Construction and Preclinical Evaluation fo an AntiCD19 Chimeric Antigen Receptor.docx

Construction and Preclinical Evaluation fo an AntiCD19 Chimeric Antigen Receptor.docx

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ConstructionandPreclinicalEvaluationfoanAntiCD19ChimericAntigenReceptor

NIHPublicAccess

AuthorManuscript

JImmunother.Authormanuscript;availableinPMC2010September1.

Publishedinfinaleditedformas:

JImmunother.2009September;32（7）:

689–702.doi:

10.1097/CJI.0b013e3181ac6138.

ConstructionandPre-clinicalEvaluationofanAnti-CD19ChimericAntigenReceptor

JamesN.Kochenderfer*,StevenA.Feldman*,YangbingZhao*,HuiXu*,MaryA.Black*,RichardA.Morgan*,WyndhamH.WilsonΨ,andStevenA.Rosenberg*

*SurgeryBranchoftheNationalCancerInstitute,NationalInstitutesofHealth,Bethesda,MD,USA

ΨMetabolismBranchoftheNationalCancerInstitute,NationalInstitutesofHealth,Bethesda,MD,USA

Abstract

Tcellscanbeengineeredtoexpressthegenesofchimericantigenreceptors（CARs）thatrecognizetumor-associatedantigens.WeconstructedandcomparedtwoCARsthatcontainedasinglechainvariableregionmoiety（scFv）thatrecognizedCD19.OneCARcontainedthesignalingmoietyofthe4-1BBmoleculeandtheotherdidnot.WeselectedtheCARthatdidnotcontainthe4-1BBmoietyforfurtherpre-clinicaldevelopment.WedemonstratedthatgammaretrovirusesencodingthisreceptorcouldtransducehumanTcells.Anti-CD19-CAR-transducedCD8+andCD4+Tcellsproducedinterferon-γandinterleukin-2specificallyinresponsetoCD19+targetcells.ThetransducedTcellsspecificallykilledprimarychroniclymphocyticleukemia（CLL）cells.WetransducedTcellsfromCLLpatientsthathadbeenpreviouslytreatedwithchemotherapy.WeinducedtheseTcellstoproliferatesufficientlytoprovideenoughcellsforclinicaladoptiveTcelltransferwithaprotocolconsistingofaninitialstimulationwithananti-CD3monoclonalantibody（OKT3）priortotransductionfollowedbyasecondOKT3stimulationsevendaysaftertransduction.ThisprotocolwassuccessfullyadaptedforuseinCLLpatientswithhighperipheralbloodleukemiacellcountsbydepletingCD19+cellspriortotheinitialOKT3stimulation.InpreparationforaclinicaltrialthatwillenrollpatientswithadvancedBcellmalignancies,wegeneratedaproducercellclonethatproducesretrovirusesencodingtheanti-CD19CAR,andweproducedsufficientretroviralsupernatantfortheproposedclinicaltrialundergoodmanufacturingpractice（GMP）conditions.

Keywords

Chimericantigenreceptor;genetherapy;CD19;Tcell;gammaretrovirus;adoptiveTcelltherapy

Introduction

Approximatelytwenty-twothousandpeopledieofBcellmalignancieseachyearintheUnitedStates1.PatientswithsomeBcellmalignanciesincludingmantlecelllymphomaandchroniclympocyticleukemia（CLL）cannotbecuredbytherapiessuchasconventionalchemotherapyandmonoclonalantibodies2,3,butsomepatientswiththesediseasescanachieveprolongeddisease-freesurvivalafterallogeneicstemcelltransplantation4-6.Unfortunately,allogeneicstemcelltransplantationislimitedbysignificanttransplant-relatedmortalityandashortageofsuitabledonors2,6,7.InpatientswithBcellmalignanciesthatrelapseafterallogeneicstem

Correspondingauthor:

J.N.KochenderferNIH10CenterDriveCRCRoom3-3888Bethesda,MD20892.Telephone:

301-451-6957Fax:

301-496-0011Email:

kochendj@mail.nih.gov.

FinancialDisclosure:

Allauthorshavedeclaredtherearenofinancialconflictsofinterestinregardstothiswork.

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celltransplantation,infusionofallogeneicdonorlymphocytescaninduceremissions8-10.TheeffectivenessoftheselymphocyteinfusionsprovidesarationaleforattemptstodevelopothercellularimmunotherapiesforBcellmalignancies.

AdoptivetransferofautologousTcellsthatareculturedfromtumorinfiltratinglympohocytescancauseregressionsofadvancedmelanomainhumans11,12.Becausetumor-reactiveTcellscannotbereliablyculturedfrommosthumantumors,methodshavebeendevelopedtoengineerTcellstoexpressgenesencodingtumor-antigen-specificTcellreceptors13,14.Adoptivetransferofthesegenetically-modifiedTcellsisapromisingapproachtocancerimmunotherapy

15.AnotherapproachtoadoptiveTcelltherapyistoengineerTcellstoexpresschimericantigenreceptors（CARs）16,17.CARsaremadeupofanantigen-recognizingreceptorcoupledtosignalingmoleculesthatcanactivateTcellsexpressingtheCAR18-20.Theantigen-receptorsmostcommonlyincorporatedintoCARsaresinglechainvariableregionmoieties（scFv）thatconsistofthelightchainandheavychainvariableregionsofamonoclonalantibodyjoinedbyapeptidelinker.MurinemodelshaveshownthatTcellstransducedwithretrovirusesencodingCARscanprotectmicefromtumorchallengesinvivo21,22.

OurgrouphascompletedaphaseIclinicaltrialinwhichpatientswithovariancarcinomaweretre