ENGINEERING CYTOLYTIC EFFECTOR CELLS FOR GLIOMA IMMUNOTHERAPY USING GENE INSERTION

AND ZINC FINGER NUCLEASE GENOMIC EDITING

22ND ANNUALiSBTc MEETING

BOSTON

DIVISION OF CANCER IMMUNOTHERAPEUTICS & TUMOR IMMUNOLOGYBECKMAN RESEARCH INSTITUTE

0102030405060708090

100

0 1 2 3 4 5 6 7 8 9 10

YEARS AFTER DIAGNOSIS

ESIT

MA

TED

PR

OPO

RTI

ON

SU

RVI

VIN

G

PILOCYTICASTROCYTOMAGLIOBLASTOMA

SEER DATA REPORTED BY DAVIS et alJ. Neurosurg 88:1-10, 1998

• Median survival duration following GBM relapse is 36-weeks

TARGET POPULATION:

fLuc+T-Cells

SubQGBM

INTRAPARECHYMAL HOMING OF T-CELLS TO ORTHOTOPIC CNS GLIOMA

XENOGRAFTS

T-CELLS USE WHITE MATTER TRACKS

GLIOMA TROPISM OF ADOPTIVELY TRANSFERRED EX VIVO PROPAGATED T-CELLS:

glioma C

SF

hydro

cephalu

s CSF

-5

0

5

10

15

20

25

30

35

% C

hem

otax

isPATIENT-DERIVED RESECTION CAVITY CSF CHEMOKINE CONTENT AND CHEMOTACTIC BIOACTIVITY

0 2500 5000 7500 10000 12500

RANTES

MIG

MCP-1

IP-10

IL-8 Glioma CSFHydroceph. CSF

Cytokine [pg/ml]

Pos

Pos

Neg

Neg

ENA-78

GCSF

GM-CSF

GRO

GRO-α

I-309

IL-1a

IL-1b

TNF-α

TNF-β

EGF

IGF-I

Ang

OSM

Tpo

VEGF

PDGF-β

Leptin

Neg

Pos

MCP-1

MCP-2

MCP-3

MCSF

MDC

MIG

MIP-1δ

RANTES

SCF

SDF-1&2

TARC

TGF-β

IL-2

IL-3

IL-4

IL-5

IL-6

IL-7

IL-8

IL-10

IL-12

IL-13

IL-15

IFN-γ

GLIOMA-DERIVED CCL2/MCP-1 DRIVES T-CELL TUMOR TROPISM

rhMCP-1

U251T T98

U87o-5

05

101520253035

% C

hem

otax

is

rhMCP-1

U251T T98

U87o

050

100150200250300350

CMCM + anti-MCP-1CM + anti-IL-8CM + anti-MCP-1 + rhMCP-1

rhM

CP-

1 (n

g/m

l)

Chemotaxis AssayMCP-1 Quantitation(CBA Analysis)

DAUDI-p DAUDI-MCP1

Quantification of T-cell Infiltration

Daudi-Parental Daudi-MCP10.0

0.5

1.0

1.5

2.0

2.5

Intracranial Tumor GroupT-

cells

per

mm

2

TUMOR SECRETED MCP-1 IS SUFFICIENT FOR IN VIVOHOMING OF ADOPTIVELY TRANSFERRED T-CELLS

TUMOR HOMING CYTOLYTIC T-CELLS: NANOSURGEONS IN THE BRAIN

EQUIPPING CTLs FOR TUMOR RECOGNITION BY GENE INSERTION

SELECTION OF A GLIOMA-SPECIFIC TARGET ANTIGEN

NORMAL BRAIN GLIOMA T-CELLSTARGET:

TnfR +/- +++ +++

EGFR ++ ++++ -EGFRvIII - ++++ (<30%) -IL-13Rα2 - ++++ (>90%) -

THE IL13-ZETAKINE CHIMERIC ANTIGEN RECEPTOR FOR RE-DIRECTING CTL EFFECTOR FUNCTION TO GBM

huIL-13(E13Y)

huIgG4 hinge-Fc

huCD4TM

huCD3-ζcyto

IL13-ZETAKINE CHIMERICANTIGEN RECEPTOR

RE-DIRECTED HLA UNRESTRICTEDZETAKINE-REGULATED GLIOMA KILLING

705 707 708 709 711 712715 719 733 710 720 734

U87 GLIOBLASTOMA(ffLucZeo/IL-2)

DAY 0: 2x105 tumor cells i.c.

