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