ARTICLE OPEN ACCESS CLASS OF EVIDENCE

NurOwn phase 2 randomized clinical trial inpatients with ALSSafety clinical and biomarker results

James D Berry MD Merit E Cudkowicz MD Anthony J Windebank MD Nathan P Staff MD

Margaret Owegi MD Katherine Nicholson MD Diane McKenna-Yasek Yossef S Levy PhD

Natalie Abramov MSc Haggai Kaspi PhD Munish Mehra PhD Revital Aricha PhD Yael Gothelf PhD and

Robert H Brown DPhil MD

Neurologyreg 201993e1-e12 doi101212WNL0000000000008620

Correspondence

Dr Brown

robertbrown

umassmededu

AbstractObjectiveTo determine the safety and efficacy of mesenchymal stem cell (MSC)-neurotrophic factor(NTF) cells (NurOwnreg autologous bone marrow-derived MSCs induced to secrete NTFs)delivered by combined intrathecal and intramuscular administration to participants withamyotrophic lateral sclerosis (ALS) in a phase 2 randomized controlled trial

MethodsThe study enrolled 48 participants randomized 31 (treatment placebo) After a 3-monthpretransplant period participants received 1 dose of MSC-NTF cells (n = 36) or placebo (n =12) and were followed for 6 months CSF was collected before and 2 weeks aftertransplantation

ResultsThe study met its primary safety endpoint The rate of disease progression (Revised ALSFunctional Rating Scale [ALSFRS-R] slope change) in the overall study population was similarin treated and placebo participants In a prespecified rapid progressor subgroup (n = 21) rate ofdisease progression was improved at early time points (p lt 005) To address heterogeneitya responder analysis showed that a higher proportion of treated participants experienced ge15pointsmonth ALSFRS-R slope improvement compared to placebo at all time points and wassignificant in rapid progressors at 4 and 12 weeks (p = 0004 and 0046 respectively) CSFneurotrophic factors increased and CSF inflammatory biomarkers decreased in treated par-ticipants (p lt 005) post-transplantation CSF monocyte chemoattractant protein-1 levelscorrelated with ALSFRS-R slope improvement up to 24 weeks (p lt 005)

ConclusionA single-dose transplantation of MSC-NTF cells is safe and demonstrated early promising signsof efficacy This establishes a clear path forward for a multidose randomized clinical trial ofintrathecal autologous MSC-NTF cell transplantation in ALS

Classification of evidenceThis phase II study provides Class I evidence

RELATED ARTICLE

EditorialStem cells in amyotrophiclateral sclerosis Hype orhope

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MORE ONLINE

Class of EvidenceCriteria for ratingtherapeutic and diagnosticstudies

NPuborgcoe

From Massachusetts General Hospital Neurological Clinical Research Institute and Harvard Medical School (JDB MEC KN) Boston Mayo Clinic (AJW NPS) Rochester MNUniversity of Massachusetts (MO DM-Y RHB) Worcester Brainstorm Cell Therapeutics (YSL NA HK RA YG) Petach Tikva Israel and Tigermed USA (MM) Somerset NJ

Go to NeurologyorgN for full disclosures Funding information and disclosures deemed relevant by the authors if any are provided at the end of the article

The Article Processing Charge was funded by Brainstorm Cell Therapeutics

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 e1

Published Ahead of Print on November 18 2019 as 101212WNL0000000000008620

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenera-tive disease characterized by degeneration and death of motorneurons in the brain and spinal cord and results in progressivemuscle weakness and respiratory failure1 The complex path-ogenesis of ALS including the prominent role of neuro-inflammation suggests that emerging ALS treatments will needto address multiple interrelated aspects of the disease

Bone marrow-derived mesenchymal stem cells (MSCs) aremultipotent progenitor cells that have demonstrated hightherapeutic potential in neurodegenerative disease MSCsmay enhance neurogenesis modulate neuroinflammationand contribute to neuroprotection2 We leveraged the thera-peutic potential of neurotrophic factors (NTFs) by inducingbone marrow-derived patient autologous MSCs into MSC-NTF cells (NurOwn) secreting high levels of multiple NTFs3

and with a unique miRNA signature4 demonstrated to besuperior to MSC of origin in several models of neurodegen-erative diseases

In open-label trials the intrathecal transplantation of Nur-Own demonstrated safety and tolerability and showed pre-liminary evidence of efficacy by slowing the rate of decline onthe Revised ALS Functional Rating Scale (ALSFRS-R) aftertransplantation5

The primary objective of this phase 2 double-blind placebo-controlled multicenter trial was to determine the safety ofMSC-NTF cells administration via combined IT and IMinjections to participants with ALS Prespecified analyses in-vestigated the effect of MSC-NTF cells on the slope of theALSFRS-R in the overall population and in a subgroup ofrapid progressors with a pretreatment decline of ge2 ALSFRS-R points Analyses of CSF pretransplantation and post-transplantation evaluated the effect of the intervention onrelevant MSC and ALS disease biomarkers

MethodsPrimary research questionWe aimed to determine the safety and efficacy of MSC-NTFcells (NurOwn autologous bone marrow-derived MSCsinduced to secrete NTFs) delivered by combined IT and IMadministration to participants with ALS in a phase 2 ran-domized controlled trial This phase II study provides Class Ievidence that MSC-NTF cell transplantation in ALS is safe

Standard protocol approvals registrationsand patient consentsThis randomized double-blind placebo-controlled phase 2trial included 3 US participating sites Massachusetts GeneralHospital (Boston) Mayo Clinic (Rochester MN) and theUniversity of Massachusetts (Worcester) The study protocolwas cleared by the Food and Drug Administration (FDA) andby the institutional review board of each participating siteThe study was conducted in Good Clinical Practice compli-ance (ClinicalTrialsgov identifier NCT02017912)

