This document discusses oncolytic virus therapies, including their mechanisms of action, preclinical studies, experiences with patients, and future outlook. It provides an overview of deletion mutant oncolytic adenoviruses that selectively replicate in cancer cells. Clinical trials have shown safety and some efficacy of these viruses as monotherapies and in combination with other treatments like radiation or chemotherapy. Oncolytic viruses show promise as a novel cancer treatment approach.

1. Onkolyyttiset virushoidot:
vaikutusmekanismit,,
prekliiniset tutkimukset,
kokemukset potilailla,
kokemukset potilailla,
tulevaisuuden näkymät
Akseli Hemminki, MD, PhD
Specialist in Oncology
Specialist in Oncology
K. Albin Johansson Research Professor,
Finnish Cancer Institute
Cancer Gene Therapy Group
Molecular Cancer Biology Program &
Transplantation Laboratory &
Haartman Institute & FIMM
University of Helsinki and Disclaimer: AH is co‐founder and shareholder of
Disclaimer AH is co founder and shareholder of
Oncos Therapeutics Inc., a company founded for
Helsinki Univ. Central Hospital
facilitating clinical trials with oncolytic viruses

2. Cancer is not a beaten
disease
CANCER
> 1/2 of people alive today will get cancer*
• 1/3 of us will die of cancer
• disseminated solid tumors cannot usually be cured
with currently available treatments
Novel treatments are needed!
* Jemal CA Cancer J Clin 2005 A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 2

3. Bert Vogelstein, ASCO 2009: Cancer
therapeutics after the cancer genome
therapeutics after the cancer genome
project (http://cgap.nci.nih.gov/)
Sequencing of complete genomes of tumors has revealed hundreds of
mutations in each tumor (Wood Science 2007, Parsons Science 2008)
However, the combination of mutations is different in each tumor
,
‐> Each tumor is an individual
‐> Difficult to use small molecular inhibitors because a different
combination of inhibitors would have to be used for each tumor
combination of inhibitors would have to be used for each tumor
(Wood Science 2007, Parsons Science 2008).
‐> For long term efficacy, each patient would have to be treated with
hundreds of inhibitors (impossible because of side effects)
hundreds of inhibitors (impossible because of side effects)
However, all of these mutations seem to fall in 12 pathways (Jones
Science 2008).
Therefore, better to use pathway selective drugs (Vogelstein ASCO
Th f b h l i d (V l i ASCO
2009).
For example, p16/Rb pathway selective oncolytic virus
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 3

4. Deletion mutant oncolytic
adenoviruses: ∆24
d i ∆24
Fueyo Oncogene 2000
Heise Nature Med 2000
E2F • S-phase
E2F Rb • Virus replication
• normal cell
• wt Ad Rb E1A & cell lysis
E1A
24 bp deletion in Rb
binding site of E1A
• normal cell E2F Rb E2F Rb • No S phase entry
S-phase
• ∆24 • No virus replication
• Replication in cells ∆24-E1A ∆24-E1A
mutant in Rb-p16
Rb p16
pathway E2F E2F
• cancer cell E2FE2F E2FE2F • S-phase
• ∆24 • Virus replication
• Includes all human ∆24 E1A
∆24-E1A ∆24-E1A
∆24 E1A & cell lysis
cancers
(Sherr Science 1996) A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 4

5. 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
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 5

6. Mutation compensation
Randomized trial: head and neck cancer
‐ 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
‐ Gendicine® for sale in China
‐ More than 10 000 patients treated
Promoter p53 gene
p53 gene pA
Press release 23 Jul 2008: Ad
Press release 23 Jul of cells p53 (Advexin )
Infection 2008: Ad‐p53 (Advexin®)
Infection of cells
Cancer cells
phase III SCCHN trial positive in US: not
Normal cells
with p53 mutation
approved by FDA
with healthy p53
Approved by EMEA for Li‐Fraumeni syndrome
Cell death, also sensitation to
(orphan drug)
( h d ) chemotherapy and radiation
h h d di i
No cell death
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 6

