Wearing-off at the end of natalizumab dosingintervals is associated with low receptoroccupancyGerd Haga Bringeland MD Nello Blaser PhD Kjell-Morten Myhr MD PhD
Christian Alexander Vedeler MD PhD and Sonia Gavasso PhD
AbstractObjectiveWe aimed to investigate whether wearing-off symptoms at the end of the natalizumab dosinginterval were associated with clinical and demographic patient characteristics or natalizumabreceptor occupancy (RO) on leukocytes
MethodsIn this cross-sectional study of 40 patients with relapsing-remitting MS (RRMS) receivingnatalizumab at the Department of Neurology Haukeland University Hospital we recordedclinical and demographic data including age body mass index (BMI) working status smokinghabits disease characteristics treatment duration vitamin D levels and wearing-off symptomsWe quantified neurofilament light chain in serum and measured natalizumab RO in leukocytesubtypes by high-parameter mass cytometry Associations with wearing-off symptoms wereanalyzed
ResultsEight (200) patients who reported regular occurrence of wearing-off symptoms 9 (225)who sometimes had wearing-off symptoms and 23 (575) who did not have wearing-offsymptoms were evaluated Patients who regularly had wearing-off symptoms had lower nata-lizumab RO than patients who reported having such symptoms sometimes or never Theformer group also had higher BMI and higher frequency of sick leave High BMI was associatedwith low RO No other demographic or disease characteristics were associated with thephenomenon
ConclusionsLow RO may explain the wearing-off phenomenon observed in some patients with RRMStreated with natalizumab and high BMI may be the underlying cause
From the Neuro-SysMed (GHB K-MM CAV SG) Department of Neurology Haukeland University Hospital Bergen Norway Department of Clinical Medicine (GHB K-MMCAV SG) University of Bergen Bergen Norway and Department of Informatics (NB) University of Bergen Bergen Norway
Go to NeurologyorgNN for full disclosures Funding information is provided at the end of the article
The Article Processing Charge was funded by the authors
This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40 (CC BY-NC-ND) which permits downloadingand sharing the work provided it is properly cited The work cannot be changed in any way or used commercially without permission from the journal
Copyright copy 2020 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1
Natalizumab (Tysabrireg Biogen Cambridge MA) is a thera-peutic monoclonal antibody used to treat patients withrelapsing-remitting MS (RRMS) It prevents leukocyte mi-gration across the blood-brain barrier into the CNS bybinding to the α4 subunit of the α4β1 integrin (α4 integrin) onleukocyte surfaces1 Natalizumab is administered IV ata standard dose of 300 mg every 4 weeks
Although highly efficacious in preventing disease activitymany patients report the so-called wearing-off symptoms atthe end of the 4-week dosing interval Although wearing-offsymptoms are often reported only a few previous studies havedescribed the phenomenon and little is known about theunderlying causes of these symptoms2ndash5
Natalizumab receptor occupancy (RO) is defined as the levelof natalizumab bound to α4 integrin on leukocytes and isa potential biomarker to monitor and individualize natalizu-mab therapy6 RO has traditionally been measured with flowcytometry Mass cytometry is a novel technology for high-parameter single-cell analysis For mass cytometry detectionantibodies are conjugated to metals instead of fluorophoresallowing analysis of over 40 parameters simultaneously onsingle cells7 This permits measurement of RO in conjunctionwith more markers and in more cell types of interest than iscurrently possible by flow cytometry We aimed to investigatewhether clinical and demographic patient characteristics ornatalizumab RO were associated with the wearing-off phe-nomenon by using high-parameter mass cytometry to mea-sure natalizumab RO in patients with RRMS treated withnatalizumab who do and do not report wearing-off symptomsat the end of dosing intervals
MethodsPatientsWe invited all patients older than 18 years with a diagnosis ofRRMS who had received a minimum of 6 natalizumab infu-sions at the Department of Neurology Haukeland UniversityHospital (n = 45) to participate in this cross-sectional study 40agreed to participate At inclusion we obtained baseline de-mographic and clinical patient characteristics from the patientsrsquomedical journal including age sex disease duration (years sincefirst MS symptoms) natalizumab treatment duration (yearssince first natalizumab infusion) numbers of new MRI lesionsand clinical relapses in the last year before inclusion serumvitamin D level Symbol Digit Modalities Test score8 andExpanded Disability Status Scale score9 Each patient filled inquestionnaires on fatigue (Fatigue Severity Scale)10 and onworking status smoking habits weight height and whether
they had wearing-off symptoms (never sometimes and regu-larly) and if applicable type of symptoms
Standard protocol approvals registrationsand patient consentsThe study was approved by the Regional Committee forMedical Research Ethics Western Norway (REK 2016579)and written informed consent was obtained from all partici-pating patients
Blood samplesAt inclusion we collected blood before and after natalizumabinfusion For mass cytometry analysis whole blood was col-lected in heparinized Vacutainer tubes (Greiner Bio-OneGmbH Kremsmunster Austria) incubated with ProteomicStabilizer (Smart Tube Inc San Carlos CA) for 10 minutesand stored at minus80degC Whole blood was then thawed and redblood cell lysis was performed with Thaw-lyse buffer I (SmartTube Inc) to obtain peripheral blood leukocytes (PBLs) Forneurofilament measurement whole blood was collected inVacutainer tubes with no additives (BD Plymouth UnitedKingdom) incubated at room temperature for 60 minutesand centrifuged at room temperature at 3200g for 13 minutesbefore the serum was retrieved and stored at minus80degC
Neurofilament measurementSerum samples were thawed and the concentration of neu-rofilament light chain (NF-L) was measured with a single-molecule array (Simoa) assay (Quanterix Billerica MA)according to the manufacturerrsquos protocol
Mass cytometry RO assayPBLs were stained with a 36-parameter mass cytometry an-tibody panel (table e-1 linkslwwcomNXIA190) Boundnatalizumab was detected with an anti-IgG4 antibody (con-jugated to 169Tm) Total α4 integrin was detected with ananti-CD49d antibody (conjugated to 141Pr) specific for a dif-ferent epitope than natalizumab The 36 metal-conjugatedantibodies were purchased preconjugated (Fluidigm SouthSan Francisco CA) or antibodies were purchased (BioL-egend San Diego CA RampD Systems Minneapolis MN andAbcam Cambridge Great Britain) and conjugated in-houseto metals with the Maxpar Antibody Labeling Kit (Fluidigm)Briefly we thawed barcoded (Cell-ID 20-Plex Pd BarcodingKit Fluidigm) and pooled PBL samples in batches of 20randomly distributed samples keeping paired samples fromthe same patients in the same batch A control PBL samplefrom 1 healthy donor was included in each batch PooledPBLs were first incubated in Maxpar cell stain buffer with 100UmL heparin (LEO Pharma AS) for 20 minutes at roomtemperature11 and then incubated with the antibody cocktail
GlossaryBMI = body mass index cDC = conventional dendritic cell EID = extended interval dosing PBL = peripheral blood leukocytePML = progressive multifocal leukoencephalopathy RRMS = relapsing-remitting MS RO = receptor occupancy
2 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
for 30 minutes at room temperature Stained PBLs werewashed fixed in fresh 2 paraformaldehyde (Thermo Sci-entific Waltham MA) in Maxpar PBS for 10 minutes at roomtemperature and then incubated in 125 nM Cell-IDTM
Intercalator-Ir in Maxpar Fix and Perm Buffer (Fluidigm) at4degC overnight We performed all centrifugation steps at roomtemperature at 800g
Before mass cytometry analysis PBLs were resuspended in01times EQ Four Element Calibration Beads (Fluidigm) inMaxpar cell acquisition solution (Fluidigm) and filtered(Corning Falcon Test Tube with Cell Strainer Snap Cap
Fisher Scientific Hampton NN) We used antibody bindingQSC beads (Bangs Laboratories Inc catalog number 815AFishers IN) to standardize signal intensities from anti-IgG4(conjugated to 169Tm) and anti-CD49d (conjugated to 141Pr)as previously described in detail12We analyzed PBL andQSCbeads with the same standard settings on a Heliosreg masscytometer (Fluidigm) after tuning (CyTOF Tuning SolutionFluidigm) and calibration (EQ Four Element CalibrationBeads Fluidigm) according to the manufacturersquos guidelinesHealthy control PBLs served as a negative control for anti-IgG4 in the absence of natalizumab and patient PBLs in-cubated in vitro with natalizumab to an expected RO of 100
Table 1 Demographic and clinical characteristics of patients with RRMS and the frequency of wearing-off symptoms
Total
Wearing-off symptoms
Never Sometimes RegularlypValuea
p Value (ageadjusted)b
Patients with RRMS n () 40 (100) 23 (575) 9 (225) 8 (200)
Age y 430 (340ndash493) 450(355ndash520)
340(310ndash510)
430(370ndash438)
0382 mdash
Sex female n () 25 (625) 15 (652) 3 (333) 7 (875) 0069 0063
Abbreviations BMI = body mass index EDSS = Expanded Disability Status Scale FSS = Fatigue Severity Scale SDMT = Symbol Digit Modalities Test NF-L =neurofilament light chain RRMS = relapsing-remitting MSNumbers are median (interquartile range) unless otherwise stateda Unadjusted p values are calculated using a Kruskal-Wallis testb Age-adjusted p values are calculated with a likelihood ratio test between a linear model with only age and a linear model with age and the relevant baselinevariable as predictors
NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 3
served as positive controls Further quality control experi-ments were performed as previously described in detail12
Data processing analysis and statisticsAfter acquisition of QSC beads we normalized (Fluidigmnormalizer) and exported FCS files to Cytobank software(Cytobank Inc Beckman Coulter Brea CA) and created QSCbead standard curves (QuickCal template Bangs Laboratories)After acquisition of PBL samples we normalized (Fluidigmnormalizer) debarcoded (Fluidigm Debarcoder) and exportedthe FSC files to Cytobank software for gating and downstreamanalysis (figure e-1 linkslwwcomNXIA190) We performedclean-up gating to obtain single PBLs and the data were arcsinhtransformed with a scale argument of 513 We then performed 2independent analyses for RO calculation in single PBLs onewas a manual analysis and one used an unsupervised approach
In the manual approach we first identified the following 11leukocyte subtypes of interest by manual gating CD8+ centralmemory (TCM) effector memory (TEM) effector memoryRA (TEMRA) T cells CD4+ TEM TCM and TEMRA cellsCD34+ cells memory B cells natural killer (NK) cellsmonocytes and conventional dendritic cells (cDCs)We thenplotted the signal intensities (dual count 90th percentiles) ofanti-IgG4 and anti-CD49d in each of these cell types with theQSC bead standard curves to obtain bead standardized valuesand calculated RO by the following formula
RO = 100 timesQSC bead standardized anti minus IgG4 eth169TmTHORNQSC bead standardized anti minusCD49d eth141PrTHORN
We compared ROs in the leukocyte subtypes in differentpatient groups using a Kruskal-Wallis test
In the unsupervised approach we used R (version 343) to addan extra variable into the FSC files the ratio between signalintensities of anti-IgG4 and anti-CD49d in each cell Thisresulted in an RO estimate for each cell For visualization ofhigh-dimensional single-cell data we performed automated di-mensionality reduction with stochastic neighborhood embed-ding (viSNE Cytobank)14 We analyzed the new RO variablewith the cluster identification characterization and regressiontool CITRUS (Cytobank) an algorithm that automatically
identifies statistically significant differences between patientgroups15We applied the correlativemodel SignificanceAnalysisof Microarrays with a false discovery rate (adjusted for multiplehypothesis testing) of 1 CITRUS was run with 10 repetitions
The relationship between baseline demographic variables andwearing-offwas compared using a Kruskal-Wallis test Statisticaldifferences with p lt 005 were considered significant usinga 2-sided comparison To test the age-corrected relationshipbetween baseline variables and wearing-off we used a likelihoodratio test between a linear model with only age and a linearmodel with age and the relevant baseline variable as predictorsWe conducted a linear regression of the association betweenRO and body mass index (BMI) and used a t test to assesswhether the slope was significantly different from zero We usedR version 34316 for statistical analysis and correlation plots
Data availabilityFCS files from anonymized patient PBL samples can beaccessed in the Flow Repository (ID FR-FCM-Z2A9)
ResultsPatient characteristicsOf the 45 patients who were eligible for inclusion 40 (89)consented to participate in the study Of 5 (11) non-consenting patients 1 refused participation and 4 had infusiontime points outside of the opening hours of the routine labo-ratory where blood samples were collected Eight (200) ofthe 40 participating patients reported having wearing-offsymptoms regularly at the end of every dosing interval 9(225) sometimes did and 23 (575) reported never havingwearing-off symptoms (table 1) Themost frequent wearing-offsymptom was fatigue (table 2) Patients who regularly hadwearing-off symptoms had significantly higher BMI and higherfrequency of sick leave than patients who never or onlysometimes experienced such symptoms (table 1) After ageadjustment weight was also significantly increased in patientswith wearing-off symptoms regularly whereas height was in-creased in patients with symptoms only sometimes None ofthe other demographic or clinical patient characteristics weresignificantly different between the groups and none of theincluded patients had clinical relapses or new lesions onMRI inthe year before inclusion in the study Age-adjusted medianserum NF-L levels were similar between groups There was noassociation between NF-L and BMI (data not shown)
Receptor occupancyManual gating of PBLs correlated well with the automatedmapping with viSNE (figure 1A) We observed a broad range ofnatalizumab RO values The median RO values in all leukocytesubtypes before infusion and in 10 of 11 leukocyte subtypesafter infusion (figure 1B) were lower in patients who regularlyexperienced wearing-off symptoms than in patients who neveror only sometimes experienced such symptoms The differenceswere statistically significant in CD8+ TEM CD4
+ TEM and
Table 2 Frequency of reported wearing-off symptoms
Symptom
Wearing-off symptoms
Sometimes Regularly
Fatigue 67 63
Psychological 33 25
Walking difficulty 11 25
Spasms 11 13
Pain 0 13
4 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
Figure 1Natalizumab receptor occupancy (RO) in patients reportingwearing-off symptoms never sometimes or regularly
(A) Manually gated PBLs visualized on a viSNEmap ROwas analyzed in 11 cell subtypes CD8+ central memory (TCM) effectormemory (TEM) effectormemoryRA (TEMRA) cells CD4
+ TEM TCM and TEMRA cells CD34+ cells memory B cells natural killer (NK) cells monocytes and conventional dendritic cells (cDCs)Neutrophils were not included in RO analysis (B) Spider plot ofmedian RO values in 11 cell subtypes in patients before and after natalizumab infusion (C) ROvalues in 11 cell subtypes before and after natalizumab infusion p values (Kruskal-Wallis test) comparing ROs in different wearing-off groups (D) Left medianRO values in cell clusters significantly different betweenwearing-off groups (SAM analysis in CITRUS) Right significant cell clusters are visualized on the viSNEmap PBL = peripheral blood leukocyte SAM = Significance Analysis of Microarrays
NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 5
CD4+ TEMRA cells before infusion and in CD8+ TCM CD8+
TEMRA CD4+ TCM cells NK cells monocytes and cDCs after
infusion (figure 1C) Furthermore in CD8+ and CD4+ T cellsnot stratified into subtypes RO was significantly lower in bothCD8+ and CD4+ T cells before infusion and in CD8+ T cellsafter infusion in patients who regularly had wearing-off symp-toms (figure e-2 linkslwwcomNXIA190) Unsupervisedanalysis of median ROs with CITRUS also showed lower me-dian natalizumab ROs in patients with regular wearing-offsymptoms (figure 1D) Neither of the 2 analysis approachesshowed significant differences between patients reportingwearing-off symptoms sometimes and never
High BMI was associated with significantly lower RO in CD8+
TEM cells and cDCs before infusion and in CD8+ TEMRA cellsCD34+ cells and monocytes after infusion (figure 2)
DiscussionNatalizumab prevents disease activity in RRMS and haspositive effects on subjective symptoms such as mood fatigueand cognitive function1718 However subjective wearing-offsymptoms at the end of the 4-week interval are frequentlyreported by patients In this study wearing-off symptomswere reported by 425 of the patients (200 regularly and225 only sometimes) We found lower natalizumab ROs inpatients who regularly experienced wearing-off symptomscompared with patients who reported such symptoms neveror only sometimes The result was replicated in 2 separatedata analysis pipelines Furthermore patients who reportedregularly experiencing wearing-off symptoms had highermedian body weight and BMI and higher sick leave frequencythan those who rarely or never experienced such symptomsMedian height was increased in patients with symptoms onlysometimes Other clinical and demographic factors were
similar between the patient groups High BMI was associatedwith low RO in several leukocyte subtypes
The main limitation of our study is the small patient cohort Asa consequence of this limited statistical power we were onlyable to detect large effects and acknowledge that there may beassociations of smaller effect size that went unnoticedWearing-off symptoms were less frequent in our study than the preva-lence of 54ndash63 reported in other studies but as previouslyreported the most frequent wearing-off symptom wasfatigue2ndash5 In contrast to our results a recent study found noassociation between the wearing-off effect and natalizumab ROor patient characteristics5 The previously reported study usedflow cytometry to measure RO in CD8+ TEM cells and CD8+