Participant selection criteriaAt screening eligible participants aged 18ndash75 years had a di-agnosis of possible probable laboratory-supported probableor definite ALS by El Escorial Criteria6 ALSFRS-R ge30 vitalcapacity (VC) ge65 of the predicted normal value for heightage and sex and symptom duration of between 1 and 2 years

Participants were either not receiving riluzole or were ona stable dose for ge30 days had the ability to lie flat for theIT cell transplant procedure and had at least some limbweakness due to ALS Potential participants were excluded forthe following reasons use of mechanical ventilation presenceof feeding tube prior treatment with stem cells pregnancyexposure to investigational agents or immunosuppressivetherapy within 4 weeks of screening active autoimmunedisease or infection (including hepatitis B hepatitis C orHIV) cancer within the previous 5 years unstable psychiatricor medical condition or clinically significant abnormal safetylaboratory values

Randomization and blindingA permuted block randomization stratified by site with a blocksize of 4 (ratio of 31 of activeplacebo) was used for eligibleparticipants to receive MSC-NTF cells or placebo (DulbeccoModified Eagle Medium) administered on a single occasionby combined IT (125 times 106 MSC-NTF cells in a 5-mL sy-ringe using a 20-G spinal needle) administration and 24 IM(48 times 106 MSC-NTF cells) injections in 1-mL syringes toa 15-cm depth (ensuring that injection is into the muscle) at24 separate sites on the participantrsquos right upper arm bicepsand triceps muscles according to a predesignedmap After theIT infusions the cells were chased with CSF withdrawn fromthe participant prior to the transplant Participants were askedto maintain a Trendelenburg position for up to 2 hours afterthe transplantation Participants trial investigators andsponsor personnel were masked to treatment assignment

GlossaryAE = adverse event ALS = amyotrophic lateral sclerosis ALSFRS-R = Revised ALS Functional Rating Scale CHIT-1 =chitotriosidase-1 CI = confidence intervalDSMB = data and safety monitoring board FDA = Food and Drug AdministrationHGF = hepatocyte growth factorHHD = hand-held dynamometry LIF = leukemia inhibitory factor LS = least squaresMCP-1 = monocyte chemoattractant protein-1MSC = mesenchymal stem cell NTF = neurotrophic factor SAE = serious adverseevent SDF-1a = stromal cell-derived factor-1a SVC = slow vital capacity TEAE = treatment-emergent adverse event VC =vital capacity VEGF = vascular endothelial growth factor

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Treatment allocation was assigned at the cell culturemanufacturing facility once the clinical site notified the cellculture facility of subject eligibility None of the study inves-tigators could have been involved in the treatment assignmentin any way The unblinded clinical teams administering theproduct (anesthesiologists or other teams) did not have anycontact with the participants prior to or after productadministration

Study overviewAfter an approximate 3-month run-in period eligible partic-ipants were randomized and their bone marrow was aspiratedfollowed by MSC-NTF cell transplantation 3 to 4 weeks laterFor transplantation all participants were admitted to thehospital for 48 hours of monitoring Subsequent outpatientvisits occurred 2 weeks after transplantation and then every 4weeks for 6 months (figure 1A)

Bone marrow aspirationAll participants regardless of randomization group underwentaspiration of 50ndash70 mL of bone marrow from the iliac crest bya hematologist per medical center standard procedures Theparticipantsrsquo aspirated bone marrow was immediately trans-ferred to the cGMP cell processing facility in a temperature-controlled shipping system per manufacturing collection andshipping procedures

Cell manufacturing and transplantationThe cell therapy product was manufactured at 2 central FDA-cleared cGMP-compliant manufacturing facilities using stan-dard operating procedures MSCs were isolated from thebone marrow expanded and differentiated to secrete NTFsusing a culture-based approach35 The MSC-NTF cells pro-duction was carried out under full environmental control ingood manufacturing practices-compliant cleanrooms Freshautologous MSC-NTF cells were released for transplantationwhen they fulfilled the cell number viability safety (sterilitymycoplasma and endotoxin) potency (using an ELISA assayfor NTF secretion) and identity (CD surface markers) re-lease criteria Potency was determined based on ELISA assaysfor NTF secretion Product was transported from themanufacturing facilities to the clinical sites in a validatedshipping system at a controlled temperature of 2degCndash8degC andadministered fresh to the participant Each shipmentrsquos tem-perature was measured by a data logger

OutcomesThe primary objective was to examine the safety of MSC-NTF cell transplant in patients with ALS Safety laboratorystudies and adverse events (AEs) were collected at each visitmonitored by the site investigator medical monitor and thedata and safety monitoring board (DSMB) which was in-dependent from the study and sponsor and analyzed ata central laboratory

The second objective was to evaluate the efficacy of MSC-NTF cell transplantation in ALS Therefore ALSFRS-R and

slow VC (SVC) were collected at all pretransplant and post-transplant visits (and at weeks 12 and 20 over the telephone)by trained evaluators who were certified by the Outcomes andMonitoring Center for the Northeast ALS Consortium atBarrow Neurological Institute

Two exploratory objectives were also examined The firsthand-held dynamometry (HHD) compared muscle strengthin the IM transplanted arm to the noninjected arm Thesecond exploratory objective examined CSF for changes inNTF biomarker and microRNA expression between treatedand placebo groups and between pretransplant and post-transplant time points This was added as a protocolamendment after the commencement of treatment and beganwith the ninth patient

