Professor Akseli Hemminki presents on gene therapy and oncolytic viruses

Treatment of human cancer with
oncolytic adenoviruses: an example
of personalized therapyof personalized therapy
Akseli Hemminki, MD, PhD
Specialist in Oncology and Radiotherapy
K. Albin Johansson Research Professor,
Finnish Cancer Institute
Cancer Gene Therapy Group
Molecular Cancer Biology Program &
Transplantation Laboratory & Haartman
Institute & FIMM
f l k Disclaimer AH is co founder and shareholder ofUniversity of Helsinki Disclaimer: AH is co‐founder and shareholder of
Oncos Therapeutics Inc., a company founded for
facilitating clinical trials with oncolytic viruses

Overview of presentation
Why new treatments ?
Gene therapy of cancer: what is the clinical evidence ?Gene therapy of cancer: what is the clinical evidence ?
Oncolytic adenoviruses in our own patients
I t f i i d t i iImportance of immune response in determining
efficacy: the next generation of oncolytic agents
Tumor targeting: the next generation of oncolytic
agents
Personalization of treatment for each patient
Questions
Akseli Hemminki | 28 Oct 2010 | 2
Questions

Cancer is not a beaten
diseasedisease
CANCER
> 1/2 of people alive today will get cancer*
• 1/3 of us will die of cancer
• few disseminated solid tumors can be cured with
currently available treatments
Novel treatments are needed!
Akseli Hemminki | 28 Oct 2010 | 3* Jemal CA Cancer J Clin 2005

Bert Vogelstein:
CancerCancer
therapeutics
ft thafter the cancer
genome project
( )(ASCO 2009)
S i f t l d h d d f t ti i h (W dSequencing of tumor genomes revealed hundreds of mutations in each (Wood
Science 2007, Parsons Science 2008)
Combination different in each tumor ‐> Each tumor is an individual
‐> Each tumor would require a different combination of inhibitors
‐> For long term efficacy, each pt would have to be treated with hundreds of inhibitors
> Impossible because of side effects‐ > Impossible because of side effects
All of these mutations seem to fall in 12 pathways (Jones Science 2008).
‐ > Use pathway selective drugs (Vogelstein ASCO 2009)
For example, p16/Rb pathway selective oncolytic virus
http://cgap.nci.nih.gov/

Deletion mutant oncolytic
d i ∆24adenoviruses: ∆24
Fueyo Oncogene 2000
Heise Nature Med 2000
• S-phase
• Virus replication
& cell lysis
E2F
Rb E1A
E2F Rb
• normal cell
• wt Ad
24 bp deletion in Rb
binding site of E1A
• No S phase entryE2F Rb
E1A
E2F Rb• normal cell
wt Ad
• Replication in cells
mutant in Rb-p16
• No S-phase entry
• No virus replication
∆24-E1A
E2F Rb
∆24-E1A
E2F Rb• normal cell
• ∆24
mutant in Rb p16
pathway
• Includes all human
• S-phase
• Virus replication
& cell lysis∆24-E1A ∆24 E1A
• cancer cell
• ∆24
E2F
E2F
E2F E2F
E2F
E2F
cancers
(Sherr Science 1996)
& cell lysis∆24 E1A ∆24-E1A

How Far is Clinical Gene Therapy ?
Phase I: Safety and toxicity ?
Phase II: Any evidence of efficacy ?Phase II: Any evidence of efficacy ?
Phase III: Proof of efficacy
(randomization)
N= 1579

Mutation compensation
Randomized ph. III trial: head & neck ca.
Ad p53 + radiation vs radiation alone‐ Ad‐p53 + radiation vs. radiation alone
‐ 67% vs. 24% CR (N= 82, P<0.01)
‐ Pan J Clin Oncol 2008
Promoter p53 gene pA
‐ Gendicine® for sale in China
‐ More than 10 000 patients treated
Infection of cells
Promoter p53 gene pA
Infection of cells
Normal cells
with healthy p53
Cancer cells
with p53 mutation
Press release 23 Jul 2008: Ad‐p53 (Advexin®)
Cell death, also sensitation to
h h d di i
phase III SCCHN trial positive in US: not
approved by FDA
chemotherapy and radiation
No cell death

