Ad3-hTERT-E1A, A Fully Serotype 3 Oncolytic Adenovirus, in Patients With Chemotherapy Refractory Cancer PDF

The American Society of Gene & Cell Therapy
original article
Ad3-hTERT-E1A, a Fully Serotype 3 Oncolytic

Adenovirus, in Patients With Chemotherapy
Refractory Cancer
Otto Hemminki1, Iulia Diaconu1, Vincenzo Cerullo1, Saila K Pesonen1, Anna Kanerva1,2, Timo Joensuu3,
Kalevi Kairemo3, Leena Laasonen3, Kaarina Partanen3, Lotta Kangasniemi4, Andre Lieber5, Sari Pesonen1
and Akseli Hemminki1,3,4
1
Cancer Gene Therapy Group, Haartman Institute and Transplantation Laboratory, University of Helsinki, Helsinki, Finland; 2Department of OB/GYN,
Helsinki University Central Hospital, Helsinki, Finland; 3International Comprehensive Cancer Center Docrates, Helsinki, Finland; 4Oncos Therapeutics
Ltd., Helsinki, Finland; 5University of Washington, Division of Medical Genetics, Seattle, Washington, USA
Twenty-five patients with chemotherapy refractory cancer were treated with a fully serotype 3-based oncolytic
adenovirus Ad3-hTERT-E1A. In mice, Ad3 induced higher
amounts of cytokines but less liver damage than Ad5 or
Ad5/3. In humans, the only grade 3 adverse reactions
were self-limiting cytopenias and generally the safety profile resembled Ad5-based oncolytic viruses. Patients that
had been previously treated with Ad5 viruses presented
longer lasting lymphocytopenia but no median increase
in Ad3-specific T-cells in blood, suggesting immunological activity against antigens other than Ad3 hexon.
Frequent alterations in antitumor T-cells in blood were
seen regardless of previous virus exposure. Neutralizing
antibodies against Ad3 increased in all patients, whereas
Ad5 neutralizing antibodies remained stable. Treatment
with Ad3-hTERT-E1A resulted in re-emergence of Ad5
viruses from previous treatments into blood and vice
versa. Signs of possible efficacy were seen in 11/15 (73%)
patients evaluable for tumor markers, four of which were
treated only intravenously. Particularly promising results
were seen in breast cancer patients and especially those
receiving concomitant trastuzumab. Taken together,
Ad3-hTERT-E1A seems safe for further clinical testing or
development of armed versions. It offers an immunologically attractive alternative, with possible pharmacodynamic differences and a different receptor compared
to Ad5.
Received 12 February 2012; accepted 12 May 2012; advance online
publication 7 August 2012. doi:10.1038/mt.2012.115
Introduction
Gene therapy is an emerging field with an increasing number of

positive clinical trials. Cancer has been the most common disease
addressed due to unmet clinical need and serotype 5 adenoviruses have been the most common vector. Oncolytic viruses are
modified so that they selectively replicate in cancer cells and cause
cell lysis. Also, it has become evident that the role of the immune
system is important in two ways. It may hinder the spread and

replication of the virus but if tumor-associated immunological
tolerance can be broken by oncolysis-mediated danger signals,
it also helps to fight the cancer.14 The immune system has many
effective ways for resisting tumor development, all of which have
to be overcome by tumors in situ. Thus, the capacity for immunoevasion is considered as a new hallmark of cancer.5 Cancer cell
lysis and inflammation caused by oncolytic virus can help present
tumor-associated antigens (TAA) to immunological cells in the
context of danger signals which can then result in reactivation of
immune responses against the tumor.6 Therefore, oncolytic viruses
can be regarded as personalized anticancer vaccines.
One evident problem with serotype 5 adenovirus (Ad5) is that
it uses coxsackie- and adenovirus receptor (CAR)known to be
downregulated in advanced tumorsas a primary entry receptor.7 In contrast, a receptor that would be regularly present in most
tumors would be optimal. The entry receptor(s) for adenovirus 3
has been debated but it seems to be amply present also in advanced
tumors, resulting in generation of Ad5/3 chimeric viruses,811
which are serotype 5 except for the knob of the fiber which is from
serotype 3. These viruses were shown to enhance the transduction
of CAR low cells.12 However, a modification in the knob region
does not overcome immune responses against other components
of the virus13 and thus the notion of an oncolytic virus based completely on a different serotype is attractive.14
Exposure to adenovirus typically results in high-neutralizing
antibody titers (NAbs) but also other elements of the immune system can recognize the virus, causing a problem for virus spreading
within tumor masses and especially in the context of hematological dissemination to distant tumors. Therefore, in the context of
patients with high pre-existing immunity against serotype 5adenovirus, due to either wild-type virus infection or subsequent
to oncolytic virus treatment, a different serotype virus would be
appealing from an immunological perspective.14
A recent publication suggests that desmoglein 2 is a primary
attachment receptor for Ad3.15 This receptor is associated with
the tight junctions and binding of Ad3 initiates a cascade resembling epithelial to mesenchymal transition (EMT) and resulting in
Correspondence:Akseli Hemminki, Molecular Cancer Biology Program, University of Helsinki, PO Box 63 (Haartmaninkatu 8) Biomedicum B506b,
University of Helsinki, Helsinki 00014. Finland. E-mail: akseli.hemminki@helsinki.fi
Molecular Therapy vol. 20 no. 9, 18211830 sep. 2012
1821
Treatment of Cancer Patients With Adenovirus 3
transient opening of tight junctions between epithelial cancer cells.

This increases access to many important receptors trapped in this
area (e.g., Her2/neu, EGFR, CAR), offering rationale for potential synergy with other cancer treatments (trastuzumab, cetuximab, Ad5).16 It was shown that Ad5/3 is capable of binding to this
receptor, but its ability to open the tight junction is compromised
presumably due to the long fiber of the serotype 5 virus. Another
interesting feature of Ad3 is that it produces a million-fold excess
of virus-like particles slightly smaller than the Ad3 virus.17 These
particles consist of the viral surface proteins but do not contain
the genetic material inside and are thought to be important in the
opening of the epithelial junctions. In addition to desmoglein 2,
Ad3 might use also other receptors such as CD46.18
To render Ad3-hTERT-E1A selectively oncolytic, the endogenous E1A promoter was replaced by a human telomerase (hTERT)
promoter enabling efficient replication of the virus only in cell with
high-telomerase activityanother hallmark of cancer.5 This virus
was then tested in vitro and in vivo and was found to behave differently than Ad5 or Ad5/3 viruses. In vitro the virus was found slower
in tumor cell killing, but in vivo the virus was always found to be
at least as potent as serotype 5 or 5/3 viruses.14 The virus retained
its oncolytic potency in the presence of anti-Ad5 neutralizing antibodies. Here, we report murine biodistribution and toxicity studies,
followed by analysis of safety, efficacy, virological, and immunological analysis of patients treated with Ad3-hTERT-E1A.
Results
Preclinical
spleen, pancreas, brain, testicle, and muscle) in mice treated with