7. Prodrug converting enzymes
(suicide gene therapy) with Ad‐TK
(suicide gene therapy) with Ad TK
and GCV
Ad coding for
TK Advantage vs.
thymidine kinase mutation
mutation
(TK) compensation:
bystander effect
via gap junctions
Non‐toxic
pro drug
pro‐drug
GCV
Activated
Activated
toxin
Cell death
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 7

8. Adjuvant Ad‐TK/GCV
CHALLENGE: even with bystander
Randomized phase 2b for glioma (N=36). effect, canA. Mol Ther 2004 penetration
Randomized phase 2b for glioma (N 36). Immonen A. Mol Ther 2004 p
Immonen we get effective
, g
Patients resected and randomized: into established tumors ?
1. follow‐up +/‐ XRT (= standard) SOLUTIONS: locally amplifying
systems
2. Standard + Ad‐TK into resection margins, ganciclovir for 14d
2 Standard + Ad TK into resection margins ganciclovir for 14d
Median survival 39.0 wk. vs. 70.6 wk. (p<0.0095)
No increase in toxicity
Similar results in hepatocellular ca. adjuvant trial (Li Clin Cancer Res 2007) d )
Si il lt i h t ll l Ph Phase dj results 22 O t 2009 (ASPECT study):
III t lt i l (Li Cli C
t Oct R 2007) t
• Cerepro vs standard care: 1.43 HR (p=0.02)
= positive primary end-point
• About 40d increase in median survival
• More temozolomide use in control group due
g p
to non-blinded Cerepro -> dilution of results
• EMEA did not approve because non-standard
end point
end-point (time to re-intervention or death)
re intervention
• Appeal ongoing
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 8

9. Oncolytic viruses
• Replication of virus
p
causes oncolytic death
of cells
• Normal cells‐ no
replication
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 9

10. Short history of oncolytic viruses
1896. 1st report of response to oncolytic virus ( influenza Dock Am J Med
1896. 1st report of response to oncolytic virus (”influenza” Dock Am J Med
Sci 1904)
1940s. 1st systematic trials with oncolytic viruses (”Egypt 101”, Hoster
Cancer Res 1949)
1950s. Adenovirus used for treatment of cervical cancer patients (Smith
Cancer 1956)
¬ Various serotypes, intratumoral, intravenous and intra‐arterial delivery
¬ Poorly characterized preparations: titers unknown
¬ Treatment with and without immune suppression
¬ Good safety, similar side effects to modern trials
¬ Frequent responses
¬ Approach was abandoned: relapses, advent of chemotherapeutics
1960s & 1970s. Rational development of oncolytic viruses in test animals,
further trials (Asada Cancer 1974)
f th t i l (A d C 1974)
1991 & 1996. Utilization of molecular features for making viruses selective
for tumor cells (Martuza Science 1991, Bischoff Science 1996)
2004. First phase III trial with oncolytic virus completed (”H101”, Yu Curr
2004 First phase III trial with oncolytic virus completed (”H101” Yu Curr
Cancer Drug Targets 2007)
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 1 0

11. H101 (Oncorine®) phase III trial in
advanced head and neck cancer
d dh d d k
H101 is almost identical to dl1520 (=ONYX‐015)
descibed earlier in the US
Randomized phase III trial (N=105)
H101 + cisplatin + 5‐FU vs. cisplatin + 5‐FU
H101 + cisplatin + 5 FU vs cisplatin + 5 FU
CR+PR = 79% vs. 38%, P<0.0001
Mild tox: flu‐like symptoms, injection site pain
p
More than 800 patients now enrolled
H101 approved in China
Yu Curr Cancer Drug Targets 2007
Yu Curr Cancer Drug Targets 2007
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 1 1

12. Cancer Gene Therapy is maturing
as a treatment approach
h
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
TUMOR PENETRATION NEEDS IMPROVEMENT
Replication competent oncolytic viruses
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
* Immonen Mol Ther 2004, Li Clin Cancer Res 2007, Yu Curr Cancer Drug
Targets 2007, Pan J Clin Oncol 2008, Ylä‐Herttuala ESGCT 2008, A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 1 2

