.001) was observed in patients with high titers of both binding and neutralizing antibodies. Dr. Perini noted the appearance of high titers of both binding and neutralizing antibodies was associated with a highly significant effect on disability. In 10 patients with high titers of both binding and neutralizing antibodies, mean EDSS score at baseline was increased from 2.2 to 3.6 at Year 2 (P<0.01), while no significant change in mean EDSS score was observed in neutralizing antibody-negative patients at Year 2.

"The study suggests that high titers of both antibodies correlate with disease activity and progression. The magnitude of anti-interferon beta response may be responsible for the diminution of therapeutic effect of interferon beta. This is in agreement with the recently published European trial on interferon beta in secondary-progressive multiple sclerosis," concluded Dr. Perini.

Predictive value of the NAB titer for the time to reversion to NAB negativity.

Gneiss C, Reindl M, Berger T, Deisenhammer F, University of Innsbruck, Innsbruck, Austria.

Neutralizing antibodies interfere with the ability of interferon beta to bind to its receptors, blocking receptor activation and the beneficial biological effects of interferon beta in treating MS. Studies show that neutralizing antibodies develop between 6 and 18 months after therapy with interferon beta is initiated.²⁹ Some patients may revert to neutralizing antibody-negative status over continued long-term treatment with interferon beta. The results of a study presented at ECTRIMS 2003 shows that the magnitude of the neutralizing antibody titer measured 2 years after initiation of beta interferon therapy predicts the time to reversion to neutralizing antibody-negative status.⁹

The retrospective study included 28 patients with clinically-definite MS who were neutralizing antibody-positive on interferon beta treatment—16 patients were being treated with Rebif 22 or 44 mcg or Avonex 30 mcg, 11 patients were receiving Betaseron, and one patient switched from interferon beta-1a to Betaseron. Neutralizing antibody titers were assayed with the MxA induction assay. Neutralizing antibody-positive status was defined as at least 2 consecutive samples with a titer of >19 neutralizing units (NU). Reversion to neutralizing antibody-negative status was defined by 2 consecutive negative samples following development of neutralizing antibody positivity. Twelve patients reverted to neutralizing antibody-negative status within 48 months and were deemed reverters (seven patients on Betaseron and five patients on Rebif 22 mcg while 16 patients remained neutralizing antibody-positive (non-reverters) after 48 months—four patients were on Betaseron, nine patients on Rebif 22 mcg, one patient on Rebif 44 mcg, one patient switched from Avonex to Rebif 22 mcg and one patient switched from Rebif 22 mcg to Betaseron.

Twenty-four months after therapy with interferon beta was initiated, mean neutralizing antibody titer was significantly lower in reverters (20 NU) compared with non-reverters (1299 NU, P = 0.04). Using a cutoff value of 65 NU, sensitivity to revert to neutralizing antibody-negative status was 81.25% and specificity was 86.6% (negative predictive value 81.25%, positive predictive value 86.6%). Mean duration of neutralizing antibody positivity until last assessment in non-reverters was 56.5 months; whereas neutralizing antibody-negative status was reached after a mean duration of 35.6 months in reverters. At last measurement, in non-reverters, a 41.6% reduction from neutralizing antibody peak titer was observed (from 4706 NU to 2748 NU).

First author, Claudia Gneiss, MD, University Clinic of Neurology, University of Innsbruck, Innsbruck, Austria, stated, "The study suggests that the time to reversion to neutralizing antibody-negative status depends on neutralizing antibody peak titer. The study also suggests that the specificity of the interferon beta product used may influence time to reversion to neutralizing antibody-negative status." Dr. Gneiss suggested that use of an interferon beta product with low immunogenicity could overcome the problems associated with the development of neutralizing antibodies in the treatment of multiple sclerosis.

Neutralizing antibodies antagonise interferon-beta suppression of matrix metalloproteinase in patients with MS.

Bertolotto A, Gilli G, Sala A, Hoffmann F, Capobianco M, Malucci S, Kappos L, Lindberg RLP, Leppert D.

Assessment of peripheral blood mononuclear cells from 39 treatment-naпve and 67 interferon beta-treated patients with MS suggests that neutralizing antibodies have a deleterious effect on therapeutic efficacy of interferon beta.¹⁰ Specifically, the study showed that interferon beta suppresses expression of matrix metalloproteinase-9 molecules involved in the pathogenesis of MS, and that this beneficial treatment effect is antagonized by the presence of neutralizing antibodies. The study was presented at a late-breaking news session by Antonio Bertolotto, MD, Centro Riferimento Regionale Sclerosi Multipla and Neurobiologia Clinica, Ospedale Universitario S. Luigi Gonzaga, Orbassano (Torino), Italy, author of a previous study that demonstrated Avonex was the least immunogenic of the interferon beta products.²⁹ In that independent study, neutralizing antibodies developed in only 2% of Avonex-treated patients, compared with 31% for Betaseron-treated and 15% for Rebif-treated patients.²⁹

Dr. Bertolotto said that the interferon beta-induced effects on matrix metalloproteinase-9 and MxA protein are different, and that neutralizing antibodies antagonize the effect of interferon beta on both of these protein markers. Expression of MxA is a sensitive measure of the biologic activity of interferon beta, and this protein is reduced in the presence of neutralizing antibodies. However, MxA protein is not involved in the pathogenesis of multiple sclerosis or the therapeutic effect of interferon beta.

