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Per Soelberg Sørensen, Poul Erik Hyldgaard Jensen, Aiden Haghikia, Malin Lundkvist, Christian ... Aiden Haghikia2, Malin Lundkvist3, Christian Vedeler4,.
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Occurrence of antibodies against natalizumab in relapsing multiple sclerosis patients treated with natalizumab Per Soelberg Sørensen, Poul Erik Hyldgaard Jensen, Aiden Haghikia, Malin Lundkvist, Christian Vedeler, Finn Sellebjerg, Nils Koch-Henriksen, Anna Fogdell-Hahn, Kjell-Morten Myhr, Jan Hillert and Ralf Gold Mult Scler 2011 17: 1074 originally published online 20 April 2011 DOI: 10.1177/1352458511404271 The online version of this article can be found at: http://msj.sagepub.com/content/17/9/1074

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Research Paper

Occurrence of antibodies against natalizumab in relapsing multiple sclerosis patients treated with natalizumab

Multiple Sclerosis Journal 17(9) 1074–1078 ! The Author(s) 2011 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1352458511404271 msj.sagepub.com

Per Soelberg Sørensen1, Poul Erik Hyldgaard Jensen1, Aiden Haghikia2, Malin Lundkvist3, Christian Vedeler4, Finn Sellebjerg1, Nils Koch-Henriksen5, Anna Fogdell-Hahn3, Kjell-Morten Myhr4, Jan Hillert3 and Ralf Gold2

Abstract Background: In the clinical trials about 9% of natalizumab treated multiple sclerosis (MS) patients generated antinatalizumab antibodies, of which 6% were persistent and 3% transient. The occurrence of antibodies reduced serum levels of natalizumab, decreased bio-efficacy, and abrogated the therapeutic efficacy. Objective: The objective was to assess the frequency of anti-natalizumab antibodies in an unselected cohort of patients from four different countries. Methods: We measured anti-natalizumab antibodies in a large cohort of 4881 unselected patients from four MS centres that systematically measured antibodies in patients treated with natalizumab. We applied the same ELISA assay developed by Biogen Idec and used in the pivotal trials of natalizumab. Results: Antibodies occurred in 4.5% (95% confidence interval, CI: 4.0–5.1%) of the patients, and were persistent in 3.5% (95% CI: 3.0–4.0%) and transient in 1.0% (95% CI: 0.7–1.3%) of the patients. The frequencies of permanently antibody positive patients did not show statistically significant differences between the four centres, whereas the frequencies of transiently antibody positive patients showed some variations. Conclusion: The frequencies of antibodies appeared to be of the same magnitude in the four centres, but might be less than in the pivotal studies of natalizumab. Keywords disease modifying therapies, immunology, multiple sclerosis, Tysabri, antibodies Date received: 4th December 2010; revised: 23rd February 2011; accepted: 25th February 2011

Introduction The beneficial effects of natalizumab in the treatment of relapsing–remitting multiple sclerosis (MS) is well documented, both in the setting of clinical trials1–4 and in general practice.5–8 Natalizumab is a monoclonal antibody directed against the a4 component of the a4b1 integrin (VLA4; CD49d) expressed on the surface of lymphocytes and monocytes.9 By binding to a4b1 integrin, which is a mediator of transendothelial leukocyte migration, natalizumab blocks the migration of T-cells into the central nervous system.10 Natalizumab was originally generated in mice, but humanized by grafting the antibody complementarity determining regions into a human IgG4 antibody frame, but is still immunogenic like

1 Danish Multiple Sclerosis Center, Department of Neurology, Copenhagen University Hospital Rigshospitalet, Denmark. 2 Department of Neurology, St Josef-Hospital, Ruhr-University Bochum, Germany. 3 Multiple Sclerosis Research Group, Department of Clinical Neuroscience, Karolinska Institutet, Sweden. 4 Department of Neurology, Haukeland University Hospital, University of Bergen, Norway. 5 The Danish MS Treatment Register, Copenhagen University Hospital Rigshospitalet, Denmark.

