lymphocyte pharmacodynamics are not associated with … · two-year lymphocyte pharmacodynamics...

ARTICLE OPEN ACCESS

Lymphocyte pharmacodynamics are notassociated with autoimmunity or efficacy afteralemtuzumabHeinz Wiendl MD PhD Matthew Carraro MD Giancarlo Comi MD Guillermo Izquierdo MD PhD

Ho Jin Kim MD PhD Basil Sharrack MD PhD FRCP FAAN Carlo Tornatore MD Nadia Daizadeh PhD

Luke Chung MD MPH Alan K Jacobs MD FAAN Richard J Hogan PhD Linda V Wychowski PhD and

Bart Van Wijmeersch MD PhD On behalf of the CARE-MS I CARE-MS II and CAMMS03409 Investigators

Neurol Neuroimmunol Neuroinflamm 20207e635 doi101212NXI0000000000000635

Correspondence

Dr Wiendl

heinzwiendlukmuensterde

AbstractObjectiveTo examine the association between peripheral blood lymphocyte pharmacodynamics andautoimmune adverse events (AEs) or return of disease activity in alemtuzumab-treated patientswith relapsing-remitting MS

MethodsPatients received 2 alemtuzumab courses (12 mgd IV 5 days at baseline 3 days 12 monthslater) in the 2-year Comparison of Alemtuzumab and Rebif Efficacy in Multiple Sclerosisstudies (NCT00530348 and NCT00548405) and could then receive as-needed alemtuzumabor other disease-modifying therapy in a 4-year extension (NCT00930553) Lymphocytes werephenotyped quarterly over 2 years using fluorescence-activated cell sorting Pharmacodynamicassessments included counts of total lymphocytes CD3+ T cells CD4+CD8+ T cells (totalnaivememoryregulatory [Treg]) and CD19

+ B cells (totalimmaturematurememory) andratios of CD19+ (totalimmaturematurememory) to Treg (CD4

+CD8+) counts Assessedautoimmune AEs included immune thrombocytopenia nephropathies and thyroid eventsEfficacy assessments included relapses 6-month confirmed disability worsening (CDW) andMRI disease activity

ResultsLymphocyte repopulation patterns including ratios between distinct lymphocyte subsets (egCD19+ to Treg cell count ratios) showed no significant differences over 2 years in patientsdevelopingnot developing autoimmune AEs relapses CDW or MRI activity through 6 yearsfollowing alemtuzumab Lymphocyte kinetics were also unrelated to multiple autoimmune AEsor extreme clinical phenotypes

ConclusionsRepopulation kinetics of the evaluated peripheral lymphocyte subsets did not predict auto-immune AE occurrence or disease activity including return of disease activity after 2 alemtu-zumab courses Further study is needed to investigate potential antigen-level markers oftreatment response

From the University of Munster (HW) Munster Germany Novant Health (MC) Charlotte NC University Vita-Salute San Raffaele (GC) Milan Italy Virgen Macarena UniversityHospital (GI) Seville Spain Research Institute and Hospital of National Cancer Center (HJK) Goyang South Korea NIHR Sheffield Biomedical Research Centre Sheffield TeachingHospitals University of Sheffield (BS) Sheffield United Kingdom Georgetown University Medical Center (CT) Washington DC Sanofi (ND LC AKJ) Cambridge MA EloquentScientific Solutions (RJH) Sydney NSW Australia Eloquent Scientific Solutions (LVW) Philadelphia PA and Rehabilitation amp MS-Centre Overpelt (BVW) BIOMED HasseltUniversity Hasselt Belgium

Go to NeurologyorgNN for full disclosures Funding information is provided at the end of the article

The Article Processing Charge was funded by Sanofi

CARE-MS I CARE-MS II and CAMMS03409 coinvestigators are listed at linkslwwcomNXIA163

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 2019 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

Alemtuzumab is a humanized monoclonal antibody that se-lectively depletes circulating CD52-expressing B and Tlymphocytes12 Following depletion a distinctive patternof lymphocyte repopulation potentially leads to a rebalancedimmune system3ndash5

In phase III trials patients with relapsing-remitting MS(RRMS) receiving alemtuzumab experienced significant clini-cal andMRI efficacy improvements vs subcutaneous interferonbeta-1a over 2 years67 Efficacy was maintained over 5 addi-tional years in 2 extension studies8ndash14 The most frequent ad-verse events (AEs) with alemtuzumab were infusion-associatedreactions autoimmune AEs also occurred including thyroidevents immune thrombocytopenia and nephropathies6ndash1315

Pharmacodynamic changes after lymphocyte depletion in-cluding different repopulation patterns among cell subsetsmay account for the overall efficacy of alemtuzumab in RRMSand its associated AE profile116ndash18 Furthermore differencesamong patientsrsquo lymphocyte repopulation patterns have beenhypothesized to explain individual differences in drug re-sponse and create the environment for autoimmune AEs insome patients19 This may include establishment of perma-nent vs more transient influences on tolerance-associatedimmune regulatory network dynamics20

However biomarkers that would predict response to alemtu-zumab or selection of patients at risk for development of au-toimmune events have not been identified21 Althoughincreased serum interleukin-21 levels before alemtuzumab havebeen associated with autoimmune disorders posttreatment thewidespread applicability of such an assay has not been estab-lished22 Furthermore no biomarkers exist for predicting re-currence of disease activity after 2 alemtuzumab courses Thecurrent post hoc analysis methodically assesses whether phar-macodynamic patterns of major peripheral blood lymphocytepopulations are associated with autoimmune AEs or MS dis-ease activity over 6 years after initiating alemtuzumab

