Materials and Methods
•Mice:6-8weekoldfemaleC57BL/6
•Tumorcellline:SyngeneicB16F0melanomainjectedscintobothflanks
•Adenovirusvector:Replication-incompetentAd5
•Vectorforassessingcellularuptake:Ad5-CMV-GFP
•VectorforinducingmIL-12:Ad5containinggeneconstructformIL-12undercontrolofRTS(Ad-RTS-mIL-12)
•Administrationofadenovectors:1010viralparticles(vp)injectedintra-tumorally(i.t.)at11D,whentumorswerepalpable
•Activatorligand(AL):1000mg/kgchow;or2-35mg/kgbodyweightinLabrasol,byoralgavageforgeneexpressionandcytokinesintumororserum
•Phase1bclinicaltrialwithDC-RTS-Il-12i.t.+AL(0.6-200mgorallydailyfor14daysfor1-5treatmentcycles
Mechanism of Action of Therapy of B16F0 Melanoma in Mice, with DC-RTS-mIL-12 i.t. + oral AL
•Conditionalinterleukin-12genetherapypromotessafeandeffectiveantitumorimmunity
HKomita,XZhao,AKKatakam,PKumar,MKawabe,HOkada,JMBraughlerandWJStorkus.CancerGeneTherapy(2009),1–9
▲DC-RTS-mIL-12injectedintratumorally(i.t.)intoB16F0+ALinducedsytemicanti-tumorimmunityandregressionofB16tumors
▲DC-RTS-mIL-12+ALhadprolongedsurvivalintumoranddraininglymphnodes
▲Treatmentefficacycorrelatedwithsystemicanti-B16CD8+TcellsinELISPOTassay
•Recent studies:MicetreatedwithDC-RTS-IL-12+ALhadincreasednumbersoftumor-specificTcellsinbothtumor-draininglymphnodesandspleencapableofsecretinggranzymeB(GzB)andIFNγinresponsetobothCD8+andCD4+tumorpeptidesmeasuredbyELISPOT.
Mechanism of Action of Therapy of Patients with Advanced Stage Melanoma, with DC-RTS-hIL-12 i.t. + oral AL
•MeasurementofseraforlevelsofIL-12andIL-12regulatedcytokines,byLuminexassay,indicatedsignificantincreaseinIL-12and/orIFNγonlyafterALat≥60mg/day,eventhoughsomepatientsshowedclinicaldiseasecontrolatsubstantiallylowerdosesofAL
•ELISPOTassaysofIFNγsecretionindicatedincreasedresponsesofbothCD8+andCD4+Tcellsafterstimulationwithmelanoma-specificpeptides
•Intumorbiopsiesaftertreatment:
▲IncreasednumberofCD4+and/orCD8+Tcells
▲NoincreaseinTregulatorycells
▲Decreaseinmyeloid-derivedsuppressorcellsinsomepatients
•DetailsofthistrialwillbepresentedattheASCOannualmeetinginJune2011
RheoSwitch® + AL Controls Timing and Level of Target Gene Expression
•TheRheoSwitchTherapeuticSystem(RTS)containsthreebasiccomponents:
▲aninduciblepromoter
▲aligand-inducibletranscriptionfactorandaco-activationpartner
▲aRheoSwitchactivatorligand(AL)
•Intheabsenceofligand,theswitchproteincomplexprovidesan“off”signal
•Inthepresenceofligand,thecomplexchangesconformationandprovidesadose=dependent“on”signalfortargetgeneexpression
•In vivo,theorallyadministeredALturnsongeneexpressionwithin24hours,anduponwithdrawaloftheAL,geneexpressionreturnstobaselinelevelswithinabout24hours
Hypothesized Therapeutic Mechanism of Action of RTS-IL-12 + AL
(1)RTS-IL-12constructisinsertedintoadenoviralvectorbackbone,(Ad-RTS-IL-12).
(2)Syngeneicmousesplenocytes,orautologousbloodmonocytesisolatedandpurifiedbyleukapheresisandelutriation,aredifferentiatedexvivointodendriticcells(DCs)
(3)DCsaretransducedexvivowithAd-RTS-IL-12.