Prodrug converting enzymes
Ad di f
Randomized Phase III trial for glioma (ASPECT):
• Ad-TK + standard care vs. standard care: 1.43
HR (p=0.02)
Ad coding for
thymidine
kinase (TK)
TK
(p )
• 40d increase in median survival
• More temozolomide use in control group due to
non-blinded gene therapy-> dilution of results
Ad t
non blinded gene therapy > dilution of results
• EMEA did not approve because non-standard
end-point (time to re-intervention or death)
Non‐toxic prodrug
Advantage vs.
mutation
compensation:
o to c p od ug
= ganciclovir
Activated
bystander
effect via gap
junctionsActivated
toxin
Cell death
CHALLENGE: even with bystander
effect, can we get effective penetration
into established tumors ?
SOLUTIONS: locally amplifying
systems

Oncolytic viruses
• Replication of virus p
causes oncolytic death
of cells
• Normal cells‐ no
replication

Phase 3 trials with oncolytic
viruses
Oncolytic adenovirus H101 (OncorineR ≈ ONYX‐015)Oncolytic adenovirus H101 (Oncorine ONYX 015)
¬ Not armed, not very potent
¬ Randomized phase III trial (N=105)
¬ Intratumoral H101 + cisplatin + 5‐FU vs. cisplatin + 5‐FU
¬ CR+PR = 79% vs. 38%, P<0.0001
Mild t fl lik t i j ti it i¬ Mild tox: flu‐like symptoms, injection site pain
¬ Approved in China in 2006 (Yu Curr Cancer Drug Targets 2007)
OncovexGMCSFOncovex
¬ Oncolytic herpes virus armed with GMCSF
¬ Previous phase 2: advanced melanoma, intratumoral injectionp , j
¬ N = 50. CR = 8 (+5), PR = 5, SD = 10. Disease control = 23/50 (46%).
Survival 58% @ 1yr, 52% @ 2yr.
Ph 3 fi i h d l 2011
¬ Phase 3 finished early 2011

Cancer Gene Therapy is maturing
has a treatment approach
Safety has been good – over 15 000 pts treated with both repli‐
cation deficient and replication competent (oncolytic) viruses
Recent randomized trials (N=5) have confirmed efficacy of even
early generation approaches*early generation approaches*
No patients w/ metastatic cancer cured: much work remains
Replication competent oncolytic viruses
TUMOR PENETRATION NEEDS IMPROVEMENT
Replication competent oncolytic viruses
Transcriptional tumor targeting (activation only in tumor)
Transductional tumor targeting (gene delivery only to tumor)g g (g y y )
Armed oncolytic viruses
Seminal phase 3 trials ongoing (Oncovex, Jennerex)
* Immonen Mol Ther 2004, Li Clin Cancer Res 2007, Yu Curr Cancer Drug
Targets 2007, Pan J Clin Oncol 2008, Ylä‐Herttuala ESGCT 2008,

Personalized oncolytic adenovirus treatments
in the Advanced Therapy Access Programin the Advanced Therapy Access Program
• 210 pts since Nov 2007. 10 different viruses
• All had metastatic solid tumors progressing after routine
treatments (chemo, radiation, etc)
• Written informed consent. Full GCP. Direct regulation by FIMEA
• Side effects: gr. 1‐2 flu‐like symptoms, fever, fatigue, pain in all ptSide effects: gr. 1 2 flu like symptoms, fever, fatigue, pain in all pt
• SAE in < 5% (eg. pain, embolus, thrombosis, cholecystitis)
• No treatment related deaths so far (compare to chemo, surgery)
Cli i l b fit (i i CR PR SD) 48% ll 77% b t i• Clinical benefit (imaging CR, PR, SD): 48% overall, 77% best virus
• Some patients have benefited for 3 years (= length of follow‐up)
• Additive/synergistic benefits from 2nd ‐ 17th treatments
• Long term (>300 d) survival in 50% with best virus, best schedule

Findings possible only in pts: Mechanisms of anti‐
tumor efficacy inflammationinflammationy
1 Killing of differentiated tumor cells
3. Induction of
cytotoxic T‐cells
against tumors1. Killing of differentiated tumor cells
CD8+
4
5
6
E+8
against tumors
vitiligovitiligo
2
3
4
0 17 41 48
10E
2. Killing of tumor initiating ”stem” cells
4. Induction
of specific
immunity
against
tumor
epitope
(survivin)
Eriksson Mol Ther 2007, Bauerschmitz Cancer Res 2008
Cerullo Cancer Res 2010