CSF analysesCSF was collected prior to cell administration and 2 weekspost-transplantation by lumbar puncture immediatelycentrifuged at 1750 g for 10 minutes aliquoted and stored atminus80degC Twenty-six sample pairs of treated and 9 sample pairsof placebo participants for whom pretransplantation and post-transplantation samples were available were analyzed usinga Multiplex immunoassay (customized Procarta immunoas-say Affymetrix Santa Clara CA) according to the manu-facturerrsquos instructions Mean fluorescence intensities ofanalyte-specific immunoassay bead sets were detected bya flow-based Luminex (Austin TX) 3D suspension arraysystem Chitotriosidase-1 (CHIT-1) was analyzed by ELISA

Statistical analysisThe primary trial outcome was safety assessed with respect tothe incidence of treatment-emergent AEs (TEAEs) and seri-ous AEs (SAEs) laboratory abnormalities vital signs ECGsand physical examinations AEs were coded using MedDRA

Secondary endpoints were comparisons of (1) the change inslopes (post-treatment slope through 24 weeks as comparedto the pretreatment slope) in ALSFRS-R and SVC betweenthe treatment and placebo groups and (2) the post-treatmentslope of decline in the ALSFRS-R and SVC at 24 weeks fol-lowing transplantation relative to the 12- to 16-week baselineperiod before transplantation (pretreatment slope) in allpatients (within each treatment group)

Prespecified efficacy analyses in the statistical analysis planincluded comparison of ALSFRS-R slopes post-treatment vspretreatment between treatment groups using model fit leastsquares (LS) means as well as responder analyses comparingthe MSC-NTF and placebo groups Responder analyses wereprespecified and defined as a 20ndash30 improvement in post-treatment slope compared to pretreatment

This was a phase 2 study for which no formal sample sizecalculation was performed and the sample size was not basedon statistical considerations The total of 36 treatment and 12placebo participants are expected to be a sufficient number to

NeurologyorgN Neurology | Volume 93 Number 24 | December 10 2019 e3

obtain adequate characterization of common TEAEs and toobserve trends for treatment effects on the efficacy measureschosen for this study In addition with 36 participants in theMSC-NTF treatment group the study has 95 probability ofseeing AEs that occur at a true rate of 8 or greater There-fore if an AE of a specific type does not occur there is 95probability that the true rate of the AE is less than 8 Efficacyanalyses were not adjusted for multiplicity

We examined 2 methods for defining cutoffs (1) defining anexact change in ALSFRS-R slope in points per month and (2)using a percent change in ALSFRS-R post-treatment slopecompared to pretreatment slope For each method at each

follow-up visit we compared the post-transplantation slopewith the pretransplantation slope and defined a range ofthresholds for responders (ge05 to ge25 pointsmonth andge25 to ge100 change) however analysis of pointsmonthimprovement in the ALSFRS-R slope was found to be moreaccurate particularly in the case of a zero pretreatment slopein which case a percent improvement analysis was not feasibleAvailable data were used to compute slopes which were as-sumed at later time points for missing data

Three prespecified subgroup analyses included ALSFRS-Roverall pretreatment score change (ltminus2 points rapid pro-gressors or geminus2 points slow progressors) ALSFRS-R motor

Figure 1 Trial design and CONSORT diagram

(A) Trial design (B) CONSORT diagram participant enrollment intervention allocation and follow-up MSC = mesenchymal stem cell NTF = neurotrophicfactor

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pretreatment score change (ltminus1 points vs geminus1 points) andbaseline SVC (ge70) Secondary and exploratory objectivesincluded analyses of SVC HHD and CSF biomarkers Sta-tistical significance for the LS means was determined if the pvalue was le005 (2-sided) and for responder analyses if the pvalue was lt005 (2-sided Fisher exact test)

The CSF data were statistically assessed for significance byeither Student t test or analysis of variance analysis as appli-cable A p value of less than 005 was considered statisticallysignificant

Data availabilityAppropriate anonymized data can be made available toqualified investigators upon submission of an acceptableanalysis plan Proposals should be directed to rkernbrain-storm-cellcom To gain access data requestors will need tosign a data access agreement

ResultsFifty-nine participants were screened in this trial There were8 screen failures and during the 3-month run-in period 3participants discontinued the study prior to randomizationForty-eight participants were randomized of whom 43completed follow-up (figure 1B)

Treatment arms showed similar baseline characteristics Themajority of the treated participants were male (35 participants729) and all were white (48 participants 100) The meanage of the participants was 511 years (range 26ndash71 table 1)

SafetyThe trial met its primary safety endpoint thus the treatmentappears to be safe Forty-three participants (90) completedthe follow-up period (33 MSC-NTF 10 placebo) Five par-ticipants discontinued in-person follow-up of these 2 com-pleted the trial by remote telephone follow-up and weretherefore included in all safety and ALSFRS-R analyses (1MSC-NTF 1 placebo figure 1B) There were no deathsduring the study no treatment-related SAEs and no AEs thatled to dropout Two participants in the MSC-NTF treatmentarm underwent tracheostomy placement for respiratory fail-ure following placement of a feeding tube approximately 4months post-treatment

Over the course of the study 11 participantsmdash936 in theMSC-NTF cells group (25) and 212 in the placebo group(17)mdashdeveloped 16 SAEs (table 2) Two SAEs occurredafter trial entry but prior to treatment All treatment-emergentSAEs (AEs that occurred after initiation of study treatment)were deemed to be related to ALS disease progression andnone was considered possibly probably or definitely relatedto study treatment The DSMB did not identify any AEslaboratory abnormalities (blood hematology chemistriesurinalysis) or substantial protocol deviations that would bea cause for concern

Table 3 summarizes the number of treatment-emergent andtreatment-related AEs or SAEs that occurred during the studyand lists all AEs that occurred in gt15 of patients from eithergroup AEs related to the delivery of MSC-NTFs or placebowere transient and included headache (75MSC-NTFs 50placebo) fever (33 MSC-NTFs 0 placebo) back pain

