Multiple Sclerosis and Related Disorders (2014) 3, 220–226

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High-dose intravenous interferon-beta in multiple sclerosis patients with high-titer neutralizing antibodies (HINABS II) – A pilot study$ H. Hegena,n, M. Gugerb, A. Harrerc, M. Hoelzla, J. Krausc, A. Skrobald, F. Schautzere, S. Schmideggb, M. Schockef, F. Deisenhammera a

Innsbruck Medical University, Department of Neurology, Anichstraße 35, 6020 Innsbruck, Austria Allgemeines Krankenhaus Linz, Department of Neurology, Krankenhausstraße 9, 4021 Linz, Austria c Paracelsus Medical University, Department of Neurology, Ignaz-Harrer-Straße 79, 5020 Salzburg, Austria d Landesklinikum Horn, Department of Neurology, Spitalgase 10, 3580 Horn, Austria e Landeskrankenhaus Villach, Department of Neurology, Nikolaigasse 43, 9500 Villach, Austria f Innsbruck Medical University, Department of Radiology, Anichstraße 35, 6020 Innsbruck, Austria b

Received 5 June 2013; received in revised form 25 July 2013; accepted 3 September 2013

KEYWORDS Multiple sclerosis (MS); Interferon-beta (IFNβ); Neutralizing antibody (NAb); Myxovirus protein A (MxA); Immune tolerance; Bioavailability

Abstract Background: Neutralizing antibodies (NAb) against interferon-beta (IFNβ) affect its treatment efficacy. So far, there are no anti-NAb strategies available.

Objectives: To investigate if the repeated administration of high-dose IFNβ-1b intravenous in NAb positive multiple sclerosis (MS) patients induces tolerance and establishes IFNβ bioavailability as measured by the induction of myxovirus protein A (MxA). Methods: Nine MS patients with NAb titers 4500 10-fold reduction units (TRU) received 1500 μg IFNβ-1b intravenously once weekly over three months. Blood samples were collected at screening, monthly during the treatment period (before and four hours after IFNβ administration), and at follow-up after 6 months for determination of NAbs and MxA expression. Results: Median NAb titer at baseline was 1429 TRU. NAb titers determined before each infusion did not significantly change over the treatment period and were not different at follow-up compared to baseline. However, NAb titers were significantly decreased four hours after IFNβ infusions (by roughly 50%) and MxA mRNA levels were significantly elevated reaching a median value of 206. Conclusions: Weekly intravenous administration of IFNβ in patients with high NAb titers established its bioavailability, but failed to induce tolerance towards IFNβ. & 2013 The Authors. Published by Elsevier B.V. All rights reserved.

$ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. n Correspondence to: Department of Neurology, Neuroimmunology Laboratory, Innsbruck Medical University, Anichstrasse 35, 6020 Innsbruck, Austria. Tel.: +43 512 504 24264; fax: +43 512 504 24266. E-mail address: [email protected] (H. Hegen).

2211-0348/$ - see front matter & 2013 The Authors. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msard.2013.09.001

HINABS II – A pilot study

1.