Ad5/3‐Cox2L‐D24 in chemotherapy
refractory OvCarefractory OvCa
• 53 yr old woman, WHO 1. Stage 3 ovarian cancer
• Progressive disease after 62 rounds of prior chemo, Progressive disease after 6 rounds of prior chemo,
including paclitaxel, paclitaxel+carbo, carbo, liposomal
doxorubicine, etoposide ...
• single intraperitoneal injection of 2x10e11 VP• single intraperitoneal injection of 2x10e11 VP
Oncolytic replication alone is usuallyOncolytic replication alone is usually
not enough to cure advanced tumors
Pesonen Gene Ther
2010
2010

Higher efficacy with a second round of
treatment: role of immune response ?treatment: role of immune response ?
• Metastatic pancreatic ca. WHO 2
• Prior gemcitabine and gemcitabine chemoradiation
• Second round of treatment with Ad5‐24‐RGD (Bauerschmitz
Cancer Res 2002) produced response
Kanerva in preparation

Improving antitumor immunity: oncolytic
adenoviruses coding for GM CSFadenoviruses coding for GM‐CSF
Cerullo Mol Ther ASGT suppl 2009
GM‐CSF
• GM‐CSF is the most potent inducer of anti‐ GM-CSF
• GM CSF is the most potent inducer of anti
tumor immunity (Dranoff Immunol Rev 2002)
• GM‐CSF in E3: expression starts at 8h
G CS
GM-CSF
⇒ GM‐CSF expressed only in cells that allow
replication of the virus
Hi h i l i• High expression at tumor, low systemic
GM-CSF
GM CSF
GM-CSF

GMCSF based approach validated 29
Apr 2010: Provenge (Sipuleucel‐T)Apr 2010: Provenge (Sipuleucel T)
HormoneHormone
refactory prostate
cancer
Collection ofCollection of
white blood cells
Incubation w/ PAP
& GMCSF to
activate antigen
presenting cells
Return cells into
ti tpatient
First
immunotherapy
product !product !
PAP = prostatic acid
phosphatase
GMCSF = granulocyte
macrophage colony
i l i f
stimulating factor
www.provenge.com

GM‐CSF can enhance antigen presentation
and induce NK and cytotoxic T‐cellsand induce NK and cytotoxic T cells
Tumor cells killed with 3 mechanisms:
- Oncolytic effect of virus replicationOncolytic effect of virus replication
- NK cell mediated direct cell killing
- DCs mediated tumor specific immunity
CD8+
CD8+CD8+CD8+
CD8+
NK
NK
NK
CD8+
NK
CD8+
CD8+
CD8+
CD8+
NK
NK
NK
CaCa
Ca
C
NK
NK
GM-CSF
= personalized
cancer vaccine
Ca
Ca
CaCa
CaCa DC
GM-CSF

Akseli Hemminki | 28 Oct 2010 | 19Treatments

Syrian hamsters cured of HapT1 tumors
with Ad5D24‐GMCSF: protection fromwith Ad5D24 GMCSF: protection from
HapT1 challenge
N=5
**
***
N=5
***
N=5
*
Akseli Hemminki | 28 Oct 2010 | 20Cerullo Cancer Res 2010

Syrian hamsters cured of HapT1 tumors
with Ad5D24 GMCSF: no protectionwith Ad5D24‐GMCSF: no protection
from HaK challenge