effector T cells By using high-parameter mass cytometry wewere able to measure RO in 11 cell subtypes simultaneouslyWe found that patients who regularly experienced wearing-offsymptoms had higher BMI than those who did not and we alsoobserved an association between high BMI and low RO Bodyweight was higher in our cohort than in the previous study5
(median 750 vsmean 729 kg) andwas evenmore pronouncedin the groupwith wearing-off symptoms regularly (median 815vs mean 746 kg) van Kempen et al5 reported nonsignificanttrends similar to our significant results Our wearing-off pop-ulation had higher body weight than theirs which may explainwhy we observed statistically significant differences despite oursmall cohort size The association between high BMI and lowRO suggests that high BMI by decreasing natalizumab ROmay be the underlying cause of the wearing-off phenomenonOthers have previously reported such an association betweenlow natalizumab RO and high body weight or BMI and somehave suggested that the dose of natalizumab should be adjustedfor body weight61920 We found no associations betweenwearing-off symptoms and disease activity markers such asclinical relapses new lesions detected by MRI in the past yearor serum NF-L levels21
Figure 2 Linear regression analysis demonstrates an association between RO and BMI
Plot of receptor occupancies (A) before and (B) after infusion for indicated cell types as a function of BMI Solid lines have slopes that are significantly differentfrom zero (p lt 005) and dashed lines have slopes that are not significantly different from zero BMI = body mass index RO = receptor occupancy
6 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
As also reported by others19 we observed large interindividualvariations in natalizumab RO and not all patients with lowRO had high BMI A previous study reported that bodyweight only partly predicts variability in natalizumab RO andsuggested other factors such as density and turnover of α4β1integrin that may drive the variability22
It has been hypothesized that cytokines could induce thewearing-off symptoms45 We speculate that lower natalizu-mab RO at the end of the dosing interval could increase themigratory capacity of cytokine-producing leukocytes into theCNS resulting in the wearing-off symptoms We found sig-nificantly lower RO in the T-cell subtypes CD8+ TEM CD4
+
TEM and CD4+ TEMRA cells before infusion in patients whoregularly had wearing-off symptoms TEM and TEMRA cellshome to the site of inflammation where they have effectorfunctions such as secretion of proinflammatory cytokines andcytotoxicity in contrast TCM cells and naive T cells home tosecondary lymphoid organs2324
Our results suggest that low RO may be a contributing factorto the wearing-off phenomenon and that higher BMI may bean underlying cause This supports the suggestion that nata-lizumab dosing should be personalized Personalization hasbeen mainly focused on extended interval dosing (EID)19
This has particularly been driven by the risk of progressivemultifocal leukoencephalopathy (PML) a serious complica-tion of natalizumab therapy in patients previously exposed toJC virus25 Retrospective studies of off-label treatment withEID have shown maintained efficacy2627 and reduced PMLrisk28 compared with standard interval dosing Howeverthese studies are limited by possible selection bias due tononrandomized design and the efficacy and safety of EID isnot fully known As patients who regularly experiencewearing-off symptoms already have lower RO and reportsymptoms at the end of dosing intervals extending dosingintervals could increase the risk of disease activity Wetherefore do not recommend EID in patients reportingwearing-off symptoms regularly as this could lead to an evenlower RO at the end of the interval than observed withstandard dosing619 Further studies should investigatewhether wearing-off symptoms are associated with increasedrisk of RRMS disease activity and whether increasing RO byreduced dosing intervals or weight loss may mitigate thesymptoms
AcknowledgmentThe authors thank Fritz og Ingrid Nilsens legat for financialresearch support and Hanne Linda Nakkestad at theNeurological Research Laboratory at Haukeland UniversityHospital for performing SiMOA analyses The masscytometry was performed at the Flow Cytometry CoreFacility Department of Clinical Science University of BergenThe Helios mass cytometer was funded by the BergenResearch Foundation Neuro-SysMed is jointly hosted byHaukeland University Hospital and University of Bergen andsupported as a Centre for Clinical Treatment Research (FKB)
by grants from The Research Council of Norway projectnumber 288164
Study fundingSupported by Fritz og Ingrid Nilsens legat for Forskning paringMultippel sklerose
DisclosureGH Bringeland has received research support from Novartisand institutional support from the Regional Heath Authorityof Western Norway N Blaser reports no disclosures K-MMyhr has received grants and personal fees from Biogen andNovartis personal fees from Genzyme Roche Almirall andMerck personal fees and nonfinancial support from Tevaand a research grant from the Norwegian Research Council(grant number 288164) CA Vedeler has received in-stitutional support from the Regional Heath Authority ofWestern Norway and research grant support from the Nor-wegian Research Council (grant number 288164) Dr Gav-asso has received institutional support from the RegionalHeath Authority of Western Norway Go to NeurologyorgNN for full disclosures
Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationOctober 22 2019 Accepted in final form December 20 2019
Appendix Authors
Name Location Role Contribution
Gerd HagaBringelandMD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studymajor role in theacquisition of datainterpretation of dataanalysis of data andrevision of themanuscript forintellectual content
NelloBlaser PhD
University of BergenBergen Norway
Author Interpretation of dataanalysis of data andrevision of themanuscript forintellectual content
Kjell-MortenMyhr MDPhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyand revision of themanuscript forintellectual content
ChristianAlexanderVedelerMD PhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyand revision of themanuscript forintellectual content
SoniaGavassoPhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyinterpretation of dataand revision of themanuscript forintellectual content
NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 7
References1 Polman CH OrsquoConnor PW Havrdova E et al A randomized placebo-controlled trial
of natalizumab for relapsing multiple sclerosis N Engl J Med 2006354899ndash9102 Katz J Lathi E Heyda L Characterizing the Natalizumab ldquoWearing offrdquo Effect [ab-
stract] ACTRIMS meeting Poster DX36 20143 Gudesblatt M Zarif M Bumstead B et al Multiple sclerosis and natalizumab ldquobe-
tween the dose symptomsrdquo [abstract 982] Mult Scler 201218(suppl 14)P9824 Ratchford JN Brock-Simmons R Augsburger A et al Multiple sclerosis symptom
recrudescence at the end of the natalizumab dosing cycle Int J MS Care 20141692ndash985 van Kempen ZLE Doesburg D Dekker I et al The natalizumab wearing-off effect end
of natalizumab cycle recurrence of MS symptoms Neurology 201993e1579ndashe15866 Punet-Ortiz J Hervas-Garcia JV Teniente-Serra A et al Monitoring CD49d receptor
occupancy a method to optimize and personalize natalizumab therapy in multiplesclerosis patients Cytometry B Clin Cytom 201894327ndash333
7 Bandura DR Baranov VI Ornatsky OI et al Mass cytometry technique for real timesingle cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry Anal Chem 2009816813ndash6822
8 Smith A Symbol digit modalities test Manual Los Angeles CA Western Psycho-logical Services 1982
9 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444ndash1452
10 Krupp LB LaRocca NG Muir-Nash J Steinberg AD The fatigue severity scaleApplication to patients with multiple sclerosis and systemic lupus erythematosusArch Neurol 1989461121ndash1123
11 Rahman AH Tordesillas L Berin MC Heparin reduces nonspecific eosinophilstaining artifacts in mass cytometry experiments Cytometry A 201689601ndash607
12 Bringeland GH Bader L Blaser N et al Optimization of receptor occupancy assays inmass cytometry standardization across channels with QSC beads Cytometry A 201995314ndash322
13 Finak G Perez JM Weng A Gottardo R Optimizing transformations for automatedhigh throughput analysis of flow cytometry data BMC Bioinformatics 201011546
14 Amir el AD Davis KL Tadmor MD et al viSNE enables visualization of highdimensional single-cell data and reveals phenotypic heterogeneity of leukemia NatBiotechnol 201331545ndash552
15 Bruggner RV Bodenmiller B Dill DL Tibshirani RJ Nolan GP Automated identi-fication of stratifying signatures in cellular subpopulations P Natl Acad Sci USA 2014111E2770ndashE2777
16 R A Language and Environment for Statistical Computing [computer program]Vienna R Foundation for Statistical Computing 2017
17 Iaffaldano P Viterbo RG Paolicelli D et al Impact of natalizumab on cognitiveperformances and fatigue in relapsing multiple sclerosis a prospective open-labeltwo years observational study PLoS One 20127e35843
18 Morrow SA OrsquoConnor PW Polman CH et al Evaluation of the symbol digit mo-dalities test (SDMT) and MS neuropsychological screening questionnaire (MSNQ)in natalizumab-treated MS patients over 48 weeks Mult Scler 2010161385ndash1392
19 Foley JF Goelz S Hoyt T Christensen A Metzger RR Evaluation of natalizumabpharmacokinetics and pharmacodynamics with standard and extended interval dos-ing Mult Scler Relat Dis 20193165ndash71
20 Tanaka M Kinoshita M Foley JF Tanaka K Kira J Carroll WM Body weight-basednatalizumab treatment in adult patientswithmultiple sclerosis JNeurol 2015262781ndash782
21 Varhaug KN Torkildsen O Myhr KM Vedeler CA Neurofilament light chain asa biomarker in multiple sclerosis Front Neurol 201910338
22 Muralidharan KK Kuesters G Plavina T et al Population pharmacokinetics andtarget engagement of natalizumab in patients with multiple sclerosis J Clin Pharmacol2017571017ndash1030
23 Mahnke YD Brodie TM Sallusto F Roederer M Lugli E The whorsquos who of T-celldifferentiation human memory T-cell subsets Eur J Immunol 2013432797ndash2809
24 Sallusto F Geginat J Lanzavecchia A Central memory and effector memory T cellsubsets function generation and maintenance Annu Rev Immunol 200422745ndash763
25 Bloomgren G Richman S Hotermans C et al Risk of natalizumab-associated pro-gressive multifocal leukoencephalopathy N Engl J Med 20123661870ndash1880
26 Bomprezzi R Pawate S Extended interval dosing of natalizumab a two-center 7-yearexperience Ther Adv Neurol Disord 20147227ndash231
27 Zhovtis Ryerson L Frohman TC Foley J et al Extended interval dosing of natali-zumab in multiple sclerosis J Neurol Neurosurg Psychiatry 201687885ndash889
28 Ryerson LZ Foley J Chang I et al Risk of natalizumab-associated PML in patientswith MS is reduced with extended interval dosing Neurology 201993e1452ndashe1462
8 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
DOI 101212NXI000000000000067820207 Neurol Neuroimmunol Neuroinflamm
Gerd Haga Bringeland Nello Blaser Kjell-Morten Myhr et al occupancy
Wearing-off at the end of natalizumab dosing intervals is associated with low receptor
This information is current as of February 4 2020
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Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2020 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
Natalizumab (Tysabrireg Biogen Cambridge MA) is a thera-peutic monoclonal antibody used to treat patients withrelapsing-remitting MS (RRMS) It prevents leukocyte mi-gration across the blood-brain barrier into the CNS bybinding to the α4 subunit of the α4β1 integrin (α4 integrin) onleukocyte surfaces1 Natalizumab is administered IV ata standard dose of 300 mg every 4 weeks
Although highly efficacious in preventing disease activitymany patients report the so-called wearing-off symptoms atthe end of the 4-week dosing interval Although wearing-offsymptoms are often reported only a few previous studies havedescribed the phenomenon and little is known about theunderlying causes of these symptoms2ndash5
Natalizumab receptor occupancy (RO) is defined as the levelof natalizumab bound to α4 integrin on leukocytes and isa potential biomarker to monitor and individualize natalizu-mab therapy6 RO has traditionally been measured with flowcytometry Mass cytometry is a novel technology for high-parameter single-cell analysis For mass cytometry detectionantibodies are conjugated to metals instead of fluorophoresallowing analysis of over 40 parameters simultaneously onsingle cells7 This permits measurement of RO in conjunctionwith more markers and in more cell types of interest than iscurrently possible by flow cytometry We aimed to investigatewhether clinical and demographic patient characteristics ornatalizumab RO were associated with the wearing-off phe-nomenon by using high-parameter mass cytometry to mea-sure natalizumab RO in patients with RRMS treated withnatalizumab who do and do not report wearing-off symptomsat the end of dosing intervals
MethodsPatientsWe invited all patients older than 18 years with a diagnosis ofRRMS who had received a minimum of 6 natalizumab infu-sions at the Department of Neurology Haukeland UniversityHospital (n = 45) to participate in this cross-sectional study 40agreed to participate At inclusion we obtained baseline de-mographic and clinical patient characteristics from the patientsrsquomedical journal including age sex disease duration (years sincefirst MS symptoms) natalizumab treatment duration (yearssince first natalizumab infusion) numbers of new MRI lesionsand clinical relapses in the last year before inclusion serumvitamin D level Symbol Digit Modalities Test score8 andExpanded Disability Status Scale score9 Each patient filled inquestionnaires on fatigue (Fatigue Severity Scale)10 and onworking status smoking habits weight height and whether
they had wearing-off symptoms (never sometimes and regu-larly) and if applicable type of symptoms
Standard protocol approvals registrationsand patient consentsThe study was approved by the Regional Committee forMedical Research Ethics Western Norway (REK 2016579)and written informed consent was obtained from all partici-pating patients
Blood samplesAt inclusion we collected blood before and after natalizumabinfusion For mass cytometry analysis whole blood was col-lected in heparinized Vacutainer tubes (Greiner Bio-OneGmbH Kremsmunster Austria) incubated with ProteomicStabilizer (Smart Tube Inc San Carlos CA) for 10 minutesand stored at minus80degC Whole blood was then thawed and redblood cell lysis was performed with Thaw-lyse buffer I (SmartTube Inc) to obtain peripheral blood leukocytes (PBLs) Forneurofilament measurement whole blood was collected inVacutainer tubes with no additives (BD Plymouth UnitedKingdom) incubated at room temperature for 60 minutesand centrifuged at room temperature at 3200g for 13 minutesbefore the serum was retrieved and stored at minus80degC
Neurofilament measurementSerum samples were thawed and the concentration of neu-rofilament light chain (NF-L) was measured with a single-molecule array (Simoa) assay (Quanterix Billerica MA)according to the manufacturerrsquos protocol
Mass cytometry RO assayPBLs were stained with a 36-parameter mass cytometry an-tibody panel (table e-1 linkslwwcomNXIA190) Boundnatalizumab was detected with an anti-IgG4 antibody (con-jugated to 169Tm) Total α4 integrin was detected with ananti-CD49d antibody (conjugated to 141Pr) specific for a dif-ferent epitope than natalizumab The 36 metal-conjugatedantibodies were purchased preconjugated (Fluidigm SouthSan Francisco CA) or antibodies were purchased (BioL-egend San Diego CA RampD Systems Minneapolis MN andAbcam Cambridge Great Britain) and conjugated in-houseto metals with the Maxpar Antibody Labeling Kit (Fluidigm)Briefly we thawed barcoded (Cell-ID 20-Plex Pd BarcodingKit Fluidigm) and pooled PBL samples in batches of 20randomly distributed samples keeping paired samples fromthe same patients in the same batch A control PBL samplefrom 1 healthy donor was included in each batch PooledPBLs were first incubated in Maxpar cell stain buffer with 100UmL heparin (LEO Pharma AS) for 20 minutes at roomtemperature11 and then incubated with the antibody cocktail
GlossaryBMI = body mass index cDC = conventional dendritic cell EID = extended interval dosing PBL = peripheral blood leukocytePML = progressive multifocal leukoencephalopathy RRMS = relapsing-remitting MS RO = receptor occupancy
2 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
for 30 minutes at room temperature Stained PBLs werewashed fixed in fresh 2 paraformaldehyde (Thermo Sci-entific Waltham MA) in Maxpar PBS for 10 minutes at roomtemperature and then incubated in 125 nM Cell-IDTM
Intercalator-Ir in Maxpar Fix and Perm Buffer (Fluidigm) at4degC overnight We performed all centrifugation steps at roomtemperature at 800g
Before mass cytometry analysis PBLs were resuspended in01times EQ Four Element Calibration Beads (Fluidigm) inMaxpar cell acquisition solution (Fluidigm) and filtered(Corning Falcon Test Tube with Cell Strainer Snap Cap
Fisher Scientific Hampton NN) We used antibody bindingQSC beads (Bangs Laboratories Inc catalog number 815AFishers IN) to standardize signal intensities from anti-IgG4(conjugated to 169Tm) and anti-CD49d (conjugated to 141Pr)as previously described in detail12We analyzed PBL andQSCbeads with the same standard settings on a Heliosreg masscytometer (Fluidigm) after tuning (CyTOF Tuning SolutionFluidigm) and calibration (EQ Four Element CalibrationBeads Fluidigm) according to the manufacturersquos guidelinesHealthy control PBLs served as a negative control for anti-IgG4 in the absence of natalizumab and patient PBLs in-cubated in vitro with natalizumab to an expected RO of 100
Table 1 Demographic and clinical characteristics of patients with RRMS and the frequency of wearing-off symptoms
Total
Wearing-off symptoms
Never Sometimes RegularlypValuea
p Value (ageadjusted)b
Patients with RRMS n () 40 (100) 23 (575) 9 (225) 8 (200)