Table 1 Demographics and baseline characteristics by treatment group

MSC-NTF (n = 36) Placebo (n = 12) All participants (n = 48)

Sex

Male 25 (694) 10 (833) 35 (729)

Female 11 (306) 2 (167) 13 (271)

Age y 503 (1190) 535 (911) 511 (1127)

El Escorial criteria

Possible 3 (83) 1 (83) 4 (83)

Laboratory-supported probable 5 (139) 1 (83) 6 (125)

Probable 16 (444) 7 (583) 23 (479)

Definite 12 (333) 3 (250) 15 (313)

ALS medical history months since diagnosis 900 (557) 901 (463) 900 (531)

ALS medical history months since first symptoma 1762 (379) 1675 (310) 1740 (362)

Abbreviations ALS = amyotrophic lateral sclerosis ALSFRS-R = Revised ALS Functional Rating Scale MSC =mesenchymal stem cell NTF = neurotrophic factorData are presented as n () or mean (plusmnSD) Percentages are based on the number of participants in a given treatment group for the population beinganalyzeda The site of ALS onset was not tracked in the course of the study

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(72 MSC-NTFs 8 placebo) and injection site bruising(306 MSC-NTF 25 placebo)

Efficacy analysesALSFRS-R LS mean slope changeAt screening ALSFRS-R scores were similar in theMSC-NTFand placebo groups (381 plusmn 36 and 386 plusmn 385 respectively)Pretreatment ALSFRS-R slopes were similar in the 2 groupsminus07 pointsmonth (MSC-NTF) and minus06 pointsmonth(placebo) The change in the ALSFRS-R slope post-transplant was +17 pointsmonth (MSC-NTF) and minus04pointsmonth (placebo) at 2 weeks (p = 0110) and +06pointsmonth (MSC-NTF) and minus003 pointsmonth (pla-cebo) at 4 weeks (p = 0368) After 8 weeks the change inslope was similar in both treatment arms (figure 2A)

In the prespecified rapid progressors subgroup (ie thosewith a decline of gt2 points in ALSFRS-R scores during the3-month pretreatment period) there were 15 participants(42) in the MSC-NTF arm and 6 (50) in the placebo armPretreatment ALSFRS-R slopes were comparable minus15

(MSC-NTF) and minus12 (placebo) (p = 0232) Comparison ofLS means of the post-transplant ALSFRS-R slope minus thepretransplantation slope between the MSC-NTF and placebogroups demonstrated a significant improvement in the MSC-NTF group at 2 and 4 weeks (+33 vs minus13 p = 0021 and +20vs minus01 p = 0033 respectively) and a continued trend forimprovement in the MSC-NTF group at all remaining timepoints (figure 2B)

ALSFRS-R responder analysesUsing the ge15 pointmonth improvement criterion(ALSFRS-R slope post-treatment compared to pretreatmentslope) a higher proportion of responders were observed inthe MSC-NTF cells group compared to placebo at all timepoints The difference was significant at week 4 (MSC-NTF47 placebo 9 p = 0033 figure 2C)

In the rapid progressors subgroup a higher proportion ofresponders (ge15 pointsmonth ALSFRS-R slope improve-ment) were observed in the MSC-NTF group compared tothe placebo group at all time points the difference was sig-nificant at week 4 (80 vs 0 95 confidence interval [CI]of difference of proportions 598ndash100 p = 0004) and

Table 2 Overall summary of adverse events (AE) and serious AEs (SAEs) by treatment group safety population

TEAEs MSC-NTF cells n = 36 n () Placebo n = 12 n ()

TEAEs 585 109

Participants with at least 1 TEAE 36 (100) 12 (100)

Treatment-related TEAEsa 197 32

Participants with at least 1 treatment-related TEAE 35 (972) 9 (750)

Treatment-related serious TEAEs 0 0

Participants with at least 1 treatment-related serious TEAE 0 0

Participants with TEAEs by maximum severity

Mild 36 (100) 12 (100)

Moderate 34 (944) 10 (833)

Severe 3 (83) 1 (83)

Potentially life-threatening 1 (28) 0

SAEs 14 2

Participants with at least 1 SAE 9 (25) 2 (166)

Participants with at least 1 treatment-emergent SAE 8 (222) 1 (83)

Participants with treatment-related SAE 0 0

Participants with TEAEs resulting in treatment withdrawal 0 0

Participants with TEAEs resulting in withdrawal from study 0 0

Participants with TEAEs resulting in death 0 0

Abbreviations MSC = mesenchymal stem cell NTF = neurotrophic factor TEAE = treatment-emergent adverse eventPercentages are based on the number of participants in a given treatment group for the population being analyzed An AE is considered a TEAE if the startdatetime of the AE is on or after the datetime of initiation of cell transplantation or if the severity worsens after the initiation of cell transplantationa Treatment-related TEAEs are TEAEs that are considered to have probable possible or definite relationship to the study drug

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week 12 (53 vs 0 95 CI of difference of proportions281ndash786 p = 0046 figure 2D)

Slow vital capacityScreening SVC was comparable in the MSC-NTF cells andplacebo groups (906 plusmn 181 and 887 plusmn 891 respectively)Analysis of the changes in post-treatment compared to pre-treatment slope and responder analyses with various thresh-olds showed no significant differences between the 2treatment groups No significant treatment effects were notedin the rapid progressors group

Hand-held dynamometryComparison of muscle strength in the IM transplanted arm tothe noninjected arm as measured by HHD did not demon-strate significant side-to-side difference in HHD musclestrength score slopes over 24 weeks

CSF analysesCSF was collected pretransplant and 2 weeks post-transplantOnly pretransplant and post-transplant paired samples wereanalyzed In total 26 treated and 9 placebo CSF sample pairswere available for analysis

