Engineering5(2019)150–155Contents lists available at ScienceDirectEngineeringResearch
Immunology—Article
Donor-DerivedCD19-TargetedTCellInfusionEliminatesBCellAcuteLymphoblasticLeukemiaMinimalResidualDiseasewithNoResponsetoDonorLymphocytesafterAllogeneicHematopoieticStemCellTransplantation
YifeiChenga,b,#,YuhongChena,b,#,ChenhuaYana,b,YuWanga,b,XiangyuZhaoa,b,YaoChena,b,
WeiHana,b,LanpingXua,b,XiaohuiZhanga,b,KaiyanLiua,b,ShashaWanga,b,LungjiChangc,LeiXiaod,XiaojunHuanga,b,?aBeijingKeyLaboratoryofHematopoieticStemCellTransplantation,PekingUniversityInstituteofHematology,PekingUniversityPeople’sHospital,Beijing100044,ChinaCollaborativeInnovationCenterofHematology,PekingUniversity,Beijing100084,ChinacShenzhenGeno-ImmuneMedicalInstitute,Shenzhen518057,ChinadInnovativeCellularTherapeuticsCo.,Ltd.,Shanghai201499,Chinabarticleinfoabstract
Leukemiarelapseisstilltheleadingcauseoftreatmentfailureafterallogeneichematopoieticstemcelltransplantation(allo-HSCT)forBcellacutelymphoblasticleukemia(B-ALL).RelapsedpatientswithB-ALLafterallo-HSCThaveaveryshortmediansurvival.Minimalresidualdisease(MRD)ispredictiveofforthcominghematologicalrelapseafterhematopoieticstemcelltransplantation(HSCT);furthermore,eliminatingMRDeffectivelypreventsrelapse.Donorlymphoblasticinfusion(DLI)isthemainestablishedapproachtotreatB-ALLwithMRDafterallo-HSCT.However,aboutone-thirdofpatientswithMRDarenon-responsivetoDLIandtheirprognosisworsens.Althoughdonor-derivedclusterofdifferentiation(CD)19-directedchimericantigenreceptor-modi?ed(CAR)Tcells(CART19s)canpotentiallycureleuke-mia,theef?ciencyandsafetyofinfusionswiththesecellshavenotyetbeeninvestigatedinpatientswithMRDafterHSCT.BetweenSeptember2014andFebruary2018,sixpatientseachreceivedoneormoreinfusionsofCART19sfromHSCTdonors.Five(83.33%)achievedMRD-negativeremission,andonecasewasnotresponsivetotheadministrationofCARTcells.Threeofthesixpatientsarecurrentlyalivewith-outleukemia.Nopatientdevelopedacutegraft-versus-hostdisease(aGVHD),andnopatientdiedofcyto-kinereleasesyndrome.Donor-derivedCARTcellinfusionsseemtobeaneffectiveandsafeinterventionforpatientswithMRDinB-ALLafterallo-HSCTandforthosewhowerenotresponsivetoDLI.ó 2019 THE AUTHORS. Published by Elsevier LTD on behalf of Chinese Academy of Engineering and
Higher Education Press Limited Company. This is an open access article under the CC BY license
Articlehistory:Received7April2018Revised23December2018Accepted26December2018Availableonline11January2019Keywords:Donor-derivedCD19-targetedTcellinfusionHematopoieticstemcelltransplantationBcellacutelymphoblasticleukemiaMinimalresidualdisease1.IntroductionBcellacutelymphoblasticleukemia(B-ALL)canbecuredbyallogeneichematopoieticstemcelltransplantation(allo-HSCT)[1,2].Hematologicalrelapseisacommoncauseoftreatmentfail-ureinpatientswithB-ALLafterallo-HSCT,andclinicaloutcomesforthesepatientsaredismal.RelapsedadultpatientswithB-ALLafterallo-HSCThadamediansurvivaltimeof5.5months,and?Correspondingauthor.#E-mailaddress:xjhrm@medmail.com.cn(X.Huang).Theseauthorscontributedequallytothiswork.