Age y 430 (340ndash493) 450(355ndash520)
340(310ndash510)
430(370ndash438)
0382 mdash
Sex female n () 25 (625) 15 (652) 3 (333) 7 (875) 0069 0063
Abbreviations BMI = body mass index EDSS = Expanded Disability Status Scale FSS = Fatigue Severity Scale SDMT = Symbol Digit Modalities Test NF-L =neurofilament light chain RRMS = relapsing-remitting MSNumbers are median (interquartile range) unless otherwise stateda Unadjusted p values are calculated using a Kruskal-Wallis testb Age-adjusted p values are calculated with a likelihood ratio test between a linear model with only age and a linear model with age and the relevant baselinevariable as predictors
NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 3
served as positive controls Further quality control experi-ments were performed as previously described in detail12
Data processing analysis and statisticsAfter acquisition of QSC beads we normalized (Fluidigmnormalizer) and exported FCS files to Cytobank software(Cytobank Inc Beckman Coulter Brea CA) and created QSCbead standard curves (QuickCal template Bangs Laboratories)After acquisition of PBL samples we normalized (Fluidigmnormalizer) debarcoded (Fluidigm Debarcoder) and exportedthe FSC files to Cytobank software for gating and downstreamanalysis (figure e-1 linkslwwcomNXIA190) We performedclean-up gating to obtain single PBLs and the data were arcsinhtransformed with a scale argument of 513 We then performed 2independent analyses for RO calculation in single PBLs onewas a manual analysis and one used an unsupervised approach
In the manual approach we first identified the following 11leukocyte subtypes of interest by manual gating CD8+ centralmemory (TCM) effector memory (TEM) effector memoryRA (TEMRA) T cells CD4+ TEM TCM and TEMRA cellsCD34+ cells memory B cells natural killer (NK) cellsmonocytes and conventional dendritic cells (cDCs)We thenplotted the signal intensities (dual count 90th percentiles) ofanti-IgG4 and anti-CD49d in each of these cell types with theQSC bead standard curves to obtain bead standardized valuesand calculated RO by the following formula
RO = 100 timesQSC bead standardized anti minus IgG4 eth169TmTHORNQSC bead standardized anti minusCD49d eth141PrTHORN
We compared ROs in the leukocyte subtypes in differentpatient groups using a Kruskal-Wallis test
In the unsupervised approach we used R (version 343) to addan extra variable into the FSC files the ratio between signalintensities of anti-IgG4 and anti-CD49d in each cell Thisresulted in an RO estimate for each cell For visualization ofhigh-dimensional single-cell data we performed automated di-mensionality reduction with stochastic neighborhood embed-ding (viSNE Cytobank)14 We analyzed the new RO variablewith the cluster identification characterization and regressiontool CITRUS (Cytobank) an algorithm that automatically
identifies statistically significant differences between patientgroups15We applied the correlativemodel SignificanceAnalysisof Microarrays with a false discovery rate (adjusted for multiplehypothesis testing) of 1 CITRUS was run with 10 repetitions
The relationship between baseline demographic variables andwearing-offwas compared using a Kruskal-Wallis test Statisticaldifferences with p lt 005 were considered significant usinga 2-sided comparison To test the age-corrected relationshipbetween baseline variables and wearing-off we used a likelihoodratio test between a linear model with only age and a linearmodel with age and the relevant baseline variable as predictorsWe conducted a linear regression of the association betweenRO and body mass index (BMI) and used a t test to assesswhether the slope was significantly different from zero We usedR version 34316 for statistical analysis and correlation plots
Data availabilityFCS files from anonymized patient PBL samples can beaccessed in the Flow Repository (ID FR-FCM-Z2A9)
ResultsPatient characteristicsOf the 45 patients who were eligible for inclusion 40 (89)consented to participate in the study Of 5 (11) non-consenting patients 1 refused participation and 4 had infusiontime points outside of the opening hours of the routine labo-ratory where blood samples were collected Eight (200) ofthe 40 participating patients reported having wearing-offsymptoms regularly at the end of every dosing interval 9(225) sometimes did and 23 (575) reported never havingwearing-off symptoms (table 1) Themost frequent wearing-offsymptom was fatigue (table 2) Patients who regularly hadwearing-off symptoms had significantly higher BMI and higherfrequency of sick leave than patients who never or onlysometimes experienced such symptoms (table 1) After ageadjustment weight was also significantly increased in patientswith wearing-off symptoms regularly whereas height was in-creased in patients with symptoms only sometimes None ofthe other demographic or clinical patient characteristics weresignificantly different between the groups and none of theincluded patients had clinical relapses or new lesions onMRI inthe year before inclusion in the study Age-adjusted medianserum NF-L levels were similar between groups There was noassociation between NF-L and BMI (data not shown)
Receptor occupancyManual gating of PBLs correlated well with the automatedmapping with viSNE (figure 1A) We observed a broad range ofnatalizumab RO values The median RO values in all leukocytesubtypes before infusion and in 10 of 11 leukocyte subtypesafter infusion (figure 1B) were lower in patients who regularlyexperienced wearing-off symptoms than in patients who neveror only sometimes experienced such symptoms The differenceswere statistically significant in CD8+ TEM CD4
+ TEM and
Table 2 Frequency of reported wearing-off symptoms
Symptom
Wearing-off symptoms
Sometimes Regularly
Fatigue 67 63
Psychological 33 25
Walking difficulty 11 25
Spasms 11 13
Pain 0 13
4 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
Figure 1Natalizumab receptor occupancy (RO) in patients reportingwearing-off symptoms never sometimes or regularly
(A) Manually gated PBLs visualized on a viSNEmap ROwas analyzed in 11 cell subtypes CD8+ central memory (TCM) effectormemory (TEM) effectormemoryRA (TEMRA) cells CD4
+ TEM TCM and TEMRA cells CD34+ cells memory B cells natural killer (NK) cells monocytes and conventional dendritic cells (cDCs)Neutrophils were not included in RO analysis (B) Spider plot ofmedian RO values in 11 cell subtypes in patients before and after natalizumab infusion (C) ROvalues in 11 cell subtypes before and after natalizumab infusion p values (Kruskal-Wallis test) comparing ROs in different wearing-off groups (D) Left medianRO values in cell clusters significantly different betweenwearing-off groups (SAM analysis in CITRUS) Right significant cell clusters are visualized on the viSNEmap PBL = peripheral blood leukocyte SAM = Significance Analysis of Microarrays
NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 5
CD4+ TEMRA cells before infusion and in CD8+ TCM CD8+
TEMRA CD4+ TCM cells NK cells monocytes and cDCs after
infusion (figure 1C) Furthermore in CD8+ and CD4+ T cellsnot stratified into subtypes RO was significantly lower in bothCD8+ and CD4+ T cells before infusion and in CD8+ T cellsafter infusion in patients who regularly had wearing-off symp-toms (figure e-2 linkslwwcomNXIA190) Unsupervisedanalysis of median ROs with CITRUS also showed lower me-dian natalizumab ROs in patients with regular wearing-offsymptoms (figure 1D) Neither of the 2 analysis approachesshowed significant differences between patients reportingwearing-off symptoms sometimes and never
High BMI was associated with significantly lower RO in CD8+
TEM cells and cDCs before infusion and in CD8+ TEMRA cellsCD34+ cells and monocytes after infusion (figure 2)
DiscussionNatalizumab prevents disease activity in RRMS and haspositive effects on subjective symptoms such as mood fatigueand cognitive function1718 However subjective wearing-offsymptoms at the end of the 4-week interval are frequentlyreported by patients In this study wearing-off symptomswere reported by 425 of the patients (200 regularly and225 only sometimes) We found lower natalizumab ROs inpatients who regularly experienced wearing-off symptomscompared with patients who reported such symptoms neveror only sometimes The result was replicated in 2 separatedata analysis pipelines Furthermore patients who reportedregularly experiencing wearing-off symptoms had highermedian body weight and BMI and higher sick leave frequencythan those who rarely or never experienced such symptomsMedian height was increased in patients with symptoms onlysometimes Other clinical and demographic factors were
similar between the patient groups High BMI was associatedwith low RO in several leukocyte subtypes
The main limitation of our study is the small patient cohort Asa consequence of this limited statistical power we were onlyable to detect large effects and acknowledge that there may beassociations of smaller effect size that went unnoticedWearing-off symptoms were less frequent in our study than the preva-lence of 54ndash63 reported in other studies but as previouslyreported the most frequent wearing-off symptom wasfatigue2ndash5 In contrast to our results a recent study found noassociation between the wearing-off effect and natalizumab ROor patient characteristics5 The previously reported study usedflow cytometry to measure RO in CD8+ TEM cells and CD8+
effector T cells By using high-parameter mass cytometry wewere able to measure RO in 11 cell subtypes simultaneouslyWe found that patients who regularly experienced wearing-offsymptoms had higher BMI than those who did not and we alsoobserved an association between high BMI and low RO Bodyweight was higher in our cohort than in the previous study5
(median 750 vsmean 729 kg) andwas evenmore pronouncedin the groupwith wearing-off symptoms regularly (median 815vs mean 746 kg) van Kempen et al5 reported nonsignificanttrends similar to our significant results Our wearing-off pop-ulation had higher body weight than theirs which may explainwhy we observed statistically significant differences despite oursmall cohort size The association between high BMI and lowRO suggests that high BMI by decreasing natalizumab ROmay be the underlying cause of the wearing-off phenomenonOthers have previously reported such an association betweenlow natalizumab RO and high body weight or BMI and somehave suggested that the dose of natalizumab should be adjustedfor body weight61920 We found no associations betweenwearing-off symptoms and disease activity markers such asclinical relapses new lesions detected by MRI in the past yearor serum NF-L levels21
Figure 2 Linear regression analysis demonstrates an association between RO and BMI
Plot of receptor occupancies (A) before and (B) after infusion for indicated cell types as a function of BMI Solid lines have slopes that are significantly differentfrom zero (p lt 005) and dashed lines have slopes that are not significantly different from zero BMI = body mass index RO = receptor occupancy
6 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
As also reported by others19 we observed large interindividualvariations in natalizumab RO and not all patients with lowRO had high BMI A previous study reported that bodyweight only partly predicts variability in natalizumab RO andsuggested other factors such as density and turnover of α4β1integrin that may drive the variability22
It has been hypothesized that cytokines could induce thewearing-off symptoms45 We speculate that lower natalizu-mab RO at the end of the dosing interval could increase themigratory capacity of cytokine-producing leukocytes into theCNS resulting in the wearing-off symptoms We found sig-nificantly lower RO in the T-cell subtypes CD8+ TEM CD4
+
TEM and CD4+ TEMRA cells before infusion in patients whoregularly had wearing-off symptoms TEM and TEMRA cellshome to the site of inflammation where they have effectorfunctions such as secretion of proinflammatory cytokines andcytotoxicity in contrast TCM cells and naive T cells home tosecondary lymphoid organs2324
Our results suggest that low RO may be a contributing factorto the wearing-off phenomenon and that higher BMI may bean underlying cause This supports the suggestion that nata-lizumab dosing should be personalized Personalization hasbeen mainly focused on extended interval dosing (EID)19
This has particularly been driven by the risk of progressivemultifocal leukoencephalopathy (PML) a serious complica-tion of natalizumab therapy in patients previously exposed toJC virus25 Retrospective studies of off-label treatment withEID have shown maintained efficacy2627 and reduced PMLrisk28 compared with standard interval dosing Howeverthese studies are limited by possible selection bias due tononrandomized design and the efficacy and safety of EID isnot fully known As patients who regularly experiencewearing-off symptoms already have lower RO and reportsymptoms at the end of dosing intervals extending dosingintervals could increase the risk of disease activity Wetherefore do not recommend EID in patients reportingwearing-off symptoms regularly as this could lead to an evenlower RO at the end of the interval than observed withstandard dosing619 Further studies should investigatewhether wearing-off symptoms are associated with increasedrisk of RRMS disease activity and whether increasing RO byreduced dosing intervals or weight loss may mitigate thesymptoms
AcknowledgmentThe authors thank Fritz og Ingrid Nilsens legat for financialresearch support and Hanne Linda Nakkestad at theNeurological Research Laboratory at Haukeland UniversityHospital for performing SiMOA analyses The masscytometry was performed at the Flow Cytometry CoreFacility Department of Clinical Science University of BergenThe Helios mass cytometer was funded by the BergenResearch Foundation Neuro-SysMed is jointly hosted byHaukeland University Hospital and University of Bergen andsupported as a Centre for Clinical Treatment Research (FKB)
by grants from The Research Council of Norway projectnumber 288164
Study fundingSupported by Fritz og Ingrid Nilsens legat for Forskning paringMultippel sklerose
DisclosureGH Bringeland has received research support from Novartisand institutional support from the Regional Heath Authorityof Western Norway N Blaser reports no disclosures K-MMyhr has received grants and personal fees from Biogen andNovartis personal fees from Genzyme Roche Almirall andMerck personal fees and nonfinancial support from Tevaand a research grant from the Norwegian Research Council(grant number 288164) CA Vedeler has received in-stitutional support from the Regional Heath Authority ofWestern Norway and research grant support from the Nor-wegian Research Council (grant number 288164) Dr Gav-asso has received institutional support from the RegionalHeath Authority of Western Norway Go to NeurologyorgNN for full disclosures
Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationOctober 22 2019 Accepted in final form December 20 2019
Appendix Authors
Name Location Role Contribution
Gerd HagaBringelandMD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studymajor role in theacquisition of datainterpretation of dataanalysis of data andrevision of themanuscript forintellectual content
NelloBlaser PhD
University of BergenBergen Norway
Author Interpretation of dataanalysis of data andrevision of themanuscript forintellectual content
Kjell-MortenMyhr MDPhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyand revision of themanuscript forintellectual content
ChristianAlexanderVedelerMD PhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyand revision of themanuscript forintellectual content
SoniaGavassoPhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyinterpretation of dataand revision of themanuscript forintellectual content
NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 7
References1 Polman CH OrsquoConnor PW Havrdova E et al A randomized placebo-controlled trial
of natalizumab for relapsing multiple sclerosis N Engl J Med 2006354899ndash9102 Katz J Lathi E Heyda L Characterizing the Natalizumab ldquoWearing offrdquo Effect [ab-
stract] ACTRIMS meeting Poster DX36 20143 Gudesblatt M Zarif M Bumstead B et al Multiple sclerosis and natalizumab ldquobe-
tween the dose symptomsrdquo [abstract 982] Mult Scler 201218(suppl 14)P9824 Ratchford JN Brock-Simmons R Augsburger A et al Multiple sclerosis symptom
recrudescence at the end of the natalizumab dosing cycle Int J MS Care 20141692ndash985 van Kempen ZLE Doesburg D Dekker I et al The natalizumab wearing-off effect end
of natalizumab cycle recurrence of MS symptoms Neurology 201993e1579ndashe15866 Punet-Ortiz J Hervas-Garcia JV Teniente-Serra A et al Monitoring CD49d receptor
occupancy a method to optimize and personalize natalizumab therapy in multiplesclerosis patients Cytometry B Clin Cytom 201894327ndash333
7 Bandura DR Baranov VI Ornatsky OI et al Mass cytometry technique for real timesingle cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry Anal Chem 2009816813ndash6822
8 Smith A Symbol digit modalities test Manual Los Angeles CA Western Psycho-logical Services 1982
9 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444ndash1452
10 Krupp LB LaRocca NG Muir-Nash J Steinberg AD The fatigue severity scaleApplication to patients with multiple sclerosis and systemic lupus erythematosusArch Neurol 1989461121ndash1123
11 Rahman AH Tordesillas L Berin MC Heparin reduces nonspecific eosinophilstaining artifacts in mass cytometry experiments Cytometry A 201689601ndash607
12 Bringeland GH Bader L Blaser N et al Optimization of receptor occupancy assays inmass cytometry standardization across channels with QSC beads Cytometry A 201995314ndash322
13 Finak G Perez JM Weng A Gottardo R Optimizing transformations for automatedhigh throughput analysis of flow cytometry data BMC Bioinformatics 201011546
14 Amir el AD Davis KL Tadmor MD et al viSNE enables visualization of highdimensional single-cell data and reveals phenotypic heterogeneity of leukemia NatBiotechnol 201331545ndash552
15 Bruggner RV Bodenmiller B Dill DL Tibshirani RJ Nolan GP Automated identi-fication of stratifying signatures in cellular subpopulations P Natl Acad Sci USA 2014111E2770ndashE2777
16 R A Language and Environment for Statistical Computing [computer program]Vienna R Foundation for Statistical Computing 2017