MethodsDesign of CARE-MS and extension studiesof alemtuzumabThe efficacy and safety of alemtuzumab were established in 2phase III studies against subcutaneous interferon beta-1a inpatients with active RRMS who were either treatment naive(Comparison of Alemtuzumab and Rebif Efficacy in Multi-ple Sclerosis [CARE-MS] I ClinicalTrialsgov trial identifierNCT00530348 aged 18ndash50 years) or had an inadequate

response to previous therapy (CARE-MS II NCT00548405aged 18ndash55 years)67 In the 2-yearCARE-MS studies (conductedat 178 academic medical centers or clinical practices in 23countries starting in September 2007) patients in the alemtu-zumab arm received 2 courses of alemtuzumab 12 mgd IV on 5consecutive days at baseline and on 3 consecutive days 12months later67 Patients who completed the phase III studiescould enter the 4-year CARE-MS extension (CAMMS03409NCT00930553) in which they could receive additionalcourses of alemtuzumab (12 mgd on 3 consecutive days ge12months after the most recent dose) as needed for relapse orMRI activity or receive other licensed disease-modifyingtherapy at the investigatorrsquos discretion1011 Patients com-pleting the CARE-MS extension study could enroll in an ad-ditional extension the 5-year long-Term follow-up study formultiple sclerOsis Patients who have completed the Alemtu-Zumab extension (TOPAZ) study (NCT02255656) in whichfurther evaluation is ongoing891213

Post hoc analysisTwo-year lymphocyte pharmacodynamics were assessed inalemtuzumab-treated patients (N = 802) stratified by whetherthey experienced autoimmune AEs relapse 6-month con-firmed disability worsening (CDW) or MRI disease activity atany time point within 6 years of follow-up

Autoimmune AEs were defined as any of the followingdocumented at any point within 6 years of follow-up thyroidAEs (excluding asymptomatic abnormal laboratory inves-tigations) immune thrombocytopenia (defined according todiagnostic criteria outlined by an international workinggroup23) or autoimmune nephropathy (in particular anti-glomerular basement membrane disease or membranous glo-merulonephropathy) Monitoring for autoimmune AEs (aspart of the safety monitoring program) included quarterlythyroid function tests monthly hematology tests for immunethrombocytopenia and monthly serum creatinine tests andurinalysis with microscopy for nephropathy as well as spon-taneous reporting of AEs and serious AEs Education on signsand symptoms of autoimmune AEs was provided to health careproviders and patients All monitoring began at baseline andcontinued until 4 years after the last alemtuzumab adminis-tration or until study end whichever occurred later The 4-yearmonitoring period restarted if patients received additionalcourses of alemtuzumab Treatment for autoimmune AEs wasat the discretion of the treating neurologist in consultation withlocal endocrinologists hematologists or nephrologists

Relapses were defined as new neurologic symptoms attrib-utable to MS lasting ge48 hours with an objective change in

GlossaryAE = adverse event Breg = regulatory B CARE-MS = Comparison of Alemtuzumab and Rebif Efficacy in Multiple SclerosisCDW = confirmed disability worsening EDSS = Expanded Disability Status ScaleMMRM = mixed-effects model for repeatedmeasures NEDA = no evidence of disease activity RRMS = relapsing-remitting MS Treg = regulatory T

2 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 1 | January 2020 NeurologyorgNN

neurologic examination Six-month CDW was defined as anincrease of ge10 Expanded Disability Status Scale (EDSS)point (or ge15 points if the baseline EDSS score = 0) con-firmed over 6 months The EDSS score was assessed quarterlyand at the time of suspected relapse by raters who wereblinded throughout the follow-up period to core studytreatment assignment and treatment history MRI diseaseactivity was defined as new gadolinium-enhancing T1 lesionson current MRI or newenlarging T2 hyperintense lesionssince last MRI MRI was assessed annually by imaging spe-cialists blinded to core study treatment assignment andtreatment history If patients experienced autoimmune eventsrelapse CDW or MRI disease activity at any time during thefollow-up period they were classified as having had that typeof event from month 0 onward

In support of the primary analyses additional analysesassessed lymphocyte pharmacodynamics in patients withmultiple autoimmune AEs and patients with various ldquoextremeefficacyrdquo phenotypes including patients with or without sus-tained ldquono evidence of disease activityrdquo (NEDA absence ofrelapse CDW and MRI disease activity sustained over years0ndash6) with or without both relapse and CDW(clinical NEDA)with or without both severe relapse and CDW and with CDW6-month confirmed disability improvement (ge10-point EDSSscore decrease from baseline [assessed in patients with baselineEDSS score ge20]) or stable EDSS score

Lymphocyte dynamicsBlood cell counts in the CARE-MS studies were performedmonthly Lymphocytes were phenotyped using fluorescence-activated cell sorting (Quest Diagnostics Exton PA) at base-line and quarterly thereafter as well as at months 1 and 13(ie 1 month after receiving alemtuzumab courses 1 and 2respectively) Peripheral blood mononuclear cells were stainedusing a T-cell panel of monoclonal antibodies againstCD45RA-FITC (clone L48) CD27-PE (clone L128) CD3-PerCP-Cy55 (clone SK7) CD25-PE-Cy7 (clone 2A3)CD127-APC (clone 40131) and CD4-APC-Cy7 (clone SK3)and a B-cell panel of monoclonal antibodies against CD27-FITC (clone L128) IgD-PE (clone IA6-2) CD19-PerCP-Cy55 (clone SJ25C1) CD10-PE-Cy7 (clone HI10a) CD38-APC (clone HB7) and CD69-APC-Cy7 (clone FN50) (allantibodies from BD Biosciences San JoseSan Diego CA[except CD127-APC from RampD Systems MinneapolisMN]) Lymphocyte data from the CARE-MS I and II studieswere pooled for analysis Absolute cell counts were analyzed fortotal levels of lymphocytes all CD3+ T lymphocytes CD4+

T lymphocytes and CD8+ T lymphocytes and CD19+

B lymphocytes

A lymphocyte substudy was conducted at select study centersin which additional lymphocyte phenotype analyses wereperformed for the naive memory and regulatory (Treg)subsets of CD4+ and CD8+ T lymphocytes and the immaturemature and memory subsets of CD19+ B lymphocytes(n = 146) The phenotypes of all cell subsets assessed are

defined in the table To control for any effect of changes inTreg cell levels over time the kinetic profiles of CD19