Neurotrophic factorsLevels of NTFs secreted by the MSC-NTF cells in vitro wereevaluated in the CSF Average basal levels of vascular endo-thelial growth factor (VEGF) were 371 plusmn 158 pgmL and 306plusmn 144 pgmL (mean plusmn SEM) in treated and placebo partic-ipants respectively Post-transplantation the mean VEGF in-crease in the treated participants was 6298 plusmn 2433 (p = 0016)and minus078 plusmn 09 (p = 045) in placebo participants Basal

hepatocyte growth factor (HGF) levels were high (treatedparticipants 3911 plusmn 1159 pgmL placebo participants 4484 plusmn1463 pgmL) Post-transplantation HGF levels significantlyincreased in the MSC-NTF-treated participants (mean change10718 plusmn 344 p = 0004) while there was no change in thosereceiving placebo (188 plusmn 222 p = 042) Leukemia inhibitoryfactor (LIF) was undetectable in the CSF prior to trans-plantation and significantly increased (1233 plusmn 323 p =00008) post-transplantation in treated participants while therewas no detectable LIF in the placebo group either pre-transplantation or post-transplantation (figure 3A)

Inflammatory biomarkersInflammatory biomarkers known to be present in the CSF ofpatients with ALS as well as cytokines chemokines and cellularfactors that reflect key processes relating to immune pathwaysCNS inflammation and cell death were selected for analysisNot all biomarkers analyzed were detectable in the CSF

Basal levels of monocyte chemoattractant protein-1 (MCP-1)were 422 plusmn 94 pgmL in treated participants and 405 plusmn 95pgmL in the placebo group Post-transplantation meanMCP-1 levels decreased significantly in the MSC-NTF-treated par-ticipants (minus173 plusmn 18 pgmL p lt 00001) while no significantchange was observed in the placebo group (minus089 plusmn 23 pgmLp = 07) The post-transplantation difference between MSC-NTF-treated participants and the placebo group was statisti-cally significant (p lt 00001) Average basal levels of stromalcell-derived factor-1a (SDF-1a) were 1956 plusmn 311 pgmL inMSC-NTF-treated participants and 2008 plusmn 149 pgmL in theplacebo group Post-transplantation SDF-1a significantly de-creased in the MSC-NTF-treated participants (minus466 plusmn 129pgmL p = 00014) while it remained unchanged in the pla-cebo group (027 plusmn 505 pgmL p = 096) The post-transplantation difference between the 2 groupswas statisticallysignificant (p = 004) CHIT-1 demonstrated a small and sig-nificant decrease in treated patients (minus1413 plusmn 601 p = 0027)and a small nonsignificant increase in placebo patients (6411 plusmn2137) post-transplantation (figure 3B) There was no signifi-cant post-transplantation change in the levels of macrophageinflammatory protein-1β or C-reactive protein in either group(data not shown) A trend towards a greater reduction of CSFinflammatory factors was observed in responder patients atsome timepoints which did not reach significance possibly dueto the small sample size

We also examined the levels of caspase-3 one of the keymediators of apoptosis Interestingly caspase-3 was signifi-cantly reduced post-transplant in MSC-NTF-treated partic-ipants (minus209 plusmn 04 p lt 00001) but not in the placebo group(minus091 plusmn 09 p = 035) A significant reduction (p =002) in responders (ge100 improvement in ALSFRS-Rslope 12 weeks post-transplantation) was observed comparedto nonresponders

Two weeks post-transplant a statistically significant inversecorrelation was observed between VEGF and MCP-1

Table 3 Percentage of participants with treatment-emergent adverse events by trial arm and type(gt15 in either group)

Adverse event MSC-NTF cells Placebo

Headache and procedural headache 806 667

Back pain 722 83

Pyrexia 333 0

Arthralgia 333 0

Injection site pain 278 83

Constipation 25 83

Pain in extremity 222 0

Neck pain 194 0

Myalgia 167 0

Cough 167 0

Nausea 167 0

Abbreviations MSC = mesenchymal stem cell NTF = neurotrophic factor

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(correlation minus056 p = 0003 figure 4A) VEGF and SDF-1a(correlation minus077 p lt 00001) HGF and SDF-1a (correla-tion minus041 p = 0004) LIF and SDF-1 (correlation minus053 p =0005) and LIF and MCP-1 (correlation minus042 p = 0034) inMSC-NTF cells treated participants but not in the placebogroup (data available on request)

A statistically significant correlation was observed between thedecrease in MCP-1 levels post-transplantation and improve-ments in the post-transplantation compared to the pre-transplantation slope Significant correlations (p lt 005) wereidentified at 4 12 16 and 24 weeks post-treatment (figure 4Billustrates the correlation at 12 weeks p lt 001) suggestingthat higher CSF MCP-1 levels may be related to ALS diseaseprogression as measured by the ALSFRS-R

miRNAWe measured the expression of miR-34a miR-132 miR-19miR376-a and miR-146a-5p which are highly expressed inMSC-NTF cells4 We found these miRNAs to be increasedpost-treatment in the CSF of MSC-NTF-treated but notplacebo participants Interestingly miR-34a miR376-a andmiR-132 basal levels are notably lower in nonresponder ascompared to responder participants In addition wemeasuredthe expression of miRNAs known to play an important role inALS including miR-9 miR-155 and miR-5777ndash9 miR-155and miR-577 were undetectable in the CSF of participantswith ALS miR-9 which is not expressed in MSC-NTF cellsdid not significantly change in either group following treat-ment (data available on request)

Figure 2 Mean change in Revised ALS Functional Rating Scale (ALSFRS-R) slope over time (top) and responder analysesge15-point ALSFRS-R slope improvement over the post-treatment follow-up period (bottom)