17 Iaffaldano P Viterbo RG Paolicelli D et al Impact of natalizumab on cognitiveperformances and fatigue in relapsing multiple sclerosis a prospective open-labeltwo years observational study PLoS One 20127e35843
18 Morrow SA OrsquoConnor PW Polman CH et al Evaluation of the symbol digit mo-dalities test (SDMT) and MS neuropsychological screening questionnaire (MSNQ)in natalizumab-treated MS patients over 48 weeks Mult Scler 2010161385ndash1392
19 Foley JF Goelz S Hoyt T Christensen A Metzger RR Evaluation of natalizumabpharmacokinetics and pharmacodynamics with standard and extended interval dos-ing Mult Scler Relat Dis 20193165ndash71
20 Tanaka M Kinoshita M Foley JF Tanaka K Kira J Carroll WM Body weight-basednatalizumab treatment in adult patientswithmultiple sclerosis JNeurol 2015262781ndash782
21 Varhaug KN Torkildsen O Myhr KM Vedeler CA Neurofilament light chain asa biomarker in multiple sclerosis Front Neurol 201910338
22 Muralidharan KK Kuesters G Plavina T et al Population pharmacokinetics andtarget engagement of natalizumab in patients with multiple sclerosis J Clin Pharmacol2017571017ndash1030
23 Mahnke YD Brodie TM Sallusto F Roederer M Lugli E The whorsquos who of T-celldifferentiation human memory T-cell subsets Eur J Immunol 2013432797ndash2809
24 Sallusto F Geginat J Lanzavecchia A Central memory and effector memory T cellsubsets function generation and maintenance Annu Rev Immunol 200422745ndash763
25 Bloomgren G Richman S Hotermans C et al Risk of natalizumab-associated pro-gressive multifocal leukoencephalopathy N Engl J Med 20123661870ndash1880
26 Bomprezzi R Pawate S Extended interval dosing of natalizumab a two-center 7-yearexperience Ther Adv Neurol Disord 20147227ndash231
27 Zhovtis Ryerson L Frohman TC Foley J et al Extended interval dosing of natali-zumab in multiple sclerosis J Neurol Neurosurg Psychiatry 201687885ndash889
28 Ryerson LZ Foley J Chang I et al Risk of natalizumab-associated PML in patientswith MS is reduced with extended interval dosing Neurology 201993e1452ndashe1462
8 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
DOI 101212NXI000000000000067820207 Neurol Neuroimmunol Neuroinflamm
Gerd Haga Bringeland Nello Blaser Kjell-Morten Myhr et al occupancy
Wearing-off at the end of natalizumab dosing intervals is associated with low receptor
This information is current as of February 4 2020
ServicesUpdated Information amp
httpnnneurologyorgcontent73e678fullhtmlincluding high resolution figures can be found at
This article has been cited by 2 HighWire-hosted articles
Subspecialty Collections
httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosisfollowing collection(s) This article along with others on similar topics appears in the
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httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
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Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2020 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
for 30 minutes at room temperature Stained PBLs werewashed fixed in fresh 2 paraformaldehyde (Thermo Sci-entific Waltham MA) in Maxpar PBS for 10 minutes at roomtemperature and then incubated in 125 nM Cell-IDTM
Intercalator-Ir in Maxpar Fix and Perm Buffer (Fluidigm) at4degC overnight We performed all centrifugation steps at roomtemperature at 800g
Before mass cytometry analysis PBLs were resuspended in01times EQ Four Element Calibration Beads (Fluidigm) inMaxpar cell acquisition solution (Fluidigm) and filtered(Corning Falcon Test Tube with Cell Strainer Snap Cap
Fisher Scientific Hampton NN) We used antibody bindingQSC beads (Bangs Laboratories Inc catalog number 815AFishers IN) to standardize signal intensities from anti-IgG4(conjugated to 169Tm) and anti-CD49d (conjugated to 141Pr)as previously described in detail12We analyzed PBL andQSCbeads with the same standard settings on a Heliosreg masscytometer (Fluidigm) after tuning (CyTOF Tuning SolutionFluidigm) and calibration (EQ Four Element CalibrationBeads Fluidigm) according to the manufacturersquos guidelinesHealthy control PBLs served as a negative control for anti-IgG4 in the absence of natalizumab and patient PBLs in-cubated in vitro with natalizumab to an expected RO of 100
Table 1 Demographic and clinical characteristics of patients with RRMS and the frequency of wearing-off symptoms
Total
Wearing-off symptoms
Never Sometimes RegularlypValuea
p Value (ageadjusted)b
Patients with RRMS n () 40 (100) 23 (575) 9 (225) 8 (200)
Age y 430 (340ndash493) 450(355ndash520)
340(310ndash510)
430(370ndash438)
0382 mdash
Sex female n () 25 (625) 15 (652) 3 (333) 7 (875) 0069 0063
Abbreviations BMI = body mass index EDSS = Expanded Disability Status Scale FSS = Fatigue Severity Scale SDMT = Symbol Digit Modalities Test NF-L =neurofilament light chain RRMS = relapsing-remitting MSNumbers are median (interquartile range) unless otherwise stateda Unadjusted p values are calculated using a Kruskal-Wallis testb Age-adjusted p values are calculated with a likelihood ratio test between a linear model with only age and a linear model with age and the relevant baselinevariable as predictors
NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 3
served as positive controls Further quality control experi-ments were performed as previously described in detail12
Data processing analysis and statisticsAfter acquisition of QSC beads we normalized (Fluidigmnormalizer) and exported FCS files to Cytobank software(Cytobank Inc Beckman Coulter Brea CA) and created QSCbead standard curves (QuickCal template Bangs Laboratories)After acquisition of PBL samples we normalized (Fluidigmnormalizer) debarcoded (Fluidigm Debarcoder) and exportedthe FSC files to Cytobank software for gating and downstreamanalysis (figure e-1 linkslwwcomNXIA190) We performedclean-up gating to obtain single PBLs and the data were arcsinhtransformed with a scale argument of 513 We then performed 2independent analyses for RO calculation in single PBLs onewas a manual analysis and one used an unsupervised approach
In the manual approach we first identified the following 11leukocyte subtypes of interest by manual gating CD8+ centralmemory (TCM) effector memory (TEM) effector memoryRA (TEMRA) T cells CD4+ TEM TCM and TEMRA cellsCD34+ cells memory B cells natural killer (NK) cellsmonocytes and conventional dendritic cells (cDCs)We thenplotted the signal intensities (dual count 90th percentiles) ofanti-IgG4 and anti-CD49d in each of these cell types with theQSC bead standard curves to obtain bead standardized valuesand calculated RO by the following formula
RO = 100 timesQSC bead standardized anti minus IgG4 eth169TmTHORNQSC bead standardized anti minusCD49d eth141PrTHORN
We compared ROs in the leukocyte subtypes in differentpatient groups using a Kruskal-Wallis test
In the unsupervised approach we used R (version 343) to addan extra variable into the FSC files the ratio between signalintensities of anti-IgG4 and anti-CD49d in each cell Thisresulted in an RO estimate for each cell For visualization ofhigh-dimensional single-cell data we performed automated di-mensionality reduction with stochastic neighborhood embed-ding (viSNE Cytobank)14 We analyzed the new RO variablewith the cluster identification characterization and regressiontool CITRUS (Cytobank) an algorithm that automatically
identifies statistically significant differences between patientgroups15We applied the correlativemodel SignificanceAnalysisof Microarrays with a false discovery rate (adjusted for multiplehypothesis testing) of 1 CITRUS was run with 10 repetitions
The relationship between baseline demographic variables andwearing-offwas compared using a Kruskal-Wallis test Statisticaldifferences with p lt 005 were considered significant usinga 2-sided comparison To test the age-corrected relationshipbetween baseline variables and wearing-off we used a likelihoodratio test between a linear model with only age and a linearmodel with age and the relevant baseline variable as predictorsWe conducted a linear regression of the association betweenRO and body mass index (BMI) and used a t test to assesswhether the slope was significantly different from zero We usedR version 34316 for statistical analysis and correlation plots
Data availabilityFCS files from anonymized patient PBL samples can beaccessed in the Flow Repository (ID FR-FCM-Z2A9)
ResultsPatient characteristicsOf the 45 patients who were eligible for inclusion 40 (89)consented to participate in the study Of 5 (11) non-consenting patients 1 refused participation and 4 had infusiontime points outside of the opening hours of the routine labo-ratory where blood samples were collected Eight (200) ofthe 40 participating patients reported having wearing-offsymptoms regularly at the end of every dosing interval 9(225) sometimes did and 23 (575) reported never havingwearing-off symptoms (table 1) Themost frequent wearing-offsymptom was fatigue (table 2) Patients who regularly hadwearing-off symptoms had significantly higher BMI and higherfrequency of sick leave than patients who never or onlysometimes experienced such symptoms (table 1) After ageadjustment weight was also significantly increased in patientswith wearing-off symptoms regularly whereas height was in-creased in patients with symptoms only sometimes None ofthe other demographic or clinical patient characteristics weresignificantly different between the groups and none of theincluded patients had clinical relapses or new lesions onMRI inthe year before inclusion in the study Age-adjusted medianserum NF-L levels were similar between groups There was noassociation between NF-L and BMI (data not shown)
Receptor occupancyManual gating of PBLs correlated well with the automatedmapping with viSNE (figure 1A) We observed a broad range ofnatalizumab RO values The median RO values in all leukocytesubtypes before infusion and in 10 of 11 leukocyte subtypesafter infusion (figure 1B) were lower in patients who regularlyexperienced wearing-off symptoms than in patients who neveror only sometimes experienced such symptoms The differenceswere statistically significant in CD8+ TEM CD4
+ TEM and
Table 2 Frequency of reported wearing-off symptoms
Symptom
Wearing-off symptoms
Sometimes Regularly
Fatigue 67 63
Psychological 33 25
Walking difficulty 11 25
Spasms 11 13
Pain 0 13
4 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
Figure 1Natalizumab receptor occupancy (RO) in patients reportingwearing-off symptoms never sometimes or regularly
(A) Manually gated PBLs visualized on a viSNEmap ROwas analyzed in 11 cell subtypes CD8+ central memory (TCM) effectormemory (TEM) effectormemoryRA (TEMRA) cells CD4
+ TEM TCM and TEMRA cells CD34+ cells memory B cells natural killer (NK) cells monocytes and conventional dendritic cells (cDCs)Neutrophils were not included in RO analysis (B) Spider plot ofmedian RO values in 11 cell subtypes in patients before and after natalizumab infusion (C) ROvalues in 11 cell subtypes before and after natalizumab infusion p values (Kruskal-Wallis test) comparing ROs in different wearing-off groups (D) Left medianRO values in cell clusters significantly different betweenwearing-off groups (SAM analysis in CITRUS) Right significant cell clusters are visualized on the viSNEmap PBL = peripheral blood leukocyte SAM = Significance Analysis of Microarrays
NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 5
CD4+ TEMRA cells before infusion and in CD8+ TCM CD8+
TEMRA CD4+ TCM cells NK cells monocytes and cDCs after
infusion (figure 1C) Furthermore in CD8+ and CD4+ T cellsnot stratified into subtypes RO was significantly lower in bothCD8+ and CD4+ T cells before infusion and in CD8+ T cellsafter infusion in patients who regularly had wearing-off symp-toms (figure e-2 linkslwwcomNXIA190) Unsupervisedanalysis of median ROs with CITRUS also showed lower me-dian natalizumab ROs in patients with regular wearing-offsymptoms (figure 1D) Neither of the 2 analysis approachesshowed significant differences between patients reportingwearing-off symptoms sometimes and never
High BMI was associated with significantly lower RO in CD8+
TEM cells and cDCs before infusion and in CD8+ TEMRA cellsCD34+ cells and monocytes after infusion (figure 2)
DiscussionNatalizumab prevents disease activity in RRMS and haspositive effects on subjective symptoms such as mood fatigueand cognitive function1718 However subjective wearing-offsymptoms at the end of the 4-week interval are frequentlyreported by patients In this study wearing-off symptomswere reported by 425 of the patients (200 regularly and225 only sometimes) We found lower natalizumab ROs inpatients who regularly experienced wearing-off symptomscompared with patients who reported such symptoms neveror only sometimes The result was replicated in 2 separatedata analysis pipelines Furthermore patients who reportedregularly experiencing wearing-off symptoms had highermedian body weight and BMI and higher sick leave frequencythan those who rarely or never experienced such symptomsMedian height was increased in patients with symptoms onlysometimes Other clinical and demographic factors were
similar between the patient groups High BMI was associatedwith low RO in several leukocyte subtypes
The main limitation of our study is the small patient cohort Asa consequence of this limited statistical power we were onlyable to detect large effects and acknowledge that there may beassociations of smaller effect size that went unnoticedWearing-off symptoms were less frequent in our study than the preva-lence of 54ndash63 reported in other studies but as previouslyreported the most frequent wearing-off symptom wasfatigue2ndash5 In contrast to our results a recent study found noassociation between the wearing-off effect and natalizumab ROor patient characteristics5 The previously reported study usedflow cytometry to measure RO in CD8+ TEM cells and CD8+
effector T cells By using high-parameter mass cytometry wewere able to measure RO in 11 cell subtypes simultaneouslyWe found that patients who regularly experienced wearing-offsymptoms had higher BMI than those who did not and we alsoobserved an association between high BMI and low RO Bodyweight was higher in our cohort than in the previous study5
(median 750 vsmean 729 kg) andwas evenmore pronouncedin the groupwith wearing-off symptoms regularly (median 815vs mean 746 kg) van Kempen et al5 reported nonsignificanttrends similar to our significant results Our wearing-off pop-ulation had higher body weight than theirs which may explainwhy we observed statistically significant differences despite oursmall cohort size The association between high BMI and lowRO suggests that high BMI by decreasing natalizumab ROmay be the underlying cause of the wearing-off phenomenonOthers have previously reported such an association betweenlow natalizumab RO and high body weight or BMI and somehave suggested that the dose of natalizumab should be adjustedfor body weight61920 We found no associations betweenwearing-off symptoms and disease activity markers such asclinical relapses new lesions detected by MRI in the past yearor serum NF-L levels21
Figure 2 Linear regression analysis demonstrates an association between RO and BMI
Plot of receptor occupancies (A) before and (B) after infusion for indicated cell types as a function of BMI Solid lines have slopes that are significantly differentfrom zero (p lt 005) and dashed lines have slopes that are not significantly different from zero BMI = body mass index RO = receptor occupancy
6 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
As also reported by others19 we observed large interindividualvariations in natalizumab RO and not all patients with lowRO had high BMI A previous study reported that bodyweight only partly predicts variability in natalizumab RO andsuggested other factors such as density and turnover of α4β1integrin that may drive the variability22
It has been hypothesized that cytokines could induce thewearing-off symptoms45 We speculate that lower natalizu-mab RO at the end of the dosing interval could increase themigratory capacity of cytokine-producing leukocytes into theCNS resulting in the wearing-off symptoms We found sig-nificantly lower RO in the T-cell subtypes CD8+ TEM CD4
+
TEM and CD4+ TEMRA cells before infusion in patients whoregularly had wearing-off symptoms TEM and TEMRA cellshome to the site of inflammation where they have effectorfunctions such as secretion of proinflammatory cytokines andcytotoxicity in contrast TCM cells and naive T cells home tosecondary lymphoid organs2324
Our results suggest that low RO may be a contributing factorto the wearing-off phenomenon and that higher BMI may bean underlying cause This supports the suggestion that nata-lizumab dosing should be personalized Personalization hasbeen mainly focused on extended interval dosing (EID)19
This has particularly been driven by the risk of progressivemultifocal leukoencephalopathy (PML) a serious complica-tion of natalizumab therapy in patients previously exposed toJC virus25 Retrospective studies of off-label treatment withEID have shown maintained efficacy2627 and reduced PMLrisk28 compared with standard interval dosing Howeverthese studies are limited by possible selection bias due tononrandomized design and the efficacy and safety of EID isnot fully known As patients who regularly experiencewearing-off symptoms already have lower RO and reportsymptoms at the end of dosing intervals extending dosingintervals could increase the risk of disease activity Wetherefore do not recommend EID in patients reportingwearing-off symptoms regularly as this could lead to an evenlower RO at the end of the interval than observed withstandard dosing619 Further studies should investigatewhether wearing-off symptoms are associated with increasedrisk of RRMS disease activity and whether increasing RO byreduced dosing intervals or weight loss may mitigate thesymptoms
AcknowledgmentThe authors thank Fritz og Ingrid Nilsens legat for financialresearch support and Hanne Linda Nakkestad at theNeurological Research Laboratory at Haukeland UniversityHospital for performing SiMOA analyses The masscytometry was performed at the Flow Cytometry CoreFacility Department of Clinical Science University of BergenThe Helios mass cytometer was funded by the BergenResearch Foundation Neuro-SysMed is jointly hosted byHaukeland University Hospital and University of Bergen andsupported as a Centre for Clinical Treatment Research (FKB)
by grants from The Research Council of Norway projectnumber 288164
Study fundingSupported by Fritz og Ingrid Nilsens legat for Forskning paringMultippel sklerose
DisclosureGH Bringeland has received research support from Novartisand institutional support from the Regional Heath Authorityof Western Norway N Blaser reports no disclosures K-MMyhr has received grants and personal fees from Biogen andNovartis personal fees from Genzyme Roche Almirall andMerck personal fees and nonfinancial support from Tevaand a research grant from the Norwegian Research Council(grant number 288164) CA Vedeler has received in-stitutional support from the Regional Heath Authority ofWestern Norway and research grant support from the Nor-wegian Research Council (grant number 288164) Dr Gav-asso has received institutional support from the RegionalHeath Authority of Western Norway Go to NeurologyorgNN for full disclosures
Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationOctober 22 2019 Accepted in final form December 20 2019
Appendix Authors
Name Location Role Contribution
Gerd HagaBringelandMD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studymajor role in theacquisition of datainterpretation of dataanalysis of data andrevision of themanuscript forintellectual content
NelloBlaser PhD
University of BergenBergen Norway
Author Interpretation of dataanalysis of data andrevision of themanuscript forintellectual content
Kjell-MortenMyhr MDPhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyand revision of themanuscript forintellectual content
ChristianAlexanderVedelerMD PhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyand revision of themanuscript forintellectual content
SoniaGavassoPhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyinterpretation of dataand revision of themanuscript forintellectual content
NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 7
References1 Polman CH OrsquoConnor PW Havrdova E et al A randomized placebo-controlled trial
of natalizumab for relapsing multiple sclerosis N Engl J Med 2006354899ndash9102 Katz J Lathi E Heyda L Characterizing the Natalizumab ldquoWearing offrdquo Effect [ab-
stract] ACTRIMS meeting Poster DX36 20143 Gudesblatt M Zarif M Bumstead B et al Multiple sclerosis and natalizumab ldquobe-
tween the dose symptomsrdquo [abstract 982] Mult Scler 201218(suppl 14)P9824 Ratchford JN Brock-Simmons R Augsburger A et al Multiple sclerosis symptom
recrudescence at the end of the natalizumab dosing cycle Int J MS Care 20141692ndash985 van Kempen ZLE Doesburg D Dekker I et al The natalizumab wearing-off effect end
of natalizumab cycle recurrence of MS symptoms Neurology 201993e1579ndashe15866 Punet-Ortiz J Hervas-Garcia JV Teniente-Serra A et al Monitoring CD49d receptor
occupancy a method to optimize and personalize natalizumab therapy in multiplesclerosis patients Cytometry B Clin Cytom 201894327ndash333
7 Bandura DR Baranov VI Ornatsky OI et al Mass cytometry technique for real timesingle cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry Anal Chem 2009816813ndash6822
8 Smith A Symbol digit modalities test Manual Los Angeles CA Western Psycho-logical Services 1982
9 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444ndash1452
10 Krupp LB LaRocca NG Muir-Nash J Steinberg AD The fatigue severity scaleApplication to patients with multiple sclerosis and systemic lupus erythematosusArch Neurol 1989461121ndash1123
11 Rahman AH Tordesillas L Berin MC Heparin reduces nonspecific eosinophilstaining artifacts in mass cytometry experiments Cytometry A 201689601ndash607
12 Bringeland GH Bader L Blaser N et al Optimization of receptor occupancy assays inmass cytometry standardization across channels with QSC beads Cytometry A 201995314ndash322
13 Finak G Perez JM Weng A Gottardo R Optimizing transformations for automatedhigh throughput analysis of flow cytometry data BMC Bioinformatics 201011546
14 Amir el AD Davis KL Tadmor MD et al viSNE enables visualization of highdimensional single-cell data and reveals phenotypic heterogeneity of leukemia NatBiotechnol 201331545ndash552
15 Bruggner RV Bodenmiller B Dill DL Tibshirani RJ Nolan GP Automated identi-fication of stratifying signatures in cellular subpopulations P Natl Acad Sci USA 2014111E2770ndashE2777
16 R A Language and Environment for Statistical Computing [computer program]Vienna R Foundation for Statistical Computing 2017
17 Iaffaldano P Viterbo RG Paolicelli D et al Impact of natalizumab on cognitiveperformances and fatigue in relapsing multiple sclerosis a prospective open-labeltwo years observational study PLoS One 20127e35843
18 Morrow SA OrsquoConnor PW Polman CH et al Evaluation of the symbol digit mo-dalities test (SDMT) and MS neuropsychological screening questionnaire (MSNQ)in natalizumab-treated MS patients over 48 weeks Mult Scler 2010161385ndash1392
19 Foley JF Goelz S Hoyt T Christensen A Metzger RR Evaluation of natalizumabpharmacokinetics and pharmacodynamics with standard and extended interval dos-ing Mult Scler Relat Dis 20193165ndash71
20 Tanaka M Kinoshita M Foley JF Tanaka K Kira J Carroll WM Body weight-basednatalizumab treatment in adult patientswithmultiple sclerosis JNeurol 2015262781ndash782
21 Varhaug KN Torkildsen O Myhr KM Vedeler CA Neurofilament light chain asa biomarker in multiple sclerosis Front Neurol 201910338
22 Muralidharan KK Kuesters G Plavina T et al Population pharmacokinetics andtarget engagement of natalizumab in patients with multiple sclerosis J Clin Pharmacol2017571017ndash1030
23 Mahnke YD Brodie TM Sallusto F Roederer M Lugli E The whorsquos who of T-celldifferentiation human memory T-cell subsets Eur J Immunol 2013432797ndash2809
24 Sallusto F Geginat J Lanzavecchia A Central memory and effector memory T cellsubsets function generation and maintenance Annu Rev Immunol 200422745ndash763
25 Bloomgren G Richman S Hotermans C et al Risk of natalizumab-associated pro-gressive multifocal leukoencephalopathy N Engl J Med 20123661870ndash1880
26 Bomprezzi R Pawate S Extended interval dosing of natalizumab a two-center 7-yearexperience Ther Adv Neurol Disord 20147227ndash231
27 Zhovtis Ryerson L Frohman TC Foley J et al Extended interval dosing of natali-zumab in multiple sclerosis J Neurol Neurosurg Psychiatry 201687885ndash889
28 Ryerson LZ Foley J Chang I et al Risk of natalizumab-associated PML in patientswith MS is reduced with extended interval dosing Neurology 201993e1452ndashe1462
8 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
DOI 101212NXI000000000000067820207 Neurol Neuroimmunol Neuroinflamm
Gerd Haga Bringeland Nello Blaser Kjell-Morten Myhr et al occupancy
Wearing-off at the end of natalizumab dosing intervals is associated with low receptor
This information is current as of February 4 2020
ServicesUpdated Information amp
httpnnneurologyorgcontent73e678fullhtmlincluding high resolution figures can be found at
This article has been cited by 2 HighWire-hosted articles
Subspecialty Collections
httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosisfollowing collection(s) This article along with others on similar topics appears in the
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httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
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Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2020 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
served as positive controls Further quality control experi-ments were performed as previously described in detail12
Data processing analysis and statisticsAfter acquisition of QSC beads we normalized (Fluidigmnormalizer) and exported FCS files to Cytobank software(Cytobank Inc Beckman Coulter Brea CA) and created QSCbead standard curves (QuickCal template Bangs Laboratories)After acquisition of PBL samples we normalized (Fluidigmnormalizer) debarcoded (Fluidigm Debarcoder) and exportedthe FSC files to Cytobank software for gating and downstreamanalysis (figure e-1 linkslwwcomNXIA190) We performedclean-up gating to obtain single PBLs and the data were arcsinhtransformed with a scale argument of 513 We then performed 2independent analyses for RO calculation in single PBLs onewas a manual analysis and one used an unsupervised approach
In the manual approach we first identified the following 11leukocyte subtypes of interest by manual gating CD8+ centralmemory (TCM) effector memory (TEM) effector memoryRA (TEMRA) T cells CD4+ TEM TCM and TEMRA cellsCD34+ cells memory B cells natural killer (NK) cellsmonocytes and conventional dendritic cells (cDCs)We thenplotted the signal intensities (dual count 90th percentiles) ofanti-IgG4 and anti-CD49d in each of these cell types with theQSC bead standard curves to obtain bead standardized valuesand calculated RO by the following formula
RO = 100 timesQSC bead standardized anti minus IgG4 eth169TmTHORNQSC bead standardized anti minusCD49d eth141PrTHORN
We compared ROs in the leukocyte subtypes in differentpatient groups using a Kruskal-Wallis test
In the unsupervised approach we used R (version 343) to addan extra variable into the FSC files the ratio between signalintensities of anti-IgG4 and anti-CD49d in each cell Thisresulted in an RO estimate for each cell For visualization ofhigh-dimensional single-cell data we performed automated di-mensionality reduction with stochastic neighborhood embed-ding (viSNE Cytobank)14 We analyzed the new RO variablewith the cluster identification characterization and regressiontool CITRUS (Cytobank) an algorithm that automatically
identifies statistically significant differences between patientgroups15We applied the correlativemodel SignificanceAnalysisof Microarrays with a false discovery rate (adjusted for multiplehypothesis testing) of 1 CITRUS was run with 10 repetitions
The relationship between baseline demographic variables andwearing-offwas compared using a Kruskal-Wallis test Statisticaldifferences with p lt 005 were considered significant usinga 2-sided comparison To test the age-corrected relationshipbetween baseline variables and wearing-off we used a likelihoodratio test between a linear model with only age and a linearmodel with age and the relevant baseline variable as predictorsWe conducted a linear regression of the association betweenRO and body mass index (BMI) and used a t test to assesswhether the slope was significantly different from zero We usedR version 34316 for statistical analysis and correlation plots
Data availabilityFCS files from anonymized patient PBL samples can beaccessed in the Flow Repository (ID FR-FCM-Z2A9)
ResultsPatient characteristicsOf the 45 patients who were eligible for inclusion 40 (89)consented to participate in the study Of 5 (11) non-consenting patients 1 refused participation and 4 had infusiontime points outside of the opening hours of the routine labo-ratory where blood samples were collected Eight (200) ofthe 40 participating patients reported having wearing-offsymptoms regularly at the end of every dosing interval 9(225) sometimes did and 23 (575) reported never havingwearing-off symptoms (table 1) Themost frequent wearing-offsymptom was fatigue (table 2) Patients who regularly hadwearing-off symptoms had significantly higher BMI and higherfrequency of sick leave than patients who never or onlysometimes experienced such symptoms (table 1) After ageadjustment weight was also significantly increased in patientswith wearing-off symptoms regularly whereas height was in-creased in patients with symptoms only sometimes None ofthe other demographic or clinical patient characteristics weresignificantly different between the groups and none of theincluded patients had clinical relapses or new lesions onMRI inthe year before inclusion in the study Age-adjusted medianserum NF-L levels were similar between groups There was noassociation between NF-L and BMI (data not shown)
Receptor occupancyManual gating of PBLs correlated well with the automatedmapping with viSNE (figure 1A) We observed a broad range ofnatalizumab RO values The median RO values in all leukocytesubtypes before infusion and in 10 of 11 leukocyte subtypesafter infusion (figure 1B) were lower in patients who regularlyexperienced wearing-off symptoms than in patients who neveror only sometimes experienced such symptoms The differenceswere statistically significant in CD8+ TEM CD4
+ TEM and
Table 2 Frequency of reported wearing-off symptoms
Symptom
Wearing-off symptoms
Sometimes Regularly
Fatigue 67 63
Psychological 33 25
Walking difficulty 11 25
Spasms 11 13
Pain 0 13
4 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
Figure 1Natalizumab receptor occupancy (RO) in patients reportingwearing-off symptoms never sometimes or regularly
(A) Manually gated PBLs visualized on a viSNEmap ROwas analyzed in 11 cell subtypes CD8+ central memory (TCM) effectormemory (TEM) effectormemoryRA (TEMRA) cells CD4
+ TEM TCM and TEMRA cells CD34+ cells memory B cells natural killer (NK) cells monocytes and conventional dendritic cells (cDCs)Neutrophils were not included in RO analysis (B) Spider plot ofmedian RO values in 11 cell subtypes in patients before and after natalizumab infusion (C) ROvalues in 11 cell subtypes before and after natalizumab infusion p values (Kruskal-Wallis test) comparing ROs in different wearing-off groups (D) Left medianRO values in cell clusters significantly different betweenwearing-off groups (SAM analysis in CITRUS) Right significant cell clusters are visualized on the viSNEmap PBL = peripheral blood leukocyte SAM = Significance Analysis of Microarrays
NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 5
CD4+ TEMRA cells before infusion and in CD8+ TCM CD8+
TEMRA CD4+ TCM cells NK cells monocytes and cDCs after
infusion (figure 1C) Furthermore in CD8+ and CD4+ T cellsnot stratified into subtypes RO was significantly lower in bothCD8+ and CD4+ T cells before infusion and in CD8+ T cellsafter infusion in patients who regularly had wearing-off symp-toms (figure e-2 linkslwwcomNXIA190) Unsupervisedanalysis of median ROs with CITRUS also showed lower me-dian natalizumab ROs in patients with regular wearing-offsymptoms (figure 1D) Neither of the 2 analysis approachesshowed significant differences between patients reportingwearing-off symptoms sometimes and never
High BMI was associated with significantly lower RO in CD8+
TEM cells and cDCs before infusion and in CD8+ TEMRA cellsCD34+ cells and monocytes after infusion (figure 2)
DiscussionNatalizumab prevents disease activity in RRMS and haspositive effects on subjective symptoms such as mood fatigueand cognitive function1718 However subjective wearing-offsymptoms at the end of the 4-week interval are frequentlyreported by patients In this study wearing-off symptomswere reported by 425 of the patients (200 regularly and225 only sometimes) We found lower natalizumab ROs inpatients who regularly experienced wearing-off symptomscompared with patients who reported such symptoms neveror only sometimes The result was replicated in 2 separatedata analysis pipelines Furthermore patients who reportedregularly experiencing wearing-off symptoms had highermedian body weight and BMI and higher sick leave frequencythan those who rarely or never experienced such symptomsMedian height was increased in patients with symptoms onlysometimes Other clinical and demographic factors were
similar between the patient groups High BMI was associatedwith low RO in several leukocyte subtypes
The main limitation of our study is the small patient cohort Asa consequence of this limited statistical power we were onlyable to detect large effects and acknowledge that there may beassociations of smaller effect size that went unnoticedWearing-off symptoms were less frequent in our study than the preva-lence of 54ndash63 reported in other studies but as previouslyreported the most frequent wearing-off symptom wasfatigue2ndash5 In contrast to our results a recent study found noassociation between the wearing-off effect and natalizumab ROor patient characteristics5 The previously reported study usedflow cytometry to measure RO in CD8+ TEM cells and CD8+
effector T cells By using high-parameter mass cytometry wewere able to measure RO in 11 cell subtypes simultaneouslyWe found that patients who regularly experienced wearing-offsymptoms had higher BMI than those who did not and we alsoobserved an association between high BMI and low RO Bodyweight was higher in our cohort than in the previous study5
(median 750 vsmean 729 kg) andwas evenmore pronouncedin the groupwith wearing-off symptoms regularly (median 815vs mean 746 kg) van Kempen et al5 reported nonsignificanttrends similar to our significant results Our wearing-off pop-ulation had higher body weight than theirs which may explainwhy we observed statistically significant differences despite oursmall cohort size The association between high BMI and lowRO suggests that high BMI by decreasing natalizumab ROmay be the underlying cause of the wearing-off phenomenonOthers have previously reported such an association betweenlow natalizumab RO and high body weight or BMI and somehave suggested that the dose of natalizumab should be adjustedfor body weight61920 We found no associations betweenwearing-off symptoms and disease activity markers such asclinical relapses new lesions detected by MRI in the past yearor serum NF-L levels21
Figure 2 Linear regression analysis demonstrates an association between RO and BMI
Plot of receptor occupancies (A) before and (B) after infusion for indicated cell types as a function of BMI Solid lines have slopes that are significantly differentfrom zero (p lt 005) and dashed lines have slopes that are not significantly different from zero BMI = body mass index RO = receptor occupancy
6 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
As also reported by others19 we observed large interindividualvariations in natalizumab RO and not all patients with lowRO had high BMI A previous study reported that bodyweight only partly predicts variability in natalizumab RO andsuggested other factors such as density and turnover of α4β1integrin that may drive the variability22