Ad3-hTERT-E1A, Ad3wt, or phosphate-buffered saline. Ad5wt
showed 36-fold and Ad5/3delta24 showed 176-fold higher liver
enzymes than mock. Other groups displayed only minor elevations in liver enzymes (Ad3wt and Ad3-hTERT-E1A threefold,
Ad5/3-hTERT-E1A fivefold) compared to the mock group.
Analysis of other blood values showed nonspecific thrombocytopenia but no changes in leukocytes or other parameters.
Clinical
Baseline characteristics. Baseline characteristics of the 25 patients treated with Ad3-hTERT-E1A can be seen in Table1. The
patients had a wide variety of cancer types and were heavily pretreated with a median of three chemotherapy regimens. About
half of them had been previously treated with serotype 5 oncolytic
adenovirus while the other half received Ad3-hTERT-E1A as a
first oncolytic virus treatment. Patient by patient cancer treatments can be found from Supplementary Table S2.
Adverse reactions. Adverse reactions are described in Table 2.
No serious adverse events leading to patient hospitalization were
encountered and the only severe events were asymptomatic and
Table 1 Patient characteristics
Characteristics
Mean age
60 years
Female
14
Ad3 biodistribution. Ad3 biodistribution assay at 6 hours following intravenous injection to immunocompetent mice
(Supplementary Figure S1, N = 5) indicated high (>60 viral particles (VP)/-actin) virus amount in lung, spleen, liver, blood clot,
and bone marrow. These results are similar to previously reports
with Ad5 and Ad5/3 and thus the biodistribution of Ad3 in rodents resembles that of Ad5 and Ad5/39,12,1921 All other organs
had low (<10 VP/-actin) amounts of virus. Without heparin, 106
VP/ml was found in the clot and only 2 104 VP/ml in the serum.
When analyzing heparin tubes 3 105 VP/ml was found from the
plasma and only 4 103 VP/ml from the erythrocytes. These findings indicate that most of the virus in mouse blood is found from
the peripheral blood mononuclear cells (PBMC) and platelets.
Male
11
Acute immunological toxicity. Acute immunological toxicity

was accessed by injecting immunocompetent mice intravenously and collecting blood samples 6 hours later (Supplementary
FigureS2). Ad3 elicited higher Rantes, tumor necrosis factor-,
and interleukin-6 proinflammatory cytokines than Ad5 or Ad5/3
control viruses suggesting that Ad3 can activate mouse macrophages and T-cells. However, the levels of these cytokines were
well below concentrations associated with toxicity.2224
Toxicity assays. Toxicity assays performed in immune competent

mice (N = 5 per group) indicated that Ad3wt and Ad3-hTERTE1A were less toxic than Ad5wt or Ad5/3delta24 (Supplementary
Figure S3). At 72 hours, mice in the latter two groups appeared ill
and livers were macroscopically yellow. No signs of toxicity were
seen in any other organs examined (heart, lung, intestine, kidney,
1822
Cancer type
NSCLC
Breast
Sarcoma
Pancreatic
Prostate
Bladder
Neuroblastoma
Ovarian
Thyroid
WHO
1
15
Median
Previous chemo regimens

Range
17
Median
Previous oncolytic adenovirus treatments

None
12
12 treatments
46 treatments
Abbreviation: NSCLC, nonsmall-cell lung carcinoma.
www.moleculartherapy.org vol. 20 no. 9 sep. 2012
Table 2 Adverse Reactions

(CTCAE 3.0)
Gr1
Gr2
Nausea
Diarrhea
Neurology
General
Gr3
Gr4
Gr5
Gastrointestinal
Pain
Constitutional symptoms
Chills
10
Fatigue
Fever
Anemia
Leucocytopenia
Lymphocytopenia
Thrombocytopenia
Blood/bone marrow
Metabolic or laboratory
Elevated AST
Abbreviation: AST, aspartate aminotransferase.

All 25 patients were evaluated. Grade 1 and 2 reactions are reported if present
in three or more patients.
self-limiting hematological findings. Therefore, no dose-limiting

toxicity was seen. Typically, the patients suffered from mild flulike symptoms: chills, fatigue, fever, and nausea were present in
about half of the patients, as was injection site (=tumor) pain.
Almost all patients displayed lymphocytopenia and some showed
minor elevation of aspartate aminotransferase. A transient decrease in hemoglobin was also seen in many patients. If the patient had a grade symptom before treatment and it worsened
to grade Y, it was scored as grade Y (not Y-X). For example, the
hemoglobin of patient P297 was 100g/l before treatment and
79g/l the day after treatment and was thus scored as grade 3. A
decrease in hemoglobin has been consistently reported in patients
treated with oncolytic adenovirus25,26 and may be a general phenomenon related to virus treatment but not to binding of the virus to erythrocytes directly since this was not seen in our patients
(Supplementary Table S1). However, many cancer patients had
baseline anemia caused by the cancer and dilution of blood from
the 3,000ml of fluids the patients received on the treatment day
may also have contributed to hemoglobin readings. Few patients
reported also mild diarrhea. No correlation of adverse reactions
with e.g. previous serotype 5 treatments, virus dose, or route of
administration was found.
Patients first treated with Ad5 and then with Ad3 experienced
prolonged lymphocytopenia (P <0.05 between Ad5 pretreated
and naive patients), which was not seen in patients that received
Ad3 as a first treatment, nor was it seen when these Ad3-treated
patients received Ad5 as a second treatment (Figure1). In attempt
to dissect reasons for the lymphocytopenia we assessed antiviral
and antitumor T-cells in the blood of the patients (Figure 1b,c).
Before Ad3-hTERT-E1A treatment, we saw some T-cell activation
against serotype 3 hexon in Ad5 pretreated patients (median 15
Molecular Therapy vol. 20 no. 9 sep. 2012
spot-forming units), but none in naive patients, perhaps suggesting

