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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.
NIH-PAAuthorManuscript
Kochenderferetal.Page2
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