Efficacy Ad5‐D24‐GMCSF: Single round of treatment
Neutralizing Antibody Titer Virus Load in Serum Responseg y p
Patient
code
Dosea
(VP)
Primary
Tumor
Week post-treatment Days post-treatment
0 1 2 4 0 1 2 3-7 8-12 21-40 RECISTa
Density/o
ther
Marker Survival
C3 8x109 Jejunum ca 0 1024 16834 0 0 <500 <500 0 MR 120
M3 1 1010 HCC 0 16384 4096 0 0 4896 0 0 0 SD ( 5 2%) 548bM3 1x1010 HCC 0 16384 4096 0 0 4896 0 0 0 SD (+5.2%) 548b
O12 3.6x1010 Ovarian ca 0 16384 16384 0 0 0 0 0 SD (+7.7.%) SD 106
O14 1x1011 Ovarian ca 64 64 0 0 0 <500 0 0 CR (-100%) CR 528b
G15 1x1011 Gastric ca 1024 16384 16384 0 0 565 <500 0 0 -4.6% 308b
K18 2x1011 NSCLC 16384 16384 16384 0 <500 0 0 856 PD (+15%) 59
T19 2x1011 Thyroid ca 0 16384 0 765 <500 <500 0 0 SD (-8.9%) MR 490b
U89 2x1011 Renal ca 64 16384 0 0 0 PD (+13%) 144
S100 2x1011 Leiomyosar
c
0 0 16384 0 <500 <500 PD (+39%) 121
S108 2x1011 Synovial
sarc
0 0 256 0 <500 <500 0 PD (+59%) 74
M50 2.5x1011 Mesothelio
ma
256 16384 0 0 <500 0 SD (-5.7%) 403b
R8 3x1011 Breast ca 64 16384 0 <500 <500 0 CR (-100%) PR 447b
M32 3x1011 Mesothelio
ma
0 256 16384 0 0 0 0 PDc 125
X49 3x1011 Cervical ca 16 4096 1024 0 4290 1211 PD (+55%) -27% 92X49 3x10 Cervical ca 16 4096 1024 0 4290 1211 PD (+55%) -27% 92
I52 3x1011 Melanoma 0 256 256 0 576 PD (+25%) 112
I78 3x1011 Choroideal
mel
0 64 0 44876 <500 63
C58 4x1011 Colon ca 256 16384 16384 0 1978 4236 PD (+37%) 118
R73 4x1011 Breast ca 0 256 1024 0 <500 25787 SD (-3.6%) 245b
O88 4 1011 O i 0 1024 0 <500 d PR 126
Overall efficacy (radiology)
- CR 2/16
O88 4x1011 Ovarian ca 0 1024 0 <500 yesd PR 126
O9e 2x1011 Ovarian ca 16384 16384 0 2133f MR (-20%) 142
Summary of side effects
CR 2/16
- MR 1/16
- SD 5/16
- PD 8/16
- All pts: gr 1-2 flu-like symptoms, fatigue, fever
- One gr 3 ileus (OvCa pt w similar previous episodes)
- Lab: gr 1-2 AST/ALT, hypo-K+, gr 1-3 hypo-Na+

Long term survival in 1/3 of patients
treated with Ad5‐D24‐GMCSFtreated with Ad5 D24 GMCSF

Systemic efficacy of Ad5‐D24‐GMCSF in
injected and non‐injected tumors: virusinjected and non injected tumors: virus
circulation, immune response
• 60 yr mesothelioma patient asbestos exposure• 60 yr mesothelioma patient, asbestos exposure
• Prior treatment with cisplatin+pemetrexed
• WHO 1
• Single intrapleural and i v injectionSingle intrapleural and i.v. injection
• More prominent reduction of non‐injected tumor than injected tumor

Improving transduction to
i l iimprove oncolysis
LOW CAR ‐LOW CAR ‐
LOW GENE
Coxsackie‐
adenovirus
receptor (CAR):
key to Ad entry
DELIVERY !
CAR IS AN
key to Ad entry
CAR IS AN
ADHESION
MOLECULE ‐
LOWLOW
EXPRESSION
IN TUMORS

Increasing infectivity of target cells:
transductional targetingtransductional targeting
T t dNon-targeted
adenovirus
Targeted
adenovirus
Adenovirus
receptor CAR
Benign cell
High
transduction
Low
transduction
receptor CAR
Tumor associated
receptor
Cancer cell
Low
transduction
High
transduction
p

Serotype chimerism for tumor targeting
Ad5
80
100
120
3x 1x108 VP i.p.CAR
Ad3 receptor
40
60
80
%Survival
Kanerva Mol Ther 2003
M1
Negative
0
20
15 25 35 45 55 65 75 85 95 105 115 125 135
CAR
Kanerva Clin Cancer Res 2002
Day
Ad3 receptor 1,E+05
1,E+06 Biodistribution
Ad5/3 1,E+02
1,E+03
1,E+04
/ mg proteinwith knob domain
from Ad3
1,E+00
1,E+01
,
RLU
*
Akseli Hemminki | 28 Oct 2010 | 27Kanerva Mol Ther 2002