(A B) ALSFRS-R least squares (LS) means of the change in slope (post-treatment minus pretreatment) for each of the post-treatment time points for the totalpopulation (A) and rapid progressors (defined as those participants with a pretreatment ALSFRS-R change geminus2 between screening and baseline) (B) Thedifference between the treated and placebo groupswas statistically significant at the 2 and 4weeks timepoints (p = 0021 and 0033 respectively indicated bya for p lt 005) (C D) The percentage of participants with a ge15-point improvement in the ALSFRS-R slope at the indicated time points as compared to theirpretreatment slope over the 12 weeks pretreatment period in the mesenchymal stem cell (MSC)ndashneurotrophic factor (NTF) cells treated and the placebogroup total population (C) and rapid progressors (defined as participants with a pretreatment ALSFRS-R change geminus2 between screening and baseline) (D) Inthe overall population the difference was statistically significant at week 4 (p = 0033) In rapid progressors the differences between the treated and placebogroups were statistically significant at the 4 and 12 weeks timepoints (p = 0004 and 0046 respectively indicated by a for p lt 005)

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Data availabilityAppropriate anonymized data can be made available toqualified investigators upon submission of an acceptableanalysis plan Proposals should be directed to rkernbrain-storm-cellcom To gain access data requestors will need tosign a data access agreement

DiscussionThis phase 2 clinical trial met its primary endpoint demon-strating the safety of MSC-NTF cells delivered by combinedIM and IT administration and demonstrated promising

Figure 3 CSF analysis pretransplantation (V5) and 2 weeks post-transplantation (V6)

(A) A significant increase in vascular endothelial growth factor (VEGF) hepatocyte growth factor (HGF) and leukemia inhibitory factor (LIF) levels is shown inthe CSF of the mesenchymal stem cell (MSC)-neurotrophic factor (NTF) cells treated group (upper panels) with no detectable change in the placebo group(lower panels) (B) A significant decrease inmonocyte chemoattractant protein-1 (MCP-1) stromal cell-derived factor-1a (SDF-1) and chitotriosidase-1 (CHIT-1)levels is shown in the CSF of the MSC-NTF cells treated group (upper panels) with no significant change in the placebo group (lower panels) p lt 005 p lt001 p lt 0001

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efficacy particularly in a prespecified subgroup of rapidprogressors

ALS is a heterogeneous disease individual patients exhibithighly variable rates of functional decline10 As a consequenceresponder analyses may more accurately capture individualtreatment responses11 and therefore provide a more in-formative comparison between treatment and placebo groupsthan changes in mean slope alone We prespecified responderanalyses using thresholds of 20ndash30 improvement anddefined a range of thresholds for responders of ge25 toge100 improvement at each follow-up visit This is consistentwith results of a survey of ALS clinicians and clinicalresearchers in which all participants endorsed a 25 or higherchange in the ALSFRS-R slope as at least somewhat clinicallymeaningful and 93 of the participants viewed a 50 changein decline as very clinically meaningful12 In addition the useof point change as a responder criterion precludes the

exclusion of participants who are stable pretreatment (zeroslope) The use of point and percentage change responderanalyses thus provides a more robust and complete ALS ef-ficacy estimate

In the responder analyses a higher proportion of MSC-NTF-treated participants achieved a ge15-point improvement permonth which may reflect disease stabilization This im-provement was most prominent immediately followingtransplantation and gradually decreased toward the end of thestudy suggesting the need for repeated treatments to main-tain a sustained therapeutic effect

To mitigate the effect of disease heterogeneity and variabilityof ALSFRS-R slope change on efficacy outcomes we per-formed a prespecified subgroup analysis based on the pre-treatment ALSFRS-R overall score change (ltminus2 points orgeminus2 points) In the rapid progressors subgroup analysis (ltminus2-

Figure 4 Monocyte chemoattractant protein-1 (MCP-1) correlation to vascular endothelial growth factor (VEGF) secretionand to disease progression

(A) A significant correlation between VEGF increase and MCP-1 decrease is shown in the CSF of the mesenchymal stem cell (MSC)ndashneurotrophic factor (NTF)cells treated group at visit six 2weeks post-transplantationwith no significant change in the placebo group No correlationwas seen between VEGF andMCP-1levels prior to treatment (V5) (B) A significant correlation betweenMCP-1 in the CSF at 2 weeks post MSC-NTF cells treatment (visit 6 right panel) and a slowerdisease progression at 12 weeks post-treatment is shown with no significant change in the placebo group (left panel) ALSFRS-R = Revised ALS FunctionalRating Scale

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point change) a statistically significant higher proportion ofMSC-NTF-treated participants experienced a ge15 pointmonth improvement compared to placebo

We did not observe a significant difference in quantitativestrength in the IM treated and untreated biceps and tricepsmuscles This might reflect the ability to detect significantHHD changes in the small trial population or asymmetricalrate of progression of each arm The small sample size mayhave also reduced the likelihood of detecting changes in SVCgiven its known high testndashretest variability in patients withALS in particular for those with bulbar onset13 In additionVC decline appears to be nonlinear in some patients with ALSwho are slow SVC progressors and experience SVC declineonly at a later stage of the disease14 which may help explainthe lack of treatment effect in this outcome measure

Alternatively a single dose may not have been sufficient toaffect this outcome measure Current clinical evaluation ofMSC-NTF cells in ALS is focused on repeat dosing by the ITroute of administration

Preclinical and clinical studies suggest that MSCs tend to beshort-lived and their beneficial effects may result from theircapacity to regulate tissue homeostasis and inflammation viaelaboration of an array of trophic factors15 In this study weobserved a significant increase in the levels of MSC-NTF cellsrsquospecific factors (eg VEGF HGF and LIF) and severalmiRNAs in the CSF of treated participants This supports theproposed paracrine mechanism of action of MSC-NTF cellsin ALS although we cannot rule out the possibility that theobserved changes may represent a secondary mechanism in-duced by the cells