+ B cells(total immature mature and memory) were also assessedrelative to CD4+ and CD8+ Treg cell counts

Statistical analysesAll data were analyzed by alemtuzumab treatment coursePatients were categorized as ldquoactiverdquo or ldquononactiverdquo based ondefined events presence or absence of autoimmune AEsrelapses CDW and MRI disease activity To compare thedepletion at month 1 and month 13 and the differential re-constitution between groups linear mixed-effects models forrepeated measures (MMRMs) were undertaken with lym-phocyte parameters as the outcome variable Explanatoryvariables included age and baseline value and either autoim-mune AEs relapse CDW or MRI disease activity SeparateMMRMs were fit for separate events as explanatory variablesMultiple hypothesis testing was adjusted using the Bonferronicorrection and the Benjamini-Hochberg procedure24

Standard protocol approvals registrationsand patient consentsThe CARE-MS trials and CAMMS03409 extension are regis-tered with ClinicalTrialsgov (NCT00530348 NCT00548405and NCT00930553) All procedures were approved by theinstitutional ethics review boards of participating sites Patientsprovided written informed consent

Data availabilityQualified researchers may request access to patient-level dataand related study documents including the clinical study re-port study protocol with any amendments blank case reportform statistical analysis plan and data set specificationsPatient-level data will be anonymized and study documentswill be redacted to protect the privacy of trial participantsFurther details on Sanofirsquos data-sharing criteria eligiblestudies and process for requesting access can be found atclinicalstudydatarequestcom

Table Lymphocyte subset phenotypes

Lymphocytedesignation

Lymphocytesubset name Phenotype

Naive CD4+ T cells Naive CD3+4+ T cells CD4+45RA+27+

Memory CD4+ T cells Memory CD3+4+ T cells CD4+45RAminus

CD4+ Treg cells CD3+4+ regulatory T cells CD4+25intbr+127lominus



CD8+ Treg cells CD3+8+ regulatory T cells CD8+25intbr127lominus

Immature B cells Immature B cells IgD+CD19+27-38+10+

Mature B cells Mature naive B cells IgD+CD19+27-38+10minus

Memory B cells Memory B cells CD19+27+

Abbreviation Treg = regulatory T

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 1 | January 2020 3

ResultsPatients and overall lymphocyte profilesA total of 811 patients were treated with alemtuzumab 12 mgin CARE-MS I and II baseline characteristics were reportedpreviously (mean age 340 [SD 824] years)67 Total lym-phocyte CD4+ and CD8+ T-lymphocyte and CD19+

B-lymphocyte counts following alemtuzumab treatment wereassessed in 802 patients (figure 1)

CD4+ CD8+ and CD19+ lymphocyte depletionand repopulation patterns over 2 years are notassociated with risk of an autoimmune eventover 6 yearsOf the 802 patients 447 (56) developed autoimmuneAEs by definition There was no significant overalldifference in depletion or repopulation patterns oftotal lymphocytes CD4+ and CD8+ T lymphocytes orCD19+ B lymphocytes (assessed over 2 years) in patientswho did or did not experience autoimmune AEs over 6years (figure 2) The timing and magnitude of the changesin total T- and B-lymphocyte levels after treatment as wellas the repopulation patterns were comparable betweenpatients with or without autoimmune AEs (figure 2)Results for CD3+ T-lymphocyte counts were similarbetween the subgroups (figure e-1 linkslwwcomNXIA160)

CD4+ CD8+ and CD19+ lymphocyte depletionand repopulation patterns over 2 years are notassociated with clinical and MRI efficacyparameters over 6 yearsTotal lymphocyte CD4+ and CD8+ T-lymphocyte andCD19+ B-lymphocyte depletion and repopulation patternswere also similar in patients who did or did not experiencerelapses CDW or MRI disease activity through 6 years fol-lowing alemtuzumab treatment (figure 3 AndashC) Among thevarious efficacy subgroups the timing and magnitude ofthe changes in total CD4+ and CD8+ T- and CD19+

B-lymphocyte levels after treatment as well as the repo-pulation pattern were comparable (figure 3 AndashC) Resultsfor CD3+ T-lymphocyte counts were similar (figure e-1linkslwwcomNXIA160)

Analysis of repopulation ofspecific lymphocyte subsets did not showassociation with the risk of an autoimmuneevent or disease activity over 6 yearsIn the expanded phenotype analysis of CD4+ and CD8+

T-lymphocyte subsets (naive memory and Treg cells) andCD19+ B-lymphocyte subsets (immature mature andmemorycells) in a subpopulation of patients (n = 146) repopulationkinetics over 2 years did not differ overall in patients with orwithout autoimmune AEs relapses CDW or MRI diseaseactivity through 6 years (figure 4 AndashC)

Figure 1 Median lymphocyte counts in patients treated with alemtuzumab 12 mg

Median total lymphocyte CD4+ and CD8+ T-lymphocyteand CD19+ B-lymphocyte counts in the overall pooledComparison of Alemtuzumab and Rebif Efficacy inMultiple Sclerosis population treated with alemtuzu-mab 12 mg N value range over 24 months 752ndash802patients Vertical lines indicate administration of alem-tuzumab Dashed horizontal lines represent LLN foreach of the lymphocyte subsets presented IQR =interquartile range LLN = lower limit of normal


For both the autoimmune AE and efficacy assessments the keypharmacodynamic characteristics of the lymphocyte subsetsfollowing alemtuzumab treatment were comparable regardlessof autoimmune event or efficacy status Absolute counts ofCD4+ and CD8+ T-lymphocyte subsets (naive memory andTreg cells) were typically lowest at the time of first

posttreatment blood count (month 1) and reconstitutedsteadily after each treatment course (figure 4 A and B) CD8+