It has been hypothesized that cytokines could induce thewearing-off symptoms45 We speculate that lower natalizu-mab RO at the end of the dosing interval could increase themigratory capacity of cytokine-producing leukocytes into theCNS resulting in the wearing-off symptoms We found sig-nificantly lower RO in the T-cell subtypes CD8+ TEM CD4
+
TEM and CD4+ TEMRA cells before infusion in patients whoregularly had wearing-off symptoms TEM and TEMRA cellshome to the site of inflammation where they have effectorfunctions such as secretion of proinflammatory cytokines andcytotoxicity in contrast TCM cells and naive T cells home tosecondary lymphoid organs2324
Our results suggest that low RO may be a contributing factorto the wearing-off phenomenon and that higher BMI may bean underlying cause This supports the suggestion that nata-lizumab dosing should be personalized Personalization hasbeen mainly focused on extended interval dosing (EID)19
This has particularly been driven by the risk of progressivemultifocal leukoencephalopathy (PML) a serious complica-tion of natalizumab therapy in patients previously exposed toJC virus25 Retrospective studies of off-label treatment withEID have shown maintained efficacy2627 and reduced PMLrisk28 compared with standard interval dosing Howeverthese studies are limited by possible selection bias due tononrandomized design and the efficacy and safety of EID isnot fully known As patients who regularly experiencewearing-off symptoms already have lower RO and reportsymptoms at the end of dosing intervals extending dosingintervals could increase the risk of disease activity Wetherefore do not recommend EID in patients reportingwearing-off symptoms regularly as this could lead to an evenlower RO at the end of the interval than observed withstandard dosing619 Further studies should investigatewhether wearing-off symptoms are associated with increasedrisk of RRMS disease activity and whether increasing RO byreduced dosing intervals or weight loss may mitigate thesymptoms
AcknowledgmentThe authors thank Fritz og Ingrid Nilsens legat for financialresearch support and Hanne Linda Nakkestad at theNeurological Research Laboratory at Haukeland UniversityHospital for performing SiMOA analyses The masscytometry was performed at the Flow Cytometry CoreFacility Department of Clinical Science University of BergenThe Helios mass cytometer was funded by the BergenResearch Foundation Neuro-SysMed is jointly hosted byHaukeland University Hospital and University of Bergen andsupported as a Centre for Clinical Treatment Research (FKB)
by grants from The Research Council of Norway projectnumber 288164
Study fundingSupported by Fritz og Ingrid Nilsens legat for Forskning paringMultippel sklerose
DisclosureGH Bringeland has received research support from Novartisand institutional support from the Regional Heath Authorityof Western Norway N Blaser reports no disclosures K-MMyhr has received grants and personal fees from Biogen andNovartis personal fees from Genzyme Roche Almirall andMerck personal fees and nonfinancial support from Tevaand a research grant from the Norwegian Research Council(grant number 288164) CA Vedeler has received in-stitutional support from the Regional Heath Authority ofWestern Norway and research grant support from the Nor-wegian Research Council (grant number 288164) Dr Gav-asso has received institutional support from the RegionalHeath Authority of Western Norway Go to NeurologyorgNN for full disclosures
Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationOctober 22 2019 Accepted in final form December 20 2019
Appendix Authors
Name Location Role Contribution
Gerd HagaBringelandMD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studymajor role in theacquisition of datainterpretation of dataanalysis of data andrevision of themanuscript forintellectual content
NelloBlaser PhD
University of BergenBergen Norway
Author Interpretation of dataanalysis of data andrevision of themanuscript forintellectual content
Kjell-MortenMyhr MDPhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyand revision of themanuscript forintellectual content
ChristianAlexanderVedelerMD PhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyand revision of themanuscript forintellectual content
SoniaGavassoPhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyinterpretation of dataand revision of themanuscript forintellectual content
NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 7
References1 Polman CH OrsquoConnor PW Havrdova E et al A randomized placebo-controlled trial
of natalizumab for relapsing multiple sclerosis N Engl J Med 2006354899ndash9102 Katz J Lathi E Heyda L Characterizing the Natalizumab ldquoWearing offrdquo Effect [ab-
stract] ACTRIMS meeting Poster DX36 20143 Gudesblatt M Zarif M Bumstead B et al Multiple sclerosis and natalizumab ldquobe-
tween the dose symptomsrdquo [abstract 982] Mult Scler 201218(suppl 14)P9824 Ratchford JN Brock-Simmons R Augsburger A et al Multiple sclerosis symptom
recrudescence at the end of the natalizumab dosing cycle Int J MS Care 20141692ndash985 van Kempen ZLE Doesburg D Dekker I et al The natalizumab wearing-off effect end
of natalizumab cycle recurrence of MS symptoms Neurology 201993e1579ndashe15866 Punet-Ortiz J Hervas-Garcia JV Teniente-Serra A et al Monitoring CD49d receptor
occupancy a method to optimize and personalize natalizumab therapy in multiplesclerosis patients Cytometry B Clin Cytom 201894327ndash333
7 Bandura DR Baranov VI Ornatsky OI et al Mass cytometry technique for real timesingle cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry Anal Chem 2009816813ndash6822
8 Smith A Symbol digit modalities test Manual Los Angeles CA Western Psycho-logical Services 1982
9 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444ndash1452
10 Krupp LB LaRocca NG Muir-Nash J Steinberg AD The fatigue severity scaleApplication to patients with multiple sclerosis and systemic lupus erythematosusArch Neurol 1989461121ndash1123
11 Rahman AH Tordesillas L Berin MC Heparin reduces nonspecific eosinophilstaining artifacts in mass cytometry experiments Cytometry A 201689601ndash607
12 Bringeland GH Bader L Blaser N et al Optimization of receptor occupancy assays inmass cytometry standardization across channels with QSC beads Cytometry A 201995314ndash322
13 Finak G Perez JM Weng A Gottardo R Optimizing transformations for automatedhigh throughput analysis of flow cytometry data BMC Bioinformatics 201011546
14 Amir el AD Davis KL Tadmor MD et al viSNE enables visualization of highdimensional single-cell data and reveals phenotypic heterogeneity of leukemia NatBiotechnol 201331545ndash552
15 Bruggner RV Bodenmiller B Dill DL Tibshirani RJ Nolan GP Automated identi-fication of stratifying signatures in cellular subpopulations P Natl Acad Sci USA 2014111E2770ndashE2777
16 R A Language and Environment for Statistical Computing [computer program]Vienna R Foundation for Statistical Computing 2017
17 Iaffaldano P Viterbo RG Paolicelli D et al Impact of natalizumab on cognitiveperformances and fatigue in relapsing multiple sclerosis a prospective open-labeltwo years observational study PLoS One 20127e35843
18 Morrow SA OrsquoConnor PW Polman CH et al Evaluation of the symbol digit mo-dalities test (SDMT) and MS neuropsychological screening questionnaire (MSNQ)in natalizumab-treated MS patients over 48 weeks Mult Scler 2010161385ndash1392
19 Foley JF Goelz S Hoyt T Christensen A Metzger RR Evaluation of natalizumabpharmacokinetics and pharmacodynamics with standard and extended interval dos-ing Mult Scler Relat Dis 20193165ndash71
20 Tanaka M Kinoshita M Foley JF Tanaka K Kira J Carroll WM Body weight-basednatalizumab treatment in adult patientswithmultiple sclerosis JNeurol 2015262781ndash782
21 Varhaug KN Torkildsen O Myhr KM Vedeler CA Neurofilament light chain asa biomarker in multiple sclerosis Front Neurol 201910338
22 Muralidharan KK Kuesters G Plavina T et al Population pharmacokinetics andtarget engagement of natalizumab in patients with multiple sclerosis J Clin Pharmacol2017571017ndash1030
23 Mahnke YD Brodie TM Sallusto F Roederer M Lugli E The whorsquos who of T-celldifferentiation human memory T-cell subsets Eur J Immunol 2013432797ndash2809
24 Sallusto F Geginat J Lanzavecchia A Central memory and effector memory T cellsubsets function generation and maintenance Annu Rev Immunol 200422745ndash763
25 Bloomgren G Richman S Hotermans C et al Risk of natalizumab-associated pro-gressive multifocal leukoencephalopathy N Engl J Med 20123661870ndash1880
26 Bomprezzi R Pawate S Extended interval dosing of natalizumab a two-center 7-yearexperience Ther Adv Neurol Disord 20147227ndash231
27 Zhovtis Ryerson L Frohman TC Foley J et al Extended interval dosing of natali-zumab in multiple sclerosis J Neurol Neurosurg Psychiatry 201687885ndash889
28 Ryerson LZ Foley J Chang I et al Risk of natalizumab-associated PML in patientswith MS is reduced with extended interval dosing Neurology 201993e1452ndashe1462
8 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
DOI 101212NXI000000000000067820207 Neurol Neuroimmunol Neuroinflamm
Gerd Haga Bringeland Nello Blaser Kjell-Morten Myhr et al occupancy
Wearing-off at the end of natalizumab dosing intervals is associated with low receptor
This information is current as of February 4 2020
ServicesUpdated Information amp
httpnnneurologyorgcontent73e678fullhtmlincluding high resolution figures can be found at
This article has been cited by 2 HighWire-hosted articles
Subspecialty Collections
httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosisfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2020 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
Figure 1Natalizumab receptor occupancy (RO) in patients reportingwearing-off symptoms never sometimes or regularly
(A) Manually gated PBLs visualized on a viSNEmap ROwas analyzed in 11 cell subtypes CD8+ central memory (TCM) effectormemory (TEM) effectormemoryRA (TEMRA) cells CD4
+ TEM TCM and TEMRA cells CD34+ cells memory B cells natural killer (NK) cells monocytes and conventional dendritic cells (cDCs)Neutrophils were not included in RO analysis (B) Spider plot ofmedian RO values in 11 cell subtypes in patients before and after natalizumab infusion (C) ROvalues in 11 cell subtypes before and after natalizumab infusion p values (Kruskal-Wallis test) comparing ROs in different wearing-off groups (D) Left medianRO values in cell clusters significantly different betweenwearing-off groups (SAM analysis in CITRUS) Right significant cell clusters are visualized on the viSNEmap PBL = peripheral blood leukocyte SAM = Significance Analysis of Microarrays
NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 5
CD4+ TEMRA cells before infusion and in CD8+ TCM CD8+
TEMRA CD4+ TCM cells NK cells monocytes and cDCs after
infusion (figure 1C) Furthermore in CD8+ and CD4+ T cellsnot stratified into subtypes RO was significantly lower in bothCD8+ and CD4+ T cells before infusion and in CD8+ T cellsafter infusion in patients who regularly had wearing-off symp-toms (figure e-2 linkslwwcomNXIA190) Unsupervisedanalysis of median ROs with CITRUS also showed lower me-dian natalizumab ROs in patients with regular wearing-offsymptoms (figure 1D) Neither of the 2 analysis approachesshowed significant differences between patients reportingwearing-off symptoms sometimes and never
High BMI was associated with significantly lower RO in CD8+
TEM cells and cDCs before infusion and in CD8+ TEMRA cellsCD34+ cells and monocytes after infusion (figure 2)
DiscussionNatalizumab prevents disease activity in RRMS and haspositive effects on subjective symptoms such as mood fatigueand cognitive function1718 However subjective wearing-offsymptoms at the end of the 4-week interval are frequentlyreported by patients In this study wearing-off symptomswere reported by 425 of the patients (200 regularly and225 only sometimes) We found lower natalizumab ROs inpatients who regularly experienced wearing-off symptomscompared with patients who reported such symptoms neveror only sometimes The result was replicated in 2 separatedata analysis pipelines Furthermore patients who reportedregularly experiencing wearing-off symptoms had highermedian body weight and BMI and higher sick leave frequencythan those who rarely or never experienced such symptomsMedian height was increased in patients with symptoms onlysometimes Other clinical and demographic factors were
similar between the patient groups High BMI was associatedwith low RO in several leukocyte subtypes
The main limitation of our study is the small patient cohort Asa consequence of this limited statistical power we were onlyable to detect large effects and acknowledge that there may beassociations of smaller effect size that went unnoticedWearing-off symptoms were less frequent in our study than the preva-lence of 54ndash63 reported in other studies but as previouslyreported the most frequent wearing-off symptom wasfatigue2ndash5 In contrast to our results a recent study found noassociation between the wearing-off effect and natalizumab ROor patient characteristics5 The previously reported study usedflow cytometry to measure RO in CD8+ TEM cells and CD8+
effector T cells By using high-parameter mass cytometry wewere able to measure RO in 11 cell subtypes simultaneouslyWe found that patients who regularly experienced wearing-offsymptoms had higher BMI than those who did not and we alsoobserved an association between high BMI and low RO Bodyweight was higher in our cohort than in the previous study5
(median 750 vsmean 729 kg) andwas evenmore pronouncedin the groupwith wearing-off symptoms regularly (median 815vs mean 746 kg) van Kempen et al5 reported nonsignificanttrends similar to our significant results Our wearing-off pop-ulation had higher body weight than theirs which may explainwhy we observed statistically significant differences despite oursmall cohort size The association between high BMI and lowRO suggests that high BMI by decreasing natalizumab ROmay be the underlying cause of the wearing-off phenomenonOthers have previously reported such an association betweenlow natalizumab RO and high body weight or BMI and somehave suggested that the dose of natalizumab should be adjustedfor body weight61920 We found no associations betweenwearing-off symptoms and disease activity markers such asclinical relapses new lesions detected by MRI in the past yearor serum NF-L levels21
Figure 2 Linear regression analysis demonstrates an association between RO and BMI
Plot of receptor occupancies (A) before and (B) after infusion for indicated cell types as a function of BMI Solid lines have slopes that are significantly differentfrom zero (p lt 005) and dashed lines have slopes that are not significantly different from zero BMI = body mass index RO = receptor occupancy
6 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
As also reported by others19 we observed large interindividualvariations in natalizumab RO and not all patients with lowRO had high BMI A previous study reported that bodyweight only partly predicts variability in natalizumab RO andsuggested other factors such as density and turnover of α4β1integrin that may drive the variability22
It has been hypothesized that cytokines could induce thewearing-off symptoms45 We speculate that lower natalizu-mab RO at the end of the dosing interval could increase themigratory capacity of cytokine-producing leukocytes into theCNS resulting in the wearing-off symptoms We found sig-nificantly lower RO in the T-cell subtypes CD8+ TEM CD4
+
TEM and CD4+ TEMRA cells before infusion in patients whoregularly had wearing-off symptoms TEM and TEMRA cellshome to the site of inflammation where they have effectorfunctions such as secretion of proinflammatory cytokines andcytotoxicity in contrast TCM cells and naive T cells home tosecondary lymphoid organs2324
Our results suggest that low RO may be a contributing factorto the wearing-off phenomenon and that higher BMI may bean underlying cause This supports the suggestion that nata-lizumab dosing should be personalized Personalization hasbeen mainly focused on extended interval dosing (EID)19
This has particularly been driven by the risk of progressivemultifocal leukoencephalopathy (PML) a serious complica-tion of natalizumab therapy in patients previously exposed toJC virus25 Retrospective studies of off-label treatment withEID have shown maintained efficacy2627 and reduced PMLrisk28 compared with standard interval dosing Howeverthese studies are limited by possible selection bias due tononrandomized design and the efficacy and safety of EID isnot fully known As patients who regularly experiencewearing-off symptoms already have lower RO and reportsymptoms at the end of dosing intervals extending dosingintervals could increase the risk of disease activity Wetherefore do not recommend EID in patients reportingwearing-off symptoms regularly as this could lead to an evenlower RO at the end of the interval than observed withstandard dosing619 Further studies should investigatewhether wearing-off symptoms are associated with increasedrisk of RRMS disease activity and whether increasing RO byreduced dosing intervals or weight loss may mitigate thesymptoms
AcknowledgmentThe authors thank Fritz og Ingrid Nilsens legat for financialresearch support and Hanne Linda Nakkestad at theNeurological Research Laboratory at Haukeland UniversityHospital for performing SiMOA analyses The masscytometry was performed at the Flow Cytometry CoreFacility Department of Clinical Science University of BergenThe Helios mass cytometer was funded by the BergenResearch Foundation Neuro-SysMed is jointly hosted byHaukeland University Hospital and University of Bergen andsupported as a Centre for Clinical Treatment Research (FKB)
by grants from The Research Council of Norway projectnumber 288164
Study fundingSupported by Fritz og Ingrid Nilsens legat for Forskning paringMultippel sklerose
DisclosureGH Bringeland has received research support from Novartisand institutional support from the Regional Heath Authorityof Western Norway N Blaser reports no disclosures K-MMyhr has received grants and personal fees from Biogen andNovartis personal fees from Genzyme Roche Almirall andMerck personal fees and nonfinancial support from Tevaand a research grant from the Norwegian Research Council(grant number 288164) CA Vedeler has received in-stitutional support from the Regional Heath Authority ofWestern Norway and research grant support from the Nor-wegian Research Council (grant number 288164) Dr Gav-asso has received institutional support from the RegionalHeath Authority of Western Norway Go to NeurologyorgNN for full disclosures
Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationOctober 22 2019 Accepted in final form December 20 2019
Appendix Authors
Name Location Role Contribution
Gerd HagaBringelandMD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studymajor role in theacquisition of datainterpretation of dataanalysis of data andrevision of themanuscript forintellectual content
NelloBlaser PhD
University of BergenBergen Norway
Author Interpretation of dataanalysis of data andrevision of themanuscript forintellectual content
Kjell-MortenMyhr MDPhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyand revision of themanuscript forintellectual content
ChristianAlexanderVedelerMD PhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyand revision of themanuscript forintellectual content
SoniaGavassoPhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyinterpretation of dataand revision of themanuscript forintellectual content
NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 7
References1 Polman CH OrsquoConnor PW Havrdova E et al A randomized placebo-controlled trial
of natalizumab for relapsing multiple sclerosis N Engl J Med 2006354899ndash9102 Katz J Lathi E Heyda L Characterizing the Natalizumab ldquoWearing offrdquo Effect [ab-
stract] ACTRIMS meeting Poster DX36 20143 Gudesblatt M Zarif M Bumstead B et al Multiple sclerosis and natalizumab ldquobe-
tween the dose symptomsrdquo [abstract 982] Mult Scler 201218(suppl 14)P9824 Ratchford JN Brock-Simmons R Augsburger A et al Multiple sclerosis symptom
recrudescence at the end of the natalizumab dosing cycle Int J MS Care 20141692ndash985 van Kempen ZLE Doesburg D Dekker I et al The natalizumab wearing-off effect end
of natalizumab cycle recurrence of MS symptoms Neurology 201993e1579ndashe15866 Punet-Ortiz J Hervas-Garcia JV Teniente-Serra A et al Monitoring CD49d receptor
occupancy a method to optimize and personalize natalizumab therapy in multiplesclerosis patients Cytometry B Clin Cytom 201894327ndash333
7 Bandura DR Baranov VI Ornatsky OI et al Mass cytometry technique for real timesingle cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry Anal Chem 2009816813ndash6822
8 Smith A Symbol digit modalities test Manual Los Angeles CA Western Psycho-logical Services 1982
9 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444ndash1452
10 Krupp LB LaRocca NG Muir-Nash J Steinberg AD The fatigue severity scaleApplication to patients with multiple sclerosis and systemic lupus erythematosusArch Neurol 1989461121ndash1123
11 Rahman AH Tordesillas L Berin MC Heparin reduces nonspecific eosinophilstaining artifacts in mass cytometry experiments Cytometry A 201689601ndash607
12 Bringeland GH Bader L Blaser N et al Optimization of receptor occupancy assays inmass cytometry standardization across channels with QSC beads Cytometry A 201995314ndash322
13 Finak G Perez JM Weng A Gottardo R Optimizing transformations for automatedhigh throughput analysis of flow cytometry data BMC Bioinformatics 201011546
14 Amir el AD Davis KL Tadmor MD et al viSNE enables visualization of highdimensional single-cell data and reveals phenotypic heterogeneity of leukemia NatBiotechnol 201331545ndash552
15 Bruggner RV Bodenmiller B Dill DL Tibshirani RJ Nolan GP Automated identi-fication of stratifying signatures in cellular subpopulations P Natl Acad Sci USA 2014111E2770ndashE2777
16 R A Language and Environment for Statistical Computing [computer program]Vienna R Foundation for Statistical Computing 2017
17 Iaffaldano P Viterbo RG Paolicelli D et al Impact of natalizumab on cognitiveperformances and fatigue in relapsing multiple sclerosis a prospective open-labeltwo years observational study PLoS One 20127e35843
18 Morrow SA OrsquoConnor PW Polman CH et al Evaluation of the symbol digit mo-dalities test (SDMT) and MS neuropsychological screening questionnaire (MSNQ)in natalizumab-treated MS patients over 48 weeks Mult Scler 2010161385ndash1392
19 Foley JF Goelz S Hoyt T Christensen A Metzger RR Evaluation of natalizumabpharmacokinetics and pharmacodynamics with standard and extended interval dos-ing Mult Scler Relat Dis 20193165ndash71
20 Tanaka M Kinoshita M Foley JF Tanaka K Kira J Carroll WM Body weight-basednatalizumab treatment in adult patientswithmultiple sclerosis JNeurol 2015262781ndash782
21 Varhaug KN Torkildsen O Myhr KM Vedeler CA Neurofilament light chain asa biomarker in multiple sclerosis Front Neurol 201910338
22 Muralidharan KK Kuesters G Plavina T et al Population pharmacokinetics andtarget engagement of natalizumab in patients with multiple sclerosis J Clin Pharmacol2017571017ndash1030
23 Mahnke YD Brodie TM Sallusto F Roederer M Lugli E The whorsquos who of T-celldifferentiation human memory T-cell subsets Eur J Immunol 2013432797ndash2809
24 Sallusto F Geginat J Lanzavecchia A Central memory and effector memory T cellsubsets function generation and maintenance Annu Rev Immunol 200422745ndash763
25 Bloomgren G Richman S Hotermans C et al Risk of natalizumab-associated pro-gressive multifocal leukoencephalopathy N Engl J Med 20123661870ndash1880
26 Bomprezzi R Pawate S Extended interval dosing of natalizumab a two-center 7-yearexperience Ther Adv Neurol Disord 20147227ndash231
27 Zhovtis Ryerson L Frohman TC Foley J et al Extended interval dosing of natali-zumab in multiple sclerosis J Neurol Neurosurg Psychiatry 201687885ndash889
28 Ryerson LZ Foley J Chang I et al Risk of natalizumab-associated PML in patientswith MS is reduced with extended interval dosing Neurology 201993e1452ndashe1462
8 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
DOI 101212NXI000000000000067820207 Neurol Neuroimmunol Neuroinflamm
Gerd Haga Bringeland Nello Blaser Kjell-Morten Myhr et al occupancy
Wearing-off at the end of natalizumab dosing intervals is associated with low receptor
This information is current as of February 4 2020
ServicesUpdated Information amp
httpnnneurologyorgcontent73e678fullhtmlincluding high resolution figures can be found at
This article has been cited by 2 HighWire-hosted articles
Subspecialty Collections
httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosisfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2020 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
CD4+ TEMRA cells before infusion and in CD8+ TCM CD8+
TEMRA CD4+ TCM cells NK cells monocytes and cDCs after
infusion (figure 1C) Furthermore in CD8+ and CD4+ T cellsnot stratified into subtypes RO was significantly lower in bothCD8+ and CD4+ T cells before infusion and in CD8+ T cellsafter infusion in patients who regularly had wearing-off symp-toms (figure e-2 linkslwwcomNXIA190) Unsupervisedanalysis of median ROs with CITRUS also showed lower me-dian natalizumab ROs in patients with regular wearing-offsymptoms (figure 1D) Neither of the 2 analysis approachesshowed significant differences between patients reportingwearing-off symptoms sometimes and never
High BMI was associated with significantly lower RO in CD8+
TEM cells and cDCs before infusion and in CD8+ TEMRA cellsCD34+ cells and monocytes after infusion (figure 2)
DiscussionNatalizumab prevents disease activity in RRMS and haspositive effects on subjective symptoms such as mood fatigueand cognitive function1718 However subjective wearing-offsymptoms at the end of the 4-week interval are frequentlyreported by patients In this study wearing-off symptomswere reported by 425 of the patients (200 regularly and225 only sometimes) We found lower natalizumab ROs inpatients who regularly experienced wearing-off symptomscompared with patients who reported such symptoms neveror only sometimes The result was replicated in 2 separatedata analysis pipelines Furthermore patients who reportedregularly experiencing wearing-off symptoms had highermedian body weight and BMI and higher sick leave frequencythan those who rarely or never experienced such symptomsMedian height was increased in patients with symptoms onlysometimes Other clinical and demographic factors were
similar between the patient groups High BMI was associatedwith low RO in several leukocyte subtypes
The main limitation of our study is the small patient cohort Asa consequence of this limited statistical power we were onlyable to detect large effects and acknowledge that there may beassociations of smaller effect size that went unnoticedWearing-off symptoms were less frequent in our study than the preva-lence of 54ndash63 reported in other studies but as previouslyreported the most frequent wearing-off symptom wasfatigue2ndash5 In contrast to our results a recent study found noassociation between the wearing-off effect and natalizumab ROor patient characteristics5 The previously reported study usedflow cytometry to measure RO in CD8+ TEM cells and CD8+
effector T cells By using high-parameter mass cytometry wewere able to measure RO in 11 cell subtypes simultaneouslyWe found that patients who regularly experienced wearing-offsymptoms had higher BMI than those who did not and we alsoobserved an association between high BMI and low RO Bodyweight was higher in our cohort than in the previous study5
(median 750 vsmean 729 kg) andwas evenmore pronouncedin the groupwith wearing-off symptoms regularly (median 815vs mean 746 kg) van Kempen et al5 reported nonsignificanttrends similar to our significant results Our wearing-off pop-ulation had higher body weight than theirs which may explainwhy we observed statistically significant differences despite oursmall cohort size The association between high BMI and lowRO suggests that high BMI by decreasing natalizumab ROmay be the underlying cause of the wearing-off phenomenonOthers have previously reported such an association betweenlow natalizumab RO and high body weight or BMI and somehave suggested that the dose of natalizumab should be adjustedfor body weight61920 We found no associations betweenwearing-off symptoms and disease activity markers such asclinical relapses new lesions detected by MRI in the past yearor serum NF-L levels21
Figure 2 Linear regression analysis demonstrates an association between RO and BMI
Plot of receptor occupancies (A) before and (B) after infusion for indicated cell types as a function of BMI Solid lines have slopes that are significantly differentfrom zero (p lt 005) and dashed lines have slopes that are not significantly different from zero BMI = body mass index RO = receptor occupancy
6 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
As also reported by others19 we observed large interindividualvariations in natalizumab RO and not all patients with lowRO had high BMI A previous study reported that bodyweight only partly predicts variability in natalizumab RO andsuggested other factors such as density and turnover of α4β1integrin that may drive the variability22
It has been hypothesized that cytokines could induce thewearing-off symptoms45 We speculate that lower natalizu-mab RO at the end of the dosing interval could increase themigratory capacity of cytokine-producing leukocytes into theCNS resulting in the wearing-off symptoms We found sig-nificantly lower RO in the T-cell subtypes CD8+ TEM CD4
+
TEM and CD4+ TEMRA cells before infusion in patients whoregularly had wearing-off symptoms TEM and TEMRA cellshome to the site of inflammation where they have effectorfunctions such as secretion of proinflammatory cytokines andcytotoxicity in contrast TCM cells and naive T cells home tosecondary lymphoid organs2324
Our results suggest that low RO may be a contributing factorto the wearing-off phenomenon and that higher BMI may bean underlying cause This supports the suggestion that nata-lizumab dosing should be personalized Personalization hasbeen mainly focused on extended interval dosing (EID)19
This has particularly been driven by the risk of progressivemultifocal leukoencephalopathy (PML) a serious complica-tion of natalizumab therapy in patients previously exposed toJC virus25 Retrospective studies of off-label treatment withEID have shown maintained efficacy2627 and reduced PMLrisk28 compared with standard interval dosing Howeverthese studies are limited by possible selection bias due tononrandomized design and the efficacy and safety of EID isnot fully known As patients who regularly experiencewearing-off symptoms already have lower RO and reportsymptoms at the end of dosing intervals extending dosingintervals could increase the risk of disease activity Wetherefore do not recommend EID in patients reportingwearing-off symptoms regularly as this could lead to an evenlower RO at the end of the interval than observed withstandard dosing619 Further studies should investigatewhether wearing-off symptoms are associated with increasedrisk of RRMS disease activity and whether increasing RO byreduced dosing intervals or weight loss may mitigate thesymptoms
AcknowledgmentThe authors thank Fritz og Ingrid Nilsens legat for financialresearch support and Hanne Linda Nakkestad at theNeurological Research Laboratory at Haukeland UniversityHospital for performing SiMOA analyses The masscytometry was performed at the Flow Cytometry CoreFacility Department of Clinical Science University of BergenThe Helios mass cytometer was funded by the BergenResearch Foundation Neuro-SysMed is jointly hosted byHaukeland University Hospital and University of Bergen andsupported as a Centre for Clinical Treatment Research (FKB)
by grants from The Research Council of Norway projectnumber 288164
Study fundingSupported by Fritz og Ingrid Nilsens legat for Forskning paringMultippel sklerose
DisclosureGH Bringeland has received research support from Novartisand institutional support from the Regional Heath Authorityof Western Norway N Blaser reports no disclosures K-MMyhr has received grants and personal fees from Biogen andNovartis personal fees from Genzyme Roche Almirall andMerck personal fees and nonfinancial support from Tevaand a research grant from the Norwegian Research Council(grant number 288164) CA Vedeler has received in-stitutional support from the Regional Heath Authority ofWestern Norway and research grant support from the Nor-wegian Research Council (grant number 288164) Dr Gav-asso has received institutional support from the RegionalHeath Authority of Western Norway Go to NeurologyorgNN for full disclosures
Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationOctober 22 2019 Accepted in final form December 20 2019
Appendix Authors
Name Location Role Contribution
Gerd HagaBringelandMD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studymajor role in theacquisition of datainterpretation of dataanalysis of data andrevision of themanuscript forintellectual content
NelloBlaser PhD
University of BergenBergen Norway
Author Interpretation of dataanalysis of data andrevision of themanuscript forintellectual content
Kjell-MortenMyhr MDPhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyand revision of themanuscript forintellectual content
ChristianAlexanderVedelerMD PhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyand revision of themanuscript forintellectual content
SoniaGavassoPhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyinterpretation of dataand revision of themanuscript forintellectual content
NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 7
References1 Polman CH OrsquoConnor PW Havrdova E et al A randomized placebo-controlled trial
of natalizumab for relapsing multiple sclerosis N Engl J Med 2006354899ndash9102 Katz J Lathi E Heyda L Characterizing the Natalizumab ldquoWearing offrdquo Effect [ab-
stract] ACTRIMS meeting Poster DX36 20143 Gudesblatt M Zarif M Bumstead B et al Multiple sclerosis and natalizumab ldquobe-
tween the dose symptomsrdquo [abstract 982] Mult Scler 201218(suppl 14)P9824 Ratchford JN Brock-Simmons R Augsburger A et al Multiple sclerosis symptom
recrudescence at the end of the natalizumab dosing cycle Int J MS Care 20141692ndash985 van Kempen ZLE Doesburg D Dekker I et al The natalizumab wearing-off effect end
of natalizumab cycle recurrence of MS symptoms Neurology 201993e1579ndashe15866 Punet-Ortiz J Hervas-Garcia JV Teniente-Serra A et al Monitoring CD49d receptor
occupancy a method to optimize and personalize natalizumab therapy in multiplesclerosis patients Cytometry B Clin Cytom 201894327ndash333
7 Bandura DR Baranov VI Ornatsky OI et al Mass cytometry technique for real timesingle cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry Anal Chem 2009816813ndash6822
8 Smith A Symbol digit modalities test Manual Los Angeles CA Western Psycho-logical Services 1982
9 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444ndash1452
10 Krupp LB LaRocca NG Muir-Nash J Steinberg AD The fatigue severity scaleApplication to patients with multiple sclerosis and systemic lupus erythematosusArch Neurol 1989461121ndash1123
11 Rahman AH Tordesillas L Berin MC Heparin reduces nonspecific eosinophilstaining artifacts in mass cytometry experiments Cytometry A 201689601ndash607
12 Bringeland GH Bader L Blaser N et al Optimization of receptor occupancy assays inmass cytometry standardization across channels with QSC beads Cytometry A 201995314ndash322
13 Finak G Perez JM Weng A Gottardo R Optimizing transformations for automatedhigh throughput analysis of flow cytometry data BMC Bioinformatics 201011546
14 Amir el AD Davis KL Tadmor MD et al viSNE enables visualization of highdimensional single-cell data and reveals phenotypic heterogeneity of leukemia NatBiotechnol 201331545ndash552
15 Bruggner RV Bodenmiller B Dill DL Tibshirani RJ Nolan GP Automated identi-fication of stratifying signatures in cellular subpopulations P Natl Acad Sci USA 2014111E2770ndashE2777
16 R A Language and Environment for Statistical Computing [computer program]Vienna R Foundation for Statistical Computing 2017
17 Iaffaldano P Viterbo RG Paolicelli D et al Impact of natalizumab on cognitiveperformances and fatigue in relapsing multiple sclerosis a prospective open-labeltwo years observational study PLoS One 20127e35843
18 Morrow SA OrsquoConnor PW Polman CH et al Evaluation of the symbol digit mo-dalities test (SDMT) and MS neuropsychological screening questionnaire (MSNQ)in natalizumab-treated MS patients over 48 weeks Mult Scler 2010161385ndash1392