Neuroinflammation is a prominent pathologic change atregions of motor neuron injury in ALS and is accompanied bymicroglial activation astrogliosis and the release of proin-flammatory cytokines16 CXCR4 and its ligand SDF-1(CXCL12) are key regulators of microglia migration and re-cruitment CXCL12CXCR4 signaling in glial networks playsa major role in enhancing and maintaining spinalneuroinflammation1718 MCP-1 a potent chemoattractantand activating peptide is expressed in astrocytes microgliaand macrophages is elevated in the CSF and spinal cord ofpatients with ALS and may be associated with more severedisease and rapid progression19

The significant decrease in MCP-1 and SDF-1 in MSC-NTF-treated participants the strong correlation between cell-specific NTF increase and inflammatory biomarker decreaseand the relationship between CSF inflammatory biomarkerreduction and the observed clinical improvement in the post-transplantation ALSFRS-R slope indicate that the cells mayalso act by modulating neuroinflammation

miRNAs are small noncoding RNAs that regulate a wide va-riety of biological processes via RNA-dependent post-

transcriptional silencing mechanisms20 Extensive down-regulation of miRNAs has been reported in spinal cord andmotor neurons from patients with ALS21 We have shown anincrease in several cell-secreted miRNAs in treated but notplacebo participants The increase of miR-146a known tomediate suppression of inflammatory response and to reduceMCP-1 levels22 in responder participants and to a lesser ex-tent in nonresponder participants is consistent with the ob-served correlation between post-transplantation decrease inCSF MCP-1 and the observed reduction in disease pro-gression in MSC-NTFndashtreated participants further support-ing the cellsrsquo anti-inflammatory mechanism of action Theincrease in miR-132-3p which is highly enriched in neuronspromotes neuronal outgrowth in vitro and in vivo and reg-ulates brain vascular integrity provides another potentialmolecular mechanism relevant to the biological effects ofMSC-NTF cells in ALS23

Taken together these exploratory analyses of NTF in-flammatory biomarker and microRNA expression levelsdemonstrate for the first time the biological effects of MSC-NTFs in patients with ALS in a randomized clinical trialThese results support the proposed paracrine mechanism ofaction of MSC-NTFs and the combined effects on bothneuroprotection and neuroinflammation We observed in-creased expression of the neuroprotection markers VEGFHGF LIF miR-132p and miR-376 and a decreased expres-sion of neuroinflammatory markers MCP-1 and SDF-1 aswell as an increase in miR-146 further supporting the hy-pothesis that MSC-NTFs operate in study participants withALS to both increase neuroprotection and decrease neuro-inflammation It is highly likely that these 2 interrelatedmechanisms may be synergistic

This randomized double-blind placebo-controlled phase 2trial of MSC-NTF cell transplant showed that administrationof bone-marrowndashderived autologous MSC-NTF cells is safeand well-tolerated In addition we have demonstrated sig-nificant efficacy in a prespecified subpopulation of rapidprogressors where meaningful changes in post-transplantALSFRS-R slope change were observed The stabilization orreversal of ALSFRS-R score in rapid progressors establishesa clear step forward for the phase III repeated-dose trial in thispredefined ALS subgroup

Author contributionsJames D Berry draftingrevising the manuscript data acqui-sition study concept or design analysis or interpretation ofdata accepts responsibility for conduct of research and finalapproval Merit E Cudkowicz draftingrevising the manu-script analysis or interpretation of data accepts responsibilityfor conduct of research and final approval acquisition of datastudy supervision Anthony JWindebank draftingrevising themanuscript data acquisition study concept or design analysisor interpretation of data accepts responsibility for conduct ofresearch and final approval study supervision Nathan P Staffdraftingrevising the manuscript data acquisition study

NeurologyorgN Neurology | Volume 93 Number 24 | December 10 2019 e11

concept or design analysis or interpretation of data acceptsresponsibility for conduct of research and final approval studysupervision Margaret Owegi data acquisition accepts re-sponsibility for conduct of research and final approval acqui-sition of data Katherine Nicholson draftingrevising themanuscript accepts responsibility for conduct of research andfinal approval acquisition of data study supervision DianeMcKenna-Yasek data acquisition accepts responsibility forconduct of research and final approval acquisition of datastudy supervision Yossef S Levy analysis or interpretation ofdata accepts responsibility for conduct of research and finalapproval contribution of vital reagentstoolspatients NatalieAbramov analysis or interpretation of data accepts re-sponsibility for conduct of research and final approval acqui-sition of data Haggai Kaspi draftingrevising the manuscriptanalysis or interpretation of data accepts responsibility forconduct of research and final approval acquisition of dataMunish Mehra draftingrevising the manuscript analysis orinterpretation of data accepts responsibility for conduct ofresearch and final approval statistical analysis Revital Arichadraftingrevising the manuscript analysis or interpretation ofdata accepts responsibility for conduct of research and finalapproval acquisition of data YaelGothelf draftingrevising themanuscript study concept or design analysis or interpretationof data accepts responsibility for conduct of research and finalapproval study supervision Robert H Brown draftingrevising the manuscript data acquisition study concept ordesign analysis or interpretation of data accepts responsibilityfor conduct of research and final approval acquisition of datastudy supervision