some crossreactivity between T-cell epitopes.27 Interestingly, after
Ad3-hTERT-E1A treatment median antiviral T-cells in the Ad5
pretreated group remained stable (13 SPU) whereas T-cells in the
non-pretreated group showed an increase (median 79 spot-forming
units). On a patient level, there was variation in the former group
whereas all patients showed an increase in the latter. With regard
to TAAs, we used survivin and one other TAA mix (selected individually for each tumor on the basis of a literature search) for each
patient. While changes in the overall activity of T-cells were seen in
most patients, suggesting immunological activation, we could not
see any clear correlation with Ad5 pretreatment.
Ad3-hTERT-E1A was detected in blood for weeks (Table3).
Patients that received >1012 VP had a median of 5,800 VP/ml in
blood the following day and 8/9 patients were positive at 3 and
6 weeks. Patients that received <1012 VP had a median of <125
VP/ml in the blood the following day, but 2/6 patients were still
detected positive at 3 and 6 weeks. Roughly 10 times more virus
was usually detected from the clot, compared with serum. This
finding however displayed considerable variance and sometimes
even with the same patient more virus was detected from the
serum than from the clot. No clear correlation between pretreatment status, neutralizing antibody titer and the amount of virus in
blood could be seen.
Neutralizing antibody assay. Neutralizing antibody assay was
performed separately against serotype 3 and 5. Interestingly most
patients had Ad3 NAbs already before treatment (median 256)
suggesting previous natural Ad3wt infection. As expected, no significant difference was found between the serotype 5 pretreated
and non-pretreated patients with regard to baseline Ad3 NAbs.
In contrast, patients previously treated with Ad5 had higher Ad5
NAbs (P < 0.05). Proving complete lack of cross-neutralization,
median Ad5 NAb titer did not increase as a result of the Ad3hTERT-E1A treatment while the Ad3 NAb titer became a median
of 64-fold higher.
Blood samples of the first two patients treated with a low
dose (1010 VP) of Ad3-hTERT-E1A were taken at 10 minutes, 2,
6, and 20 hours (Supplementary Table S1) and different blood
compartments were analyzed. While most of the virus seemed to
be cleared from the blood in minutes, no virus was ever detected
in red blood cells. At 10-minute virus was detected in PBMC,
plasma and platelet compartments while later it was mainly seen
in PBMCs and plasma.
Objective evidence of possible antitumor activity (stable
disease or better in RECIST, positron emission tomography
(PET) criteria or tumor markers) were seen in 15 out of 23 (=
65%) evaluable patients. Most patients (22/25) received a serial
treatment (three different viruses at a 3-week interval followed
by imaging with the same method as done at baseline) and thus
were not radiological evaluable for Ad3-hTERT-E1A effect
only (Table4). Of the 16 patients that could be evaluated for
Ad3-hTERT-E1A only, 12 (75%) showed disease control. In 15
cases tumor markers were measured immediately before and
3 weeks after treatment while patient S171 was imaged with
computer tomography (CT) immediately before and 1 month
after the virus.
1823
3.0
lymphocytes x109/ml blood
2.5
2.0
Ad3-hTERT-E1A to treatment naive patients
Ad5 to Ad3-hTERT-E1A pretreated patients
40
40
20
20
Ad3-hTERT-E1A to Ad3 pretreated patient
1.5
60
1.0
0.5
140
90
40
60
V2
64
K3
04
P2
84
H
30
9
K2
53
R
2
M 18
ed
ia
n
K2
S1
71
T1
81
01
4
S1
19
H
30
5*
R
M 8
ed
ia
n
-10
Pretreated with serotype 5 virus
No previous virus treatments
0 39
0 39
0 39
0
60
T181
40
20
20
60
Pre Ad3-hTERT-E1A treatment

Post Ad3-hTERT-E1A treatment
S171
40
21
INF- (SFU/200,000 cells)
Ad3 hexon
INF- (SFU/200,000 cells)
1
Days after treatment
240
190
No previous virus treatments

60
Ad3-hTERT-E1A to Ad5 pretreated patients
0.0
Pretreated with serotype 5 virus

60
0 3
0 3
0 3
0
60
O14
40
40
20
20
0
60
0 33
0 33
0 33
0
60
S119
40
40
20
20
0
60
0 6
0 6
0 6
0
60
H305*
40
40
20
20
0
60
0 3
0 3
0 3
0
60
R8
40
40
20
20
0 3
0 3
0 3
TAA (Survivin)
TAA (SSX2, CEA or PSA)
Unstimulated
0
60
K260
0 4
0 4
0 4
0 3
0 3
0 3
0 3
0 3
0 3
0 3
0 3
0 10
0 10
0 5
0 5
V264
0 3
K304
0 3
P284
0 3
H309
0 3
K253
0 10
R218
40
20
0
0 5
Weeks after treatment
Figure 1 White blood cell changes in treated patients. (a) Ad5 pretreated patients get longer lasting lymphocytopenia after Ad3-hTERT-E1A treatment
P < 0.05. Please note that Ad3-hTERT-E1A to Ad3 pretreated patient consists from only one patient and thus little conclusions can be drown from this
patient. (b) Ad3 hexon enzyme-linked immunosorbent spot (ELISPOT) of peripheral blood mononuclear cells. Ad5 pretreated patients have lymphocytes
that are activated when stimulated by serotype 3 hexon [median 15 spot-forming units (SFU)], this is not seen with non-pretreated patients (median 2
SFU, P = 0.06). After Ad3-hTERT-E1A treatment the lymphocytes of the pretreated patients do not show big changes against serotype 3 hexon (median
13 SFU), whereas the non-pretreated patients show high activity (median 79 SFU, P = 0.10). (ab) A long-lasting lymphocytopenia is generated when
patients are primed by Ad5 and then treated by Ad3 but the immune reaction is not against serotype 3 hexon. Unstimulated background is subtracted.
(c)Tumor-associated antigen (TAA) ELISPOT of peripheral blood mononuclear cells. Clear change in lymphocyte behavior is seen after Ad3-hTERT-E1A
treatment. No clear correlation with the pretreated or non-pretreated is seen. No prestimulation or clonal expansion of PBMCs was done in these assays
and thus the results indicate the actual frequency of these cells in blood. Survivin was analyzed from all patients. SSX2 was analyzed from S171, T181,
O14, S119, H309. CEA from H305, K260, V264, K253, K304, R218, R8, and PSA from P284. Unstimulated is shown. Bars mean + SD.
11 out of 15 patients (= 73%) analyzed for tumor markers

had signs of antitumor activity. Two complete responses (mCR)
were noted via normalization of CEA and CA15-3 in patients
R217 and R263, respectively. Patient K260 had a high CEA level of
854 before treatment and was after treatment graded as a partial
response (mPR) due to a drop to 460. CA12-5 of R218 had previously been elevated and changes had correlated with treatment
efficacy. However, before treatment with Ad3-hTERT-E1A, her
CA12-5 was within normal limits but decreased further (15>10)
1824
and this was therefore scored as a partial marker response (mPR).

H309 was graded as minor response (mMR) in CA19-9. Patients
O14, R8, R277, K236, P297 were graded stable disease (mSD) and
rest as progressive disease (mPD).
Correlation between objective signs of antitumor activity and
survival. If we focus on patients that survived <100 days (N =4),
all evaluable cases had progressive disease. In contrast, all patients that survived over 300 days (N = 8) showed objective signs
Table 3 Virus amounts in blood and neutralizing antibody titers.