Expression of matrix metalloproteinase and MxA mRNA was quantified by real-time polymerase chain reaction (PCR) in peripheral blood mononuclear cells of study participants. Neutralizing antibody status was evaluated every three months with CPE. Patients were categorized as neutralizing antibody-negative, persistent neutralizing antibody-positive (two or more positive tests), or isolated neutralizing antibody-positive (only one positive test).

Interferon beta therapy with any of the four available treatment regimens (Avonex 30 mcg, Betaseron 250 mcg, Rebif 22 or 44 mcg) significantly downregulated the expression of matrix metalloproteinase-9 and abolished expression of matrix metalloproteinase-2 in patients treated with interferon beta (P<0.0001); this effect was not observed in treatment-naпve patients. By contrast, levels of MxA protein were increased with both acute (treatment-naпve patients) and long-term interferon beta treatment compared with baseline. In the interferon beta-treated patients, 71% were neutralizing antibody-negative, 22% were persistent neutralizing antibody-positive, and 7% were isolated neutralizing antibody-positive.

Persistent neutralizing antibodies reversed the effects of interferon beta on both MxA and matrix metalloproteinase expression. Patients with persistent neutralizing antibodies had significantly reduced MxA expression (P<0.0001) and significantly increased matrix metalloproteinase-9 expression (P<0.044) compared with neutralizing antibody-negative patients.

Dr. Bertolotto indicated, "These findings support the concept of the significant role of neutralizing antibodies in reducing the therapeutic efficacy of interferon beta."

Neutralizing antibody formation in MS patients treated with a new liquid formulation of interferon beta-1a (Avonex).

Goelz SE, Phillips JT, Jethwa V.

A multicenter, single-arm, open-label study has demonstrated that a new liquid formulation of Avonex available in a prefilled syringe is safe and has a similarly low level of immunogenicity as the standard lyophilized formulation of Avonex injection.¹¹ Susan Goelz, PhD, Biogen Idec™, Cambridge, Massachusetts, who presented the results at a late-breaking news session, noted that human serum albumin, often added to interferon products for stabilization, was omitted from the new liquid formulation. Until recently, Avonex was only available in a vial containing lyophilized product; before injecting themselves, the patient had to reconstitute the product with water and draw it into a syringe. The availability of a prefilled syringe should translate to improved convenience for patients, which will be important for those patients whose manual dexterity is impaired by MS or comorbidity.

The study enrolled 153 patients with relapsing-remitting MS, EDSS scores between 0-5.5, and were between the ages of 18 and 60 years (mean age 39.6 years). Mean disease duration was 4.1 years and mean EDSS score was 2.4; 86% of patients had an EDSS score of 3.5 or less, 14% had EDSS between 4.0-5.5, and no patients had a score of 6.0 or higher at baseline. Mean pre-study relapse rate was 2.4. All patients were treated with the new prefilled syringe liquid formulation of intramuscular Avonex 30 mcg once weekly for up to 24 months. Patients were excluded if they experienced a relapse within 2 months prior to receiving the first dose of study drug or if they were treated previously with any other drug for MS. Safety and immunogenicity were evaluated at baseline and every 3 months.

Neutralizing antibodies were assayed with a two-step test. Serum samples were screened for binding antibodies using ELISA, and ELISA-positive samples were screened for neutralizing antibodies using CPE. Two levels of neutralizing antibody positivity were determined—patients with at least 2 consecutive titers ≥20 were identified and those with any titer of ≥5 were also identified. These levels were chosen to reflect the levels of neutralizing antibodies likely to have clinical impact and to demonstrate the immunogenicity of the product. Safety was assessed by the incidence of adverse events and the results of blood chemistry, hematology, and urine testing.

Safety assessment showed that the new liquid formulation of Avonex in a prefilled syringe was well tolerated. There were 19 (12%) withdrawals due to adverse events, nine (6%) of which were attributed to flu-like symptoms. The most common adverse event was flu-like symptoms (88%), followed by headache (45%), asthenia (26%), paresthesia (22%), myalgia (20%), accidental injury (20%), and pharyngitis (20%).

Twenty-three (15%) patients reported depression of which 19 were deemed to be unrelated or likely to be unrelated to study drug by the investigators. Fourteen patients (9%) had serious adverse events, but none of these was considered likely or definitely related to the study drug. No new treatment-related adverse events were reported.

Immunogenicity assessment included 147 patients who were neutralizing antibody-negative at baseline and had one or more post-baseline neutralizing antibody results. Two patients (1.7%) had at least two consecutive titers of ≥20, which Dr. Goelz said is the level associated with the likelihood of clinical import (Table 2). At Month 18, a total of five patients (4%) had at least one neutralizing antibody titer of ≥5 at any one time during the study. Over the next 6 months, only one additional patient became neutralizing antibody-positive, for a total of 6 patients (5%) who had a neutralizing antibody titer of ≥5 at 24 months.

Dr. Goelz noted that neutralizing antibodies did not begin to emerge until Month 2 and the incidence of neutralizing antibodies appeared to reach a plateau after Month 18. This suggested to Dr. Goelz that the kinetics of the new liquid formulation of Avonex are somewhat slower than with the lyophilized formulation of Avonex. The mean time for the first neutralizing antibody titer of ≥5 was 16 months. Binding antibodies were present on at least two consecutive ELISA tests in 16% of the 147 patients assessed for antibody status.

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