Study fundingStudy supported by Brainstorm Cell Therapeutics

DisclosureJ Berry has no financial conflicts has acted as a consultant foran advisory panel for Denali Therapeutics and has researchfunding andor acts as an investigator for studies funded byMT Pharma Brainstorm Cell Therapeutics Amylyx Phar-maceuticals Genentech Anelixis Pharmaceuticals ALS As-sociation Muscular Dystrophy Association ALS Findinga Cure and ALS One M Cudkowicz is a consultant forBiohaven Takeda MT pharma Aclipse and Avexis was ona DSMB for Lilly and receives grant support from NINDSALSFAC and the ALS Association A Windeank is a consul-tant for the Federal Trade Commission on stem cell issues(those consulting fees go to his research budget at MayoClinic) and serves on the external advisory boards of 2 re-search institutes in Europe CURAM in Ireland and SCI-TReCS in Austria (he derives no compensation for thesepositions other than for travel and accommodation) N Staffhas no financial conflicts has research funding andor acts asan investigator for studies funded by Orion PharmaceuticalsCytokinetics Regenerative Medicine Minnesota and Brain-storm Therapeutics and has research funding from the NIH(R01 CA 211887) M Owegi has no financial conflicts withBrainstorm and no relevant financial interestsdisclosures K

Nicholson has no financial conflicts with the BrainstormStudy has acted as a consultant for an advisory panel forAvanir Pharmaceuticals and has research funding andor actsas an investigator for studies funded by Brainstorm CellTherapeutics Amylyx Pharmaceuticals ALS AssociationMuscular Dystrophy Association ALS Finding a Cure TargetALS and the Salah Foundation D McKenna-Yasek has noconflict of interests regarding the Brainstorm Therapeuticsclinical trial YS Levy is a Brainstorm employee N Abramovis a Brainstorm employee H Kaspi is a Brainstorm employeeM Mehra received consulting fees for the analyses fromBrainstorm Cell Therapeutics R Aricha is a Brainstormemployee Y Gothelf is a Brainstorm employee R Brown hasno financial conflicts with the Brainstorm study is a scientificfounder of Apic-Bio and holds equity in Amlyx and Imstarand has research funding andor acts as an investigatorwithout personal financial compensation for studies fundedby Brainstorm Cell Therapeutics the Angel Fund ALS As-sociation Muscular Dystrophy Association ALS Findinga Cure Project ALS Target ALS and ALS One Go toNeurologyorgN for full disclosures

Publication historyReceived by Neurology January 30 2019 Accepted in final formJuly 30 2019

References1 Brown RH Al-Chalabi A Amyotrophic lateral sclerosis N Engl J Med 2017377

162ndash1722 Uccelli A Moretta L Pistoia VMesenchymal stem cells in health and disease Nat Rev

Immunol 20088726ndash7363 Gothelf Y Abramov N Harel A Offen D Safety of repeated transplantations of

neurotrophic factors-secreting human mesenchymal stromal stem cells Clin TranslMed 2014321

4 Gothelf Y Kaspi H Abramov N Aricha R miRNA profiling of NurOwnreg mesen-chymal stem cells secreting neurotrophic factors Stem Cell Res Ther 20178249

5 Petrou P Gothelf Y Argov Z et al Safety and clinical effects of mesenchymal stemcells secreting neurotrophic factor transplantation in patients with amyotrophic lateralsclerosis results of phase 12 and 2a clinical trials JAMA Neurol 201673337ndash344

6 Brooks BR Miller RG Swash M Munsat TL El Escorial revisited revised criteria forthe diagnosis of amyotrophic lateral sclerosis Amyotroph Lateral Scler Other MotorNeuron Disord 20001293ndash299

7 Zhang Z Almeida S Lu Y et al Downregulation of microRNA-9 in iPSC-derivedneurons of FTDALS patients with TDP-43 mutations PLoS One 20138e76055

8 Butovsky O Jedrychowski MP Cialic R et al Targeting miR-155 restores abnormalmicroglia and attenuates disease in SOD1 mice Ann Neurol 20157775ndash99

9 Figueroa-Romero C Hur J Lunn JS et al Expression of microRNAs in human post-mortem amyotrophic lateral sclerosis spinal cords provides insight into diseasemechanisms Mol Cell Neurosci 20167134ndash45

10 Kiernan MC Vucic S Cheah BC et al Amyotrophic lateral sclerosis Lancet 2011377942ndash955

11 Finkel RS Mercuri E Darras BT et al Nusinersen versus sham control in infantile-onset spinal muscular atrophy N Engl J Med 20173771723ndash1732

12 Castrillo-Viguera C Grasso DL Simpson E Shefner J Cudkowicz ME Clinicalsignificance in the change of decline in ALSFRS-R Amyotroph Lateral Scler 201011178ndash180

13 Banno H Schoenfeld D Cudkowicz ME Atassi N Vital capacity high test-retestvariability in ALS patients with bulbar weakness J Neurol Sci 2017381

14 Polkey MI Lyall RA Yang K Johnson E Leigh PN Moxham J Respiratory musclestrength as a predictive biomarker for survival in amyotrophic lateral sclerosis Am JRespir Crit Care Med 201719586ndash95

15 Wang Y Chen X Cao W Shi Y Plasticity of mesenchymal stem cells in immuno-modulation pathological and therapeutic implications Nat Immunol 2014151009ndash1016

16 Zhao W Beers DR Appel SH Immune-mediated mechanisms in the pathoprog-ression of amyotrophic lateral sclerosis J Neuroimmune Pharmacol 20138888ndash899

17 Rabinovich-Nikitin I Ezra A Barbiro B Rabinovich-Toidman P Solomon B Chronicadministration of AMD3100 increases survival and alleviates pathology inSOD1(G93A) mice model of ALS J Neuroinflammation 201613123

e12 Neurology | Volume 93 Number 24 | December 10 2019 NeurologyorgN

DOI 101212WNL0000000000008620 published online November 18 2019Neurology

James D Berry Merit E Cudkowicz Anthony J Windebank et al biomarker results

NurOwn phase 2 randomized clinical trial in patients with ALS Safety clinical and

This information is current as of November 18 2019

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