Ad5/3‐D24‐GMCSF = CGTG‐102
Fiber chimerism for enhanced
transduction of cancer cells
Replication  in cells mutant in Rb‐p16
CD8+
NK
NK
CGTG‐102: 76% clinical
benefit in advanced ca ptspathy
Includes most human cancers
CD8+
NK
CD8+
CD8+
CD8+
CD8+
CD8+
CD8+
CD8+
NK
NK
NK
benefit in advanced ca. pts
GM‐CSF  can enhance antigen
presentation and induce NK and
cytotoxic CD8+ T‐cells
CaCa
Ca
Ca
NK
Ca
Ca DC
NK
GM-CSF
Expressed under the control of E3
Starts at 8h
Expression coupled to virus
replication
Ca Ca
Ca
Ca
GM-CSF
= personalized
cancer vaccine
p
Koski Mol Ther 2010

Overall survival of
patients treated
50% survival =  320 days
Survival at 200 days = 66%
patients treated
with CGTG‐102
(Ad5/3‐D24‐
N= 19
(Ad5/3 D24
GMCSF)
Overall survival
All patients were chemo refractory and
All treatments
N= 155
All patients were chemo refractory and
progressing at treatment
Overall clinical benefit  in imaging = 76%
Criteria: death due to any cause
N= 144
Censoring: alive at last follow‐up
Median overall survival of chemotherapy
resistant patients 30‐115 days (eg.
Vigano Palliat Med 2000 Llobera Eur J of
Vigano Palliat Med 2000, Llobera Eur J of
Cancer 2000)

Effect of serial
treatment on
50% survival
277 d treatment on
overall survival
of CGTG‐102
112 d
109 d
300 day survival of CGTG 102
patients
Violet= CGTG‐102 serial
y
48 %
33 %
7 %
treatment  (Kanerva,
Nokisalmi in
preparation)
Green = CGTG‐102
7 %
rols
Green = CGTG‐102
single treatment  (Koski
Mol Ther 2010)
Blue = our first ATAP
i (P G
al contr
virus (Pesonen Gene
Ther 2010)
Non‐randomised
istorica
series but inclusion
criteria and patient
characteristics are
identical
30    115
H
Survival rates between 30‐115 d have been reported for this patient population in
historical control series (Vigano Palliat Med 2000, Llobera Eur J of Cancer 2000)

Inclusion and exclusion criteria,
personalization of oncolytic virus treatmentpersonalization of oncolytic virus treatment
Inclusion criteria Exclusion criteriac us o c te a
Refractory solid tumor
Failed treatments for which there is
c us o c te a
confirmed brain met. or glioma
organ transplant, HIV
severe comorbiditystrong scientific evidence*
Good performance status: WHO  0‐
2. (WHO 3‐4 safe but less efficacy)
severe comorbidity
Elevated serum bilirubin
Serum AST or ALT > 3 x normal
Personalization:
( y)
Written informed consent Thrombocytes  < 75.
Personalization:
Selection of virus (out of 10): existing preclinical data on capsids, promoters,
arming, pretreatment efficacy prediction
Dose: tumor burden, comorbidities
Route:  i.t (ultrasound or CT‐guided), intraperitoneal, intrapleural, i.v.
Vi iti T R t h TH2 >TH1
* In most cases this means 1st line chemotherapy
f t t ti di d i l
Virus sensitizers: T‐Reg, autophagy, TH2‐>TH1
Seroswitching when intravenous efficacy sought
for metastatic disease, and in some cases several
lines of chemotherapy (eg. breast, ovarian and
colorectal cancer)
In practice, the median number of prior chemo
regimens is 4 –> heavily pretreated

Systemic efficacy of Ad5/3‐Cox2L‐D24
in chemo refractory neuroblastomay
• 6 yr old boy, WHO 1
• Heavily pretreated: 5 lines of
h t ll t l tchemo, stem cell transplant
• Metastases in bone marrow
and near kidney
T i l difi d i• Triple‐modified virus was
selected for intravenous efficacy
• Cox2 expression confirmed  in
bone marrow biopsy
0
bone marrow  biopsy
• Gr. 1 stomach pain, diarrhea,
flu‐like symptoms, liver enzymes
• 4 wk later: complete response
6540
500
• 4 wk later: complete response
in bone marrow, partial
response in primary
Pesonen Acta Oncol 2010