Introduction

Interferon-beta (IFNβ) is associated with the development of neutralizing antibodies (NAb) in up to 44% of treated multiple sclerosis (MS) patients (Deisenhammer, 2009). NAbs prevent the biological effects of IFNβ resulting in the reduced expression of IFN-stimulated gene products such as myxovirus protein A (MxA) (Deisenhammer et al., 1999; Sominanda et al., 2008) and, if persistent, in loss of treatment effects (Francis et al., 2005; Group, 1996). MxA mRNA levels inversely correlate with NAb titers. Whereas MxA is fully abrogated in patients with high NAb titers, it might range from absent to fully expressed in patients with low NAb titers (Sominanda et al., 2008). Therefore, current recommendations suggest to stop IFNβ therapy in case of two consecutive high NAb titer positive test results or in case of persistent low NAb titers and lost MxA bioactivity (Polman et al., 2010). So far, there are no anti-NAb strategies available. Switching to another IFNβ preparation does not restore treatment effects, as NAbs cross-react between IFNβ-1a and IFNβ-1b (Khan and Dhib-Jalbut, 1998), and switching from a high to a low immunogenic IFNβ preparation does not influence the course of NAb titers (Gneiss et al., 2009). Although reversion to NAb negativity has been reported with continuous IFNβ treatment, this occurs mostly in IFNβ-1b treated patients with low NAb titers after several years (Hegen et al., 2012b; Sorensen et al., 2005). NAb reversion can neither be accelerated by the concomitant treatment with corticosteroids nor by the combined treatment with azathioprine and corticosteroids (Hesse et al., 2009; Ravnborg et al., 2009). However, induction of immune tolerance has been successfully achieved in patients with hemophilia A and NAbs against recombinant factor VIII (FVIII) using repeated administration of high-dose intravenous FVIII which results in reversion of NAbs in the majority of patients (Astermark et al., 2006). In a previous pilot study in NAb positive MS patients we have shown that a single intravenous administration of IFNβ results in a short-term decrease of NAbs and a significant increase of MxA gene expression (Millonig et al., 2009). Based on these findings, we aimed to investigate whether the repeated highdose intravenous IFNβ administration in patients with high NAb titers leads to a sustained reversion of NAbs and an increase of MxA expression.

2. 2.1.

Materials and methods Study design

This was a prospective, multicenter, open-label, interventional, proof-of-concept study conducted at five clinical

221 centers in Austria. After screening, patients entered the treatment period and received 1500 μg IFNβ-1b reconstituted in 100 ml 0.9% NaCl intravenously once a week over 3 months (i.e. a total of 13 infusions). To reduce side effects such as flu-like symptoms (FLS), 1000 mg paracetamol were administered intravenously prior to each IFNβ infusion. Clinical assessment, recording of adverse events, evaluation of the expanded disability status scale (EDSS) score and blood collections were performed at screening, then monthly at baseline, week 4, 8 and 12, as well as at follow-up after 24 weeks. At baseline and weeks 4, 8 and 12 blood samples were withdrawn immediately before and 4 h after IFNβ administration. Serum was isolated by centrifugation after blood samples were allowed to clot for at least 30 min. Whole blood samples were kept at room temperature for at least 4 h after their withdrawal. All samples were stored at 20 1C until analysis. Safety lab investigations were performed monthly during the treatment period and included blood count, liver enzymes and pregnancy testing if appropriate. Brain MRI was done at baseline before the first IFNβ infusion and at week 12 after the last infusion (Fig. 1).

2.2.

Patients

Patients were eligible if they were diagnosed as relapsing MS or clinically isolated syndrome (CIS) according to the revised McDonalds criteria 2005 (Polman et al., 2005), were at least 18 years old, received previous treatment with any IFNβ preparation: intramuscular (IM) IFNβ-1a 30 μg once weekly (Avonexs, Biogen Idec, Cambridge, MA, USA), subcutaneous (SC) IFNβ-1a 22 or 44 mg three times weekly (Rebifs, Merck Serono, Geneva, Switzerland) or SC IFNβ-1b 250 mg every other day (Betaferons, Bayer Schering, Berlin, Germany), and were NAb positive with a titer 4500 TRU at screening. Patients during pregnancy or lactation were not included into the study. Previous IFNβ treatment had to be interrupted for at least 7 days before baseline visit. Patients, who did not show sufficient MxA gene expression after administration of the study dose of 1500 μg IFNβ-1b at baseline or at week 1, were withdrawn from the study. For determination of sufficient MxA gene expression, i.e. to calculate a cut-off MxA level discriminating between patients with and without IFNβ treatment, we used a cohort of 159 patients with relapsing MS or CIS recruited within the prospective European multicentre study NABINMS. These patients received de novo IFNβ treatment and had MxA mRNA measurement before and 4 h after the first IFNβ injection. ROC analysis revealed a cut-off MxA value of

Fig. 1 Study design. IFNβ-1b, interferon beta-1b; wk, week; MRI, magnetic resonance imaging.

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24.66 with a sensitivity of 96.2% and specificity of 96.9% (AUC= 0.997, po0.001).

Table 1

Demographic and baseline clinical data.

Female/male

2.3.