+1

+4

DAY +5:10x105 CD8+ CTL i.c.

+6

+8

+11

AntiCD19-CAR+ CD8+

CTL Clone E8IL13-zetakine+ CD8+

CTL Clone 2D7

EGF/FGF/LIF/NO SERUM DMEM 10%FCS

CD133+/GFAP- CD133/GFAP+

SOCS2

CD133

GLIOMA STEM-LIKE TUMOR PROGENITOR CELLS

PROTOCOL 01020 UPN-033: TARGET VALIDATION/T-CELL KILLING

OF AUTOLOGOUS TUMOR/TUMOR STEM CELLS

IL13Ra2010 110 210 310 410

Cou

nt

45

40

35

30

25

20

15

10

5

0

81 .56

IL13Ra2010 110 210 310 410

300

225

150

75

0

96 .38UPN033-IL13z @ S8D13

25:1 5:1 1:10

20406080

100120 PBT015 NS

PBT015 AdhDaudi

E:T ratio%

Spe

cific

Lys

is

GFAP+/CD133- DIFFERENTIATED TUMOR

GFAP-/CD133+ TUMOR PROGENITORS

CLINICALAPPLICATIONS

PLASMID EXPRESSION VECTORIL13 ZETAKINE/HyTK-pMG

6785 bp

HyTKIL13zetakine

SpAn

late SV40pAN

bGh pA

h CMV-1Aprom

hEF1p

ori ColE1

PacI (3240)

IL13zetakine/HyTK-pMG

SCHEMA FOR T-CELL GENETIC MODIFICATION, CLONING, AND EX VIVO EXPANSION

SCHEMA FOR T-CELL GENETIC MODIFICATION, CLONING, AND EX VIVO EXPANSION

PlasmidDNA

Vector

Day 0PolyclonalActivationof T Cellswith OKT3

Day 3Electroporationof OKT3-ActivatedPBMC

Day 5Addition of SelectionDrug

Day 28Cloning of SurvivingT Cells

Day 42Expansionof Drug-ResistantClones

Day 56Large ScaleExpansionof Zetakine+ Clones forRe-Infusion

PROCESS DEVELOPMENT

T-CELL BIOREACTORSCLOSED SYSTEM CELL PROCESSINGIMMUNOMAGNETIC SELECTIONENGINEERED FEEDER CELLSOPTIMIZED CYTOKINE COMBINATIONS

THE CENTER FOR BIOMEDICINE AND

GENETICSAT CITY OF HOPE

cGMP-MANUFACTURING FACILITY:

PLASMID DNA, VIRAL VECTORS, MONOCLONAL ANTIBODIES, AUTOLOGOUS CELL PRODUCTS

CD3-FITC CD45 RO-FITC CD27-PE CD28-PE

CXCR3-P CXCR4-P CCR2-PE CCR5-PE

CD3 CD45RO CD27 CD28

CXCR3 CXCR4 CCR2 CCR5

Glioma-Stimulated Cytokine Production

0 10000 20000 30000 40000 50000

TNF-alpha

IL-5

IL-10

IL-4

IFN-gamma

IL-2

Cytokine (pg/ml)

COHNMC PROTOCOL IRB#01020:(BB-IND #10109)

A PILOT FEASIBILITY/SAFETY STUDY OF CELLULAR IMMUNOTHERAPY FOR RECURRENT/REFRACTORY

MALIGNANT GLIOMA USING GENETICALLY MODIFIED AUTOLOGOUS CD8+ T-CELL CLONES

INTRACAVITARYCYCLE: WEEK: DAY: CELL DOSE:

1 1 1 107

3 5x107

5 108

2 2 1 108

3 108

5 108

3 REST/RESTAGING

3 4 1 108

3 108

5 108

4 5 1 108

3 108

5 108

6 REST/RESTAGING

PROTOCOL #01020CELL DOSING SCHEDULE

PLACEMENT OF RICKHAM SHUNT VAD IN RESECTION CAVITY

PRE-ADOPTIVE TRANSFER POST-COURSE #1

T1-GADOLINIUM MRI PULSE SEQUENCE

POST-COURSE #2 4-WKS POST RX

PROTOCOL 01020 SAFETY DATACell Dose Level

Number of Doses

Observed Side-Effects Grade 3 or higher Attributable to Cell Doses

•Intra-cavitary107 2 None

5x107 2 None

108 19 Headache 2x – only on UPN 028

•Intra-parenchyma25 x 106 1 None

50 x 106 1 WBC, Headache, Pain – localized to scalp/catheter site

108 3 Hypoxia, Headache (3x), Fatigue

•Cumulative Cell DoseUPN-028 - 9.6 x 108 cells over 21-days (Intra-cavitary)UPN-033 - 10.6 x 108 cells over 21-days (Intra-cavitary)