Corresponding author: Professor Per Soelberg Sørensen, MD, DMSc, Danish Multiple Sclerosis Center, Department of Neurology 2082, Copenhagen University Hospital Rigshospitalet, DK-2100 Copenhagen, Denmark Email: [email protected]

Sørensen et al. other biopharmaceuticals.11 In the clinical trials about 9% of natalizumab-treated MS patients generated antinatalizumab antibodies, and the presence of these resulted in reduced serum levels of natalizumab, decreased bio-efficacy, and abrogation of the therapeutic efficacy.3,4,12 In the clinical trials, binding anti-natalizumab antibodies were measured using a sandwich/bridging ELISA method12 and a flow-cytometric assay to detect blocking anti-natalizumab antibodies. The blocking assay assessed the ability of natalizumab specific antibodies to block the binding of biotinylated natalizumab to its cognate a4 receptor.12 All patients with antibodies measured by ELISA were also antibodypositive in the blocking assay.12 The anti-natalizumab antibodies in MS patients typically had developed 12 weeks after initiation of treatment.3,4,12 The AFFIRM study showed that 88% of the eventually antibody-positive patients had developed antibodies when tested 12 weeks after initiation of natalizumab therapy.3,4,12 About 6% of the patients with antibodies to natalizumab were persistently antibody positive, defined as antibodies detected at two or more times with an interval of at least 42 days,3,4,12 whereas about 3% of the antibodygenerating patients were transiently antibody positive and in these patients the antibodies normally disappeared after a few months of treatment. The occurrence of natalizumab-binding antibodies outside the setting of a clinical trial is unknown. Therefore we decided to investigate the occurrence of natalizumab antibodies in a large cohort of unselected patients from four MS centres that are systematically testing all local patients treated with natalizumab for anti-natalizumab antibodies.

Patients The study comprised consecutive patients from four large MS centres at which all patients starting therapy with natalizumab routinely are tested for the presence of anti-natalizumab antibodies in the blood. Eligible patients had been treated with natalizumab for at least 48 weeks and had had at least two tests for antibodies performed with an interval of at least 12 weeks, of which one test had been performed six months or later after start of natalizumab therapy. Blood samples for antibody measurements were obtained after 12, 24 and 48 weeks or after 24, 36 and 48 weeks from initiation of natalizumab therapy. Patients were included from June 2006 to March 2010.

Methods All four centres used the same ELISA developed by Biogen Idec (Maine Biotechnology Services Inc.,

1075 Portland, ME, USA). All laboratories had to analyse high positive and low positive quality control samples distributed by Biogen Idec. All patient serum samples were tested for antibodies against natalizumab using the ELISA procedure as prescribed by Biogen Idec. Biogen Idec provided all material for control of the assay (natalizumab reference standard, biotinylatednatalizumab, high positive quality control 1 (QC1) (3 mg/ml of the monoclonal antibody 12C4) and low positive quality control 2 (QC2) (0.5 mg/ml of the monoclonal antibody 12C4). The negative control (NC) was derived from a pool of non-treated healthy people serum samples (Pel-freez Clinical Systems, VI, USA [cat. no. 34005/100]). Microtitre plates (cat. no. 3590) were from Corning Laboratories, NY, USA. The buffers used in the assay were: 0.1 M sodium phosphate, pH 8.5 with 2 mg/ml bovine serum albumin (BSA) as plate coating buffer; 0.1 M Tris, pH 7.7 with 20% sucrose as plate blocking buffer; Tris-buffered saline with 0.05% Tween-20 as plate washing buffer; and phosphate-buffered saline with 0.5% BSA and 0.05% Tween-20 as assay diluent. Sucrose (cat. no. 84097), Tween-20 (cat. no. P2287), and BSA (cat. no. A3059) were obtained from Sigma Life Science. Tris-HCl, pH 7.5 and PBS (10 times concentrated) were from Gibco (cat. no. 15567027 and cat. no. 14200, respectively). Streptavidinhorseradish peroxidase was from Pierce, IL, USA (cat. no. 21127), TMB 1 ready to use substrate from KEM-EN-TEC Diagnostics (cat. no. 4380H) and STOP solution H2SO4 was from Bie & Berntsen, Denmark (cat. no. 1.00731). We used Thermo Wellwash AC (Thermo Electron Corporation, USA) for washing the plates and Biotech Synergy HT (Biotech, USA) to measure absorbance at 400 or 450 nm. All materials and chemicals are described in the Biogen Idec protocol: ‘Assay procedure to determine natalizumab (Tysabri) immunogenicity (CST02180AP-R.3)’. All samples and controls were tested at 1:10 dilutions in the ELISA. The requirement for a positive sample by screening was that the analysed sample’s optical density (OD) value was greater than or equal to 0.5 mg/ml (QC2) of the monoclonal anti-natalizumab antibody (12C4) and the ratio of OD or the competition result divided by the OD of the screening result was lower than or equal to 0.5. Persistently positive patients were defined as patients with at least two positive samples taken at an interval of at least 12 weeks and no following negative samples. A transiently positive patient was defined as a patient with a positive sample followed by a subsequent negative sample taken at an interval of at least 12 weeks and no following positive samples.

