German Cancer Research Center ( DKFZ )Division of Tumor Virology

French National Institute of Health and Medical Research ( INSERM)Cancer Virotherapy Unit

Heidelberg , Germany

Rodent Parvoviruses as Anti-cancer Agents

University of HeidelbergDepartment of NeurosurgeryDepartment of SurgeryDepartment of Hematology,

Oncology & Rheumatology

Heidelberg , Germany

Viral replicationinhibited

Normalcell spared

Normalcell

Cancer Cell

(genetic target)

Viral replicationproceeds

Tumor lysis(virus spread)

Schematic representation of tumor-selective viral replication and oncolysis (Kirn et al., 2001)

(Autonomous) Parvoviruses : Structure

Icosahedral capsid

non enveloped

diameter ~25 nm

ssDNA

~ 5100 bases

Modified from Sgro and Spencer, Encyclopedia of Virology

Autonomous Parvoviruses: Lytic Cycle

VIRUSPRODUCTION

VIRAL SS DNADISPLACEMENTSYNTHESIS

PRODUCTION OF VIRALCAPSID PROTEINS

PRODUCTION OF VIRAL REGULATORY PROTEINSVIRAL DNA AMPLIFICATION

VIRAL SS DS DNACONVERSION

S PHASE

NUCLEAR TRANS-LOCATIONDECAPSIDATION

UPTAKE

(Autonomous) Parvoviruses : Biology

REQUIREMENTS TOWARDS HOST CELLS:Common:cell proliferation(S factor)

Specific: animal species

cell differentiation

IN VIVO BIOLOGYCytopathic effect restricted to proliferating tissues

Pathogenesis in adult < new-born < foetus

Antineoplastic effect

SUSCEPTIBLE SPECIES:

Mammals (rodents, human, felines, canines, porcines, bovines,...)

Birds(?)

tumor

peritumoral

cerebellum

contralateral

pos

neg

Oncotropism of H-1PV in i.t. Injected RG-2 Glioma-b earing Immunocompetent Rats

Brain Periphery

pos

neg

tumor

peritumoral

contralateral

cerebellum

kidney

pos

heartliverlung

spleenne

g

DAPI α−NS-1 DAPI α−NS-1

Virus multiplication

lung

NS1 protein (Western Blotting)

Viral DNA (PCR)

BrainNS1 protein (IF)

Viral RNA (RT-PCR)

1

100

Inoculum

2 d p.i.

2

Pos

tumor

tumor

tumor

tumor

Pos liv

erliver

spleen

spleen

spleen

lungN

eg

NS1

Infe

ctio

us U

nits

(10

-6)

Regulation of Parvovirus DNA Replication and Gene E xpression by Proliferation/Transformation-dependent Cellular Fac tors

Examples

Cyclin A activates the conversion of parvovirus ss DNA into ds replicative form

The early promoter P4 is activated by E2F/ATFfamilies of transcription factors

cdk2

Phosphorylation of RPA p32?

?

RFC

PCNA

Pol δδδδRPA

MVM P4 promoter

EBSE2F GC TATA

EY CRECRE

CREB1

E2F 'free'

G1 / StransitionG0/G1

Y

p27

p130

X

ATF1

-+

+

+

E2FPhosphorylation and/or presence in the complex?

cyclin A

NORMAL FIBROBLAST

CY

TO

PLA

SM

NU

CLE

US

MVMp

type-I IFN genes

MVMp

PRRs

EX

TR

AC

ELL

ULA

R S

PA

CE

MVMp

MVMp

MVMp

MVMp

MVMp

MVMp

MVMp

antiviral genes

type-IIFNs

ANTIVIRALSTATE

PV multiplication

αIFN Ab

TRANSFORMED FIBROBLAST

CY

TO

PLA

SM

NU

CLE

US

MVMp

type-I IFN genes

MVMp

PRRs

EX

TR

AC

ELL

ULA

R S

PA

CE

MVMp

MVMp

MVMp

MVMp

MVMp

MVMp

MVMp

antiviral genes

type-IIFNs

ANTIVIRALSTATE

PV multiplication

αIFN Ab

Searching for Viral Oncotoxins

NORMAL CELL

NORMAL NS TRANSFECTANT

TRANSFORMEDNS TRANSFECTANT

TRANSFORMEDCELL

SURVIVAL+ind

+ind

+ind NS - 1

NS - 1

SURVIVAL

DEATH

The Parvoviral NS1 Product Is Sufficient to Kill Oncogene-transformed Cells

NS1 Interaction with CKII α:α:α:α:a Determinant of Parvovirus Cytotoxicity

Oligo-merization

NTP-Binding

CytotoxicityNS1:S473A

F/TNS1:T363A

F/T

WB CKIIααααCKIIαααα

P473P473PP

A9 cells

A9-E81A(P38:dnCKII α)α)α)α)

Mock MVM 24 h p.i. MVM 48 h p.i.