19 Foley JF Goelz S Hoyt T Christensen A Metzger RR Evaluation of natalizumabpharmacokinetics and pharmacodynamics with standard and extended interval dos-ing Mult Scler Relat Dis 20193165ndash71
20 Tanaka M Kinoshita M Foley JF Tanaka K Kira J Carroll WM Body weight-basednatalizumab treatment in adult patientswithmultiple sclerosis JNeurol 2015262781ndash782
21 Varhaug KN Torkildsen O Myhr KM Vedeler CA Neurofilament light chain asa biomarker in multiple sclerosis Front Neurol 201910338
22 Muralidharan KK Kuesters G Plavina T et al Population pharmacokinetics andtarget engagement of natalizumab in patients with multiple sclerosis J Clin Pharmacol2017571017ndash1030
23 Mahnke YD Brodie TM Sallusto F Roederer M Lugli E The whorsquos who of T-celldifferentiation human memory T-cell subsets Eur J Immunol 2013432797ndash2809
24 Sallusto F Geginat J Lanzavecchia A Central memory and effector memory T cellsubsets function generation and maintenance Annu Rev Immunol 200422745ndash763
25 Bloomgren G Richman S Hotermans C et al Risk of natalizumab-associated pro-gressive multifocal leukoencephalopathy N Engl J Med 20123661870ndash1880
26 Bomprezzi R Pawate S Extended interval dosing of natalizumab a two-center 7-yearexperience Ther Adv Neurol Disord 20147227ndash231
27 Zhovtis Ryerson L Frohman TC Foley J et al Extended interval dosing of natali-zumab in multiple sclerosis J Neurol Neurosurg Psychiatry 201687885ndash889
28 Ryerson LZ Foley J Chang I et al Risk of natalizumab-associated PML in patientswith MS is reduced with extended interval dosing Neurology 201993e1452ndashe1462
8 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
DOI 101212NXI000000000000067820207 Neurol Neuroimmunol Neuroinflamm
Gerd Haga Bringeland Nello Blaser Kjell-Morten Myhr et al occupancy
Wearing-off at the end of natalizumab dosing intervals is associated with low receptor
This information is current as of February 4 2020
ServicesUpdated Information amp
httpnnneurologyorgcontent73e678fullhtmlincluding high resolution figures can be found at
This article has been cited by 2 HighWire-hosted articles
Subspecialty Collections
httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosisfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2020 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
As also reported by others19 we observed large interindividualvariations in natalizumab RO and not all patients with lowRO had high BMI A previous study reported that bodyweight only partly predicts variability in natalizumab RO andsuggested other factors such as density and turnover of α4β1integrin that may drive the variability22
It has been hypothesized that cytokines could induce thewearing-off symptoms45 We speculate that lower natalizu-mab RO at the end of the dosing interval could increase themigratory capacity of cytokine-producing leukocytes into theCNS resulting in the wearing-off symptoms We found sig-nificantly lower RO in the T-cell subtypes CD8+ TEM CD4
+
TEM and CD4+ TEMRA cells before infusion in patients whoregularly had wearing-off symptoms TEM and TEMRA cellshome to the site of inflammation where they have effectorfunctions such as secretion of proinflammatory cytokines andcytotoxicity in contrast TCM cells and naive T cells home tosecondary lymphoid organs2324
Our results suggest that low RO may be a contributing factorto the wearing-off phenomenon and that higher BMI may bean underlying cause This supports the suggestion that nata-lizumab dosing should be personalized Personalization hasbeen mainly focused on extended interval dosing (EID)19
This has particularly been driven by the risk of progressivemultifocal leukoencephalopathy (PML) a serious complica-tion of natalizumab therapy in patients previously exposed toJC virus25 Retrospective studies of off-label treatment withEID have shown maintained efficacy2627 and reduced PMLrisk28 compared with standard interval dosing Howeverthese studies are limited by possible selection bias due tononrandomized design and the efficacy and safety of EID isnot fully known As patients who regularly experiencewearing-off symptoms already have lower RO and reportsymptoms at the end of dosing intervals extending dosingintervals could increase the risk of disease activity Wetherefore do not recommend EID in patients reportingwearing-off symptoms regularly as this could lead to an evenlower RO at the end of the interval than observed withstandard dosing619 Further studies should investigatewhether wearing-off symptoms are associated with increasedrisk of RRMS disease activity and whether increasing RO byreduced dosing intervals or weight loss may mitigate thesymptoms
AcknowledgmentThe authors thank Fritz og Ingrid Nilsens legat for financialresearch support and Hanne Linda Nakkestad at theNeurological Research Laboratory at Haukeland UniversityHospital for performing SiMOA analyses The masscytometry was performed at the Flow Cytometry CoreFacility Department of Clinical Science University of BergenThe Helios mass cytometer was funded by the BergenResearch Foundation Neuro-SysMed is jointly hosted byHaukeland University Hospital and University of Bergen andsupported as a Centre for Clinical Treatment Research (FKB)
by grants from The Research Council of Norway projectnumber 288164
Study fundingSupported by Fritz og Ingrid Nilsens legat for Forskning paringMultippel sklerose
DisclosureGH Bringeland has received research support from Novartisand institutional support from the Regional Heath Authorityof Western Norway N Blaser reports no disclosures K-MMyhr has received grants and personal fees from Biogen andNovartis personal fees from Genzyme Roche Almirall andMerck personal fees and nonfinancial support from Tevaand a research grant from the Norwegian Research Council(grant number 288164) CA Vedeler has received in-stitutional support from the Regional Heath Authority ofWestern Norway and research grant support from the Nor-wegian Research Council (grant number 288164) Dr Gav-asso has received institutional support from the RegionalHeath Authority of Western Norway Go to NeurologyorgNN for full disclosures
Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationOctober 22 2019 Accepted in final form December 20 2019
Appendix Authors
Name Location Role Contribution
Gerd HagaBringelandMD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studymajor role in theacquisition of datainterpretation of dataanalysis of data andrevision of themanuscript forintellectual content
NelloBlaser PhD
University of BergenBergen Norway
Author Interpretation of dataanalysis of data andrevision of themanuscript forintellectual content
Kjell-MortenMyhr MDPhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyand revision of themanuscript forintellectual content
ChristianAlexanderVedelerMD PhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyand revision of themanuscript forintellectual content
SoniaGavassoPhD
University of Bergenand HaukelandUniversity HospitalBergen Norway
Author Conception anddesign of the studyinterpretation of dataand revision of themanuscript forintellectual content
NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 7
References1 Polman CH OrsquoConnor PW Havrdova E et al A randomized placebo-controlled trial
of natalizumab for relapsing multiple sclerosis N Engl J Med 2006354899ndash9102 Katz J Lathi E Heyda L Characterizing the Natalizumab ldquoWearing offrdquo Effect [ab-
stract] ACTRIMS meeting Poster DX36 20143 Gudesblatt M Zarif M Bumstead B et al Multiple sclerosis and natalizumab ldquobe-
tween the dose symptomsrdquo [abstract 982] Mult Scler 201218(suppl 14)P9824 Ratchford JN Brock-Simmons R Augsburger A et al Multiple sclerosis symptom
recrudescence at the end of the natalizumab dosing cycle Int J MS Care 20141692ndash985 van Kempen ZLE Doesburg D Dekker I et al The natalizumab wearing-off effect end
of natalizumab cycle recurrence of MS symptoms Neurology 201993e1579ndashe15866 Punet-Ortiz J Hervas-Garcia JV Teniente-Serra A et al Monitoring CD49d receptor
occupancy a method to optimize and personalize natalizumab therapy in multiplesclerosis patients Cytometry B Clin Cytom 201894327ndash333
7 Bandura DR Baranov VI Ornatsky OI et al Mass cytometry technique for real timesingle cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry Anal Chem 2009816813ndash6822
8 Smith A Symbol digit modalities test Manual Los Angeles CA Western Psycho-logical Services 1982
9 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444ndash1452
10 Krupp LB LaRocca NG Muir-Nash J Steinberg AD The fatigue severity scaleApplication to patients with multiple sclerosis and systemic lupus erythematosusArch Neurol 1989461121ndash1123
11 Rahman AH Tordesillas L Berin MC Heparin reduces nonspecific eosinophilstaining artifacts in mass cytometry experiments Cytometry A 201689601ndash607
12 Bringeland GH Bader L Blaser N et al Optimization of receptor occupancy assays inmass cytometry standardization across channels with QSC beads Cytometry A 201995314ndash322
13 Finak G Perez JM Weng A Gottardo R Optimizing transformations for automatedhigh throughput analysis of flow cytometry data BMC Bioinformatics 201011546
14 Amir el AD Davis KL Tadmor MD et al viSNE enables visualization of highdimensional single-cell data and reveals phenotypic heterogeneity of leukemia NatBiotechnol 201331545ndash552
15 Bruggner RV Bodenmiller B Dill DL Tibshirani RJ Nolan GP Automated identi-fication of stratifying signatures in cellular subpopulations P Natl Acad Sci USA 2014111E2770ndashE2777
16 R A Language and Environment for Statistical Computing [computer program]Vienna R Foundation for Statistical Computing 2017
17 Iaffaldano P Viterbo RG Paolicelli D et al Impact of natalizumab on cognitiveperformances and fatigue in relapsing multiple sclerosis a prospective open-labeltwo years observational study PLoS One 20127e35843
18 Morrow SA OrsquoConnor PW Polman CH et al Evaluation of the symbol digit mo-dalities test (SDMT) and MS neuropsychological screening questionnaire (MSNQ)in natalizumab-treated MS patients over 48 weeks Mult Scler 2010161385ndash1392
19 Foley JF Goelz S Hoyt T Christensen A Metzger RR Evaluation of natalizumabpharmacokinetics and pharmacodynamics with standard and extended interval dos-ing Mult Scler Relat Dis 20193165ndash71
20 Tanaka M Kinoshita M Foley JF Tanaka K Kira J Carroll WM Body weight-basednatalizumab treatment in adult patientswithmultiple sclerosis JNeurol 2015262781ndash782
21 Varhaug KN Torkildsen O Myhr KM Vedeler CA Neurofilament light chain asa biomarker in multiple sclerosis Front Neurol 201910338
22 Muralidharan KK Kuesters G Plavina T et al Population pharmacokinetics andtarget engagement of natalizumab in patients with multiple sclerosis J Clin Pharmacol2017571017ndash1030
23 Mahnke YD Brodie TM Sallusto F Roederer M Lugli E The whorsquos who of T-celldifferentiation human memory T-cell subsets Eur J Immunol 2013432797ndash2809
24 Sallusto F Geginat J Lanzavecchia A Central memory and effector memory T cellsubsets function generation and maintenance Annu Rev Immunol 200422745ndash763
25 Bloomgren G Richman S Hotermans C et al Risk of natalizumab-associated pro-gressive multifocal leukoencephalopathy N Engl J Med 20123661870ndash1880
26 Bomprezzi R Pawate S Extended interval dosing of natalizumab a two-center 7-yearexperience Ther Adv Neurol Disord 20147227ndash231
27 Zhovtis Ryerson L Frohman TC Foley J et al Extended interval dosing of natali-zumab in multiple sclerosis J Neurol Neurosurg Psychiatry 201687885ndash889
28 Ryerson LZ Foley J Chang I et al Risk of natalizumab-associated PML in patientswith MS is reduced with extended interval dosing Neurology 201993e1452ndashe1462
8 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
DOI 101212NXI000000000000067820207 Neurol Neuroimmunol Neuroinflamm
Gerd Haga Bringeland Nello Blaser Kjell-Morten Myhr et al occupancy
Wearing-off at the end of natalizumab dosing intervals is associated with low receptor
This information is current as of February 4 2020
ServicesUpdated Information amp
httpnnneurologyorgcontent73e678fullhtmlincluding high resolution figures can be found at
This article has been cited by 2 HighWire-hosted articles
Subspecialty Collections
httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosisfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2020 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
References1 Polman CH OrsquoConnor PW Havrdova E et al A randomized placebo-controlled trial
of natalizumab for relapsing multiple sclerosis N Engl J Med 2006354899ndash9102 Katz J Lathi E Heyda L Characterizing the Natalizumab ldquoWearing offrdquo Effect [ab-
stract] ACTRIMS meeting Poster DX36 20143 Gudesblatt M Zarif M Bumstead B et al Multiple sclerosis and natalizumab ldquobe-
tween the dose symptomsrdquo [abstract 982] Mult Scler 201218(suppl 14)P9824 Ratchford JN Brock-Simmons R Augsburger A et al Multiple sclerosis symptom
recrudescence at the end of the natalizumab dosing cycle Int J MS Care 20141692ndash985 van Kempen ZLE Doesburg D Dekker I et al The natalizumab wearing-off effect end
of natalizumab cycle recurrence of MS symptoms Neurology 201993e1579ndashe15866 Punet-Ortiz J Hervas-Garcia JV Teniente-Serra A et al Monitoring CD49d receptor
occupancy a method to optimize and personalize natalizumab therapy in multiplesclerosis patients Cytometry B Clin Cytom 201894327ndash333
7 Bandura DR Baranov VI Ornatsky OI et al Mass cytometry technique for real timesingle cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry Anal Chem 2009816813ndash6822
8 Smith A Symbol digit modalities test Manual Los Angeles CA Western Psycho-logical Services 1982
9 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444ndash1452
10 Krupp LB LaRocca NG Muir-Nash J Steinberg AD The fatigue severity scaleApplication to patients with multiple sclerosis and systemic lupus erythematosusArch Neurol 1989461121ndash1123
11 Rahman AH Tordesillas L Berin MC Heparin reduces nonspecific eosinophilstaining artifacts in mass cytometry experiments Cytometry A 201689601ndash607
12 Bringeland GH Bader L Blaser N et al Optimization of receptor occupancy assays inmass cytometry standardization across channels with QSC beads Cytometry A 201995314ndash322
13 Finak G Perez JM Weng A Gottardo R Optimizing transformations for automatedhigh throughput analysis of flow cytometry data BMC Bioinformatics 201011546
14 Amir el AD Davis KL Tadmor MD et al viSNE enables visualization of highdimensional single-cell data and reveals phenotypic heterogeneity of leukemia NatBiotechnol 201331545ndash552
15 Bruggner RV Bodenmiller B Dill DL Tibshirani RJ Nolan GP Automated identi-fication of stratifying signatures in cellular subpopulations P Natl Acad Sci USA 2014111E2770ndashE2777
16 R A Language and Environment for Statistical Computing [computer program]Vienna R Foundation for Statistical Computing 2017
17 Iaffaldano P Viterbo RG Paolicelli D et al Impact of natalizumab on cognitiveperformances and fatigue in relapsing multiple sclerosis a prospective open-labeltwo years observational study PLoS One 20127e35843
18 Morrow SA OrsquoConnor PW Polman CH et al Evaluation of the symbol digit mo-dalities test (SDMT) and MS neuropsychological screening questionnaire (MSNQ)in natalizumab-treated MS patients over 48 weeks Mult Scler 2010161385ndash1392
19 Foley JF Goelz S Hoyt T Christensen A Metzger RR Evaluation of natalizumabpharmacokinetics and pharmacodynamics with standard and extended interval dos-ing Mult Scler Relat Dis 20193165ndash71
20 Tanaka M Kinoshita M Foley JF Tanaka K Kira J Carroll WM Body weight-basednatalizumab treatment in adult patientswithmultiple sclerosis JNeurol 2015262781ndash782
21 Varhaug KN Torkildsen O Myhr KM Vedeler CA Neurofilament light chain asa biomarker in multiple sclerosis Front Neurol 201910338
22 Muralidharan KK Kuesters G Plavina T et al Population pharmacokinetics andtarget engagement of natalizumab in patients with multiple sclerosis J Clin Pharmacol2017571017ndash1030
23 Mahnke YD Brodie TM Sallusto F Roederer M Lugli E The whorsquos who of T-celldifferentiation human memory T-cell subsets Eur J Immunol 2013432797ndash2809
24 Sallusto F Geginat J Lanzavecchia A Central memory and effector memory T cellsubsets function generation and maintenance Annu Rev Immunol 200422745ndash763
25 Bloomgren G Richman S Hotermans C et al Risk of natalizumab-associated pro-gressive multifocal leukoencephalopathy N Engl J Med 20123661870ndash1880
26 Bomprezzi R Pawate S Extended interval dosing of natalizumab a two-center 7-yearexperience Ther Adv Neurol Disord 20147227ndash231
27 Zhovtis Ryerson L Frohman TC Foley J et al Extended interval dosing of natali-zumab in multiple sclerosis J Neurol Neurosurg Psychiatry 201687885ndash889
28 Ryerson LZ Foley J Chang I et al Risk of natalizumab-associated PML in patientswith MS is reduced with extended interval dosing Neurology 201993e1452ndashe1462
8 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN
DOI 101212NXI000000000000067820207 Neurol Neuroimmunol Neuroinflamm
Gerd Haga Bringeland Nello Blaser Kjell-Morten Myhr et al occupancy
Wearing-off at the end of natalizumab dosing intervals is associated with low receptor
This information is current as of February 4 2020
ServicesUpdated Information amp
httpnnneurologyorgcontent73e678fullhtmlincluding high resolution figures can be found at
This article has been cited by 2 HighWire-hosted articles
Subspecialty Collections
httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosisfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2020 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
DOI 101212NXI000000000000067820207 Neurol Neuroimmunol Neuroinflamm
Gerd Haga Bringeland Nello Blaser Kjell-Morten Myhr et al occupancy
Wearing-off at the end of natalizumab dosing intervals is associated with low receptor
This information is current as of February 4 2020
ServicesUpdated Information amp
httpnnneurologyorgcontent73e678fullhtmlincluding high resolution figures can be found at
This article has been cited by 2 HighWire-hosted articles
Subspecialty Collections
httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosisfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2020 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