Treg cells showed less pronounced depletion compared withCD4+ Treg cells (figure 4 A and B) Absolute counts of matureand memory CD19+ B lymphocytes decreased posttreatmentand were lowest at month 1 whereas absolute counts of

Figure 2 Median lymphocyte counts in alemtuzumab-treated patients with or without autoimmune AEs

(A) Median total lymphocyte (B) CD4+ and (C) CD8+ T-lymphocyte and(D) CD19+ B-lymphocyte counts in patients from the pooled Compari-son of Alemtuzumab and Rebif Efficacy in Multiple Sclerosis trialstreated with alemtuzumab 12 mg stratified by the presence or ab-sence of autoimmune events N value range over 24 months 415ndash447patientswithout autoimmuneAEs 337ndash355 patients with autoimmuneAEs Vertical lines indicate administration of alemtuzumab AE = ad-verse event IQR = interquartile range LLN = lower limit of normal


immature B lymphocytes increased posttreatment and werehighest at month 3 (figure 4C) Mature B cells repopulated toapproach baseline levels by month 6 followed by a rise abovebaseline levels memory B cells repopulated more slowly(figure 4C)

In light of a recently put forward hypothesis that variations inthe efficacy and safety of alemtuzumab may reflect B-cellhyper-repopulation in the absence of effective T-cell regula-tion in some patients19 we additionally visualized our data inan alternative manner by normalizing the kinetic profiles ofB cells against CD4+ and CD8+ Treg cell levels No differencesin depletion or repopulation kinetics were seen in patientswith or without autoimmune events relapse CDW or MRIactivity through 6 years (figure e-2 A and B linkslwwcomNXIA161)

Additional analyses in patients with or without multiple au-toimmune AEs or ldquoextreme efficacyrdquo phenotypes (eg with orwithout sustained NEDA) also failed to find any significantoverall differences in depletion or repopulation profiles (datanot shown) Of note our analysis did not reveal a specificlymphocyte pattern in the first 2 years that would allowa prediction of the return of disease activity after the secondcourse of alemtuzumab

DiscussionB and T lymphocytes play an integral role in the diseaseprocess in MS25 By selectively depleting B andT lymphocytes alemtuzumab induces a lymphopenia that ismeasurable in the peripheral blood The distinct repopulationpatterns of B and T subsets following alemtuzumab

Figure 3 Median lymphocyte counts in alemtuzumab-treated patients with or without disease activity

Median total lymphocyte CD4+ and CD8+ T-lymphocyte and CD19+ B-lymphocyte counts in Comparison of Alemtuzumab and Rebif Efficacy in MultipleSclerosis patients treatedwith alemtuzumab12mg stratified by the presence or absence of (A) relapse (B) 6-month CDW and (C)MRI disease activity N valuerange over 24months 356ndash388 patientswithout relapse 395ndash414 patientswith relapse 567ndash606 patientswithout CDW 184ndash196 patientswith CDW 263ndash280patients without MRI activity 485ndash516 patients with MRI activity Vertical lines indicate administration of alemtuzumab CDW = confirmed disability wors-ening IQR = interquartile range LLN = lower limit of normal


treatment which have been described previously2627 lead toquantitative and qualitative changes in immune regulatorynetworks These changes include suppression of memoryB cells which may play several roles in the pathogenesis of

MS2829 a relative increase in Treg and memory T-cell countsand a potential shift from a pro- to anti-inflammatory envi-ronment (driven by differential reconstitution of T-cellsubsets)26273031 Despite this knowledge few studies have

Figure 4 Lymphocyte subset counts in alemtuzumab-treated patients with or without autoimmune AEs or disease activity

(A) CD4+ T- (B) CD8+ T- and (C) CD19+ B-lymphocyte subset counts in Comparison of Alemtuzumab and Rebif Efficacy in Multiple Sclerosis patients treatedwith alemtuzumab 12mg stratified by the presence or absence of autoimmune AEs relapse 6-month CDW and MRI disease activity N value range over 24months 29ndash72 patients without autoimmune AEs 30ndash76 patients with autoimmune AEs 23ndash72 patients without relapse 27ndash75 patients with relapse 44ndash108patients without CDW 14ndash38 patients with CDW 23ndash63 patients without MRI activity 35ndash84 patients with MRI activity AE = adverse event CDW = confirmeddisability worsening IQR = interquartile range Treg = regulatory T


attempted to correlate aspects of these pharmacodynamicchanges with either safety or efficacy or the need for additionalcourses of alemtuzumab

The present study assessed lymphocyte dynamics over thefirst 2 years following alemtuzumab initiation in relation to thedevelopment of autoimmune events or the return of diseaseactivity over 6 years This enabled a methodical evaluation ofthe prognostic value of lymphocyte depletion and repopula-tion patterns specifically focusing on the putatively mostimportant populations (CD4+CD8+ T cells and CD19+

B cells) including key subsets such as Treg cells The questionsaddressed were whether differences in the depletion orrepopulation patterns of these cell types are associated with(1) development of autoimmune AEs or (2) efficacy (in-cluding the relevant question of the need for retreatment) Inanswer to the first question our analysis demonstrated therepopulation kinetics of the tested lymphocyte populationsdid not differ in alemtuzumab-treated patients with or withoutautoimmune AEs over 6 years Our findings are consistentwith a previous study demonstrating that T-cell regulationdoes not play a role in the development of autoimmunity inalemtuzumab-treated patients instead the autoimmunitymay be largely driven by reduced thymopoiesis a restrictedT-cell repertoire and homeostatic expansion of T cells thathave escaped depletion32 This is also in line with a study byJones et al33 which suggested that increased levels ofinterleukin-21 may drive cycles of T-cell expansion and apo-ptosis after alemtuzumab thus increasing the opportunitiesfor T cells to encounter self-antigen that leads to autoim-mune AEs