(4)~5x107transducedDCsareinjectedintooneormoretargettumorlesions.
(5)Alternatively,Ad-RTS-IL-12Iviralconstructfrom#1abovecanbedirectlyinjectedintotargettumors.
(6)OralALisadministeredatdose(s)toregulatedesiredtimingandlevelofexpressionofIL-12andIL-12associatedgenesintumor.
(7) IL-12drivesDCsintumortodevelopintoDC1s,whichpreferentiallypresenttumorantigenstoTH1cells
(8)Thisinducesspecificanti-tumorCD4+TH1cellsandCD8+CTL,locallyandthensystemically
Other Poster Presentation on this Therapeutic Approach During 2011 Annual ASGCT Meeting:
Poster 883 (May 19, 2011): Murugesan et al, Rheoswitch-Mediated Regulation of IL-12 Protein Delivered Using an Adenoviral Vector Results in Anti-Tumor Effects Across a Spectrum of Tumor Types
ThatposterwasfocusedonthetherapeuticefficacyofAd-RTS-mIL-12+AL,andis
complementarytothisposter,whichfocusesontheimmunologicmechanismofaction
(MOA)ofthistherapeuticapproach,andontheanalogousMOAstudiesinB16-tumor-
bearingmiceandmelanomapatientswithDCstransducedwithAd-RTS-IL-12+AL
Intratumoral Injection of Ad-RTS-IL-12 + AL Treatment Modulates Lymphoid and Myelomonocytic Phenotypes within B16F0 Tumors at Day 7
Cell TypeTumor Treatment
Ad-RTS-mIL-12 %+
Ad-RTS-mIL-12 + AL%+
CD3+, CD4+ 4.7 10.9
CD3+, CD8+ 10.5 26.5
CD3-, NK1.1+ 0 7.9
B200+ 9.9 4.2
CD19+ 6.6 2.2
CD14+ 3.1 18.1
F480+ 19.0 28.7
CD11c+ 21.7 19.9
Local and Systemic Anti-Tumor Immunity is Induced by Rheoswitch Regulated IL-12 Production After Intra-Tumoral Injection of Adenovirus Vector as Well as Vector-Transduced Dendritic Cells
ZIOPHARM Oncology, Inc. 1FirstAvenue,ParrisBuilding#34,NavyYardPlaza,Boston,MA02129Main617-259-1970Fax617-241-2855www.ziopharm.com
Cellular Uptake of Ad-CMV-GFP Directly Injected into Subcutaneous B16F0 Tumors
% GFP+ Cells
Intra-tumoral injection with Ad-CMV-GFP
(Time after injection)Tumor (CD44+) Cells Host infiltrating (CD45+) Cells
None 0 0
12 h 3.5 2.5
36 h 14.5 2.7
7 days 0 0
mIL12 and mIFNγ RNA Expression in B16 Melanoma After AL + i.t. Injection with Ad-RTS-mIL-12
mIL12 and mIL12 Regulated Cytokine Levels in B16 Tumor After AL + i.t. Injection with Ad-RTS-mIL-12
mIL12 and mIL12 Regulated Cytokine Levels in Serum After Treatment of B16 Tumor-Bearing Mice with AL + i.t. Ad-RTS-mIL-12
Granzyme B ELISPOT Assays in Response to Melanoma-Specific CD8+ Restricted Peptides at 7 Days After Treatment of B16 Tumor-Bearing Mice with Ad-RTS-mIL-12 + AL
IFNγ ELISPOT Assays in Response to Melanoma-Specific CD8+ or CD4+ (TRP-1) Restricted Peptides at 7 Days After Treatment of B16 Tumor-Bearing Mice with Ad-RTS-mIL-12 + AL
IFNγ ELISPOT Assays in Response to Melanoma-Specific CD8+ or CD4+ (TRP-1) Restricted Peptides at 7 Days After Treatment of B16 Tumor-Bearing Mice with Ad-RTS-mIL-12 + AL
•TreatmentofB16F0-bearingmicewithAL+i.t.injectionwithDC-RTS-IL-12inducessystemicanti-melanomaimmunity,includingCTL