Patient
Ad5
pretreated Virus dose qPCR
K152
Yes
K214
S171
Yes
Yes
T181
Yes
O14
Yes
S212
Yes
R217
R8
Yes
Yes
S119
Yes
N227
Yes
Pretreated
median
V264
K260
No
Day 1
3 weeks
6 weeks NAbs
Titer
change
Day 0
3 weeks
Ad3
1,024
16,384
Ad5
4,096
1,024
-1
Low
Serum
e10
Clot
<250
Low
Serum
e10
Clot
Low
Serum
e10
Clot
Low
Serum
Ad3
1,024
4,096
e11
Clot
Ad5
4,096
16,384
Low
Serum
e11
Clot
Low
Serum
6,400
e11
Clot
8,300
Low
Serum
<250
<250
Ad3
16
e11
Clot
400
Ad5
4,096
4,096
High
Serum
1,100
Ad3
4,096
16,384
e12
Clot
<250
Ad5
16,384
16,384
High
Serum
Ad3
1,024
16,384
e12
Clot
309
Ad5
1,024
1,024
High
Serum
600
Ad3
16,384
256
<250
0
0
11,900
63,000
1200
e12
Clot
Low
Serum
Clot
<250
High
Serum
e11
Clot
High
Serum
1,100
e12
Clot
10,900
High
Serum
1,900
6,600
e11
No
Day 0
534,900
250
7400
Ad5
1,024
Ad3
1,024
16,384
Ad5
4,096
2,560
<250
Ad3
256
16,384
5,800
<250
Ad5
64
64
Ad3
1,024
Ad5
Ad3
256
16,384
500
400
Ad5
64
256
Ad3
256
16,384
280
P284
No
e12
Clot
H309
No
High
Serum
e12
Clot
260
<250
Ad5
H305
Noa
High
Serum
200
7,800
900
Ad3
4,096a
16,384
e12
Clot
200
561,000
600
400
Ad5
High
Serum
5,700
e12
Clot
19,200
High
Serum
4,600
400
Ad3
16
4,096
e12
Clot
3,700
<250
Ad5
16
High
Serum
<250
Ad3
64
4,096
K301
K304
K253
No
No
No
e12
Clot
49,000
Ad5
1,024
1 024
Nonpretreated
median
High
Serum
1,900
Ad3
160
16,384
e12
Clot
10,900
260
<250
Ad5
16
Median all
patients
High
Serum
675
Ad3
256
16,384
e12
Clot
5,800
<250
250
Ad5
1,024
256
Abbreviations: NAb, neutralizing antibody titer; qPCR, quantitative PCR.

a
Pretreated with Ad3-hTERT-E1A 3 weeks before. bPatient 3 years old.
1825
Table 4 Patients treated with Ad3-hTERT-E1A

Ad5 treated
before
baselineimaging
RECIST/PET Markers 3
criteria, after weeks after
Survival days
treatmenta
treatment
post-treatment
Dose (VP)
%
given i.v.
K152
37 (M)
NSCLC
Yes
1 1010
33
PD
135
K214
70 (F)
NSCLC
Yes
1 1010
50
PMDe
124
S171
67 (M)
MFH sarcoma
Yes
5 1010
20
SDf
449
T181
60 (M)
Thyroidea ca.
Yes
11
1 10
25
SD
O14
60 (F)
Ovario ca.
Yes
1 1011
20
PDe
S212
15 (F)
Rhabdomyosarcoma
Yes
1 1011
25
PMD
R217
48 (F)
Breast ca.
Yes
3 1011
75
PMD
S241
36 (F)
MFH sarcoma
Yes
1 1012
50
PMDe
R8
63 (F)
Breast ca.
Yes
2 1012
20
SD
S119
19 (M)
Sarcoma
Yes
12
3 10
100
SMD
N227
3 (F)
Neuroblastoma
Yes
7 1011
100
Responsec
R277
43 (F)
Breast ca.
Yes
4 1012
40
R218
48 (F)
Breast ca.t
No
5 1011
100
CMRd
H233
61 (F)
Pancreatic ca.
No
7 1011
67
PMDe
K236
69 (F)
NSCLC
No
11
7 10
50
SMD
mSD
295
K260
66 (F)
NSCLC
No
2 1012
20
PMDe
mPR
108
P284
73 (M)
Prostate ca.
No
2 1012
100
mPD
221
K253
55 (M)
NSCLC
No
12
2 10
100
P297
60 (M)
Prostate ca.
No
3 1012
100
SD
mSD
Alive 366
R262
67 (F)
Breast ca.t
No
3 1012
100
CMRd
mCR
Alive 310
V264
70 (M)
Bladder ca.
No
4 1012
20
MMR
mPD
107
12
PMD
mMR
ID
Age (sex)
Tumor type
WHO
402
mSD
360
51
mCR
181
104
mSD
255
Alive 442
mMR
208
mSD
138
mPRn
Alive 630
92
Alive 436
H309
68 (M)
Pancreatic ca.
No
4 10
20
K301
58 (M)
NSCLC
No
4 1012
67
K304
67 (F)
NSCLC
No
4 1012
80
PMD
mPD
112
H305
58 (M)
Pancreatic ca.
Nob
4 1012
65
PMD
mPD
76
Disease control 15
CR/CMR 3d
mCR 2
N/A 2
PR/PMR 0
mPR 2
No objective sign of
response 8
MR/MMR 1
mMR 2
SD/SMD 6
mSD 5
N/A 5
N/A 10
139
51
PD/PMD5+6e mPD 4
Abbreviations: CR/CMR/mCR, complete response/complete metabolic response/complete marker response; MR/MMR/mMR, minor response/minor metabolic
response/minor marker response, 1229% reduction; PR/PMR/mPR, Partial response/partial metabolic response/partial marker response, >30% reduction; N/A, not
assessable; NSCLC, nonsmall-cell lung carcinoma; i.v., intravenous; VP virus particles; PD/PMD/mPD, progressive disease/progressive metabolic disease/progressive
disease in tumor markers >30% increase; SD/SMD/mSD, stable disease/stable metabolic disease/stable disease with tumor markers.
a
Mostly not evaluative for Ad3-hTERT-E1A only due to serial treatment; other adenovirus treatments 3 weeks before and/or after. bPretreated with Ad3-hTERT-E1A.
c
Post-treatment bone marrow aspirate and biopsy tumor free, scored as CR on the summary line. dCR continues, i.e., patient had measurable metastases earlier but
was at CMR already prior to Ad3-hTERT-E1A. However, lack of progression could be a sign of antitumor activity. eSome sign of efficacy (e.g., some tumors responded
but progression in other sites) was seen in 6 of the 11 progressing patients. fCT immediately before and 1 month after Ad3-hTERT-E1A; not a serial treatment. nCA12-5
had previously been elevated and changes had correlated with treatment efficacy. Therefore, even though it was within normal limits before Ad3-hTERT-E1A the 30%
decrease (15>10) seen is compatible with antitumor efficacy. tTrastuzumab (Herceptin) treatment ongoing.
of antitumor activity in efficacy evaluations. Obviously larger

patient numbers are needed to reliably study possible surrogate
endpoints. Nevertheless, another tantalizing finding is that 5 out
of 6 evaluable patients that received 100% of the virus through the
intravenous route featured disease control.
only the marker responders (N = 11, mSD or better) into account

the median survival is 255 days and for nonresponders (N = 4) it
is 110 days, (P < 0.05). No clear correlation between WHO, pretreatment status, virus dose, route of administration, and the patient responses or survival was observed.
Median survival. Median survival for patients that had any objective sign of antitumor activity (N = 15) is 295 days while for
nonresponders (N = 8) it is 108 days (P < 0.001). When taking
Patient examples
All five breast cancer patients showed objective evidence of possible anticancer activity (markers: mCR 2, mPR 1, mSD 2, Table4).
1826
b
S 171
Week before Ad3-hTERT-E1A treatment
Tumor volume= 314cm3
1.0
Overall survival
0.8
Censored
0.6
0.4
0.2
0.0
0
200
400
600
Time (days)
Month after Ad3-hTERT-E1A treatment
Tumor volume=219cm3= 30% reduction
4 months after Ad3-hTERT-E1A treatment