Importantly our study could not confirm what one of theprevailing hypotheses in this area of research would suggestnamely that an overshoot of B cells is a likely contributor tothe risk of disease exacerbation andor autoimmune de-regulation That hypothesis is based on the observation thatafter initial depletion with alemtuzumab treatment B cells inmany patients begin to repopulate to levels exceeding thoseobserved before treatment with implications for safety basedon B-cell hyper-repopulation in the absence of effective T-cellregulation19 The results from our analysis demonstrated thatthe repopulation kinetics of B cells including memory B cellsdid not differ in alemtuzumab-treated patients with or withoutautoimmune AEs over 6 years Furthermore there were nodifferences either in the B to Treg cell ratios or the repopu-lation patterns of T-cell subsets between patients with orwithout autoimmune AEs

Our study also provides evidence that more robust oraccelerated lymphocyte repopulation does not predict thereturn of clinical or MRI disease activity in alemtuzumab-treated patients over 6 years These findings extend thosefrom a previous analysis that examined the relationshipbetween lymphocyte pharmacodynamics and clinical efficacyover the course of the CARE-MS core (2-year) studies In thatanalysis median cell counts for total lymphocytes

T lymphocytes (CD3+ CD4+ and CD8+) and CD19+

B lymphocytes were similar at months 1 and 13 in patientsubgroups stratified by whether they experienced a relapse orCDW event subsequent to the time point being assessed Therate of lymphocyte repopulation was also similar betweenpatients who did or did not experience a relapse or CDW afterreceiving alemtuzumab34

The findings of this study imply that the pharmacodynamicpatterns of major lymphocyte populations in the peripheralblood within the 2 years following alemtuzumab initiation donot have prognostic value Peripheral immune phenotypingmay be too superficial or not sufficiently sensitive to detectdifferences among individuals in immune regulatory and tol-erance networks that likely occur after alemtuzumab treat-ment Of particular interest a recent study of patients withMSdemonstrated that some self-antigens that are expressed onperipheral B memory cells are also expressed in the brain andT cells that become autoreactive to the antigen can also mi-grate to brain MS lesions35 Further research on the effects ofalemtuzumab at the antigen-specific level and within the CNSwill likely provide additional insights into its overall mecha-nism of action and underlying mechanisms of autoimmuneAEs clinical response and potential prognosis

Limitations of our study include the caveats that are inherentin any post hoc analysis In addition the lymphocyte data inthis study only extend for the first 2 years after initiation ofalemtuzumab treatment longer-term follow-up may haveprovided additional insights As the depth of immune phe-notyping in our study was limited the pharmacodynamics ofother leukocyte subsets that are linked to response to alem-tuzumab (ie natural killer cells dendritic cells and regula-tory B [Breg] cells) need to be further investigated Forexample relative increases in CD56bright natural killercells3637 and Breg cells have been reported following initiationof alemtuzumab along with decreased proportions of den-dritic cell subsets that are capable of eliciting detrimentalimmune responses in MS37 Differences in the pharmacody-namics of these subsets could also underlie autoimmuneevents or differences in efficacy responses in patients treatedwith alemtuzumab Of particular interest in the case of pe-ripheral Breg cells a recent study showed levels of theCD19+CD24hiCD38hi cell subset to deplete with the onset ofsevere relapses and repopulate during recovery although thisobservation was based on data from a single alemtuzumab-treated patient38 Further analysis of the pharmacodynamicsof B-cell clones may provide additional insight into autoim-mune events in alemtuzumab-treated patients Finally inaddition to studying peripheral blood further research onlymphoid organs and the CSF is needed to improve un-derstanding of the interrelation of different compartmentscontributing to immune regulation after alemtuzumabtreatment

The current analyses suggest that differences in depletionor repopulation kinetics of the major subpopulations of


peripheral blood lymphocytes have no prognostic value inalemtuzumab-treated patients with RRMS Howeverrepopulation patterns of specific B-cell clones with thepotential to express autoimmune antibodies and the rele-vance of those patterns to autoimmune AE occurrenceremain to be determined Lymphocyte repopulation ki-netics at the level in the present analysis likely cannot beused to predict the need for administration of additionalcourses of alemtuzumab

AcknowledgmentThe authors and Sanofi thank the patients for theirparticipation in the CARE-MS I CARE-MS II andCAMMS03409 studies and the steering committees and theinvestigators Critical review of the manuscript for medicalaccuracy was provided by Darren P Baker PhD Ericka MBueno PhD and Colin P Mitchell PhD of Sanofi

Study fundingThis study was supported by Sanofi and Bayer HealthCarePharmaceuticals Prof H Wiendl was supported by theDeutsche Forschungsgemeinschaft (DFG) Grant CRC128Project A09 and the Kompetenznetz Multiple Sklerose(Competence Network for Multiple Sclerosis) funded by theFederal Ministry of Education and Research (FKZ01GI1308B 01GI0907)

DisclosuresHWiendl reports receiving consulting andor speaking fees andgrantresearch support from Bayer Bayer Schering PharmaBiogen Elan Corporation Lilly Lundbeck Merck SeronoNovartis Novo Nordisk Sanofi and Teva Neuroscience MCarraro reports receiving speaking and consulting fees and ad-visory honoraria from Biogen Genentech Genzyme and Mal-linckrodt G Comi reports receiving consulting fees fromActelion Bayer Merck Serono Novartis Sanofi and Teva andlecture fees from Bayer Biogen Dompe Merck SeronoNovartis Sanofi Serono Symposia International Foundationand Teva G Izquierdo reports receiving speaking and advisoryfees from Almirall Bayer Biogen Merck Serono NovartisRoche Sanofi and Teva HJ Kim reports receiving consultingandor speaking fees from Bayer Biogen Celltrion Eisai Gen-zyme HanAll BioPharma MedImmune Merck SeronoNovartis Teva-Handok and UCB research support fromGenzyme Merck Serono Ministry of Science amp ICT Teva-Handok and UCB serving as a steering committee member forMedImmune and serving as a coeditor for Multiple SclerosisJournalmdashExperimental Translational and Clinical and as an as-sociate editor for the Journal of Clinical Neurology B Sharrackreports receiving research and travel grants honoraria for expertadvice on MS and speaking fees from Biogen Merck NovartisRoche Sanofi and Teva C Tornatore reports receiving hono-raria for attending advisory boards and research funding fromBayer Biogen Novartis and Sanofi N Daizadeh L Chung andAK Jacobs report receiving personal compensation as employ-ees of Sanofi RJ Hogan and LV Wychowski report receivingpersonal compensation as employees of Envision Pharma