13. Our own experience with oncolytic
adenovirus (update 5 Feb 2010)
adenovirus (update 5 Feb 2010)
• 171 patients since Nov 07. 8 different viruses
• All had metastatic solid tumors progressing after routine
All had metastatic solid tumors progressing after routine
treatments (chemo, radiation, etc)
• Written informed consent. Full GCP implemented.
• Side effects: gr. 1‐2 flu‐like symptoms, fever, fatigue, pain in all pt
Side 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)
• Clinical benefit (imaging CR, PR, SD): 61% overall, 76% best virus
• Some patients have benefited for > 2 years (= length of follow‐up)
• Additive/synergistic benefits from 2nd ‐ 10th treatments
Additive/synergistic benefits from 2 treatments
• Long term (>500 d) survival in 48% with best virus
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 1 3

14. Inclusion and exclusion criteria for
oncolytic virus treatment
oncolytic virus treatment
Inclusion criteria Exclusion criteria
Solid tumor (not leukemia or confirmed brain metastases or glioma
lymphoma) organ transplant, HIV
Refractory disease = previously severe cardiovascular, metabolic or
d l b l
treated with oncology treatments for pulmonary disease
which there is strong scientific Other diseases that prevent oncolytic
evidence* virus treatment
i t t t
Good performance status: WHO 0‐2. Elevated serum bilirubin
(
(WHO 3 is safe but less efficacy)
y) Serum AST or ALT > 3 x upper limit of
Written informed consent normal
No major organ function deficiencies Thrombocytes < 75.
* In most cases this means 1st line chemotherapy for metastatic
disease, and in some cases several lines of chemotherapy (eg.
di di l li f h th (
breast, ovarian and colorectal cancer)
In practice, the median number of prior chemo regimens is 3 A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 1 4

15. Findings possible only in pts: Mechanisms of anti‐
tumor efficacy
y inflammation
3. Induction of
cytotoxic T‐cells
1. Killing of differentiated tumor cells
1 Killing of differentiated tumor cells against tumors
against tumors
6 CD8+
5
vitiligo
E+8
4
10E
3
2
0 17 41 48
2. Killing of tumor initiating ”stem” cells
4. Induction
of specific
immunity
against
tumor
epitope
(survivin)
Cerullo Cancer Res in press 2010 A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 1 5
Eriksson Mol Ther 2007, Bauerschmitz Cancer Res 2008

16. Case: Systemic efficacy of Ad5/3‐Cox2L‐D24 in
chemo refractory neuroblastoma
chemo refractory neuroblastoma
• Ad5/3‐Cox2L‐D24 replicates in cells overexpressing Cox2 and
defective in the Rb/p16 pathway
Previous
Previous
treatments: • 6 yr old boy, WHO 1
Vincristine + • Progressive disease in bone marrow, left kidney, lymph nodes.
cis/carboplatin • Single oncolytic adenovirus treatment: i v intratumoral
cis/carboplatin Single oncolytic adenovirus treatment: i.v., intratumoral.
+ etoposide + • Gr. 1 stomach pain, diarrhea, flu‐like symptoms, liver enzymes
cyclophospham • 4 wk later: complete response in bone marrow, partial
ide response in primary
i i
Doxorubicin +
etoposide +
iphosphamide;
iphosphamide;
Intensive chemo
and autologous
stem cell
stem cell
transplant;
Oral 13‐cis‐retinoic
acid
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 1 6

17. Ad5/3‐
Cox2L‐D24
in neuro‐
blastoma
→ CD56 staining (brown) for
tumor cells in bone
marrow
→ Imaging of primary before
and after treatment
→ Increase in cytotoxic T‐cells
→→ Increase in virus
neutralizing antibodies
→→→ Extended presence of
virus in blood
0
→→→→ Cox2 expression in
6540
tumor (reason for
( f
500
selectivity and efficacy)
Pesonen Submitted 2008| 1 7
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0
Pesonen Acta Oncol 2010

18. Higher efficacy with a second round of
treatment
• Metastatic pancreatic ca. WHO 2
• Prior gemcitabine and gemcitabine chemoradiation
• Second round of treatment with Ad5‐24‐RGD (Bauerschmitz
Cancer Res 2002) produced response
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 1 8
Pesonen in preparation