•NS1 cytotoxicity mutant S473A is defective in CKIIαααα binding

•CKIIαααα is involved in the induction of cytopathic effects

NS1wt bound NS1wt bound

Adaptor Model of NS1 Cytotoxic Functions

CKIIααααNS1(235-379) GFP

NS1

CKII-B

NS1(235-379) GFP

mCKII ααααNS1(235-379) GFP

CKII

GFP CKIIααααNS1(235-379)

XNS1(235-379)

CKIIαααα

CKII-B

GFP

Colony Formation Inhibition Assay

0%

20%

40%

60%

80%

100%

120%

K H-1PV H-1GC H-1CG

120100806040200

PAMP

CpG amplificationand

oncolytic release

vaccinationeffect

Improvement of the Antitumor Immunomodulating Effect of H-1PV through Arming with CpG Elements

I.V.

TARGETS

MH3924Ahepatoma

cells

AUTOLOGOUS TUMOR CELL VACCINE

γγγγH-1PV

S.C.

Suppression of lung metastases

IFNγCD80CD86

ß-actin

Immunostimulation (mediastinal lymph nodes)

Mock H-1 wt H-1GC H-1CGVaccine treatment

2w 2w

lung metastasesM

etas

tase

s / l

ung

(%no

n-va

ccin

ated

rat

s)

Oncolytic Parvovirotherapy of Non-Hodgkin

B-cell Lymphomas

Why Burkitt´s Lymphoma ?

•Form of non-Hodgkin lymphoma, divided in endemic, sporadic and immunodeficiency-associated types

•Initiating role for Epstein-Barr Virus in tumorigenesis (in partic. endemic type)

•An extremely aggressive human cancer : among the fastest growing tumors

•The 1-year survival rate for adult patients with advanced disease is 15%

•Current treatment : standard chemotherapy in combination with rituximab

�short- and long-term side effects�drug resistance�early or late relapse : durable remission may not be achieved

Burkitt’s Lymphoma-derived Cell Lines Are Sensitive to H-1PV-induced Killing

� Irrespective of EBV presence

� Through necrotic cell deathElijah

EBV - EBV +

Mock

H-1PV(10 pfu/cell)

� Irrespective of rituximab resistance

CD

20+

cells

(%

)

0

20

40

60

80

100

0

20

40

60

80

100

RAMOS Namalwa

Cel

l dea

th (

%)

*

* 10 pfu/cell, 72 hpi

RAMOS Namalwa

CD20 expression(rituximabsensitivity)

H-1PV-inducedcell killing

R1: apoptotic cell fractionR2: necrotic cell fraction

Annexin-V

Mock H-1PV

2.3

0.5

R1

R2

1.1

0.3

R1

R2

27.7

0.9

R1

R2

80.8

1.3

R1

R2

PI

24 hpi 48 hpi

H-1

PV

Mo

ck

Early treatment(14 d post tumor initiation)

Late treatment (28 d post tumor initiation)

H-1PV Suppresses Burkitt’s Lymphomas in a SCID Mous e Model

Days post tumor initiation

*

*

*

0

1

2

3

4

5

0 3 7 10 17 23 300 3 7 10 17 23 30

H-1PV

0

1

2

3

4

0 3 6 8 10 16 18 20 23 25 27 330 3 6 8 10 16 18 20 23 25 27 33

H-1PV H-1PV

Tum

or s

ize

(cm

3 )T

umor

siz

e (c

m3 )

Sur

viva

l rat

eS

urvi

val r

ate

Days post i.t. injection of H-1PVH-1PV

* Sacrificed

* Sacrificed

UV H-1PV H-1PVUV H-1PV H-1PV

Tumor growth Animal survival

H-1PV-induced Regression of BLs Correlates with i.t . Expression of the Viral Cytotoxic Protein NS1 ( SCID mouse model )

IHC detection of NS1 protein in necrotic areas of treated

tumors

Single tumors Double tumors

UV.H-1PV H-1PV H-1PV Untreated

RT-PCR detection of NS1 transcripts in treated and (contralateral) untreated

tumors

Oncolytic Parvovirotherapyof Pancreatic Ductal

Adenocarcinoma

Why Pancreatic Ductal Adenocarcinoma (PDAC)?