Group B Van Wijmeersch reports receiving research and travelgrants honoraria for MS-expert advice and speaking fees fromBayer Schering Biogen Merck Serono Novartis Roche SanofiandTeva Go toNeurologyorgNN for full disclosures Fundinginformation is provided at the end of the article

Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationMay 15 2019 Accepted in final form September 5 2019

Appendix Authors

Name Location Role Contribution

HeinzWiendl MDPhD

University ofMunster MunsterGermany

Author Acquisition andinterpretation ofthe data and criticalrevision of themanuscript

MatthewCarraro MD

Novant HealthCharlotte NC


GiancarloComi MD

University Vita-Salute San RaffaeleMilan Italy


GuillermoIzquierdoMD PhD

Virgen MacarenaUniversity HospitalSeville Spain


Ho Jin KimMD PhD

Research Instituteand Hospital ofNational CancerCenter GoyangSouth Korea

Author Acquisition andinterpretation of thedata and criticalrevision of themanuscript

BasilSharrackMD PhDFRCP FAAN

NIHR SheffieldBiomedicalResearchCentre SheffieldTeaching HospitalsUniversity ofSheffield SheffieldUnited Kingdom


CarloTornatoreMD

GeorgetownUniversity MedicalCenterWashington DC


NadiaDaizadehPhD

Sanofi CambridgeMA

Authorandstatistician

Design andconceptualizationof the analysisanalysis of the datainterpretation ofthe data andcritical revision ofthe manuscript

Luke ChungMD MPH

Sanofi CambridgeMA

Author Design andconceptualizationof the analysisinterpretation ofthe data andcritical revision ofthe manuscript

Continued


References1 Freedman MS Kaplan JM Markovic-Plese S Insights into the mechanisms of the

therapeutic efficacy of alemtuzumab inmultiple sclerosis J Clin Cell Immunol 201341000152

2 Hu Y Turner MJ Shields J et al Investigation of the mechanism of action of alemtu-zumab in a human CD52 transgenic mouse model Immunology 2009128260ndash270

3 Cox AL Thompson SA Jones JL et al Lymphocyte homeostasis followingtherapeutic lymphocyte depletion in multiple sclerosis Eur J Immunol 2005353332ndash3342

4 Havari E Turner MJ Campos-Rivera J et al Impact of alemtuzumab treatment on thesurvival and function of human regulatory T cells in vitro Immunology 2014141123ndash131

5 Wiendl H Kieseier B Multiple sclerosis reprogramming the immune repertoire withalemtuzumab in MS Nat Rev Neurol 20139125ndash126

6 Cohen JA Coles AJ Arnold DL et al Alemtuzumab versus interferon beta 1a as first-line treatment for patients with relapsing-remitting multiple sclerosis a randomisedcontrolled phase 3 trial Lancet 20123801819ndash1828

7 Coles AJ Twyman CL Arnold DL et al Alemtuzumab for patients with relapsingmultiple sclerosis after disease-modifying therapy a randomised controlled phase 3trial Lancet 20123801829ndash1839

8 Arnold DL Barnett M Comi G et al Durable reduction in MRI disease activity andslowing of brain volume loss with alemtuzumab in patients with active RRMS 7-yearfollow-up of CARE-MS I patients (TOPAZ study) Mult Scler J 201723(suppl 3)P1189

9 Coles AJ Boyko AN De Seze J et al Alemtuzumab durably improves clinical out-comes in patients with active RRMS in the absence of continuous treatment 7-yearfollow-up of CARE-MS I patients (TOPAZ study) Mult Scler J 201723(suppl 3)P1188

10 Coles AJ Cohen JA Fox EJ et al Alemtuzumab CARE-MS II 5-year follow-upefficacy and safety findings Neurology 2017891117ndash1126

11 Havrdova E Arnold DL Cohen JA et al Alemtuzumab CARE-MS I 5-year follow-updurable efficacy in the absence of continuous MS therapy Neurology 2017891107ndash1116

12 Pelletier D Traboulsee A Barnett M et al Patients with active RRMS experiencedurable reductions in MRI disease activity and slowing of brain volume loss withalemtuzumab 7-year follow-up of CARE-MS II patients (TOPAZ study) Mult Scler J201723(suppl 3)P741

13 Singer BA Alroughani R Brassat D et al Durable improvements in clinical outcomeswith alemtuzumab in patients with active RRMS in the absence of continuoustreatment 7-year follow-up of CARE-MS II patients (TOPAZ study) Mult Scler J201723(suppl 3)P736

14 Ziemssen T Thomas K Alemtuzumab in the long-term treatment of relapsing-remitting multiple sclerosis an update on the clinical trial evidence and data from thereal world Ther Adv Neurol Disord 201710343ndash359

15 Wiendl H Bourdette D Ciccarelli O Can immune reprogramming with alemtuzu-mab induce permanent remission in multiple sclerosis Neurology 2017891098ndash1100

16 Genzyme Therapeutics Ltd LEMTRADA (alemtuzumab) summary of productcharacteristics 2017 Available at emaeuropaeudocsen_GBdocument_libraryEPAR_-_Product_Informationhuman003718WC500150521pdf Accessed Feb-ruary 23 2018

17 Fox EJ Alemtuzumab in the treatment of relapsing-remitting multiple sclerosis Ex-pert Rev Neurother 2010101789ndash1797