relapsedchildrenwithB-ALLafterallo-HSCThadathree-yearevent-freesurvival(EFS)of15%andanoverallsurvival(OS)of20%[3,4].Minimalresidualdisease(MRD)afterhematopoieticstemcelltransplantation(HSCT)isareliablemarkerforimpendinghemato-logicalrelapseandcanthusserveasatriggerforpre-emptivetherapy[5–7].Ingeneral,reduction/withdrawaloftheimmunosup-pressant,theadministrationofinterferon,anddonorlymphoblasticinfusion(DLI)arethemainestablishedapproachestopreventrelapseinpatientswithB-ALLandpositiveMRDafterallo-HSCT[8–10].Severalstudieshaveinvestigatedtheeffectivenessofinter-feronandDLIintreatingMRDinB-ALLafterallo-HSCT.DLIisanY.Chengetal./Engineering5(2019)150–155151importantoptionfortherelapseofB-ALLafterHSCT.However,responseratesafterDLIhavebeenlow.Yanetal.[11]foundthatapproximately64%patientswithrelapsedacuteleukemiaafterhap-loidenticalhematopoieticstemcelltransplantation(haplo-HSCT)wereabletoachievecompleteremission(CR)afterDLI,andthattheone-yeardisease-freesurvival(DFS)ratewas36%.Yanetal.[12]alsofoundthatMRD-andgraft-versus-hostdisease(GVHD)-guidedmultipleconsolidationchemotherapyandDLIreducedthecumulativeincidenceofrelapse(CIR)andincreasedleukemia-freesurvival(LFS)andthesurvivalrateinpatientswhohadrelapsedafterallo-HSCTforacuteleukemia,incomparisonwiththecontrols.However,approximatelyone-thirdofpatientswhowerenon-responsivetoDLIhadworseprognoses,andinnovativeapproachesareurgentlyneededtoimprovetheOSofthesepatients.Leukemiccellscanbeeliminatedef?cientlybyclusterofdiffer-entiation(CD)19-directedchimericantigenreceptor-modi?ed(CAR)Tcells(CART19s)inrelapsedpatientstreatedwithchemotherapy;theyalsoplayanimportantroleinpatientsafterHSCT.Historically,CART19scanbedevelopedfromeitherthepatientoradonor.AlthoughautologousCART19scanexhibitpotentanti-leukemiaactivitybeforeorafterHSCTwithoutGVHD,itmaybedif?culttoobtainsuf?cienthigh-qualityTcellsfrompatientswithpreviousHSCT,thosehavingundergonechemother-apy,andthosewithdiseaserecurrence.Donor-derivedCART19swieldpotentgraft-versus-leukemia(GVL)activitywithoutaffect-ingthecytotoxicactivityfromCART19s[13],andthesafetyandeffectivenessofdonor-derivedCART19swerethefocusofattentioninthisstudy.Brudnoetal.[14]evaluatedtheeffectofCART19sinmorphologicallyrelapsedpatientsafterhumanleukocyteantigen(HLA)-matchedsiblingdonorandunrelateddonorallogeneicbloodormarrowtransplantationwiththeabsenceofGVHD.Subse-quently,donor-derivedCD19-targetedTcellinfusionsinducedMRD-negativeremissioninpatientswithrelapsedB-ALLwhohadnoresponsetoDLIafterhaplo-HSCT[15].Theseclinicalstudiesindicatethatdonor-derivedCART19scaneliminateleukemiccellsinrelapsedpatientsafterHSCT,eveninpatientswithnoresponsetoDLI.Preliminaryresultsalsoindicatethatdonor-derivedanti-CD19CARTcellinfusionissafeforpatientswithrelapsedBcellmalignanciesafterHSCT.Whetherornotdonor-derivedCARTcellinfusionscanbeusedasaneffec-tiveinterventiontotreatMRDinBcellmalignanciesafterHSCThasnotbeendetermined.Inthepresentstudy,weevaluatedprepara-toryclinicaloutcomesfollowingdonor-derivedCARTcellinfusioninpatientswithMRDofBcelllineagemalignancieswhowerenon-responsivetoDLIafterallo-HSCT.2.Methods2.1.PatientsPatientswhohadreceivedaCARTcellinfusionbetweenSeptem-ber2014andFebruary2018atthePekingUniversityInstituteofHematologywereenrolledinthepresentstudyiftheymetthefol-lowingcriteria:①TheywerediagnosedwithB-ALL;②theyhadMRDafterallo-HSCT,theirMRDwasde?nedasmorphologicremis-sion,andtheyreceivedpositiveresultsonanyMRDtest,including?owcytometry,WT1expression,andfusiongenedetectionbyquan-titativereal-timepolymerasechainreaction(qPCR)inpatientswithdetectablefusiongenes;③theyshowednoresponsetooneormoreDLIs(noresponsewasde?nedasMRD-positivebasedontestingofbonemarrowaspirate25–30dafterDLI);and④inthesepatients,CD19expressiononleukemiccellswascon?rmedby?owcytometry.TheprotocolwasevaluatedandallowedbythePekingUniver-sityPeople’sHospitalreviewboard.AllenrolledpatientsgaveinformedconsentinaccordancewiththeDeclarationofHelsinki.2.2.Haplo-HSCTprocedureThehaplo-HSCTprocedure,includingtheconditioningregimen,GVHDprophylaxis,stemcellcollection,andsupportivecare,isdescribedinourpreviousreport[16].Thefollowingdrugswereadministeredduringtheconditioningregimen:cytosinearabi-noside(4gá(m2ád)à1for2d);busulfan(Bu)(3.2mgá(kgád)à1for3d);cyclophosphamide(Cy)(1.8gá(m2ád)à1for2d);semustine(250mgámà2for1d);andanti-humanthymocyteimmunoglobulin(2.5mgá(kgád)à1for4d;Thymoglobulin,GenzymeCorporation,Boston,MA,USA).Allpatientsreceivedgranulocytecolony-stimulatingfactor-mobilizedbonemarrowcellsplusperipheralbloodstemcells.AlltransplantrecipientsreceivedcyclosporineA(CsA),mycophenolatemofetil,andshort-termmethotrexate(MTX)forprophylaxisagainstacutepost-transplantationGVHD.2.3.HLA-matchedsiblingdonorHSCTprocedureAllpatientsweretreatedwithamodi?edBuCy2regimenconsistingofthefollowing:hydroxyurea(80mgákgà1,orallyintwodosesondayà10);Ara-C(2gámà2,intravenousinjectionondayà9);andBu(3.2mgá(kgád)à1administeredintravenouslyondaysà8toà6),Cy(1.8gá(m2ád)à1,daysà5toà4),andsemustine(250mgámà2,dayà3).ForGVHDprophylaxis,CsAwasusedasdescribedabove;mycophenolatemofetilwasdiscontinuedonthedayofmyeloidrecovery,andMTXwasadministeredondays1,3,and6[17].2.4.DLIprocedureInthepresentstudy,DLIconsistedofaninfusionofgranulocytecolony-stimulatingfactor-mobilizedperipheralbloodstemcellsafterchemotherapyandadministrationofimmunosuppressiveagentpost-infusiontopreventGVHD[18].Themediandoseofmononucleatedcellswas1?108kgà1.AfterDLI,allpatientsreceivedimmunosuppressiveagentssuchasCsAtopreventGVHDfor4–6weeksatthediscretionoftheattendingphysicians(usuallydependingonthepatient’sGVHDstatusafterDLI).TheinitialdoseofCsAwas2.5mgá(kgád)à1,andthedosewasadjustedtomaintainaplasmaconcentrationof150–250ngámLà1.NoresponsetoDLIwasde?nedasthedetectionofMRDonemonthafterDLI.2.5.CellproductionWeproducedanti-CD19CARTcellsderivedfromperipheralbloodmononuclearcells(PBMCs)fromtheallogeneictransplantdonorsofeachpatientviaapheresisorperipheralblood.Thedura-tionofCARTcellproductionwas5–15d.Tcellswereactivatedandmodi?edtoexpressthe4SCAR19geneafterPBMCswereobtained.Ondaysà5toà7,PBMCswereactivatedandenrichedforTcells,followedby4SCAR19lentiviraltransduction.PriortoCART19infu-sion,theCART19sforeachpatientweresubjectedtoa?uorescence-activatedcell-sorting(FACS)analysisoftransductionef?ciencyandinvitrocytotoxicityassays.Inaddition,possiblefun-gal,bacterial,mycoplasma,chlamydia,andendotoxincontamina-tionwasevaluatedduringCART19cultures.TheotherdetailsofCART19sareprovidedintheAppendixA.2.6.CARTcellinfusionprotocolPatientswereadministeredaconditioningtreatmentforlym-phodepletion.Mostreceiveda?udarabine-orcyclophosphamide-basedconditioningtreatmentaccordingtothetumorburdenandthediscretionoftheattendingphysicians(detailsaredescribedinSection3.2).CART19sweretransfuseddirectlytopatientswithoutpremedication;thedosagesandcharacteristicsoftheinfusedcells152Y.Chengetal./Engineering5(2019)150–155areprovidedinSection3.4.PatientsreceivedthesecondCARTcellinfusiondosewhenoneofthefollowingcriteriawasful?lled:①PositiveresultsofMRDweredetectedinthepatientsafterthe?rstinfusion;or②thepatientssufferedfrommorphologicalrelapse.3.Results3.1.CharacteristicsofpatientsSixB-ALL(threefemales,threemales;agerange:12–46yearsold)withMRDafterallo-HSCT(fourhaplo-HSCTandtwoHLA-matchedsiblingdonorHSCTcases)wereenrolledinthisstudy.AllpatientsexperiencedMRDandwerenon-responsivetoatleastonecourseofDLI(Table1).Fivepatientsreceivedmorethanoneinfusioneach(totalinfusions:15),andthemedianCARTcellinfu-siondosewas1.51?108cellsákgà1(range:2?107–3.66?108cellsákgà1).AllpatientswereobserveduntilFebruary2018.3.2.CRrateandclinicalresultsFiveofsix(83.33%)patientsachievedMRD-negativeCRafterthe?rstinfusion,and?vepatientsremainedhealthyforamedianof3months(range2–17months)afterCARTcellinfusion.Patients1–4developedrecurrentMRD.Patient1receivedasecondinfu-sion,butfailedtoachieveremission.Patient2receivedasecondinfusionandagainachievedremission,butsufferedMRDrecur-rence2monthslater.Subsequently,patient2wasadministeredfourinfusionsofCART19sandfailedtoattainremission.Patients3and4receivedsecondinfusionsandcontinuetolivewithoutleu-kemia.Patient5didnotrespondtothe?rstandthesecondCARTcellinfusion,developedmorphologicrelapse,andthendiedofleu-kemia.Patient6achievedremissionandcontinuestolivewithoutdisease.AlldetailsaresummarizedinTables2and3.3.3.Graft-versus-hostdiseaseNopatientdevelopedaGVHDduringthefollow-upCARTcellinfusion.3.4.CytokinereleasesyndromeThetoxicitiesobservedafterCARTcellinfusion,whichincludedfever,hypotension,hypoxemia,andelevatedalaninetransaminase(ALT),wereclassi?edasCRSafterrulingoutothercauses.CRSwasscoredonthebasisofarevisedgradingsystem[21].ThetoxicitiesthatoccurredineachpatientarelistedinTable4.Sixpatientsexperiencedatotalof?ve(83.33%)CRSevents,andthese?veeventsoccurredafterthe?rstinfusion(grade1,twoevents;grade2,oneevent;grade3,twoevents).Onepatientrequiredmedicationintervention(tocilizumab):Patient6developedarash,fever,andhypotensiononthe8th2.7.ToxicityoftheinfusionofCARTcellsandGVHDToxicitywasgradedusingthecommoncriteriaforcytokinereleasesyndrome(CRS)afterCARTcellinfusion[19,20].Thepos-sibilityofinfectionwasconsideredinallpatientspresentingwithCRSsymptomsafterobtainingtheappropriateculturesandinitiat-ingempiricantibiotictreatment.GVHDwasdiagnosedasacuteorchronicaccordingtotheclinicalfeaturesoftheaffectedorgans,andacutegraft-versus-hostdisease(aGVHD)wasgradedaccordingtopreviouslypublishedcriteria[21].Primarytherapyconsistingofmethyl-prednisoloneoradexamethasoneequivalentwasadministeredwhenGVHDcouldnotberuledout.Methylprednisolonewasadministeredintravenouslyatadoseof1–2mgá(kgád)à1.CsAwasadministeredandthenadjustedtomaintainabloodconcen-trationgreaterthan150ngámLà1inpatientswhoseGVHDwasnotcontrolled.2.8.ResponseevaluationAbonemarrowexaminationwasperformedonthe15thand/or30thdayafterCARTcellinfusiontoassesstheclinicaleffect,orsoonerifclinicallyindicated.Subsequently,bonemarrowexami-nationwasperformedevery1–3months.AbonemarrowsmearandMRDdetectionby?owcytometry,fusiongenedetectionbyqPCR,andmeasurementofWT1geneexpressionlevelswereper-formedforallpatients.MRD-negativeCRwasde?nedasmorphologicremissionandnegativeresultsonallMRDtests,includingWT1expression,?owcytometry,andfusiongenedetectionbyqPCRinpatientswithdetectablefusiongenes.2.9.StatisticalanalysisThepatientcharacteristicswereevaluatedusingdescriptivestatistics.MRDrateswerecomparedusingFisher’sexacttest,andqualitativevariableswereassessedusingStudent’st-test.AllanalyseswereperformedusingSPSSsoftwareversion19(SPSSInc.,Chicago,IL,USA).Table1
Characteristicsofpatients.PatientAge(yearsold)16SexDonor/typeoftransplantFather/haplo-HSCT(4/6)MalignancyDetectionofMRDStatusbeforeCARTcellinfusionMRD+,CNSLDLIbeforeCARTcellinfusion6FrequencyofCARTcellinfusion1+11M234561240281846FMFMFFather/haplo-HSCT(3/6)Brother/haplo-HSCT(4/6)Father/haplo-HSCT(3/6)ThesecondHSCT,CP3Sibling(6/6)Sibling(6/6)B-ALLph+(T315I)CNSLTLB-ALL,TEL/AML1+B-ALLph+CNSLCMLlymphoblasticcrisisB-ALLB-ALLph+FCM(0.02%),WT1,BCR/ABL=1.4üM,WT1FCM,WT1,BCR/ABLFCM,WT1,BCR/ABLFCM,WT1FCM,WT1,BCR/ABLMRD+MRD+,CNSLMRD+MRD+MRD+122211+51+11+11+11CML:chronicmyelogenousleukemia;CNSL:centralnervoussystemleukemia;F:female;FCM:?owcytometry;M:male;ph+:philadelphiachromosome-positive;sibling:HLA-matchedsiblingdonor;TL:testicularleukemia.Y.Chengetal./Engineering5(2019)150–155Table2