19. Higher efficacy with retreatment
Adenovirus replication is
very immunogenic
Second round of injection
results in enhanced
immune attack on tumor ?
i tt k t ?
Immunity against
adenovirus or also tumor ?
adenovirus or also tumor ?
Cytotoxic
Cytotoxic
lymphocytes
in pt treated
with Ad5/3‐
with Ad5/3
Cox2L‐D24
Cytotoxic T‐lymphocytes approaching
tumor cells (pic from Natl Geographic) A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 1 9

20. Improving antitumor immunity: oncolytic
adenoviruses coding for GM‐CSF
adenoviruses 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 anti G CS
tumor immunity (Dranoff Immunol Rev 2002)
GM-CSF
• GM‐CSF in E3: expression starts at 8h
⇒ GM‐CSF expressed only in cells that allow
replication of the virus
• Hi h expression at tumor, l systemic
High i low i
GM CSF
GM-CSF
GM-CSF
Cerullo Cancer Res in press 2010 A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 2 0

21. GM‐CSF can enhance antigen presentation
and induce NK and cytotoxic T cells
and induce NK and cytotoxic T‐cells
Tumor cells killed with 3 mechanisms:
- Oncolytic effect of virus replication
- NK cell mediated direct cell killing
- DCs mediated tumor specific immunity
NK NK CD8+ CD8+ CD8+
NK NK CD8+ CD8+
CD8+
NK CD8+ CD8+
NK
= personalized
NK Ca Ca cancer vaccine
GM-CSF Ca
Ca Ca Ca
C DC
Ca Ca Ca
GM-CSF A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 2 1

22. Cerullo Cancer Res in press 2010
Treatments A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 2 2

23. Syrian hamsters cured of HapT1 tumors
with Ad5D24 GMCSF: protection from
with Ad5D24‐GMCSF: protection from
HapT1 challenge
N=5
**
***
***
N=5
*
N=5
Cerullo Cancer Res in press 2010 A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 2 3

24. Syrian hamsters cured of HapT1 tumors
with Ad5D24‐GMCSF: no protection
with Ad5D24 GMCSF: no protection
from HaK challenge
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 2 4
Cerullo Cancer Res in press 2010

25. Efficacy Ad5‐D24‐GMCSF: Single round of treatment
Neutralizing Antibody Titer
g y Virus Load in Serum Response
p
Patient Dosea Primary Week post-treatment Days post-treatment
code (VP) Tumor RECISTa
0 1 2 4 0 1 2 3-7 8-12 21-40 Density/o Marker Survival
ther
C3 8x109 Jejunum ca 0 1024 16834 0 0 <500 <500 0 MR 120
M3 1 1010
1x10 HCC 0 16384 4096 0 0 4896 0 0 0 SD (+5.2%)
( 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 0 0 16384 0 <500 <500 PD (+39%) 121
c
S108 2x1011 Synovial 0 0 256 0 <500 <500 0 PD (+59%) 74
sarc
M50 2.5x1011 Mesothelio 256 16384 0 0 <500 0 SD (-5.7%) 403b
ma
R8 3x1011 Breast ca 64 16384 0 <500 <500 0 CR (-100%) PR 447b
M32 3x1011 Mesothelio 0 256 16384 0 0 0 0 PDc 125
ma
X49 3x1011 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 0 64 0 44876 <500 63
mel
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
4x10 Ovarian
O i ca 0 1024 0 <500 yesd PR 126
O9e 2x1011 Ovarian ca 16384 16384 0 2133f MR (-20%) 142
Overall efficacy (radiology)
Summary of side effects
- CR 2/16
- All pts: gr 1-2 flu-like symptoms, fatigue, fever
- MR 1/16
- One gr 3 ileus (OvCa pt w similar previous episodes)
- SD 5/16 Cerullo Cancer Res in press 2010
- Lab: gr 1-2 AST/ALT, hypo-K+,mgrk1-3 hypo-Na+ | 2 5
A k s e l i H e m i n i | 2 3 F e b 2 0 1 0
- PD 8/16

