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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