Laboratory procedures

NAbs were measured in serum by a luciferase assay using the human fibrosarcoma cells. These cells express IFNβ receptors on their surface and have been stably transfected with a luciferase reporter gene cassette. Upon binding of IFNβ, luciferase is produced in a predictable dose-dependent manner and diminished in the presence of NAbs (Farrell et al., 2008; Lam et al., 2008) (for detailed assay description see Supplemental material). Titers were calculated using the Kawade method (Grossberg et al., 2001a, 2001b; Kawade, 1986) and expressed in 10-fold reduction units (TRU). Samples with a titer Z 20 TRU were considered as NAb positive. MxA was determined at the mRNA level as previously described. Briefly, RNA was extracted from whole blood samples, transcribed to cDNA, and subjected to real-time polymerase chain reaction for MxA quantification (Millonig et al., 2009).

2.4.

Statistics

Statistical analysis was performed using SPSS 18.0 (SPSS Inc, Chicago, IL) and GraphPad Prism 5.03 (GraphPad Inc, La Jolla, CA). Wilcoxon or Friedman tests were used for analyzing repeated measurements such as of NAb titers or MxA mRNA levels. For correlation analysis Spearman coefficient was used. Two-tailed P values of o0.05 were considered statistically significant.

2.5. Standard protocol approvals and patient consents The study protocol was approved by the ethics committee of Innsbruck Medical University (study no. UN3703 279/2.7, 22.10.2009) and each participating center. All patients gave written informed consent before any study-related procedures.

2.6.

Goal

The primary outcome of the study was the proportion of patients who showed a reduction of NAb titer less than 100 TRU at the end of treatment period and an established IFNβ bioavailability as measured by MxA gene expression. The secondary outcomes included the change of NAb titer at the end of treatment period, NAb titers at follow-up, as well as change of T2 lesion volume on brain MRI scans.

3. 3.1.

Disease type, n CIS RRMS SPMS with relapses Age, yearsa EDSS scorea Previous IFNβ therapy, yearsa Previous IFNβ-1a therapy, yearsa Previous IFNβ-1b therapy, yearsa Treatment-free period before BL, monthsa Pre-treatment NAb titer, TRUa Pre-treatment MxA expression, NRa

4/5

1 7 1 46.9 (39.6–55.3) 1.0 (1.0–2.75) 3.1 (2.5–10.2) 4.1 (0–9.8) 1.4 (0–2.0) 3.2 (1.5–5.6) 1429 (902–2995) 1.4 (0.8–2.6)

a Values are shown as median and interquartile range; CIS, clinically isolated syndrome; RRMS, relapsing-remitting multiple sclerosis; SPMS, secondary progressive multiple sclerosis; EDSS, expanded disability status scale; IFNβ, interferonbeta; BL, baseline; NAb, neutralizing antibody; TRU, 10-fold reduction unit; NR, normalized ratio; MxA, myxovirus protein A.

(week 1) due to lacking MxA induction and, thus, was not included in further statistical analysis. Demographic and baseline clinical data of the nine patients completing the study are shown in Table 1. First IFNβ infusion was administered at baseline, 39 days (median) after screening visit and, further, every 7 days. Follow-up visits were performed 25.0 weeks (median) after baseline. Blood samples of all nine patients were drawn per protocol with the single exception of a missed serum sample of patient 4 at week 12.

3.2. NAb titers are decreased shortly after IFNβ infusion Median pre-treatment NAb titer at baseline was 1429 TRU. NAb titers determined monthly before each IFNβ infusion did not significantly change over the treatment period (p= 0.79, Fig. 2). Median NAb titer at follow-up was 2175 TRU (IQR 1803–2519) and was not significantly different compared to baseline (p= 0.23) or week 12 (p= 0.24). None of the patients showed a NAb titer decrease below 100 TRU. However, NAb titers were decreased after each IFNβ infusion; the ratios of pre-/post-infusion NAb titers are shown in Fig. 2. Individual NAb titer development of all nine patients is shown in Fig. 3.