26. Efficacy of Ad5‐D24‐GMCSF in injected and
non injected tumors: mesothelioma patient
non‐injected tumors: mesothelioma patient
• 60 yr old man, asbestos exposure
• Prior treatment with cisplatin+pemetrexed
Prior treatment with cisplatin+pemetrexed
• WHO 1
• Single intrapleural and i.v. injection
• More prominent reduction of non‐injected tumor than
More prominent reduction of non injected tumor than
injected tumor
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 2 6
Cerullo Cancer Res in press 2010

27. Complete response in OvCa pt
with small disease burden
ith ll di b d
Metastatic ovarian ca. 2002
M t t ti i 2002
Operation, adjuvant CEF x6, taxol+carbo x6, docetaxel,
bevacizumab, topotecan, erlotinib, aromatase inhibitor
Progressive disease, WHO 1
Single intraperitoneal treatment
Complete response (CT, markers) for 9 mo
Cerullo Cancer
Cerullo Cancer
Res in press
2010
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 2 7

28. Rapid response upon re‐treatment with
GM CSF coding oncolytic
GM‐CSF coding oncolytic adenovirus
• Peritoneally metastatic ovarian cancer since 2005.
• 5 lines of chemo (paclitaxel‐carbo, liposomal doxorubicine,
gemcitabine+carbo, gemcitabine, topotecan)
• Progressive disease, WHO 1
• 52.5% tumor size reduction in 17 days after 2nd treatment
y
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 2 8
Cerullo Cancer Res in press 2010

29. Long term survival in 1/3 of patients
treated with Ad5 D24 GMCSF
treated with Ad5‐D24‐GMCSF
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 2 9
Cerullo Cancer Res in press 2010

30. Immunological response to GM‐CSF
coding oncolytic adenovirus
coding oncolytic adenovirus
Adenovirus: T cell response
towards adenovirus
towards adenovirus
components (Hexon, Penton,
Fiber etc.)
)
Tumor: T cell response to
tumor specific epitopes
tumor specific epitopes
6 CD8+
5
10E+8
8
4
3
2
0 17 41 48
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 3 0

31. CT guided injection displays
immunological response to adenovirus
immunological response to adenovirus
3 h before oncolytic virus. 10 min after CT guided
3 h before oncolytic virus. 10 min after CT guided
injection (3 ml)
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 3 1

32. eceptor
king of T‐Cell re
Block
Ad5 (hexon) Specific
Immunity
Cerullo Cancer Res in press 2010 A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 3 2

33. eceptor
king of T‐Cell re
Block
Tumor‐specific
Immunity y
(Survivin)
Cerullo Cancer Res in press 2010 A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 3 3

34. Inflammation due to virus and/or GM‐
CSF can falsely increase tumor markers
CSF can falsely increase tumor markers
and enlarge tumors
Virus replication • 59 old man with esophageal cancer
59 old man with esophageal cancer
activates tumor
cell metabolism • Prior chemo: oxaliplatin+capesitabine,
oxaliplatin, docetaxel, irinotecan‐
paclitaxel, cyklo‐doxo‐cisplatin, oxali‐
irino‐cetuximab, capecitabine
• Progressive disease WHO 1
• Intratumoral and i.v. virus injection
Necrosis
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 3 4

35. Baseline 3 months 6 months
Senzer J Clin Oncol 2009
Effects of
Effects of
oncolytic
inflammation
inflammation
on tumor size
Reid Cancer Res 2002 A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 3 5

36. PET‐CT more useful for oncolytic viruses ?
Park Lancet Oncol 2008
Senzer J Clin Oncol 2009
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 3 6

37. Improving transduction to
improve oncolysis
i l i
Coxsackie‐ LOW CAR ‐
LOW CAR ‐
adenovirus
receptor (CAR):
key to Ad entry
key to Ad entry
LOW GENE
DELIVERY !
CAR IS AN
CAR IS AN
ADHESION
MOLECULE ‐
LOW
LOW
EXPRESSION
IN TUMORS
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 3 7