• A highly malignant disease (almost 100% lethal)

• The 4th ( Western Europe ) and 5th( North America ) leading cause of

cancer-related deaths

•The worst 5-year survival rate of all human cancers: <5% of all PDAC

patients survive 5 years

• Surgery is the only curative treatment currently available

• Most patients present with advanced PDAC : > 80% have

unresectable disease at the time of diagnosis

• Highly resistant to conventional cytotoxic agents (e.g. Gemcitabine )

Local tumor development T Lymph node metastases N Liver metastases M 1

2

3

Therapeutic Effect of H -1PV in a Rat Orthotopic PDAC Model

Features

� Tumor progression

� Immunocompetent model

� Stromal reaction present

Animals survival

at treatment

8 weeks after mock treatment

8 weeks afterH-1PV treatment

1

2

3

0%

20%

40%

60%

80%

100%

1 to 600 0,875 1 to 2400 1 to 4800 1 to 9600

Serum dilutions

Livi

ng c

ells

(%

unt

reat

ed c

ultu

res)

Survival of Recipients of splenocytes from H-1PV vs mock-treated Donors

Neutralizing Ab response in H-1PV-treated Donors but not in Recipients therefrom

Orthotopic PDAC Suppression through Adoptive Immune Cell Transfer (Rat Model)

days post tumor initiation

Recipients from mock-treated Donors

Recipients from H-1PV-treated Donors

Mock-treated DonorsH-1PV-treated DonorsRecipients from H-1PV-treated Donors

transfer

1:600 1:1200 1:2400 1:4800 1:9600

splenocytes

020406080

100

Panc-1

Panc-1R

H-1PV gemcitabine

020406080

100BxPC-3

BxPC-3R

Cel

l dea

th (

%)

02040

6080

100

Colo 357

Colo 357R

H-1PV gemcitabine

020406080

100

T3M-4

T3M-4R

Cel

l dea

th (

%)

H-1PV Kills Human PDAC Cells Irrespective of their Resistance to Gemcitabine

Panc-1

BxPC-3

Colo 357

T3M-4

Treatment Treatment

Wild-type SMAD4 Expression:a Predictive Marker of PDAC Cell Permissiveness for H-1P V Infection

Changes in SMAD4 status during PDAC progression

SMAD4-mediated signalling

Cell line Origin SMAD4

Panc-1 Primary PDAC wt

MiaPaCa-2 Primary PDAC wt

AsPC-1 Metastasis mt

CFPac-1 Metastasis loss

Stimulation of H-1PV production by SMAD4

Panc-1

functional knock-down

Correlation of wt SMAD4 status with enhanced PDAC cell sensitivity to H-1PV

wt mt/loss0

50

100

Sur

viva

l(%

livi

ng c

ells

, d3

p.i.,

MO

I=10

)

cell line

short-term culture

SMAD4

(1) Survival and death pathways

(2) Anticancer immune responses

(4) Cytoskeleton dynamics

(3) Cell cycle checkpoints

Neoplastic Cell

PV*

PROSPECTS OF CANCER PARVOVIROTHERAPY

Conclusion

Arming withregulatory elements

Mutagenesis Transgene addition

Acknowledgements

Glioma , NS1Laurent Daeffler

Laurent Deleu

Matteo Di Piazza

Irina Kiprijanova

Ute Koch

Jürg Nüesch

Jörg Schlehofer

Lymphoma ,Pancreatic cancer

Assia Angelova

Marc Aprahamian

Rauf Baht

Sebastian Dempe

Christiane Dinsart

Svitlana Grekova

Zahary Raykov

Nathalia Giese , University Hosp. HeidelbergAnthony Ho , University Hosp. HeidelbergMathias Witzens-Harig , University Hosp. Heidelberg

Karsten Geletneky , University Hosp. HeidelbergMarta Herrero y Calle , University Hosp. FreiburgEkkehard Weber , University Halle

Barbara Leuchs , DKFZ Virus Production Unit

SMAD4: a Positive Regulator of H-1PV Gene expression

Presence of putative SMAD-binding elements (SBE) in the H-1PV P4 promoter

Activation of H-1PV P4 promoter by SMAD4

RLU

Panc-1 (wt SMAD4)

0369

pSMAD4

pCtrl

AsPC-1 (mt SMAD4)wt-pP4LucH1

+

wt-pP4LucH1

+

pdn Smad4

pCtrl

3210

Correlation of NS1 accumulation with wt SMAD4 expression

H1 M

NS1

SMAD4

14-3-3

H1 M

Panc-1 AsPC-1

Oncolytic Parvovirotherapy of Glioblastoma Multiforme

Why Glioma ?