- 3.75 x 108 cells over 5-days (Intra-parenchymal)

•Best Response to therapyUPN-028 Radiographic CR Survival Duration post Relapse: 12 monthsUPN-033 Radiographic CR Survival Duration post Relapse: 11 Months

PROTOCOL 01020 UPN-033: RADIOGRAPHIC COMPLETE RESPONSE FOLLOWING

ADOPTIVE THERAPY

Bx. Proven Progressive GBM Spectroscopy=NecrosisFDG PET = Cold

5 T-CELLDOSES @

10e8

PRE-RX 14-WEEKS POST-RX

INITIAL CLINICAL IMAGING EXPERIENCEUSING [18F]-FHBG PET ON COHNMC

PROTOCOL #01020

PET-BASEDMOLECULAR IMAGING OF HyTK

EXPRESSING T-CELLS

P-FHBG*P-FHBG*

P-FHBG*

HSV TK- HSV TK+

[18F] FHBG*

Pre-Adoptive Rx Post-Adoptive RxAx Flair irFSE Ax Flair irFSE 18FHBG PET

Tumor ProgressionVersus T-Cell Mediated

Inflammation

FHBG SignalFrom TK Reporter

Expressed in T-Cells

UPN-033: Correlative Imaging of CNS Changes Observed Following Intracranial Adoptive Transfer Of Autologous IL13-zetakine+/HyTK+ CD8+ CTL Clone

Ax Flair irFSE 18FHBG PET

Tumor ProgressionVersus T-Cell Mediated

Inflammation

FHBG CD8+ T CELLPET SIGNAL INFILTRATE

UPN-033: Correlative Imaging of CNS Changes Observed Following Intracranial Adoptive Transfer Of

Autologous IL13-zetakine+/HyTK+ CD8+ CTL Clone

CHALLENGES TO BROADER CLINICAL UTILITY:

• PATIENTS FREQUENTLY NEED TO TAKE GLUCOCORTICOIDS (DEXAMETHASONE) TO MANAGE CERBRAL EDEMA

• TIME TO PRODUCE AUTOLOGOUS PRODUCT NOT COMPATIBLE WITH RECURRENT DISEASE PROGRESSION KINETICS

Potential Advantagesof Steroid-Resistant Alloclone:

• READY-TO-USE PRODUCT AVAILABLE AT TIME OF NEED

• FUNCTIONAL IN PATIENTS RECEIVING DEXAMETHASONE

• CONCURRENT USE OF DEXAMETHASONE LIMITS CNS INFLAMMATORY REACTION TO ADOPTIVE THERAPY/ PROLONGS SURVIVAL OF T-CELL ALLOGRAFT BY INHIBITION OF REJECTION RESPONSE

Grp.A 10^6 NSO IL2(2)_ffluc-Hytk-pMG,GFP-DHFRdm-pEK s.c.

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.03

4

5

6440441442443444445

Day post tumor injection

Flux

(pho

tons

/sec

)

Grp.B 25,000 cells NSO IL2(2)_ffluc-Hytk-pMG,GFP-DHFRdm-pEK i.c.

0 1 2 3 4 5 6 7 8 9 101.0×103

1.0×104

1.0×105

1.0×106

1.0×107

1.0×108

446447448449450451

Day post tumor injection

Flux

(pho

tons

/sec

)

Grp.C 25,000 cells NSO IL2(2)_ffluc-Hytk-pMG,GFP-DHFRdm-pEK i.c.& 1x10^6 s.c. - Flank only

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.01.0×103

1.0×104

1.0×105

1.0×106

452453454455456457

Day post tumor injection

Flux

(pho

tons

/sec

)

Grp.C 25,000 cells NSO IL2(2)_ffluc-Hytk-pMG,GFP-DHFRdm-pEK i.c.& 1x10^6 s.c. - Head only

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.01.0×103

1.0×104

1.0×105

1.0×106

1.0×107

452453454455456457

Day post tumor injection

Flux

(pho

tons

/sec

)

INTRAPARENCHYMAL CNS LYMPHOIDALLOGRAFTS ARE NOT REJECTED

FLA

NK

FLA

NK

BR

AIN

BR

AIN

+

ZFP-Fok I Fusion Proteins

ZFN-Mediated Targeted Gene Disruption (TGD)

x

1.