Preclinical data suggests correlation between
gene delivery and oncolytic potency:
h l l l d d d ffArchival receptor levels did not predict efficacy
CAR staining – to +++                                                          CAR ++ , CEA MR               CAR ++, CT SD
Archival tumors are
typically operated
primariesprimaries
They may be quite
different from
CAR+ CT SD CAR negative CT CR CAR+ Choi resp
metastatic and
relapsing tumors
treated with many
CAR+, CT SD                    CAR negative, CT CR          CAR+, Choi resp.
y
different therapies
Eg CAR level is
known to correlate CAR CT CR CAR CT PD CAR CT MRknown to correlate
with tumor
aggressiveness ‐>
h i CAR
CAR+, CT CR                       CAR ++ CT PD                   CAR‐, CT MR
change in CAR
Haavisto  et al. unpublished

Tumor biopsies may be able to measure
gene delivery to relevant tumor substrates
←   Pre‐treatment biopsies
infected with virus with
gene delivery to relevant tumor substrates
same capsid as oncolytic
virus
• O12: SD in imaging and
k
O12
Biopsy
C3
Biopsy
markers
• C3: MR in markers
• Cerullo Cancer Res 2010
←    Malignant pleural
ff i d i2 5E+07 V136
1 6E 05 K75 effusion and ascites
• V136: SD in imaging, CR
in non‐injected liver
metastasis1,5E+07
2,0E+07
2,5E+07
ssion(RLU)
Pleural effusion cells
1,0E+05
1,2E+05
1,4E+05
1,6E+05
ression(RLU)
K75
Ascites cells
metastasis
• K75: CR in ascites
formation
• Koski Mol Ther 20105,0E+06
1,0E+07
,
nsgene expres
2 0E+04
4,0E+04
6,0E+04
8,0E+04
ransgene expr
Koski Mol Ther 2010
0,0E+00
Ad5luc1 Ad5/3luc1
Tran
0,0E+00
2,0E+04
Ad5luc1 Ad/3luc1
Tr

Pre‐treatment prediction of Ad5/3‐D24‐GMCSF
efficacy: killing of pleural effusion cells
160
180
200
V136
200
250
M137
efficacy: killing of pleural effusion cells
- SD in CT scan
- CR in liver
t t i
- SD in CT scan
- CR in pleural
100
120
140
yof(%)
150
ty(%)
metastasis
p
effusion
- Survival > 700d
(ongoing)
20
40
60
80
Viability
50
100
Viabilit
***
***
0
20
Uninfected
cells
Ad5luc1 Ad5/3-d24-
GMCSF
0
Uninfected
cells
Ad5luc1 Ad5/3-d24-
GMCSF
Koski Mol Ther 2010
R73
SD i CT- SD in CT scan
- Survival >800d
(ongoing)
Cerullo Cancer
Res 2010

Virus circulates for
extended times inextended times in
most patients – no
correlation to
Last d38 Last d28
correlation to
efficacy or adverse
events
Last d40 Last d63
Escutenaire Ann Med in
ll
Last d52
Last d64
press, Cerullo Cancer Res
2010, Nokisalmi CCR 2010,
Koski Mol Ther 2010,
Pesonen Gene Ther 2010,
Last d52
Pesonen Gene Ther 2010,
Pesonen submitted

Neutralizing antibodies are present in some
patients before treatment. They are induced rapidly p y p y
in most patients – neither correlates with efficacy
Intriguing case with no NAb induction
• Heavily pre treated 9yr old Wilms tumor patient:
4 096
16 384
• Heavily pre‐treated  9yr old Wilms tumor patient:
Multiple operations, 6 lines of chemotherapy, radiation
• Progressive disease in several abdominal locations
• ICOVIR‐7 1x10e11 VP; 80% i.t.,20% i.v.
• gr 2 stomach pain fatigue fever gr 1 AST nausea
256
1 024
er
• gr 2 stomach pain, fatigue, fever. gr 1 AST, nausea
• Pre‐treat Nab titer 4, no increase in 4 weeks
Before oncolytic virus         Partial response 36d after
4
16
64
Nab tite
0
1
Koski  Mol Ther 2010
0
0 1 2 3‐4 > 4
Time (weeks)
Y62 H64 C66 S67 S70 P74
K75 O79 I80 O82 H83 I87
C95 H96 I98 N110 O113 S119
X122 O129 V136 M137
Nokisalmi Clin Cancer Res 2010

Anti‐adenoviral and anti‐tumor immunity
are induced in most patients: noare induced in most patients: no
correlation to efficacy.
Decrease might also relate to efficacy ?Decrease might also relate to efficacy ?
Anti‐Ad Anti‐Survivin
Akseli Hemminki | 28 Oct 2010 | 38Koski Mol Ther 2010