38. Increasing infectivity of target cells:
transductional targeting
Non-targeted Targeted
T t d
adenovirus adenovirus
Adenovirus
receptor CAR
High Low
transduction Benign cell transduction
Tumor associated
receptor
p
Low High
transduction Cancer cell transduction
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 3 8

39. Serotype chimerism for tumor targeting
120
Ad5 CAR 100 3x 1x108 VP i.p.
Ad3 receptor
80
% Survival
Negative
M1
60
40 Kanerva Mol Ther 2003
20
0
15 25 35 45 55 65 75 85 95 105 115 125 135
Day
Kanerva Clin Cancer Res 2002
1,E+06 Biodistribution
Ad3 receptor CAR 1,E+05
RLU / mg protein
1,E+04
1,E+03
Ad5/3 1,E+02
,
with knob domain
1,E+01 *
from Ad3
1,E+00
Kanerva Mol Ther 2002 s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 3 9
Ak

40. Cancer stem cell (CSC) hypothesis
CSC Committed progenitors cells:
Rapid replication
PCa Limited lifespan
Self-renewal: fibro
CSC Slow replication other
Unlimited lifespan inflam vasc
Ca Ca
Most ca. treatments select target Ca
cells based on higher replication Ca Ca Ca Ca
Ca stem cells may not actively
y y
replicate: not killed Ca
C Ca Ca
Differentiated Ca
Ion transporters remove drugs ca. cells
from cells: not killed CSC Ca
Tumors
T mors are mixed
mi ed
Clinical research may have missed populations of cells
CSC specific agents A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 4 0

41. Cancer stem cells can be killed with
oncolytic adenoviruses
oncolytic adenoviruses
Eriksson Mol Ther
2007
Bauerschmitz
Cancer Res 2008 A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 4 1

42. Ad5/3‐D24‐GMCSF
Fiber chimerism for enhanced
transduction of cancer cells
Replication in cells mutant in Rb‐p16 NK
NK
CD8+
pathy CD8+
CD8+
NK CD8+ CD8+
Includes most human cancers NK
CD8+ CD8+
CD8+
NK NK
GM‐CSF can enhance antigen
NK Ca Ca
presentation and induce NK and
GM-CSF Ca
cytotoxic CD8+ T‐cells Ca Ca DC
Ca
Expressed under the control of E3
Ca Ca
Starts at 8h Ca
= personalized
Expression coupled to virus GM-CSF cancer vaccine
replication
p
Koski Submitted 2010 A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 4 2

43. Ad5/3‐D24‐GMCSF effective in vitro
MDA-MB-436 A549
A549
As effective as wild type
GMCSF secretion verified
GMCSF secretion verified
Koski Submitted 2010
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 4 3

44. Benefits of treatment (single injection
of Ad5/3 D24 GMCSF)
of Ad5/3‐D24‐GMCSF)
Objective benefit in Computer Scans (RECIST 1.1):
Objective benefit in Computer Scans (RECIST 1 1):
Minor Response (MR) 17% (best ‐15%) Long term survival of
Stable Disease (SD) 50%
y
>250 days in circa 40%
Progressive Disease (PD) 33%
Clinical benefit (imaging): 67%
(ongoing)
Other benefits:
Other benefits: Adverse events
d e se e e ts
CR: ascites and pleural effusion Monitored for 4 weeks
CR: pleural effusion No grade 4‐5 events
CR: non‐injected liver metastasis
CR i j d li i One grade 3 cholecystitis
1% reduction in injected tumor Mild to moderate flu‐like
Injected tumor 6% smaller
j symptoms, fever, fatigue,
symptoms fever fatigue
Overall biological activity injection site pain common
(intent to treat population): 13/21= 62% No elevations in cytokine
levels
l l
Koski Submitted 2010 A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 4 4

45. Examples of Ad5/3‐D24‐GMCSF
nt
Before treatmen
t efficacy in patients
K75 NSCLC: CR in O129 ovarian cancer: I98 melanoma: - 39% (v/v)
p
pleural eff. & ascites -17% (v/v)
(v/v)
)
eatment
2 mo. after tre
Koski Submitted 2010 2 3 F e b 2 0 1 0 | 4 5
Akseli Hemminki |