18 Turner MJ Lamorte MJ Chretien N et al Immune status following alemtuzumabtreatment in human CD52 transgenic mice J Neuroimmunol 201326129ndash36

19 Baker D Herrod SS Alvarez-Gonzalez C Giovannoni G Schmierer K Interpretinglymphocyte reconstitution data from the pivotal phase 3 trials of alemtuzumab JAMANeurol 201774961ndash969

20 Wiendl H Calabresi PA Meuth SG Defining response profiles after alemtuzumabrare paradoxical disease exacerbation Neurology 201890309ndash311

21 Dorr J Baum K Alemtuzumab in the treatment of multiple sclerosis patient selectionand special considerations Drug Des Devel Ther 2016103379ndash3386

22 Azzopardi L Thompson SA Harding KE et al Predicting autoimmunity afteralemtuzumab treatment of multiple sclerosis J Neurol Neurosurg Psychiatry 201485795ndash798

23 Rodeghiero F Stasi R Gernsheimer T et al Standardization of terminologydefinitions and outcome criteria in immune thrombocytopenic purpura of adultsand children report from an international working group Blood 20091132386ndash2393

24 Benjamini Y Hochberg Y Controlling the false discovery rate a practical andpowerful approach to multiple testing J R Stat Soc Ser B Stat Methodol 199557289ndash300

25 Frohman EM Eagar T Monson N Stuve O Karandikar N Immunologic mecha-nisms of multiple sclerosis Neuroimaging Clin N Am 200818577ndash588

26 Hartung HP Arnold DL Cohen JA et al Lymphocyte subset dynamics followingalemtuzumab treatment in the CARE-MS I study Presented at the 28th Congress ofthe European Committee for Treatment and Research in Multiple Sclerosis October10ndash13 2012 Lyon France

27 Kasper LH Arnold DL Coles AJ et al Lymphocyte subset dynamics followingalemtuzumab treatment in the CARE-MS II study Presented at the 29th Congress ofthe European Committee for Treatment and Research in Multiple Sclerosis October2ndash5 2013 Copenhagen Denmark

28 Blauth K Owens GP Bennett JL The ins and outs of B cells in multiple sclerosisFront Immunol 20156565

29 von Budingen HC Palanichamy A Lehmann-Horn K Michel BA Zamvil SS Updateon the autoimmune pathology of multiple sclerosis B-cells as disease-drivers andtherapeutic targets Eur Neurol 201573238ndash246

30 De Mercanti S Rolla S Cucci A et al Alemtuzumab long-term immunologic effectTreg suppressor function increases up to 24 months Neurol Neuroimmunol Neu-roinflamm 20163e194 doi101212NXI0000000000000194

31 Durelli L DeMercanti S Rolla S et al Alemtuzumab long term immunological studythe immunosuppressive effect does not last more than 48 months Neurology 201686(suppl 16)S2008

32 Jones JL Thompson SA Loh P et al Human autoimmunity after lymphocyte de-pletion is caused by homeostatic T-cell proliferation Proc Natl Acad Sci U S A 201311020200ndash20205

33 Jones JL Phuah CL Cox AL et al IL-21 drives secondary autoimmunity in patientswith multiple sclerosis following therapeutic lymphocyte depletion with alemtuzu-mab (Campath-1H) J Clin Invest 20091192052ndash2061

34 Coles AJ Palmer J Margolin DH Lymphocyte counts do not predict risk of sub-sequent relapse or disability accumulation in alemtuzumab-treated relapsing-remitting multiple sclerosis patients an analysis of the CARE-MS studies Neurol-ogy 201482(suppl 10)P3181

35 Jelcic I Al Nimer F Wang J et al Memory B cells activate brain-homing autoreactiveCD4+ T cells in multiple sclerosis Cell 201817585ndash100

36 Gilmore W Lund BT Traboulsee A et al Leukocyte repopulation following alem-tuzumab treatment in relapsing-remitting MS contains multiple regulatory immunecell types Mult Scler 201723(suppl 3)P979

37 Gross CC Ahmetspahic D Ruck T et al Alemtuzumab treatment alters circulatinginnate immune cells in multiple sclerosis Neurol Neuroimmunol Neuroinflamm20163e289 doi101212NXI0000000000000289

38 Kim Y Kim G Shin HJ et al Restoration of regulatory B cell deficiency followingalemtuzumab therapy in patients with relapsing multiple sclerosis J Neuroinflammation201815300

Appendix (continued)


Alan KJacobs MDFAAN

Sanofi CambridgeMA


Richard JHogan PhD

Eloquent ScientificSolutions SydneyNSW Australia

Author Interpretation ofthe data anddrafted themanuscript forintellectual content

Linda VWychowskiPhD

Eloquent ScientificSolutionsPhiladelphia PA


Bart VanWijmeerschMD PhD

Rehabilitation ampMS-CentreOverpelt BIOMEDHasselt UniversityHasselt Belgium



DOI 101212NXI000000000000063520207 Neurol Neuroimmunol Neuroinflamm

Heinz Wiendl Matthew Carraro Giancarlo Comi et al alemtuzumab

Lymphocyte pharmacodynamics are not associated with autoimmunity or efficacy after

This information is current as of October 29 2019

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Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

Alemtuzumab is a humanized monoclonal antibody that se-lectively depletes circulating CD52-expressing B and Tlymphocytes12 Following depletion a distinctive patternof lymphocyte repopulation potentially leads to a rebalancedimmune system3ndash5

In phase III trials patients with relapsing-remitting MS(RRMS) receiving alemtuzumab experienced significant clini-cal andMRI efficacy improvements vs subcutaneous interferonbeta-1a over 2 years67 Efficacy was maintained over 5 addi-tional years in 2 extension studies8ndash14 The most frequent ad-verse events (AEs) with alemtuzumab were infusion-associatedreactions autoimmune AEs also occurred including thyroidevents immune thrombocytopenia and nephropathies6ndash1315