Tumor specific T‐cell responses might require
HLA However HLA staining of archivalHLA. However, HLA staining of archival
specimens does not predict efficacy
HLA t i i ( l t ++)HLA staining (scale – to ++)
G15 + R8 negative R73 +
Efficacy
-Choi response, - CR in imaging, markers - SD in CT scan
- Survival > 400d - Survival > 1000d, ongoing - Survival >800d(ongoing)
Akseli Hemminki | 28 Oct 2010 | 39Diaconu, Cerullo, unpublished

Metronomic cyclophosphamide for down‐
regulation of T‐Regregulation of T Reg
Background: T‐Regs
TReg
g g
inhibit NK and cytotoxic
T‐cells
CC
CD8+
NK
CaCa
Ca
Ca
CCa
Ca Ca
Ca
CaCa

Cyclophosphamide for reducing T‐Regs and
enhancing the effect of oncolyticenhancing the effect of oncolytic
virotherapy
TReg Cyclophosphamide
CC
CD8+
NK
CaCa
Ca
Ca
CCa
Ca Ca
Ca
CaCa

GM‐CSF can enhance antigen presentation
and induce NK and cytotoxic T‐cellsand induce NK and cytotoxic T cells
TReg TReg TRegTReg TReg TReg
CD8+
CD8+CD8+CD8+
CD8+
NK
NK
NK
CD8+
NK
CD8+
CD8+
CD8+
NK
NK
NK
CaCa
Ca
C
NK
NK
GM-CSF
Ca
Ca
CaCa
CaCa DC
GM-CSF

Cyclophosphamide can enhance the effect of
GM‐CSF induced NK and cytotoxic T‐cellsGM CSF induced NK and cytotoxic T cells
TReg TRegTReg TReg
C clophosphamide
Cyclophosphamide
CD8+
CD8+CD8+CD8+
CD8+
NK
NK
NK
Cyclophosphamide
CD8+
NK
CD8+
CD8+
CD8+
NK
NK
NK
Ca
NK
NK
GM-CSF Dying
tumor cells
Ca CaCa
Ca DCtumor cells
GM-CSF

Oral or intravenous low‐dose
l h h id b th ?cyclophosphamide or both ?
Metronomic cyclophosphamide useful for reducing T‐Reg
I.v. low dose bolus cyclo useful for Th2‐>Th1 switch
Combination useful for both ?Combination useful for both ?
Cyclo also synergistic for oncolytic cell killing
High doses could be antagonistic for anti‐tumor immune responses
Hypothesis: medium dose i.v.+p.o. optimal for oncolytic Ad treatment ?
Cerullo Submitted

Low dose cyclophosphamide reduces T‐reg in
most but not all patients Most effective inmost but not all patients. Most effective in
patients with high T‐Reg counts?
Akseli Hemminki | 28 Oct 2010 | 45Cerullo Submitted

Possible clinical predictors of
benefit from oncolytic Ad: tumorbenefit from oncolytic Ad: tumor
burden, performance score, age
57 yr old woman with Stage 4 ovarian cancer57 yr old woman with Stage 4 ovarian cancer
Operation, adjuvant CEF x6, taxol+carbo x6, docetaxel,
bevacizumab, topotecan, erlotinib, aromatase inhib.
WHO 1 Progressive disease but low tumor burdenWHO 1. Progressive disease, but low tumor burden
Single intraperitoneal treatment Ad5‐D24‐GMCSF
Complete response (CT, markers) for 9 mo. Survival >1000d (ongoing)
Immunotherapy works best in good perf score patients as seen for ipilimumab (HodiImmunotherapy works best in good perf. score patients, as seen for ipilimumab (Hodi
NEJM 2010), sipuleucel‐T (Kantoff NEJM 2010)
Cerullo Cancer
Res 2010
WHO
performance
score:score:
0 no symptoms
1 symptoms
2 needs rest <50%
3 d t >50%
3 needs rest >50%
4 needs rest 100%
5 dead