Results Study population

A total of 10 patients were enrolled into the study from January until September 2010. One female patient with relapsing-remitting MS and a screening NAb titer of 4610 TRU had to be excluded after the second IFNβ infusion

3.3. IFNβ bioavailability is established in all nine patients MxA mRNA levels were significantly increased in all nine patients after IFNβ infusions (p= 0.008) showing values ranging from 38.0 to 837.5. MxA levels of all patients at all sampling time points are shown in Table 2.

HINABS II – A pilot study

223

3.4. High-dose IFNβ does not influence T2 lesion volume Baseline MRI was performed 2 days (median) before the first IFNβ infusion and follow-up MRI 1 day (median) after the last infusion (week 12). Median T2 lesion volume slightly decreased from 2250.2 mm3 (IQR 2059.4–3078.2) at baseline to 1962.0 mm3 (IQR 940.9–3687.0) at week 12 (p= 0.767). There was no correlation between T2 lesion volume change and post-infusion MxA levels.

3.5. Fig. 2 NAb titer development during treatment period. Data are shown as median and 10.-90. percentile. aNAb titer ratio calculated as NAb titer after/ before IFNβ-1b administration. NAb, neutralizing antibody; TRU, 10-fold reduction unit; IFNβ1b, interferon beta-1b.

Adverse events

IFNβ infusions were well tolerated if administered slowly. There were two infusion-related adverse events. One patient experienced significant flu-like symptoms (week 10) that resolved after symptomatic treatment. In another patient transient hypotonia was observed that resolved

Fig. 3 Individual NAb titer development. NAb, neutralizing antibody; TRU, 10-fold reduction unit; IFNβ, interferon-beta; +, first NAb positive test; ↓, adverse event.

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Table 2

MxA mRNA levels before and after IFNβ-1b infusions.

Patient

1 2 3 4 5 6 7 8 9 10 All patientsa a

Week 0

Week 4

Week 12

Before IFNβ-1b

After IFNβ-1b

Before IFNβ-1b

After IFNβ-1b

Before IFNβ-1b

After IFNβ-1b

Before IFNβ-1b

After IFNβ-1b

0.68 2.79 1.68 1.11 0.84 2.79 0.83 2.36 1.4 0.71 1.4

199.29 372.54 175.92 115.16 38.02 546.38 62.68 349.71 106.71 0.98 175.92

0.95 1.81 4.54 0.93 0.56 1.48 0.75 1.76 1.11 0.33 1.11

313.81 149.73 186.59 110.85 164.71 248.78 140.07 634.73 183.86 0.47 183.86

0.95 1.68 0.73 0.69 0.99 1.61 1.09 2.77 0.77

472.36 170.22 67.83 60.86 200.68 253.13 221.32 65.12 211.94

1.01 2.64 1.54 4.63 0.64 1.74 0.81 1.57 0.76

274.14 289.27 228.14 146.27 295.86 756.79 401.71 837.53 255.56

0.99

200.68

1.54

289.27

Median MxA mRNA levels were calculated excluding patient 10. IFNβ-1b; interferon beta-1b; MxA, myxovirus protein A.

after administration of 1000 ml of isotone cristalloid fluid. Three non-infusion-related adverse events occurred during the treatment period: respiratory tract infections in two patients and a relapse in another patient. EDSS scores did not change at the end of treatment period or at follow-up as compared to baseline. Safety lab results were within normal ranges for all patients throughout the whole study.

4.

Week 8

Discussion

Here, we showed that the repeated intravenous administration of high-dose IFNβ-1b (1500 μg) in patients with high titer NAbs (4500 TRU) establishes IFNβ bioavailability as measured by elevated MxA mRNA levels in 9 out of 10 patients. We observed a considerable short-term decrease of NAb titers by roughly 50% after IFNβ infusions, however, induction of immune tolerance to IFNβ as indicated by a long-term reduction of NAb titers was not achieved. In a previous study, we have shown that the intravenous infusion of 250 or 500 μg IFNβ-1b in patients with a median NAb titer of 426 TRU resulted in a significant increase of MxA mRNA levels, while the SC injection of 250 μg IFNβ-1b did not establish its bioavailability. The amount of MxA expression (after the intravenous IFNβ application) varied depending on the baseline NAb titer: whereas MxA mRNA levels markedly increased in patients with low NAb titers (median 118 TRU), they did not change in patients with high titers (median 3550 TRU) (Millonig et al., 2009). In the present study, we confirmed that intravenous high-dose IFNβ establishes its bioavailability in NAb positive patients. The observation that MxA mRNA levels were clearly increased after the intravenous application of IFNβ might be explained by all NAbs are bound and excess IFNβ activates IFNβ receptors. Yet, NAb titers were only decreased but did not disappear after IFNβ infusions, which requires alternative considerations than simple saturation of circulating NAbs by IFNβ. Pharmacokinetic studies in healthy volunteers revealed that IFNβ serum concentrations are markedly increased already 5 min after the intravenous bolus administration of IFNβ (Chiang et al., 1993). One might speculate