•TreatmentofB16F0-bearingmicewithdirecti.t.injectionwithAd-RTS-IL-12vectoralsoinducessystemicanti-melanomaimmunity,withsimilarifnotidenticalmechanismofaction:
▲Macrophagesandplasmacytoiddendritic/myeloidcellsaswellasTcellsandmela-nomatakeupAdvectorwithin12hoursafteri.t.injection
▲By7daysafteri.t.Ad-RTS-IL-12+AL,thereisashifttoincreasedpercentagesofCD4+&CD8+Tcells,NKcells,andmacrophages
▲By1dayaftertreatmentwithALatintermediatedoseaswellashighdose,expressionofgeneforIL-12isincreasedandisstillincreasedatday7,andIFNγgeneexpressionincreasesbyday2andishighatday7
▲IL-12intheinjectedtumorandinserumismainlyobservedafterhighdosedoseofAL,whereasotherIL-12associatedcytokinesareobservedwithevenlowdoseofAL
•Similarmechanismofactionaswellastherapeuticefficacyfori.t.treatmentwithAd-RTS-IL-12+ALorDC-RTS-IL-12indicatesagoodbasisforclinicaltrialwithAd-RTS-IL-12+AL,whichisplannedforinitiationinJune2011
Detection by PCR of Ad-RTS-mIL-12 DNA in Murine Immune Tissues, at 7 Days after Intra-Tumoral Injection of Ad-RTS-mIL-12
TissueTreatment
Sham (PBS) Injection Intra-Tumorally
Intra-TumoralAd-RTS-mIL-12
Tumor – +
Spleen – –
Tumor-Draining Lymph Nodes – +
Non-Draining Lymph Nodes – –
1IntrexonCorp,Germantown,MD;2UniversityofPittsburghCancerInstitute,Pittsburgh,PA;3ZIOPHARMOncology,Inc.,Boston,MA.
Ronald B. Herberman1, Meixia Bi1, Mario Moreno1, Lisa Butterfield², Mary Jo Buffo², Mark O. Thornton3, and Kimberly A. Shafer-Weaver1.
Poster#899
Abstract
Interleukin-12(IL-12)hasbeenshowntobeakeycytokinethatenhancestheability
ofdendriticcells(DCs)toelicitanti-tumorTcellsincludingTH1cellsandcytotoxic
Tlymphocytes(CTLs)aswellasactivationofnaturalkiller(NK)cells.Althoughthis
cytokineisapotentmediatorofanti-tumorimmunity,itsuseinimmunotherapy
hasbeenimpededbysubstantialtoxicityduetoadministrationoftherecombinant
protein.Komitaetal[CancerGeneTherapy(2009)]reportedthatgenetherapywith
intra-tumoralinjectionofsyngeneicDCstransducedbyanadenovirusvector,
Ad-RTS-mIL-12,withthemIL-12geneunderthecontroloftheRheoswitch
TherapeuticSystem(RTS)™],ledtotherapeuticefficacyagainstB16melanoma.The
mechanismofactionappearedtobeinductionofsystemicanti-tumorimmunity,with
CD8+TcellssecretingincreasedlevelsofIFNγinresponsetospecifictumorcells,
andthepersistenceandaccumulationofDCsintreatedtumors.Ourrecentstudies
haveindicatedthatmicetreatedwithDC-RTS-IL-12+anoralactivatorligand(AL)
hadincreasednumbersoftumor-specificTcellsinbothtumor-draininglymphnodes
andspleencapableofsecretinggranzymeB(GzB)andIFNγinresponsetobothCD8+andCD4+tumorpeptidesmeasuredbyELISPOT.Sincestudiesfromourteam
(Murugesanetal)haveshownthatdirectintra-tumoralinjectionofAd-RTS-mIL-12+
oralALalsoledtotherapeuticefficacyagainstB16andothermurinetumorstypes,
studieshavebeenperformedtodeterminewhetherthemechanismofactionis
similartothatofDC-RTS-mIL-12+AL.Toevaluatecelltypestransducedbydirect