Summary
Clinical proof‐of‐principle available for oncolytic virusesp p p y
Anti‐viral and anti‐tumoral immunity key in efficacy
Clinical benefit 76% (radiology) with CGTG‐102 (N=110)
50% overall survival at 300d with CGTG‐102 (serial treatments)50% overall survival at 300d with CGTG 102 (serial treatments)
CGTG‐102 now being tested in clinical trial (Oncos Therapeutics)
Clinical trials very expensive (3.5 mil€ for phase 1‐2 with 40 pts)
For personalization archival specimens not useful Fresh biopsiesFor personalization archival specimens not useful. Fresh biopsies,
effusion or ascites seem more promising
Virus replication, antibodies or T‐cells do not seem to predict efficacy
T‐Reg modulation and autophagy induction can be tailoredT‐Reg modulation and autophagy induction can be tailored
Some clinical parameters seem to predict efficacy
Personalized therapy in an advanced therapy access program can give
patients not eligible for trials access to therapypatients not eligible for trials access to therapy
Personalized therapy can help answer scientific questions pave the way
for trials, which are ultimately needed (and help design succesful trials)
Every patient and tumor is different ‐ > therapy should also be
Every patient and tumor is different > therapy should also be

Acknowledgements
Akseli Hemminki
Sari Pesonen
Sophie Esc tenaire
Marko Ahonen
Karoliina Autio
I lia Diacon
Institut Catala
d’Oncologica:
Ramon Alemany
Univ. Helsinki & HUCH:
Petteri Arstila
Pekka Häyry
K i Hö k d
Suvi Parviainen
Maria Rajecki
Noora Ro inenSophie Escutenaire
Vincenzo Cerullo
Anna Kanerva
Minna Oksanen
Iulia Diaconu
João Dias
Otto Hemminki
Mari Hirvinen
Anniina Koski
a o e a y
U. Washington
Andre Lieber
U. Ottawa
Krister Höckerstedt
Helena Isoniemi
Tuula Kiviluoto
Jorma Paavonen
Risto Renkonen
Ari Ristimäki
Noora Rouvinen
Kikka Holm
Eerika Karli
Saila PesonenAnniina Koski
Ilkka Liikanen
Petri Nokisalmi
John Bell Ari Ristimäki
Mirja Ruutu
Jarmo Salo
Kalle Saksela
Ulf‐Håkan Stenman
Mikko Tenhunen
Saila Pesonen
Mikko Tenhunen
Pekka Virkkunen
Grant support:
Timo Joensuu
Tuomo Alanko
Pekka Simula
Timo Ahopelto
Charlotta Backman
The Patients
Grant support:
ERC
Academy of Finland
ASCO
Biocentrum Helsinki
Bi t Fi l d
Tuomo Alanko
Saila Eksymä‐Sillman
Kalevi Kairemo
Jenni Kylä‐Kause
Leena Laasonen
Satu Kauppinen
Charlotta Backman
Elina Haavisto
Lotta Kangasniemi
Aila Karioja‐Kallio
Heli Nyrhinen
Biocenter Finland
Sigrid Juselius Foundation
University of Helsinki
HUCH Research Funds (EVO)
Satu Kauppinen
Kaarina Partanen
Marina Rosliakova
Tuuli Ranki
Maija Salo
Mikko Salo
Antti Vuolanto

Immunological response to GM‐CSF coding
oncolytic adenovirus: against virus or tumor?oncolytic adenovirus: against virus or tumor?
3 h before CT guided injection (3ml 10 min after
CD8+
5
6
8
2
3
4
10E+8
2
0 17 41 48

king of MHC I
Ad5 (hexon) Specific
Immunity
Block

king of MHC I
Tumor‐specific
Immunity
Block
y
(Survivin)

Cancer stem cell (CSC) hypothesis
CSCCSC Committed progenitors cells:
PCa
CSCCSC Committed progenitors cells:
Rapid replication
Limited lifespan
CSCCSC
Self-renewal:
Slow replication other
fibro
CSCCSC
CaCa
Slow replication
Unlimited lifespan
othervascinflam
Ca
Ca
C
Ca
Ca
Most ca. treatments select target
cells based on higher replication
Ca stem cells may not actively
CaCa
Ca
CaCa Ca
CSCCSC
T mors are mi ed
y y
replicate: not killed
Ion transporters remove drugs
from cells: not killed
CaDifferentiated
ca. cells
CaTumors are mixed
populations of cellsClinical research may have missed
CSC specific agents

Breast cancer stem cells can be
killed with oncolytic adenoviruseskilled with oncolytic adenoviruses
Eriksson Mol Ther
Eriksson Mol Ther
2007
Bauerschmitz
Cancer Res 2008

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