The?rstinfusionofCARTcellsandpatientoutcomes.PatientChemotherapybeforeinfusionCODPCyCOMPNoneCHOPFlu+CyChimericCARTcellsinfused(kg)1.10?108chimerachimerachimerachimera2.00?1072.24?1089.00?1071.30?1083.66?108CRSEvaluationofMRDCRCRCRCRNR0.07%(3%)CRGVHDGluVirusBasMRtime(month)5234—17ClinicaloutcomeRelapse(death)Relapse(death)MR(CNSL-CR)MRRelapse(death)MR153Survivaltimeafterinfusion(month)121215(alive)16(alive)517(alive)123456—FullFullFull—Full2311—3a————————————Herpessimplex(12thmonth)———————————Bas:basiliximab;CHOP:cyclophosphamide(750mgámà2onday1)+vincristine(1.4mgámà2ondays1,8,15,21)+adriamycin(50mgámà2onday1)+prednisone(60mgámà2ondays1–21);CODP:cyclophosphamide(750mgámà2onday1)+vincristine(1.8mgámà2ondays1,8,15,21)+daunorubicin(40–60mgámà2ondays1–3)+prednisone(60mgámà2ondays1–21);COMP:cyclophosphamide(400mgámà2ondays1,8)+vincristine(1.4mgámà2ondays1,8)+mitoxantrone(5mgámà2ondays1–4)+prednisone(60mgámà2ondays1–21);Cy:cyclophosphamide(500mgámà2ondays1–3);Flu:?udarabine(30mgámà2ondays1–3);Glu:glucocorticoid;MR:molecularremission.NR:noresponse.aPatient6requiredmedicationintervention(tocilizumab?2ondays8and9).Table3
ThesecondinfusionofCARTcellsandpatientoutcomes.Patient12345ChemotherapyCODPAra-CFluNoneFlu+CyEvaluationbeforeCARTcellfusionMRD+MRD+MRD+,CNSLRelapseMRD+CARTcellsinfused(kg)Cy2.60?10—8.00?1076.20?1071.70?1088CRS00000EvaluationMRD+MRD+CRCRRelapseClinicaloutcomesRelapse(death),7monthsRelapse(death),12monthsAliveAliveRelapse(death),3monthsdayafterinfusionandreceivedtwodosesoftocilizumabonthe8thand9thdaysafterinfusion.3.5.PeripheralbloodCART19sDuringfourinfusioncourses,CART19sintheperipheralbloodweremeasuredbyqPCRonthe7th,14th,and21stdaysafterinfu-sion.ThenumberofCART19sintheperipheralbloodpeakedonthe7thdayanddecreasedbythe21stdayafterinfusion(Table4).4.DiscussionMRDisagoodmethodtopredicthematologicalrelapseafterHSCT[22,23],andeliminatingMRDcanpreventforthcomingrelapse.SeveralmeasureswereadoptedtotreatMRD.InterferontherapyandchemotherapyfollowedbyDLIconstituteanimportantinterventionforrelapsedacutelymphoblasticleukemiaafterHSCT.Moetal.[24]haveindicatedthatpreemptiveinterferon-a(IFN-a)therapymaybeanalternativeforMRD-positivepatientswhocan-notreceivepreemptiveDLIafterHSCT,basedonacomparisonoftheresultsofDLIandIFN-atreatmentinacutelymphoblasticleu-kemiapatientswithpositiveMRDafterHSCT.Inthiscomparison,theone-yearprobabilitiesofDFSafterinterventionwere68.2%and60.0%forpatientsintheIFN-aandDLIgroups,respectively.Yanetal.[12]havesuggestedthatMRD-andGVHD-guidedmulti-pleconsolidationchemotherapyandDLIcouldreduceCIRandincreaseLFSandsurvivalincomparisonwiththecontrolsinper-sonsrelapsingafterallo-HSCTwithacuteleukemia.Thethree-yearCIR,LFS,andsurvivalpost-transplantwere32.4%,50.3%,and51.4%,respectively.Maetal.