Tumor volume=175cm3= 44% reduction
Figure 2Example of imaging result and survival graph of patients. (a) Sarcoma patient that was treated with a single dose of Ad3-hTERT-E1A and
followed by CT. (b) Cumulative survival of Ad3-hTERT-E1A-treated patients.
No other tumor types yielded such provocative data but the number of patients was smaller for many of them. Two patients who
received concurrent trastuzumab showed a drop in tumor markers (CEA 11>5 and CA12-5 15>10, and the former was graded
as mCR) after the administration of the Ad3-hTERT-E1A virus.
This was the first virus treatment these patients had ever received
and both had progressed on trastuzumab previously. Both patients
received a fully intravenous high dose of Ad3-hTERT-E1A and
experienced prolonged survival (both alive at 310 and 630 days,
respectively). Of all of the 25 patients treated with Ad3-hTERTE1A, only these two received trastuzumab as a concominant therapy so no conclusions can be drawn but the data is compatible
with an additive effect between Ad3 and trastuzumab as proposed

in preclinical studies.15
For patient S171 long-term radiological follow up was possible without confounding from other treatments. The patient had
metastatic malignant fibrous histocytoma and had been heavily
pretreated with multiple operations and chemotherapeutics, but
the disease was progressing before virus therapy. He was initially treated with five Ad5-based treatments over 6 months but
CT scans suggested progression. He was then treated with a single round of Ad3-hTERT-E1A and CT scans were taken before,
1 month and 4 months after the Ad3-hTERT-E1A treatment
(Figure2). Reduction of the largest injected tumor was seen and
1827
the overall situation was stable according to RECIST. At 4 months,

the injected tumors were still stable (N = 5, 0.4%) but noninjected tumors had grown and progressive disease was diagnosed.
Patient N227 showed high blood virus titers for up to 6weeks
suggesting emphatic replication. The patient was a 3-year-old
female with neuroblastoma. Before virus treatment, she had
received seven rounds of chemotherapy and radiation therapy but
the NSE tumor marker was rising and bone marrow immunofluorescence showed more malignant cells than before. Progressive
disease was therefore present and she was treated first with an
Ad5-based oncolytic adenovirus. After administration of Ad3hTERT-E1A (her second oncolytic virus), high-virus titer was
found in blood on day 1 (Table3). On day 3, 310,000 VP/ml from
serum and 261,000 VP/ml from blood clot were detected suggesting replication of the virus. Virus was detected at 3 weeks and
even at 6 weeks 600 and 7,400 VP/ml were present in the serum
and clot, respectively. At 6 weeks bone marrow aspirate immunofluorescence and biopsy were performed and found free of malignant cells, in contrast to the samples taken before oncolytic virus
treatment. Tumor marker NSEprogressing prior to therapy
decreased from 2521 in 3 weeks which was graded as mMR.
Patient H305 is a 58-year-old male with pancreatic cancer
who had been operated a year before and multiple lines of chemotherapy had been tried. He received Ad3-hTERT-E1A as a first
and also as a second virus treatment three weeks later. Low but
persistent virus was still seen in the serum before the second treatment. Before the third treatment [Ad5-based Icovir-7 (ref. 28)] no
Ad3-hTERT-E1A could be detected from serum but 3 and 19 days
later Ad3-hTERT-E1A re-emerged with titers of 210 and 900 VP/
ml, respectively (Supplementary Figure S4).
Ad3-hTERT-E1A increased in six out of nine evaluable patients
in serum and/or clot after Ad5 treatment (K304, H305, N227,
O14, S171, H309). Motivated by this finding we also assessed if
Ad3-hTERT-E1A treatment would result in re-emergence of serotype 5 virus into blood. Interestingly, five out of the seven evaluable patients previously treated with an Ad5-based virus showed
reappearance of the Ad5 virus to the clot and/or serum after Ad3hTERT-E1A treatment (K214, S212, R217, S119, N227).
Discussion
In biodistribution assays in mice at 6 hours Ad3 was still present

in blood. High amounts of proinflammatory cytokines were seen
suggesting that serotype 3 is potent in activating T-lymphocytes
(RANTES, interleukin-6) and macrophages (tumor necrosis
factor-, interleukin-6). Activation of multiple proinflammatory
cytokines has been suggested to correlate with cytolytic function
and in vivo activity.29,30 Whether serotype 3 truly is more immunogenic in mice or whether the higher amount of cytokines is due to
the prolonged presence in blood due to lack of high-avidity entry
receptors, resulting in eventual uptake by e.g., macrophages, are
interesting subjects for further study.
According to the murine toxicology results at 72 hours, serotype 3 seems to be safer than serotype 5 or 5/3 viruses. When comparing Ad5/3delta24 and Ad5/3-hTERT-E1A, which are identical
except for the E1 region, the hTERT promoter seems to protect
the liver from damage, perhaps through downregulation of E1A
expression in nontarget tissues, as the selectivity of delta-24 type
1828
viruses occurs post-E1A-expression. This finding has relevance

for Ad3-hTERT-E1 as the same promoter and virus design are
used. Taking the mouse cytokine and toxicity data together with
our previous data14 and adding the finding that Ad3,16 in contrary
to Ad5 (ref. 31) and Ad5/3 (ref. 32), does not seem to bind into
erythrocytes in mice (Supplementary Figure S1) or in patients
(Supplementary Table S1) it seems that Ad5/3 resembles more
Ad5 than Ad3 from the point of view of toxicity in mice and binding to blood cells.
In oncolytic virus-treated patients, blood lymphocytes tend to
drop to one-third of the pretreatment value in 1 day (Figure1 and
previous reports2526,33). One explanation for this is that these lymphocytes traffic to lymph nodes, spleen and to the tumor where the
virus is injected. Thus, lymphocytopenia in blood could reflect
redistribution of cells to tissues where they are needed for mounting an immune response, and could in fact constitute a surrogate for
immunological activity, as reported in mice or in humans treated
with another immunotherapeutic.3436 When measuring lymphocytes
3 weeks later they are normally back to pretreatment values2526,33 but
this was not the case with Ad3-hTERT-E1A-treated patients that had
received previous Ad5 treatments (Figure1a). These patients had
some T-cells reactive against Ad3-hTERT-E1A already before treatment, but the median amount in blood did not change after the treatment (Figure1b). When antitumor T-cells were analyzed, changes
compatible with trafficking to tumor or clonal replication were seen
in many patients. Taken together, these finding raise the possibility
that priming advanced cancer patients with Ad5 viruses and then
boosting with Ad3 leads to immunological changes sufficiently
prominent to be seen even in blood lymphocyte counts and that the
effect is not only against the Ad3 virus (Figure1ac). However, the
right place to assess this would be the tumor, not the blood, which is
important to keep in mind for future clinical approaches.
We did not see a trend between an increase in antitumor T-cells
and treatment benefits. This may relate to (i) the blood being the
wrong place to look at antitumor T-cells, (ii) us not knowing what
the most relevant epitope is for each patient, (iii) the lack of surrogate endpoints for treatment benefits (the biggest impact of
immunotherapy will ultimately be on survival but a randomized
setting would be needed to measure this. Tumor size is not a good
endpoint due to inflammatory swelling), (iv) a possible disconnect between induction of antitumor immunity and clinical benefit, perhaps mediated by immunosuppression, as proposed in the
cancer vaccine field.37
Preclinical studies have indicated that despite the presence of
viral DNA in cancer cells, in vivo cell lysis and antitumor effect
may be disabled. This phenomenon is not well understood but
proposals implicate induction of antiviral interferon pathways38 or
intratumoral antibodies.39 These phenomena might relate also to
attenuation of cell-to-cell spread of the virus. Therefore, reactivation of virus residing dormant in tumors could result in therapeutic effects. In six out of nine patients Ad3-hTERT-E1A increased
in serum and/or clot after Ad5 treatment. This suggests reactivation of replication in cancer cells or destruction of cancer cells
containing Ad3 viruses or possibly both. In theory, Ad3which
is only 67% similar to Ad5 on the genomic level40might have
mechanisms for counteracting resistance mechanisms resulting in reactivation of the oncolytic effect. Alternatively, the high
local concentration of virus resulting from intratumoral injection