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Multiple Sclerosis Journal 17(9)

In order to validate the inter-assay variation between the four laboratories 20 samples chosen from patients tested for anti-natalizumab antibodies at the Neuroimmunology Laboratory, Danish Multiple Sclerosis Center, Copenhagen were evaluated blindly in all four laboratories. The blood sampling had been performed between June 2006 and June 2009 and had been selected to include both negative samples (N ¼ 10) and positive samples (N ¼ 10) with low and high concentrations of anti-natalizumab antibodies.

Results A total of 4873 patients fulfilled the inclusion criteria: The Norwegian MS Competence Centre, Haukeland University Hospital, Bergen, Norway: 235 patients; Bochum MS Clinic, Bochum, Germany: 3526 patients; Danish Multiple Sclerosis Center, Copenhagen, Denmark: 399 patients; and NAb lab, MS Research Group, Department of Clinical Neuroscience, Stockholm, Sweden: 713 patients. Patient demographics are shown in Table 1. Information about the anti-IFN-b neutralizing antibody (NAb) status was available in the Danish patient cohort: 98 out of 298 patients (32.9%) had developed NAbs during IFN-b treatment (defined as at least one positive NAb test), of whom seven (7.6%) developed

anti-natalizumab antibodies. In comparison, 14 out of 199 (7.0%) IFN-b NAb negative patients developed anti-natalizumab antibodies. In all, 95.5% (95% confidence interval, CI: 94.9– 96.1%) of the patients remained persistently antibody negative (Table 2), whereas on average 4.5% (95% CI: 4.0 - 5.1%) were antibody positive at anytime (range 4.0–7.7%). The antibody positive patients included 3.5% (95% CI: 3.0–4.0%) (range 2.4–5.1%) persistently antibody positive, and 1.0% (95% CI: 0.7– 1.3%) (range 0.4–3.0%) transiently antibody positive (Table 2). The 20 blindly evaluated samples were classified identically as positive or negative in all cases but one. This was a sample with low-level antibodies that tested positive in Copenhagen but negative in the other three laboratories.

Discussion The occurrence of antibodies against natalizumab did not vary significantly across the four participating centres (Table 1). The same validated assay was used in all four laboratories in order to minimize the inter-laboratory variation. All four laboratories had blindly been analysing the same 20 quality control samples from the Danish Multiple Sclerosis Center, Copenhagen. Indeed,

Table 1. Patient demographics

Age (years), median (range) Sex (men/women) Race (Caucasian/other) Disease duration (years), median (range) Previous IFN-b treatment, proportion (%)

Bergen N ¼ 235

Bochum N ¼ 3526

Copenhagen N ¼ 399

Stockholm N ¼ 713

All N ¼ 4873

40 (14–68) 70/165 233/2 NA NA

40 (15–75) 1011/2515 3524/2 NA NA

40 (16–65) 128/271 399/0 9.3 (0–37) 355/399 (89.0%)

39 (14–67) 212/501 674/39 9.0 (1–37) 575/713 (80.8 %)

40 (14–75) 1421/3452 4,830/43 9.1 (0–37)* 930/1112 (83.6)*

*N ¼ 1112.

Table 2. Percentage antibody-positive and antibody-negative patients with 95% confidence intervals (CI) in consecutive patients who after at least 3 months of natalizumab therapy had two or more tests for antibodies against natalizumab performed with an interval of at least 3 months

Bergen (N ¼ 235) Bochum (N ¼ 3526) Copenhagen (N ¼ 399) Stockholm (N ¼ 713) All (N ¼ 4873)

Start of antibody testing

Persistently antibody-negative patients N (%; 95% CI)

Antibody-positive patients N (%; 95% CI)

Persistently antibody-positive patients N (%; 95% CI)

Transiently antibody-positive patients N (%; 95% CI)

October 2006 June 2006 August 2006 August 2006 June–October 2006

217 3384 371 680 4660

18 142 28 33 221

12 127 16 17 172

6 15 12 16 49

(92.3; (96.0; (93,0; (95.4; (95.5;