[25]havereportedthat19patientswithahighriskfordiseasewhorelapsedafterallo-HSCTweretrea-tedwithDLIandmonitoredforMRD.Sixofthesepatients(31.58%)showednoleukemicprogressionafterDLI.Thus,itcanbeseenthattheDLIapproachwasoflimitedef?cacyintreatingacutelym-phoblasticleukemiaandcouldresultinlife-threateningGVHD.Moreover,approximatelyone-thirdofpatientswithMRDmaybenon-responsivetoDLIandhavetheworstprognosis.Thesepatientsshouldbetreatedusinginnovativeapproaches.CAR-engineeredTcellspresentanovelandpromisingimmunotherapy[26].Infusionofdonor-derivedCART19safterHLA-matchedsiblingdonor/unrelateddonortransplantationwasinvestigated,andfourof?ve(80%)patientswithB-ALLachievedremissionwithouttheoccurrenceofaGVHD[14].Wealsocon-ductedaclinicalstudyfordonor-derivedCD19-targetedTcellinfu-sionandfoundthatthistreatmentinducedMRD-negativeremissioninrelapsedB-ALLwithnoresponsetodonorlymphocyteinfusionsafterhaplo-HSCT.Fiveofsix(83.33%)patientsachievedMRD-negativeremission;furthermore,CRSandGVHDwerecon-trolled[15].Insummary,donor-derivedCD19-targetedTcellinfu-sionseemstobeeffectiveforrelapsedpatientswithB-ALLafterallo-HSCT.However,itremainsunknownwhetherCARTcellinfu-sionscanbeeffectiveinpatientswithMRDwhoareresistanttoDLIafterHSCT.Inthepresentstudy,theresultsindicatedthatdonor-derivedCART19scanalsobeeffectiveintreatingpatientswithMRDafterHSCT.Asfarasweknow,thepresentstudycomprisesthelargestinvestigationofdonor-derivedCART19infusioninrelapsedpatientswithB-ALLafterallo-HSCT.TheresultsofthisstudyofferpreliminaryevidenceoftheeffectivenessofeliminatingMRDbymeansofdonor-derivedCART19infusion.Thesafetyofdonor-derivedCARTcellinfusionhaslongbeenaconcern.Ininvitroexperiments,donorCART19scouldpromoteeffectiveGVLactivityintheabsenceofdamagingGVHDactivitywithoutaffectingthecytotoxicactivityofCART19s[27].Inthepre-sentstudy,thesafetyofdonor-derivedCARTcellinfusionsforHSCTrecipientswasalsomainlyevaluatedwithrespecttoaGVHDandCRS.NopatientdevelopedaGVHD,whichwasthecomplica-tionofprimaryconcern.TheincidenceandintensityofGVHDinthepresentstudywerelessthanthoseresultingfromtheCARTcellinfusionsthatwereusedtotreatrelapsepatientspreviously[15].ThisclinicalphenomenonwasconsistentwiththeoutcomereportedbyJacobyetal.[28].Invitro,murineallogeneicCD19Tcellsdisplaypotentantileukemicactivity,butdemonstratepoten-tiallylethalGVHD.However,CAR-inducedGVHDoccurredonlywiththeappearanceofleukemiccells.CRSwasanothermajorcon-cern.Inthepresentstudy,nopatientdieddirectlyofCRS.More-over,nocaseofCRS-relatedcerebraledemaoccurred,despitethe154Y.Chengetal./Engineering5(2019)150–155inclusionofcentralnervoussystemleukemia(CNSL)patients.Therefore,donor-derivedCARTcellinfusionsseemtobesafeforMRDinB-ALLafterHSCT.Inthepresentstudy,?veofsix(83.33%)patientswithMRDbeforeCARTcellinfusionattainedmolecularCRafterthe?rstinfu-sion,andthreepatientshavesurvivedtothepresentday.Inourpreviousstudy,fourof?veresponsivepatientsshowedhematolog-icalrelapseafter2–7months[15].Althoughthenumberofcaseswaslessinthisstudy,determiningwhethertheeffectofMRDtreatmentwasadequateinrelapsepatientswarrantedthisevaluation.Fever,hypotension,dysfunctionalbloodcoagulation—Fever,hypotension,hypoxemiaFever—Fever—Fever—Fever,hypotension,dysfunctionalbloodcoagulationCRSsymptoms))II)AIAn)IADo))))iAID)IIIID)IAhAAAAhttAtcDehDt0DDDDs0tt1Dettthd0s2hst2h3(01(ts18(td12801225478)r((222((((01(...0em30040000001itno))))IIIIitAAAAceDDDDthhhhettttd7777(((((dAAAAooNNNNlDDDDb1111laàààà))))reggggááááhggggplllli((((rááááe))sssspIAeeee)IInAiiii)nppppIAoiiD)DooooADtccccdchID)IheetAht5555tt8D20000hAttce31117D7(ed(h11(tted871????(h2t68dn8(3000617(........3s2001126400191TRA)))IICnIAAoA))fi)otDIDII)))DctAhAAIIIdtnestDt7DDAAADDDnee1(t(hh2ttt(td1sttsssnt11441112os0(2(((((3...C10000—00000nisll4DedcCe/8sTuDRfA2.CniC1—————————+65D1C1—————————+8D8C4—————————)%(eg+a4tnD0eC4—————————crep+ll3.se8eDiCC8—————————ticixo)tsgl8878777888dlkee(0000000000tcd1111111111aiTecs??????????oRu0004000006sAfsn1602002376..........aCi1222896113dnasnnconnnnnnnioitsoooiooioonioiiiiiissussssssssiurfuuufuufuufufenfffitnnnnfeninnfinincgiiiiiiaatdrnttdtdtdtsssnsnsntsaS1211212121hclletcn4deieetlsabuP123456afTnI.noisufniretfayad:IAD5.SummaryBasedonclinicalobservation,weproposethatdonor-derivedCARTcellinfusionisaneffectiveandsafeinterventionforelimi-natingMRDinpatientswithBcellmalignanciesafterHSCT.ItwasevenmoreimpressivethatnopatientdevelopedGVHDduringtheperiodunderobservationinthepresentstudy.However,themechanismremainsunclear.Furtherexperimentsandlargerscaleclinicaltrialsarerequiredtocon?rmwhetherCARTcellinfusioncanbeappliedasa?rst-lineinterventionmeasuretoeliminateMRDinpatientswithnoresponsetoDLIandinpatientswithMRD.AcknowledgementThisworkwassupportedbytheScienti?cResearchFoundationforCapitalMedicineDevelopment(2018-2-4084).AuthorcontributionsYifeiChengandYuhongChenanalyzedthematerialandwrotethepaper.ShashaWangcollectedthedata.XiangyuZhao,ChenhuaYan,YuWang,YaoChen,WeiHan,LanpingXu,andXiaohuiZhangperformedtheresearch.LungjiChangandLeiXiaocontributedtocellproduction.KaiyanLiudesignedtheresearch.XiaojunHuangdesignedtheresearchandeditedthemanuscript.CompliancewithethicsguidelinesThepresentprotocolwasevaluatedandallowedbythePekingUniversityPeople’sHospitalreviewboard.AllenrolledpatientsgaveinformedconsentinaccordancewiththeDeclarationofHelsinki.YifeiCheng,YuhongChen,ChenhuaYan,YuWang,XiangyuZhao,YaoChen,WeiHan,LanpingXu,XiaohuiZhang,KaiyanLiu,ShashaWang,LungjiChang,LeiXiao,andXiaojunHuangdeclarethattheyhavenocon?ictofinterestor?nancialcon?ictstodis-close.Nomenclature
aGVHDacutegraft-versus-hostdiseaseallo-HSCTallogeneichematopoieticstemcelltransplantationALTelevatedalaninetransaminaseB-ALLBcellacutelymphoblasticleukemiaBubusulfanCARchimericantigenreceptorCART19CD19-directedchimericantigenreceptor-modi?edTcellCDclusterofdifferentiationCIRcumulativeincidenceofrelapse
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