Glioblastoma multiforme

is the most common ofprimary brain tumors

2–3% of all human malignancies

Malignant gliomas have an extremely bad prognosis:

50% of patients die within12-16 months after diagnosis (grade IV-gliomas)

< 5% long-time survivors

from: Kleihues, P., Kiessling, M., Wiestler, O.D. (2004)

Human Glioma Cells Are Targets for H-1PV Oncolytic Activity

H-1PV kills human glioma cells resisting DNA-damagingagents and/or soluble death-ligands

Cathepsins are involved in H-1PV-induced glioma cell death

Cell Lysis

1

1,25

1,5

1,75

2

2,25

2,5

NCH82 NCH82 gfp NCH82 StefinB

Rel

ativ

e G

6PD

rele

ase

(H-1

PV

vs.

moc

k)

-

Luc

CathB

CathL

CathB + L

siRNA

Rescue of NCH82 cells from H-1PV infection (5 pfu/cell) (cell lysis)

0

20

40

60

80

100S

urvi

val (

%)

NCH 89

NCH 82

U 138

astro

cytes

cisplatin 5

trail 50

H1 5

Cisplatin 5 µg/ml

Trail 50 ng/ml

H-1PV 5 pfu/cell

norm

al

astro

cyte

s

Cytosolic cathepsin-inhibitors are down-regulatedAcidic vesicles accumulatein the cytosol

Cathepsins relocatein the cytosol

Acridine orange staining Cathepsin B staining

NCH82 cells

Mock

H-1PV5pfu/cell24h p.i.

Early infection

H-1PV-induced Suppression of Human Gliomas in Immunod eficient Rats

H-1PV-

NS1 expression in tumor

Late infectionH-1PV-

Days post- implantation

7

19

10

17

Start of treatment

U87 human glioma :105 cells/rat

H-1PV (i.c./i.v. combination):6x109 pfu/rat

Sur

viva

l pro

babi

lity

Days after implantation

control

early infection

late infection

0,0

0,

2

0

,4

0,6

0,

8

1,0 censoring

P<0,002

P<0,002

Survival

PopulationPopulation• up to 25 patients• histologically confirmed glioblastoma, WHO grade IV• single center (University of Heidelberg)

Further Further main inclusion criteriamain inclusion criteria• recurrent and resectable GBM progressing (MRI-Scan) in spite of radio-

and/or chemotherapy• chemotherapy stop for at least 4 weeks prior to treatment with H1PV

Main Main exclusion criteriaexclusion criteria• multifocal disease• poor Karnofsky-Score (<70)• severe systemic infection• pregnant or breast feeding women

Administration of virusAdministration of virusescalated dosage; i.t. (pre-/intra-OP); i.v. (pre-OP)

Study design developed with the advice of NCT and KKS,

with provisional approval of the National Authority (Paul-Ehrlich Institute)

Phase I/II Study Design for H-1PV Treatment of Gli oma Patients

Enhancing Potencyof Oncolytic Parvoviruses

-H-1PV hgH-1PV

110

100

110

100

110

100

110

100

rat RG-2

hum NCH 89

hum NCH 82

hum NCH 37

5 1.5 0.5 0.150.05

H-1PV

H-1PV

H-1PV

H-1PV

hgH-1PV

hgH-1PV

hgH-1PV

hgH-1PV

m.o.i

fold

inpu

t

Selection of H -1PV Variants Adapted for Multiplication in Human Glioma Cells

large production

RG2(rat glioma)

rat H-1PV rat H-1PV

( )little/no production

NCH(hum glioma)

hgH-1PV

large production

NCH149(hum glioma)

> 25 passages

rat H-1PVVirus selection

Enhanced fitness of hgH-1PV to propagate in human glioma cell cultures

Production of recombinant parvoviral

vectors

co-transfectionP38P4 P VPCMV

VP-expression

NS

packaging helper plasmidrecombinant parvoviralDNA plasmid

capsid formation

amplification

packaging

release of virus

293T cells

excision andamplification

IP-10 (human) /CRG-2 (mouse)Interferon-inducible protein of 10kD

•Cellular sources: monocytes, T cells, fibroblasts, osteoblasts, keratinocytes and endothelial cells

•Member of the CXC family of chemokines

•Chemoattractant for activated monocytes, T cells and NK cells

•Inducer of lymphocyte adhesion to endothelium

•Activator of NK cells

•(In)direct antiangiogenic effects

•Potent suppressor of vascular(ized) tumours

• Cytokine, 2 receptors (TNF-R1, -R2)• Pro-apoptotic factor

• Anti-angiogenic properties• Immunostimulant (DC maturation, macrophage

activation)

→ use of TNF-α in combination with chemokines which can recruit but not always activate immune cells

Tumor Necrosis Factor- α (TNF- α)

Tumour growth inhibition following infection of glioma cells with recombinant parvoviruses

GL 261 cells, C57/Bl6 mice

control

MVM ∆800MVM/ IP-10 MVM/TNF

MVM/ IP-10/TNF0

200

400

600

0 10 20 30 40 50 60 70 80

days post inoculation

tum

our

volu

me

(mm

3)

140