中小学生道德行为规范

ProtocolforgenetransductionandexpansionofhumanTlymphocytesforclinicalimmunogenetherapyofcancerCorHJLamers,RalphAWillemsen,BarbaraALuider,RenoDebets,andReinderLHBolhuisDepartmentofMedicalOncology,SubdivisionofClinicalandTumorImmunology,ErasmusMedicalCenter-Daniel,Rotterdam,TheNetherlands.InpreparationofaclinicalphaseI/IIstudyinrenalcellcarcinoma(RCC)patients,wedevelopedaclinicallyapplicableprotocolthatmeetsgoodclinicalpractice(GCP)criteriaregardingthegenetransductionandexpansionofprimaryhumanTlymphocytes.Wepreviouslydesignedatransgenethatencodesasinglechain(sc)FvG250antibodychimericreceptor(ch-Rec),specificforaRCCtumor-associatedantigen(TAA),andthatgeneticallyprogramshumanTlymphocyteswithRCCimmunespecificity.Herewedescribetheconditionsforactivation,genetransduction,andproliferationforprimaryhumanTlymphocytestoyield:(a)optimalfunctionalexpressionofthetransgene;(b)ch-Rec–mediatedcytokineproduction,and(c)cytolysisofG250-TAAPOSRCCbytheT-lymphocytetransductants.Moreover,theseparametersweretestedatclinicalscale,i.e.,yieldingupto5–10
109T-celltransductants,definedasthetreatmentdoseaccordingtoourclinicalprotocol.Thefollowingparameterswere,forthefirsttime,testedinaninteractiveway:(1)mediacompositionsforproductionofvirusbythestablePG13packagingcell;(2)T-lymphocyteactivationconditionsandreagents(anti-CD3mAb;anti-CD3+anti-CD28mAbs;andPHA);(3)kineticsofT-lymphocyteactivationpriortogenetransduction;(4)(i)T-lymphocytedensity,and(ii)volumeofvirus-containingsupernatantpersurfaceunitduringgenetransduction;and(5)mediumcompositionforT-lymphocytemaintenance(i)in-betweengenetransductioncycles,and(ii)duringinvitroT-lymphocyteexpansion.CriticaltogenetransductionofhumanTlymphocytesatclinicalscaleappearedtobetheuseofthefibronectinfragmentCH-296(Retronectin2)aswellasLifecell1X-fold2cellculturebags.InordertocomplywithGCPrequirements,weused:(a)bovineserum-freehumanT-lymphocytetransductionsystem,i.e.,mediasupplementedwithautologouspatients’plasma,and(b)aclosedcellculturesystemforalllymphocyteprocessing.Thisclinicalprotocolroutinelyyields30–65%scFvG250ch-RecPOSTlymphocytesinbothhealthydonorsandRCCpatients.CancerGeneTherapy(2002)9,613–623doi:10.1038/sj.cgt.7700477Keywords:clinicalprotocol;immunogenetherapy;humanTlymphocytes;singlechainchimericreceptor;renalcellcarcinomaThesuccessfulclinicaladoptivetransferoftumor-specificcytotoxicTlymphocytes(CTLs)forcancertreatmentremainshamperedbythedifficultytoreproduciblyisolateandexpandsuchhumanTlymphocytesinvitroorinvivo.1,2Incontrast,alibraryofmonoclonalantibodies(mAbs)specificfortumor-associatedantigens(TAA)isavailable.ThetumorcellkillingpotentialofanyCTLcanbetargetedtotheseTAA,whensensitizedwithbi-specificmAbsspecificforbothTlymphocyteandTAA.3Weandothers4–8haveindeedusedsuchbi-specificmAbs-sensitizedhumanTlymphocytesinclinicalstudies.Inspiteofobjectiveclinicalresponses,itsuseiscomplicatedby(a)limitedaccessibilityofsolidtumorsto(bi-specific)mAb;9(b)dissociationofbi-specificmAbfromCTL;10limitedrecyclingcapacityofthosehumanTlymphocytes;(c)developmentofhumananti-mouseantibody(HAMA)responses;11and(d)inactivationofthebi-specificmAb-redirectedTlymphocytesfollowingtumortargetantigenbinding.3,10Moreover,theclinicalanti-tumoreffectswerelocoregional,notsystemic.8Recently,humanTlymphocytescanalsobegeneticallyprogrammedwithmAb-dictatedspecificities.12-14Wehavechosenforrenalcellcarcinoma(RCC)patientsbecausenoadequatetherapyisavailableforthesepatients.15,16TheG250mAbrecognizestheG250TAAexpressedonthecellmembraneofover90%ofprimaryRCCandover80%ofRCCmetastases,17,18andisRCC-selective:itsligandisnotexpressedonthemembraneofnormalrenalcells(orcellsderivedfromothertissues)inadensitysuf-ficienttoelicitcellularorhumoralimmuneresponses.17Moreover,theG250mAbhasextensivelybeentestedinclinicalstudies.18–20WeearlierreportedonthegeneticprogrammingofhumanTlymphocytesyieldingRCCkillinghumanTcellsfollowingretroviraltransductionwiththetransgeneencodingforthesinglechain(sc)G250mAbReceivedApril10,2002.Addresscorrespondenceandreprintrequeststo:DrCorHJLamers,DepartmentofMedicalOncology,SubdivisionofClinicalandTumorImmunology,ErasmusMedicalCenter-Daniel,POBox5201,3008AERotterdam,TheNetherlands.E-mail:[e-mail](2002)9,613–623D2002NaturePublishingGroupAllrightsreserved0929-1903/02$25.00(scFvG250)ch-Rec.21–24Further,studiesfromourlabo-ratoryhaveshownthatscFvG250-CD4/
POSTlympho-cytesthatselectivelyandeffectivelykillG250-TAAPOSRCCalsoarespecificallytriggeredbyG250-TAAPOSRCCtoproducecytokines.Moreover,theT-celltransductantscanrecycletheirtumorcellkillingcapacity.21Becausethech-RecismAb-based,TAArecognitionandcytolysismediatedbyscFvG250POSTlymphocytesareMHC-unrestricted.ThescFvG250ch-Recdoesactincontextwithaccessorymolecules,suchasCD11a/CD18,toallowlysisofeven‘‘lowTAAPOS’’tumorcells.23,24WesucceededtooptimizetheconditionsforhumanT-lymphocyteactivation,genetransduction,andexpansiontoyield:(a)optimalfunctionalexpressionofthescFvG250-CD4/
transgene;(b)ch-Rec–mediatedcytokineproduc-tion;and(c)cytolysis