88.9–95.7) 95.3–96.6) 90.5–95.5) 93.9–97.0) 94.9–96.1)

(7.7; (4.0; (7,0; (4.6; (4.5;

4.4–11.1) 3.4–4.7) 4.5–9.5) 3.1–6.1) 4.0–5.1)

(5.1; (3.6; (4,0; (2.4; (3.5;

2.3–7.9) 3.0–4.2) 2.1–5.9) 1.3–3.5) 3.0–4.0)

(2.6; (0.4; (3,0; (2.2; (1.0;

0.3–4.0) 0.2–0.7) 1.3–4.7) 1.1–3.3) 0.7–1.3)

Sørensen et al. the concurrent results of antibody measurement of these samples indicated that a satisfactory standardization was achieved. The discrepancy in the test result of one test sample close to the OD value of QC2 might be due to variations between laboratories in the QC2 batches used or different storing and transportation conditions of reagents and test samples. Our finding of 4.5% anti-natalizumab antibody positive patients is lower than the frequencies reported in the AFFIRM and SENTINEL studies.12 In the AFFIRM study 57 out of 625 patients (9.1%; 95% CI: 6.9–11.4%) tested positive of whom 37 patients (5.9%; 95% CI: 4.1–7.8%) were persistently positive and 20 patients (3.2%; 95% CI: 1.8–4.6%) were transiently positive.3 In the SENTINEL study 12% of the patients tested positive for antibodies at any time during the study; approximately 6.5% were persistently and 5.5% transiently positive.12 However, the 95% CI for persistently antibody positive patients in our study (3.5%; 95% CI: 3.0–4.0%) and in the AFFIRM study (5.9%; 95% CI: 4.1–7.8%) were almost overlapping3. We were able to corroborate the previously reported observations from two of the participating centres, Stockholm and Copenhagen, that there seems to be no association between previous development of NAbs against IFN-b and occurrence of antibodies to natalizumab.13,14 In the majority of patients in both the AFFIRM study (88%) and SENTINEL study (96%) anti-natalizumab antibodies were detectable by 12 weeks and the remaining antibody positive patients exhibited detectable antibodies by week 24 apart from one single patient, who first became antibody positive by week 36 after initiation of natalizumab therapy.12 In our study eligible patients should have a test performed at week 24 or later after treatment start, and all permanently antibody positive patients should in all probability have been detected and classified as antibody positive patients. However, if some patients were tested only once in the first year with a negative test, and had a second negative test at a time point after 12 months, these persistently antibody negative patients would not be included in our study. This could introduce a bias towards enrichment of the study with antibody positive patients. Contrary, if some patients had their first anti-natalizumab antibody test performed at six months or later after the start of natalizumab therapy, these patients may have been antibody positive before and have reverted to antibody negativity. We did not require systematically testing for antibodies at three months after initiation of natalizumab treatment, and some patients might have been antibody positive at three months and antibody negative at six months. These patients would be false negative patients and should correctly have

1077 been classified as transiently antibody positive. This is in accordance with our findings of lower frequencies of transient antibody positive patients, in particular in Bochum where testing at three months was not routinely used. Further, in daily practice natalizumab is discontinued in patients with two positive antibody tests separated by an interval of at least 12 weeks, whereas in the AFFIRM study antibody positive patients continued therapy and had the possibility of reverting to antibody negative status.12 In the AFFIRM study 19 persistently positive patients were treated for the full two years of the study at which time 10 patients (53%) had reverted to antibody negative status, some of them during the second year of treatment.12 In our study such patients would have been classified as persistently antibody positive, and natalizumab treatment would have been terminated. Discontinuation of treatment in persistent antibody positive patients is recommended based on the observation in the AFFIRM trial that showed that the relapse rate in persistent antibody positive patients was similar to that seen in the placebo treated patients3. Hence, it cannot be recommended to continue natalizumab therapy in these patients, who are virtually untreated, when other effective treatments, e.g. fingolimod and, in some countries, cladribine, are available. In conclusion, the results from our study of close to 5000 patients systematically tested for anti-natalizumab antibodies confirm the occurrence of antibodies in patients treated with natalizumab. The frequency of persistent antibodies might be lower than that of 6% reported in the pivotal trials of natalizumab. Acknowledgement The authors wish to acknowledge the support of Susan Goelz, PhD, Director, Neurology at Biogen Idec in establishing the ELISA assay for natalizumab antibodies.

Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflict of interest statement None declared.

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