2.

3.

4.

Endogenous gene targeted for disruption

ZFNs dimerize and introducea double stranded DNA break in the gene

Break repaired by non-homologous end-joining (NHEJ) – resulting in loss of genetic information

Gene disruptedx

Western Blot Shows Reduction of GR Protein Levels in GR-ZFN Treated CD8+ T-cells

GR (N-terminus)

TFIIB

GFP10 30 100

ZFN10 30 100

IL-13 ZKPool 10A1

CD8 CTL Clone

RT-PCR Analysis Shows Loss of GR Target Gene Transcriptional Regulation in ZFN Modified T-Cells

IFNg Expression

-

1.0

2.0

3.0

4.0

un dex un dex un dex un dex

GFP 30 ZFN 100 10A1

IFN

g/G

AP

DH

GILZ Expression

-

0.5

1.0

1.5

2.0

2.5

un dex un dex un dex un dex

GFP 30 ZFN 100 10A1

GIL

Z/G

AP

DH

IL-13ZKPool

IL-13ZKPool

0102030405060708090

100

WTGCR

WTGCR

KOGCR

KOGCR

NO DEXDEX

% V

IAB

ILIT

Y

ADENO MOI: 100 30 100 30

ZFN-MEDIATED GLUCOCORTICOID DISRUPTIONOF ZETAKINE+ CTLs RESULTS IN RESISTANCE TO

DEXAMETHASONE-TRIGGERED APOPTOSIS

GR-KO CTLs RETAIN ZETAKINE-REGULATED LYTIC ACTIVITY

Cytotoxicity: cJ04274 UPN-036 - ZFN, MOI 100, DEX

0102030405060708090

100

50:1 25:1 5:1 1:1Effector:Target

% C

hrom

ium

Rel

ease

U251(T)

Daudi-IL13Ra2Daudi

TM-LCL-OKT3hygro

Cytotoxicity: cJ04271 UPN-036 - GFP, MOI 100, DEX

0102030405060708090

100

50:1 25:1 5:1 1:1Effector:Target

% C

hrom

ium

Rel

ease

U251(T)Daudi-IL13Ra2DaudiTM-LCL-OKT3hygro

Dex 10e-4M

GR-ZFN Adeno TreatedMOI 100

GFP Adeno ControlMOI 100

0.2 x10^6 U87O-ffluczeo-IL2 i.c. Day 0PBS control i.c. Day 5 & 8

0.0 2.5 5.0 7.5 10.0 12.51.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

1.0×1010

668568698271

Day post tumor injection

Flux

(pho

tons

/sec

)

0.2 x10^6 U87O-ffluczeo-IL2 i.c. Day 0PBS control i.c. Day 5 & 84mg/kg Dex i.p. @ Day 5

0.0 2.5 5.0 7.5 10.0 12.51.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

1.0×1010

727386757689

Day post tumor injection

Flux

(pho

tons

/sec

)

0.2 x10^6 U87O-ffluczeo-IL2 i.c. Day 02x10^6 UPN-036 - IL13-zeta_ GR adeno

@ MOI 100 i.c. Day 5 & 8

0.0 2.5 5.0 7.5 10.0 12.51.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

1.0×1010

787980817083

Day post tumor injection

Flux

(pho

tons

/sec

)

0.2 x10^6 U87O-ffluczeo-IL2 i.c. Day 02x10^6 UPN-036 - IL13-zeta_ GR adeno

@ MOI 100 i.c. Day 5 & 84mg/kg Dex i.p. @ Day 5

0.0 2.5 5.0 7.5 10.0 12.51.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

1.0×1010

846774878877

Day post tumor injection

Flux

(pho

tons

/sec

)

GR- ZETAKINE+ CTL ARE DEX RESISTANT IN VIVO

GR-KO IL13-ZETAKINE ALLOCLONE CLINICAL TRIAL

• UNRESECTABLE TUMORS• DECADRON DEPENDENT PATIENTS• INTRAPARENCHYMAL T-CELL

INFUSIONS• CONVECTION ENHANCED rHuIL-2

DELIVERY

ReenaVishwanathCOH Grad.

StudentTumor Tropism

Project

Jensen Lab/Translational Research Team

COH Collaborators-Behnam Badie, MDDavid DiGiusto, PhDAndrew Raubitschek, MDStephen Forman, MDMichael Kalos, PhD

Collaborators-Stanley Riddell FHCRCSam Gambhir StanfordSangamo Biosciences