免疫基因治疗讲课讲稿.ppt

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1、免疫基因治疗免疫基因治疗CD8+activatedCTL cellsInduction of ImmunityTumor cellMHC II/AgCD4+Th cellsCD8+CTL cellsAntigen presenting cellCD28B7MHC I/AgTCRTCRIL-2IL-6IL-2T CellAPCTCR/CD3MHC-I/IIAgCD4/CD8LFA-2LFA-3ICAM-1ICAM-2ICAM-3LFA-1CD28CTLA-4B7-1/B7-2CD40LCD40Signal Transduction of T Cell ActivationSignal 1Sign

2、al 2Tumor Immunology(I)Low MHC I expressionIncreasing MHC expression on tumor cells by introduction of MHC I/II geneCD8+activatedCTL cellsTumor cellCD8+CTL cellsTumor cellLack of costimulatory molecules(B7)Inactivationor AnergyIntroducing costimulatory molecule B7 into tumor cells CD8+CTL cellsCD28C

3、TLA-4CD8+activatedCTL cellsCD8+CTL cellsTumor Immunology(II)Tumor cellTumor cellTumor cellTumor cellImmune suppressive factors Supernatant(Tumor cells)Sera(Cancer patients,animal)Generation of LAK,TIL and CTLCytokine productionLymphocyte proliferationAntibody synthesisMacrophage accumulation and che

4、motaxisTGF-b bIL-10(cytokine synthesis inhibitory factor)LBIF(lymphocyte blastogenesis-inhibiting factor)IGF-1Angiogenesis factorsSER(suppressive E-receptor)Engineering tumor cells,fiboblasts or TIL with cytokine genes IL-1,IL-2,IL-4,IL-5,IL-6,IL-7,IL-12,INF-a,INF-r,TNF-a,GM-CSFTumor Immunology(III)

5、(-)(+)Immuno-stimulatory intervention Tumor vaccines-Engineer tumor cells to express cytokines-Engineer fibroblasts to express cytokines-Introduction of genes encoding tumor antigens In vivo gene transfer of cytokines Dendritic cellsAPCCD8+T cellCD4+T cellTh2Th1NK cellIL-12IL-12IL-12IFN-ggIFN-ggIFN-

6、ggIFN-ggInfectionTumorVEGF+Ag+APCIL-2IFN-ggTNF-aaTNF-bbGM-CSFIL-12 mediated antitumor immunityStructure of Retroviral vectorsLNC/B7LNC/B7-IL12LNC/IL12LTRCMVNeoB7LTRLTRCMVNeop35 IRES p40LTRLTRCMVNeoB7LTRIRES p35 IRES p40LTRCMVNeoLTRLNCX Expression of mB7-1,hB7-1 and mIL-12 on genetically Modified BNL

7、 Cells Relative Cell NumberFluorescence IntensityNegativeControlmB7-1hB7-1mIL-12(ng/5x105cells/24hr)none none none 79 63 BNL BNL/Neo BNL/B7-1 BNL/IL-12 BNL/B7-1-IL-123H-TdR uptake(x103 cpm)Proliferation of genetically Modified BNL Cells BNLBNL/NeoBNL/B7-1BNL/IL-12BNL/B7-1-IL-12Tumorigenicity of Gene

8、tically Modified BNL Cells in Immunocompetent Mice(n=22)(n=17)(n=20)(n=20)(n=20)Days after tumor cell inoculationTumor free mice%Therapeutic cell inoculationDays after tumor cell inoculationTumor free mice(%)Mean tumor size(mm3)Distant Therapeutic Effect of Irradiated Modified BNL CellsTherapeutic E

9、ffect of Retrovirus-producing CellsTherapeutic cell inoculationMean tumor size(mm3)Days after tumor cell inoculationTumor free mice(%)Tumor vaccines(IL-12,B7-1)Efficiency?Immuno-stimulatory intervention Tumor vaccines-Engineer tumor cells to express cytokines-Engineer fibroblasts to express cytokine