46. Pretreatment prediction of CGTG‐102
efficacy
2,5E+07
, V136 1,6E+05
K75
Pleural effusion cells *** ***
sion (RLU)
1,4E+05 Ascites cells
ene expression (RLU)
2,0E+07
1,2E+05
1,0E+05
gene express
1,5E+07
8,0E+04
1,0E+07 6,0E+04
Transge
4,0E+04
4 0E+04
Transg
5,0E+06
2,0E+04
0,0E+00 0,0E+00
Ad5luc1 Ad5/3luc1 Ad5luc1 Ad5/3luc1
/
Ad/3luc1
***p<0.005 against Ad5luc1
Pre‐treatments samples of malignant pleural effusion and ascites
Pre‐treatments samples of malignant pleural effusion and ascites
Luciferase expression of cells infected with 5000 vp/cell
V136: SD in CT scan, liver metastasis disappeared
K75: CR in ascites formation
K75: CR in ascites formation
Koski Submitted 2010 A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 4 6

47. Ad5/3‐D24‐GMCSF is able to kill
pleural effusion cells
pleural effusion cells
200 250
V136 M137
180
160 200
140
bility of (%)
)
120 150
ability (%)
100
80 100
***
Via
60
Viab
40 50 ***
20
0 0
Uninfected Ad5luc1 Ad5/3-d24- Uninfected Ad5luc1 Ad5/3-d24-
cells GMCSF cells GMCSF
***p<0.005 against uninfected cells
Pre‐treatment samples of malignant pleural effusions
Pre‐treatment samples of malignant pleural effusions
Cells from sample infected with 100 vp/cell
V136: SD in CT scan, liver metastasis disappeared
M137: SD in CT scan, CR in pleural effusion formation
M137: SD in CT scan CR in pleural effusion formation
Koski Submitted 2010 A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 4 7

48. Higher amounts of virus at the tumor than
in blood
8000
6851
7000 Serum
6000 Ascites Virus in ascites sample
r (vp / ml)
5000
able to produce
bl d
cytopathic effect in 70%
4000 of wells in cell culture
(293 cells)
(293 ll )
Titer
3000
2000
Functional virus present
1000 <500
0
0 NA NA
0
Day 0 1 Day 1 Week
Patient (O82) had MR in tumor marker
levels during follow up
Koski Submitted 2010 A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 4 8

49. Induction of anti‐adenoviral and anti‐
tumoral immunity in blood
tumoral immunity in blood
ELISPOT- patient I80
(had vitiligo)
Increase of CD8+ T lymphocytes after
of CD8+ T lymphocytes
treatment in 10/14 patients
an SFC per million cells
120
Adenovirus
Average CD8+ cell count increases 100
1,60
80
CD cells x10e10/l
1,40 60
1,20 40
1,00 N 10
N=10 20
x
Mea
0,80 0
0,60 0 35
0,40 Days (post‐treatment)
D8+
Mean SFC per million cells
0,20
60
0,00 Survivin
Pre-treatment After treatment 50
40
30
Analysis of T cells specific for
of T cells for 20
adenovirus and tumor epitopes with 10
0
interferon gamma ELISPOT 0 35
Days (post‐treatment)
Koski Submitted 2010 A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 4 9

50. Summary Overall survival w/ CGTG‐102
50% survival = 315d
Survival at 200d = 75%
Survival at 500d = 48%
N=25
Clinical proof‐of‐principle available
for many ca. gene therapy approaches
y g py pp
Safety has generally been excellent
Effective gene delivery continues to be key to efficacy
Oncolytic viruses amplify and help in tumor penetration
Anti‐viral and anti‐tumoral immunity key in efficacy
Multiple injections more effective than single
Multiple injections more effective than single
Overall clinical benefit 76% in 110 treatments with our best
virus CGTG‐102 (now being tested in a clinical trial)
48% overall survival at 500d with CGTG‐102
Earlier treatment and smaller tumor load increase benefits
Formal clinical trials are needed ! (€€€)
Formal clinical trials are needed ! (€€€)
A k s e l i H e m m i n k i | 2 3 F e b 2 0 1 0 | 5 0