adenovirusinjection,adenovirusexpressinggreenfluorescentprotein(GFP)under
astrongconstitutivepromoterwasinjectedintoestablishedB16F0melanoma
tumors,anduptakeofthevectorafterintratumoralinjectionwasmainlyinDCs
andtumorcells.Atday7afterintra-tumoralinjectionofAd-RTS-mIL-12+dailyAL,
increasedpercentagesofTcellsubsets,NKcellsandDCswereobservedinthe
tumormicroenvironment,andcellsisolatedfromtheDLNandspleensofthetreated
animalsshowedincreasednumberofGzBandIFNγ-secretingTcellsinresponsetoCD8+andCD4+melanomapeptidesbyELISPOTanalysis.Takentogether,these
dataindicatethatintra-tumoralinjectionofeitherAd-RTS-mIL-12orDCstransduced
exvivobythisvector,incombinationwithdailyoralAL,inducedchangesinthe
tumormicro-environmentthatfavoranti-tumorimmuneresponsesandsystemic
anti-tumorimmunity.Initialresultsfromaphase1bclinicaltrialwithDC-RTS-IL-12+
ALinpatientswithadvancedstagesofmelanomaindicatedinductionofmelanoma-
specificCD4+andCD8+peripheralbloodTcells,byELISPOT,in2of4melanoma
patients.TheanalogousimmunologicalmechanismofactionwithAd-RTS-IL-12in
themousetumormodelprovidesagoodrationalefornowtranslatingthelogistically
simplerapproachofdirectinjectionoftheAdvector,comparedtogenerationofthe
patient-specifictransducedDCproduct,intoaccessibletumorlesionsincombination
withALforclinicaltherapy.
RheoSwitchProtein 1
RheoSwitch Components
RheoSwitchProtein 2
InduciblePromoter
TargetGene
RheoSwitch Components Co-ActivatorProtein
Ligand
BasalTranscriptionProteins
Therapeutic Gene Expression is OFF
Therapeutic Gene Expression is ON
RheoSwitchProtein 1
RheoSwitch Components
RheoSwitchProtein 2
InduciblePromoter
TargetGene
RheoSwitch Components Co-ActivatorProtein
Ligand
BasalTranscriptionProteins
Therapeutic Gene Expression is OFF
Therapeutic Gene Expression is ON
RheoSwitchProtein 1
RheoSwitch Components
RheoSwitchProtein 2
InduciblePromoter
TargetGene
RheoSwitch Components Co-ActivatorProtein
Ligand
BasalTranscriptionProteins
Therapeutic Gene Expression is OFF
Therapeutic Gene Expression is ON
Not actual activator (1)
(2)
(3)
(4)
(5)
(6)
(6)
(7)
(4)(5)
(7)
(6)
7)(7
(44)(5)
Macrophages, Plasmacytoid Dendritic/yeloid, and T Cells are the Primary Leukocyte Subpopulation Transduced by Ad-CMV-GFP Directly Injected into Subcutaneous B16f0 Tumors
Cell Type
Time After Intra-Tumoral Injection
12 Hours 36 Hours
% of Total Cell in the Tumor
% GFP+ (within cell type) % of Total Cells % GFP+
T(CD3+) Cells 9.9 0.3 9.9 0.3
NK(NK1.1+) Cells 0 0 0.2 0.1
B or DC (B220+) Cells 2.0 0 2.4 0.1
Macrophages (CD14+) 0.1 0 18.1 2.1
Macrophages (F480+) 2.2 0.3 2.7 0.1
Dendritic (CD11c+) Cells 0.3 0 0.6 0.1
Plamacytoid Dendritic/My-eloid (CD317+) Cells 36.7 1.7 21.2 2.4
mlFNgmlFNg
0 10 20 30 40 50 60 70 80 90
100
0 1 2 4 7
Tum
or m
IFN
g ex
pre
ssio
n(N
orm
aliz
ed t
o H
PR
T1)
Time point(days)
PBS
AdmIL12
AdmIL12+2mg ligand
AdmIL12+10mg
ligand
AdmIL12+35mg