10、s-Introduction of genes encoding tumor antigens In vivo gene transfer of cytokines Dendritic cellsConstruction of AdCMVmIL-12Homologous recombinationAd5Therapeutic gene(mIL-12)Adenovirus genome0100E1 deletedE3 deletedAdCMVmIL-12293 cellsCMV p35 IRES p40 pAExperimental Protocol(I)Injection of tumor c

11、ells(BNL)to miceIntratumoralgene transferHepatocellular carcinomainduction periodTreatment periodSalineAdCMVlacZAdCMVIL-12s.c.3-4 weeks(tumor 5-6 mm)Days after treatmentSize of tumor(cm3)Treatment of HCC by AdCMVIL-12109108107109Treatment of established HCC with AdCMVIL-12Injection of tumor cells(MC

12、A-RH7777)to buffalo ratIntratumoralgene transferHCCinduction periodTreatment periodSalineAdCMVlacZAdCMVIL-1214 days(tumor 5-7 mm)Treatment of established HCC with AdCMVIL-12Before treatmentAdCMVlacZAdCMVIL-12 Before Aftertreatment treatmentSize of tumor(cm)5x1091x1091x1010Effect of AdCMVIL-12 on HCC

13、 in liverTreatment of established HCCs with AdCMVIL-12Injection of tumor cells(MCA-RH7777)to buffalo ratIntratumoralgene transferHCCinduction periodTreatment periodSalineAdCMVIL-1214 days(tumor 5-8 mm)Size of tumor(mm)Treated tumor Before Aftertreatment treatmentEffect of AdCMVIL-12 on HCCs in liver

14、 Before Aftertreatment treatmentUntreated tumorEffect of AdCMVIL-12 on HCCs in liverDays after treatmentSurvival(%)Protective immunity induced by AdCMVIL-12 treatmentDays after tumor cell rechallengeAnimals free of tumor(%)Anti-tumor effect of IL-12Activation of NK cellsActivation of CTL cellsInduct

15、ion of IFN-g gInduction of chemokinesInduction of adhesion molecules and increased traffic of CTL to tumorAnti-angiogenesisApplication of AdCMVIL-12I.Intratumoral injection 1.HCC models(mouse,rat)2.Colon cancer model(mouse)3.Pancreatic cancer model(mouse)II.ex vivo infection of dentritic cells 1.Col

16、on cancer models(mouse)III.Co-administration with adenovirus expressing HCV for vaccinationT helperT killer co-stimulationNew Model of the Delivery of T Helper to T KillerAPCActivated APCCD40LCD40T killer co-stimulationAPCActivated APCCD40LCD40Anti-tumor Immunity Induced by CD40LTumor cellWeeks afte

17、r tumor cell inoculationTumor size(mm)Tumorigenicity of ex vivo gene transfer of CD40L in HCC cellsTreatment of established HCC with AdCMVCD40LInjection of tumor cells(MCA-RH7777)to buffalo ratIntratumoralgene transferHCCinduction periodTreatment periodAdCMVlacZAdCMVCD40L7 days(tumor 5-7 mm)Weeks af

18、ter treatmentEffect of AdCMVCD40L on HCC in liverInvolvement of CD8+T cells in antitumor effect induced by AdCMVCD40LDays after treatmentTumor size(mm)Protective anti-tumor immunity induced by AdCMVCD40L (15 days after tumor cell inoculation)Groups Size of tumor(mm)Incidence(%)Control(n=6)10 mm 100%

19、AdCMVCD40L(n=10)0 mm0%Combined therapy for cancerIL-12CD40LMIP-3a aIP-10MDCFLT3LCombination of intratumoral AdCMVIP-10 and AdCMVIL12AdCMVIL-120/10 (0%)AdCMVIP-10+AdCMVLacZTumor diameter(mm)10/10 (100%)AdCMVIP-10+AdCMVIL-12Tumor free mice0/6 (0%)SalineTumor free mice4/11(36%)Days after treatment onse