that the speed of its increase in concentration might lead to a transient excess of IFNβ allowing IFNβ molecules to bind with high affinity to the receptors on circulating lymphocytes. The high affinity binding might be an advantage of the IFNβ–receptor interaction. Circulating NAbs are probably polyclonal with a range of different affinities. Therefore, the formation of immune complexes will take longer and once the sample is collected a state of antibody excess has been reached which leads to detection of NAbs in the assay. However, to prove this hypothesis, further studies would be required with short interval blood collections after intravenous IFNβ administration for monitoring changes in NAb titers, IFNβ concentrations and MxA mRNA levels. In the present study, blood sampling was performed 4 h after IFNβ infusion. Although the reported peak MxA mRNA levels occur between 3 and 12 h after conventional IFNβ injection, it is difficult to determine the ideal time point as there is no plateau phase but a short time when RNA levels reach peak expression (Gilli et al., 2006, 2005; Millonig et al., 2009; Pachner et al., 2005). The 4 h time point was chosen because we used it successfully in a previous study on intravenous IFNβ-1b (HINABS I) (Millonig et al., 2009). Furthermore, one study reported earlier peak MxA expression in IFNβ-1b than IFNβ-1a treated patients (Gilli et al., 2005). The rationale for exclusion of the one patient with insufficient MxA expression after the administration of the study dose of 1500 μg IFNβ (predefined scenario) was the hypothesis that immune tolerance to IFNβ might be only induced, if there is excess IFNβ after all NAbs have been bound, which can then impact on the patients' immune system. Immune tolerance induction (ITI) to a protein-based drug has been reported more than 30 years ago in hemophilia patients with NAbs against FVIII (Brackmann and Gormsen, 1977). Despite obvious differences between the FVIII and IFNβ protein molecules, there are some similarities between the anti-drug antibodies. Both, anti-FVIII and anti-IFNβ antibodies undergo affinity maturation, class switch (towards IgG4) and finally develop neutralizing activity (Deisenhammer et al., 2001; Gibbs and Oger, 2008; Waters and Lillicrap, 2009). Accordingly, one might still speculate about analogies in terms of ITI. Hausl et al. revealed that