Pharmacodynamic changes after lymphocyte depletion in-cluding different repopulation patterns among cell subsetsmay account for the overall efficacy of alemtuzumab in RRMSand its associated AE profile116ndash18 Furthermore differencesamong patientsrsquo lymphocyte repopulation patterns have beenhypothesized to explain individual differences in drug re-sponse and create the environment for autoimmune AEs insome patients19 This may include establishment of perma-nent vs more transient influences on tolerance-associatedimmune regulatory network dynamics20

However biomarkers that would predict response to alemtu-zumab or selection of patients at risk for development of au-toimmune events have not been identified21 Althoughincreased serum interleukin-21 levels before alemtuzumab havebeen associated with autoimmune disorders posttreatment thewidespread applicability of such an assay has not been estab-lished22 Furthermore no biomarkers exist for predicting re-currence of disease activity after 2 alemtuzumab courses Thecurrent post hoc analysis methodically assesses whether phar-macodynamic patterns of major peripheral blood lymphocytepopulations are associated with autoimmune AEs or MS dis-ease activity over 6 years after initiating alemtuzumab

MethodsDesign of CARE-MS and extension studiesof alemtuzumabThe efficacy and safety of alemtuzumab were established in 2phase III studies against subcutaneous interferon beta-1a inpatients with active RRMS who were either treatment naive(Comparison of Alemtuzumab and Rebif Efficacy in Multi-ple Sclerosis [CARE-MS] I ClinicalTrialsgov trial identifierNCT00530348 aged 18ndash50 years) or had an inadequate

response to previous therapy (CARE-MS II NCT00548405aged 18ndash55 years)67 In the 2-yearCARE-MS studies (conductedat 178 academic medical centers or clinical practices in 23countries starting in September 2007) patients in the alemtu-zumab arm received 2 courses of alemtuzumab 12 mgd IV on 5consecutive days at baseline and on 3 consecutive days 12months later67 Patients who completed the phase III studiescould enter the 4-year CARE-MS extension (CAMMS03409NCT00930553) in which they could receive additionalcourses of alemtuzumab (12 mgd on 3 consecutive days ge12months after the most recent dose) as needed for relapse orMRI activity or receive other licensed disease-modifyingtherapy at the investigatorrsquos discretion1011 Patients com-pleting the CARE-MS extension study could enroll in an ad-ditional extension the 5-year long-Term follow-up study formultiple sclerOsis Patients who have completed the Alemtu-Zumab extension (TOPAZ) study (NCT02255656) in whichfurther evaluation is ongoing891213

Post hoc analysisTwo-year lymphocyte pharmacodynamics were assessed inalemtuzumab-treated patients (N = 802) stratified by whetherthey experienced autoimmune AEs relapse 6-month con-firmed disability worsening (CDW) or MRI disease activity atany time point within 6 years of follow-up

Autoimmune AEs were defined as any of the followingdocumented at any point within 6 years of follow-up thyroidAEs (excluding asymptomatic abnormal laboratory inves-tigations) immune thrombocytopenia (defined according todiagnostic criteria outlined by an international workinggroup23) or autoimmune nephropathy (in particular anti-glomerular basement membrane disease or membranous glo-merulonephropathy) Monitoring for autoimmune AEs (aspart of the safety monitoring program) included quarterlythyroid function tests monthly hematology tests for immunethrombocytopenia and monthly serum creatinine tests andurinalysis with microscopy for nephropathy as well as spon-taneous reporting of AEs and serious AEs Education on signsand symptoms of autoimmune AEs was provided to health careproviders and patients All monitoring began at baseline andcontinued until 4 years after the last alemtuzumab adminis-tration or until study end whichever occurred later The 4-yearmonitoring period restarted if patients received additionalcourses of alemtuzumab Treatment for autoimmune AEs wasat the discretion of the treating neurologist in consultation withlocal endocrinologists hematologists or nephrologists

Relapses were defined as new neurologic symptoms attrib-utable to MS lasting ge48 hours with an objective change in

GlossaryAE = adverse event Breg = regulatory B CARE-MS = Comparison of Alemtuzumab and Rebif Efficacy in Multiple SclerosisCDW = confirmed disability worsening EDSS = Expanded Disability Status ScaleMMRM = mixed-effects model for repeatedmeasures NEDA = no evidence of disease activity RRMS = relapsing-remitting MS Treg = regulatory T






B lymphocytes


































































Appendix Authors


HeinzWiendl MDPhD



MatthewCarraro MD



GiancarloComi MD






Ho Jin KimMD PhD






CarloTornatoreMD



NadiaDaizadehPhD

Sanofi CambridgeMA



Luke ChungMD MPH

Sanofi CambridgeMA


Continued











































Alan KJacobs MDFAAN

Sanofi CambridgeMA


Richard JHogan PhD



Linda VWychowskiPhD






















Reprints








B lymphocytes


































































Appendix Authors


HeinzWiendl MDPhD



MatthewCarraro MD



GiancarloComi MD






Ho Jin KimMD PhD






CarloTornatoreMD



NadiaDaizadehPhD

Sanofi CambridgeMA



Luke ChungMD MPH

Sanofi CambridgeMA


Continued











































Alan KJacobs MDFAAN

Sanofi CambridgeMA


Richard JHogan PhD



Linda VWychowskiPhD






















Reprints

















































Appendix Authors


HeinzWiendl MDPhD



MatthewCarraro MD



GiancarloComi MD






Ho Jin KimMD PhD






CarloTornatoreMD



NadiaDaizadehPhD

Sanofi CambridgeMA



Luke ChungMD MPH

Sanofi CambridgeMA


Continued











































Alan KJacobs MDFAAN

Sanofi CambridgeMA


Richard JHogan PhD



Linda VWychowskiPhD






















Reprints













































Alan KJacobs MDFAAN

Sanofi CambridgeMA


Richard JHogan PhD



Linda VWychowskiPhD






















Reprints



















Reprints




lymphocyte pharmacodynamics are not associated with … · two-year lymphocyte pharmacodynamics...

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