could overwhelm antiviral mechanisms. To investigate whether
this finding was specific for Ad3 we analyzed Ad5 quantitative
PCR before and after Ad3-hTERT-E1A treatment in patients who
had previously been treated with an Ad5 virus and made the same
observation suggesting a general phenomenon.
For effective transduction of distant metastases extended seroprevalance might be beneficial. If the dose was over 1012 VP ample
virus was usually detected in blood on the following day and it often
persisted for at least 6 weeks. No correlation between dose and
adverse reactions was seen. As dose-limiting toxicity was not seen
either, even higher doses than 4 1012 VP might be considered.
Seven patients were safely treated by an intravenous injection (without intratumoral delivery) of Ad3-hTERT-E1A. Six
were evaluable to efficacy and five of them showed signs of possible benefit. If this is confirmed in larger patient populations,
one explanation could be that a large virus dose can overwhelm
NAbs and other antiviral immunity. After treatment the NAb
titer however increased exponentially and such response might
compromise further systemic treatments with the same virus
unless the virus is able to e.g., hitchhike on cells to avoid NAbs.32
Alternatively, if opsonization by NAbs is a dynamic process, some
virus might nevertheless have access to target receptors despite
high NAb titers. When treating intratumorally NAbs are probably
not an issue, but changing the serotype might nevertheless give an
additive boost to the immune system (Figure1).
Roughly two thirds of the patients show some kind of objective
evidence of possible antitumor activity. Trying to adopt RECIST or
PET response criteria to oncolytic virus treatments likely underestimates the actual antitumor effect because of inflammation and
tumor swelling, resulting in false diagnosis of tumor progression.41
With regard to tumor markers, virus-induced S-phase can increase
marker peptide production.42 Following lysis, these proteins are
released into blood, and therefore the amount of marker peptide in
blood might no longer correlate with the number of viable tumor
cells. Also increased metabolism due to virus infection might cause
stronger PET signals, especially as activated lymphocytes preferentially use glucose for fuel, and PET imaging is based on uptake of
labeled glucose. However, these evaluation criteria may not be completely useless as tumor control with these methods was associated
with a survival benefit in our patient series.
In summary, Ad3-hTERT-E1A seems safe for treatment of
cancer patients and therefore further clinical development seems
indicated. Of note, the patients reported here were treated in a personalized therapy scheme and thus it would be important to confirm
and expand the data in trials featuring more homogeneous patient
populations. Immunologically the virus differs from serotype 5
viruses and changing the serotype might reactivate replication of the
original treatment virus. Also, the ability of Ad3 to use desmoglein 2,
found abundantly in advanced tumors, as a primary entry receptor,
could be a benefit over CAR binding viruses. Combining serotype 3
viruses with trastuzumab, anti-EGFR or CAR dependent viruses is
appealing due to the ability of Ad3 to open intercellular tight junctions. To further enhance the immunological effects of the virus arming with immunostimulatory molecules such as GM-CSF or CD40L
might be beneficial. The apparently good safety of Ad3-hTERT-E1A
provides a good starting point for development of such viruses.
Materials and Methods
Treatment protocol. Twenty-five cancer patients with advanced solid
tumors, progressing after routine therapies, were treated. Patients with

other severe diseases, brain tumors, severe thrombocytopenia (< lower
normal limit) or clearly elevated liver enzymes (>3 upper normal limit)
were not treated. All patients signed written informed consent. Ad3-hTERTE1A14 was manufactured by Oncos Therapeutics (Helsinki, Finland). It
was administered intravenously and usually also in ultrasound guidance
in a personalized scheme (Table4). Time-lapse dose escalation from 1010
VP was utilized to maximize patient safety but minimize delays in enrolling new patients at potentially more effective higher doses. Dose could be
escalated when sufficient time (typically 2 weeks) had lapsed (and relevant
safety information collected) from the treatment of the first patient at that
dose. Most patients were treated in a serial mannerthree treatments with
different viruses, every three weeks and many were also treated with concomitant low-dose cyclophosphamide43 and/or low-dose pulse temozolomide.44 This Advanced Therapy Access Program (ATAP) is regulated by
Finnish Medicines Agency FIMEA as determined by EC/1394/2007, Good
Clinical Practice and the Declaration of Helsinki. Side effects were graded
according to CTCAE v.3.0. Patients were monitored overnight at the clinic
and then as outpatients for 28d for adverse events. Survival and late adverse
events were followed ad infinitum.
Human interferon- enzyme-linked immunosorbent spot. PBMCs were
isolated from the blood by Percoll (Sigma, St Louis, MO) gradient. Cells
were frozen in CTL-CryoABCTM serum-free media (Cellular Technology,
Cleveland, OH). Enzyme-linked immunosorbent spot Pro was performed
as instructed in the manufacturers (Mabtech, Nacka strand, Sweden) protocol. 200,000 PBMCs per well was used. For adenovirus enzyme-linked
immunosorbent spot, cells were stimulated with HAdV-3 Hexon protein
(ProImmune, Oxford, UK). For tumor enzyme-linked immunosorbent
spot we used the following peptides: Survivin (BIRC5_PONAB), SSX2,
CEA or PSA (ProImmune). PBMCs of each patient were stimulated with
survivin and one rationally chosen peptide mix. No prestimulation or
clonal expansion of PBMCs was done in this assay and thus the results
indicate the actual frequency of these cells in blood.
NAb. A549 (ATCC, Manassas, VA) cells were seeded at 1 104 cells per
well on 96-well plates and cultured overnight in Dulbeccos modified Eagle