HINABS II – A pilot study physiological protein concentration (of FVIII) leads to memory B cell differentiation, whereas the stimulation by a high protein load inhibits the transformation into plasma cells and, subsequently, antibody production (Hausl et al., 2005). Several different ITI protocols have been used in hemophilia patients applying different protein doses and dosing intervals. The normal therapeutic dose of recombinant factor VIII varies depending on the severity of FVIII deficiency and the patient's body weight. Assuming a mean dose of 20 IE/kg, ITI protocols apply double up to roughly 7-fold (or even higher) doses (Coppola et al., 2010). According to our own previous findings that 500 μg IFNβ-1b neutralize NAbs at a titer of roughly 400 TRU (Millonig et al., 2009) and the exponential dependency of NAb titer and the amount of IFNβ antigen (i. e. 1000 μg IFNβ-1b would neutralize 4000 TRU and 1500 μg up to 40,000 TRU) (Grossberg et al., 2001b), we decided to use an 8-fold higher dose of IFNβ-1b. However, neither NAb reversion nor an impressive reduction of NAb titer has been achieved in the present study. There were decreasing NAb titers observed in five patients possibly due to the long treatment duration before study entry (median 10.0 years). In contrast, in four patients with increasing NAb titers the median duration of IFNβ therapy was only 2.5 years. In comparison to hemophilia patients, one might hypothesize that FVIII (2351 amino acid, 280 kDa) as a much bigger protein than IFNβ (166 amino acids, 22.5 kDa) (Schroder et al., 2004; Wood et al., 1984) is per se not only more immunogenic (NAbs against FVIII occur in approximately 30% of intravenously treated patients and against IFNβ in around 20% of SC/IM treated patients; whereas the intravenous route of administration is generally considered as less immunogenic (Schellekens, 2002)), but probably also be more capable to re-induce immune tolerance. Evidence for this hypothesis comes from the observation that NAbs against IFNβ-1b show earlier and more frequent reversion to negativity than those against IFNβ-1a (during continuous treatment within normal therapeutic doses) (Hegen et al., 2012b), possibly due to the higher protein load of IFNβ-1b (250 μg versus 22/44 μg for SC IFNβ-1a and 30 μg for IM IFNβ1a). Another reason for failing immune tolerance induction to IFNβ might be the short treatment period, as well as longer dosing intervals compared to protocols used in hemophilia patients (Coppola et al., 2010). It seems unlikely that the type of IFNβ used before study entry negatively influences the outcome of this trial, as NAbs against IFNβ cross-react between IFNβ-1a and IFNβ-1b preparations (Khan and Dhib-Jalbut, 1998). In hemophilia patients, plasma-derived or different recombinant FVIII products are successfully used for ITI irrespective of the previously applied product and NAbs against FVIII are also crossreactive between different FVIII preparations (Astermark et al., 2006; Lusher, 2000; Rocino et al., 2001). Infusion-related side effects were all transient and resolved after symptomatic treatment, i.e. acetaminophen for flu-like symptoms and cristalloids for hypotonia. These findings are in line with several studies using high doses of intravenous IFNβ (up to 720 MIU) showing generally good tolerance with little changes in rate and severity of adverse events without any allergic reactions (Goldstein et al., 1989; Larocca et al., 1989; Millonig et al., 2009). Finally, although NAbs did not revert in this study, MxA gene expression was successfully induced and, therefore, IFNβ

225 bioavailability established. Apart from a general interest in inducing tolerance towards biopharmaceuticals there is a particular interest in IFNβ because there is evidence that eventually NAb positive patients show a better clinical response to IFNβ before NAb evolution as compared to always NAb negative patients (Sorensen et al., 2007). Furthermore, as a recent study demonstrated early prediction of NAbs (Hegen et al., 2012a), it might be of interest to apply immune tolerance induction methods or other strategies before the development of NAbs for prevention rather than reversion as demonstrated in enzyme replacement therapies (Lacana et al., 2012).

Conflict of interest statement H. Hegen received honoraria (lectures) from pharmaceutical companies marketing treatment for multiple sclerosis (Bayer Schering, Biogen Idec, Merck Serono). M. Guger reports no disclosures. A. Harrer reports no disclosures. M. Hoelzl reports no disclosures. J. Kraus received financial support for research activities from Bayer, Biogen Idec, Genzyme, Sanofi-Aventis, Merck Serono, and Novartis. JK received personal compensation from Bayer, Biogen Idec, Merck Serono, Novartis, and Sanofi-Aventis, for lectures, advisory board participations, and consultations. A. Skrobal reports no disclosures. F. Schautzer reports no disclosures. S. Schmidegg reports no disclosures. F. Deisenhammer has participated in meetings sponsored by, or received honoraria for acting as an advisor for, Biogen Idec, Merck Serono, Bayer and Sanofi. His institution has received financial support for participation in randomized controlled trials of INFb-1b (Betaferon, Bayer Schering Pharma), INFb-1a (Avonex, Biogen Idec; Rebif, Merck Serono), glatiramer acetate (Copaxone, Teva Pharmaceuticals) and natalizumab (Tysabri, Biogen Idec) in multiple sclerosis.

Funding The study medication was provided free of charge by Bayer Schering.

Appendix A.

Supporting information

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/ j.msard.2013.09.001.

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