20、tDays after treatment onset Immuno-stimulatory intervention Tumor vaccines-Engineer tumor cells to express cytokines-Engineer fibroblasts to express cytokines-Introduction of genes encoding tumor antigens In vivo gene transfer of cytokines Dendritic cellsBMPBPeptidesTumor AgTumor cellsGM-CSF+IL-4 or

21、 TNFDCs based on immunotherapyTUMORMALIGNANTCELLSAG TRANSFERMIGRATIONLYMPH NODEThCTLPRECURSORNKsTRANSFECTEDCYTOKINEEFFECTORCTLsPERIPHERALBLOOD ORLEUKOAPHERESISPRODUCTVIRAL VECTORENCODINGIMMUNOSTIMULATINGFACTORDCsIN VITRODIFFERENTIATIONDIRECTULTRASOUND-GUIDED INJECTIONCytokine gene transfer into dend

22、ritic cells for cancer treatmentTirapu et al.Cur Gene Ther 2:79-89(2002)%of b b-gal+cellsIL-12(pg/ml)104 5x103 3x103 103 102104 5x103 3x103 103 102 0Infection of DC by adenoviral vectorsMOIMOITumor regression induced by DC infected with AdCMVIL-12Tumor modelsAdCMVIL-12 AdCMVlacZ None HBSSSyngeneic A

23、llogeneic Fibroblasts Syngeneic Syngeneic DC DC DC DCCT-2616/190/90/112/151/15 0/17MC-38 3/60/50/50/50/5 0/5%Specific lysis100 50 25 12 6 3E:T ratioInduction of tumor-specific CTLsClinical trials in CUN1.Intratumoral injection of AdhIL-12(Sangro et al.Journal of clinical Oncology 22:1389-1397(2004)2

24、.Intratumoral injection of dendritic cells with AdhIL-12(Mazzolini et al.Journal of Clinical Oncology 23:999-1010(2005)3.Intratumoral injection Adtk(Peuelas et al.Gatroenterology 128:1787-1795(2005)Phase I Clinical Trial(scalating doses)of i.t.Ad.IL-12 in Advanced HCC and GI Tumors(21 patients with

25、HCC or liver metastases from CRC or PC)7 dose steps(3 patients in each step and 3 equal doses per patient with 30 days separation):step 1:2.5x1010 viral particles step 2:1011 viral particles step 3:2.5x1011 viral particles step 4:5x1011 viral particles step 5:1012 viral particles step 6:2.5x1012 vir

26、al particles step 7:3x1012 viral particles (20 viral particles=1pfu)Patient no.10(51011 vp)Hepatocellular carcinomaInj TInj TAFPAFPSangro et al.Journal of clinical Oncology 22:1389-1397(2004)1,0E+11,0E+21,0E+31,0E+41,0E+518/0608/0728/0717/0806/0926/09 16/10HCV viral loadPatient with HCC and chronic

27、HCV infection4th dose step with AdIL-121st dose3rd dose2nd doseSangro et al.Journal of clinical Oncology 22:1389-1397(2004)In vivo imaging of vectors1.Bioluminescence imaging(Luciferase/luciferin)2.Fluorescence imaging(GFP,RFP)3.Positron Emission Tomography(PET)(HSV-tk/18 FHBG)Imaging of Gene Transf

28、er Using Imaging of Gene Transfer Using Positron Emission Tomography(PET)Positron Emission Tomography(PET)Ad.lacZFHBGFHBGTk geneTk geneAd.tk(tk substrate;iv injection)Active uptakeNo uptakeFHBGFHBGlacZ genelacZ geneRadioactive signal(positive PET image)No signal(negative PET image)PET monitoring of

29、tk activity(using 18FHBG as substrate for tk)in rats injected with Ad.tk or Ad.LacZkidneysbladderliverAd.LacZAd.tkAd.tk,1012 vp501636Peuelas et al.Gastroenterology 128:1787-1795.(2005)Immuno-stimulatory intervention Tumor vaccines-Engineer tumor cells to express cytokines-Engineer fibroblasts to express cytokines-Introduction of genes encoding tumor antigens In vivo gene transfer of cytokines Dendritic cells结束结束