medium without fetal calf serum. Next day, human serum samples were
incubated at 56C for 90 minutes to inactivate complement, and a fourfold dilution series (1:11:16 384) was prepared in serum-free Dulbeccos
modified Eagle medium45. Ad5-luc8 and Ad3-luc45 was mixed with serum
dilutions and incubated at room temperature for 30 minutes. Thereafter,
cells in duplicates were infected with 100 VP per cell in 50l of mix, and
100l of growth medium with 10% fetal calf serum was added 1 hour later.
Twenty four hours postinfection, cells were lysed and luciferase activity
was measured with Luciferase Assay System (Promega, Madison, WI)
using TopCount luminometer (PerkinElmer, Waltham, MA). Luciferase
readings were plotted relative to gene transfer achieved with Ad5luc1 or
Ad3luc1 alone. The NAb titer was determined as the lowest degree of dilution that blocked gene transfer >80%.
Evaluation of antitumor efficacy. CT and/or PET imaging was done
before and after a serial treatment (three virus injections) or after a single
treatment (S171). RECIST1.1 criteria are: PR (>30% reduction in the sum
of tumor diameters), SD (no response/progression), PD (>20% increase25).
In addition MR was scored to indicate 1229% reduction. For tumor
markers the same percentages were used. For PET the following criteria
were used 25: PR (30% decrease in summed SUVmax, up to five lesions
counted, max. 2/organ), MR (1029% decrease in summed SUVmax), SD
(9 to 29% change in summed SUVmax), PD (30% increase in summed
SUVmax or 2cm PET positive new lesion, except local lymph nodes
whose signal might indicate immune reaction).
1829
The following experiments are described in Supplementary

Materials and Methods: Ad3-hTERT-EIA qPCR, biodistribution and
cytokine experiments, toxicity experiment and the Ad3-hTERT-E1A
biodistribution.
SUPPLEMENTARY MATERIAL
Figure S1. Ad3 biodistribution in mice.
Figure S2. Cytokines.
Figure S3. Ad3 toxicity in mice.
Figure S4. Reappearance of Ad3-hTERT-E1A in serum after Ad5
treatment.
Table S1. For the first two patients treated with Ad3-hTERT-E1A
blood samples were taken as indicated to get preliminary information
about the in vivo human biodistribution of the virus.
Table S2. Previous cancer treatments of the patients.
Materials and Methods.
ACKNOWLEDGMENTS
We thank the National Graduate School of Clinical Investigation
(CLIGS, University of Helsinki, Finland) for financial support. Minna
Oksanen, Sirkka-Liisa Holm, Gerd Bauerschmitz (CGTG, University of
Helsinki, Helsinki, Finland), Silvio Hemmi (University of Zurich, Zurich,
Switzerland), Maija Lappalainen (HUCH, Finland) for expert advice and
help and Ari Ristimki (Department of Pathology, HUCH, Finland) for
histological analysis. We thank Saila Eksym-Sillman, Marina Rosliakova
and other personnel at International Comprehensive Cancer Center
Docrates, and at Eira Hospital, Helsinki, Finland. Mikael von Euler (Oncos
Therapeutics) is thanked for critical commentary. Akseli Hemminki is K.
Albin Johansson Research Professor of the Foundation for the Finnish
Cancer Institute. O.H. is shareholder in Oncos Therapeutics. A.H. is
shareholder in and consultant for Oncos Therapeutics. The work has
been done in Helsinki, Finland.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Edukulla, R, Ramakrishna, E, Woller, N, Mundt, B, Knocke, S, Grlevik, E et al. (2009).

Antitumoral immune response by recruitment and expansion of dendritic cells in tumors
infected with telomerase-dependent oncolytic viruses. Cancer Res 69: 14481458.
Grlevik, E, Woller, N, Strver, N, Schache, P, Kloos, A, Manns, MP et al. (2010).
Selectivity of oncolytic viral replication prevents antiviral immune response and
toxicity, but does not improve antitumoral immunity. Mol Ther 18: 19721982.
Bernt, KM, Ni, S, Tieu, AT and Lieber, A (2005). Assessment of a combined,
adenovirus-mediated oncolytic and immunostimulatory tumor therapy. Cancer Res
65: 43434352.
Choi, KJ, Kim, JH, Lee, YS, Kim, J, Suh, BS, Kim, H et al. (2006). Concurrent delivery of
GM-CSF and B7-1 using an oncolytic adenovirus elicits potent antitumor effect. Gene
Ther 13: 10101020.
Hanahan, D and Weinberg, RA (2011). Hallmarks of cancer: the next generation. Cell
144: 646674.
Mellman, I, Coukos, G and Dranoff, G (2011). Cancer immunotherapy comes of age.
Nature 480: 480489.
Kanerva, A and Hemminki, A (2004). Modified adenoviruses for cancer gene therapy.
Int J Cancer 110: 475480.
Kanerva, A, Mikheeva, GV, Krasnykh, V, Coolidge, CJ, Lam, JT, Mahasreshti, PJ et
al. (2002). Targeting adenovirus to the serotype 3 receptor increases gene transfer
efficiency to ovarian cancer cells. Clin Cancer Res 8: 275280.
Kangasniemi, L, Kiviluoto, T, Kanerva, A, Raki, M, Ranki, T, Sarkioja, M et al. (2006).
Infectivity-enhanced adenoviruses deliver efficacy in clinical samples and orthotopic
models of disseminated gastric cancer. Clin Cancer Res 12: 31373144.
Tsuruta, Y, Pereboeva, L, Glasgow, JN, Rein, DT, Kawakami, Y, Alvarez, RD et al.
(2007). A mosaic fiber adenovirus serotype 5 vector containing reovirus sigma 1 and
adenovirus serotype 3 knob fibers increases transduction in an ovarian cancer ex vivo
system via a coxsackie and adenovirus receptor-independent pathway. Clin Cancer Res
13: 27772783.
Zheng, S, Ulasov, IV, Han, Y, Tyler, MA, Zhu, ZB and Lesniak, MS (2007). Fiber-knob
modifications enhance adenoviral tropism and gene transfer in malignant glioma.
JGene Med 9: 151160.
Kanerva, A, Wang, M, Bauerschmitz, GJ, Lam, JT, Desmond, RA, Bhoola, SM et al.
(2002). Gene transfer to ovarian cancer versus normal tissues with fiber-modified
adenoviruses. Mol Ther 5: 695704.
Hemminki, A, Wang, M, Desmond, RA, Strong, TV, Alvarez, RD and Curiel, DT (2002).
Serum and ascites neutralizing antibodies in ovarian cancer patients treated with
intraperitoneal adenoviral gene therapy. Hum Gene Ther 13: 15051514.
Hemminki, O, Bauerschmitz, G, Hemmi, S, Lavilla-Alonso, S, Diaconu, I, Guse, K et al.
(2011). Oncolytic adenovirus based on serotype 3. Cancer Gene Ther 18: 288296.
Wang, H, Li, ZY, Liu, Y, Persson, J, Beyer, I, Mller, T et al. (2011). Desmoglein 2 is a
receptor for adenovirus serotypes 3, 7, 11 and 14. Nat Med 17: 96104.
Wang, H, Li, Z, Yumul, R, Lara, S, Hemminki, A, Fender, P et al. (2011).
Multimerization of adenovirus serotype 3 fiber knob domains is required for efficient
1830
binding of virus to desmoglein 2 and subsequent opening of epithelial junctions.

JVirol 85: 63906402.
17. Fender, P, Boussaid, A, Mezin, P and Chroboczek, J (2005). Synthesis, cellular
localization, and quantification of penton-dodecahedron in serotype 3 adenovirusinfected cells. Virology 340: 167173.
18. Trinh, HV, Lesage, G, Chennamparampil, V, Vollenweider, B, Burckhardt, CJ, Schauer,
S et al. (2012). Avidity binding of human adenovirus serotypes 3 and 7 to the
membrane cofactor CD46 triggers infection. J Virol 86: 16231637.
19. Kim, KH, Ryan, MJ, Estep, JE, Miniard, BM, Rudge, TL, Peggins, JO et al. (2011). A
new generation of serotype chimeric infectivity-enhanced conditionally replicative
adenovirals: the safety profile of ad5/3-?24 in advance of a phase I clinical trial in
ovarian cancer patients. Hum Gene Ther 22: 821828.
20. Sarkioja, M, Kanerva, A, Salo, J, Kangasniemi, L, Eriksson, M, Raki, M et al. (2006).
Noninvasive imaging for evaluation of the systemic delivery of capsid-modified
adenoviruses in an orthotopic model of advanced lung cancer. Cancer 107:
15781588.
21. Ranki, T, Srkioja, M, Hakkarainen, T, von Smitten, K, Kanerva, A and Hemminki, A
(2007). Systemic efficacy of oncolytic adenoviruses in imagable orthotopic models of
hormone refractory metastatic breast cancer. Int J Cancer 121: 165174.
22. Brunetti-Pierri, N, Palmer, DJ, Beaudet, AL, Carey, KD, Finegold, M and Ng, P (2004).
Acute toxicity after high-dose systemic injection of helper-dependent adenoviral
vectors into nonhuman primates. Hum Gene Ther 15: 3546.
23. Morral, N, ONeal, WK, Rice, K, Leland, MM, Piedra, PA, Aguilar-Crdova, E et al.
(2002). Lethal toxicity, severe endothelial injury, and a threshold effect with high
doses of an adenoviral vector in baboons. Hum Gene Ther 13: 143154.
24. Cerullo, V, Seiler, MP, Mane, V, Brunetti-Pierri, N, Clarke, C, Bertin, TK et al. (2007).
Toll-like receptor 9 triggers an innate immune response to helper-dependent
adenoviral vectors. Mol Ther 15: 378385.
25. Pesonen, S, Diaconu, I, Cerullo, V, Escutenaire, S, Raki, M, Kangasniemi, L
etal.(2012). Integrin targeted oncolytic adenoviruses Ad5-D24-RGD and Ad5-RGDD24-GMCSF for treatment of patients with advanced chemotherapy refractory solid
tumors. Int J Cancer 130: 19371947.
26. Koski, A, Kangasniemi, L, Escutenaire, S, Pesonen, S, Cerullo, V, Diaconu, I etal.
(2010). Treatment of cancer patients with a serotype 5/3 chimeric oncolytic
adenovirus expressing GMCSF. Mol Ther 18: 18741884.
27. Leen, AM, Sili, U, Vanin, EF, Jewell, AM, Xie, W, Vignali, D et al. (2004). Conserved
CTL epitopes on the adenovirus hexon protein expand subgroup cross-reactive and
subgroup-specific CD8+ T cells. Blood 104: 24322440.
28. Nokisalmi, P, Pesonen, S, Escutenaire, S, Srkioja, M, Raki, M, Cerullo, V et al. (2010).
Oncolytic adenovirus ICOVIR-7 in patients with advanced and refractory solid tumors.
Clin Cancer Res 16: 30353043.
29. Kannanganat, S, Ibegbu, C, Chennareddi, L, Robinson, HL and Amara, RR (2007).
Multiple-cytokine-producing antiviral CD4 T cells are functionally superior to singlecytokine-producing cells. J Virol 81: 84688476.
30. Badr, G, Bdard, N, Abdel-Hakeem, MS, Trautmann, L, Willems, B, Villeneuve, JP et al.
(2008). Early interferon therapy for hepatitis C virus infection rescues polyfunctional,
long-lived CD8+ memory T cells. J Virol 82: 1001710031.
31. Carlisle, RC, Di, Y, Cerny, AM, Sonnen, AF, Sim, RB, Green, NK et al. (2009). Human
erythrocytes bind and inactivate type 5 adenovirus by presenting Coxsackie virusadenovirus receptor and complement receptor 1. Blood 113: 19091918.
32. Escutenaire, S, Cerullo, V, Diaconu, I, Ahtiainen, L, Hannuksela, P, Oksanen, M et
al. (2011). In vivo and in vitro distribution of type 5 and fiber-modified oncolytic
adenoviruses in human blood compartments. Ann Med 43: 151163.
33. Cerullo, V, Pesonen, S, Diaconu, I, Escutenaire, S, Arstila, PT, Ugolini, M et al. (2010).
Oncolytic adenovirus coding for granulocyte macrophage colony-stimulating factor
induces antitumoral immunity in cancer patients. Cancer Res 70: 42974309.
34. Kanerva, A. American Society of Gene and Cell Therapy. Philadelphia, 2012.
35. Chen, J, Zajac, AJ, McPherson, SA, Hsu, HC, Yang, P, Wu, Q et al. (2005). Primary
adenovirus-specific cytotoxic T lymphocyte response occurs after viral clearance and
liver enzyme elevation. Gene Ther 12: 10791088.
36. Brahmer, JR, Drake, CG, Wollner, I, Powderly, JD, Picus, J, Sharfman, WH et al. (2010).
Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory
solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates.
J Clin Oncol 28: 31673175.
37. Postow, M, Callahan, MK and Wolchok, JD (2011). Beyond cancer vaccines: a reason
for future optimism with immunomodulatory therapy. Cancer J 17: 372378.
38. Liikanen, I, Monsurr, V, Ahtiainen, L, Raki, M, Hakkarainen, T, Diaconu, I et al. (2011).
Induction of interferon pathways mediates in vivo resistance to oncolytic adenovirus.
Mol Ther 19: 18581866.
39. Dhar, D, Spencer, JF, Toth, K and Wold, WS (2009). Effect of preexisting immunity on
oncolytic adenovirus vector INGN 007 antitumor efficacy in immunocompetent and
immunosuppressed Syrian hamsters. J Virol 83: 21302139.
40. Sirena, D, Ruzsics, Z, Schaffner, W, Greber, UF and Hemmi, S (2005). The nucleotide
sequence and a first generation gene transfer vector of species B human adenovirus
serotype 3. Virology 343: 283298.
41. Reid, TR, Freeman, S, Post, L, McCormick, F and Sze, DY (2005). Effects of Onyx-015
among metastatic colorectal cancer patients that have failed prior treatment with
5-FU/leucovorin. Cancer Gene Ther 12: 673681.
42. Pesonen, S, Nokisalmi, P, Escutenaire, S, Srkioja, M, Raki, M, Cerullo, V et al. (2010).
Prolonged systemic circulation of chimeric oncolytic adenovirus Ad5/3-Cox2L-D24 in
patients with metastatic and refractory solid tumors. Gene Ther 17: 892904.
43. Cerullo, V, Diaconu, I, Kangasniemi, L, Rajecki, M, Escutenaire, S, Koski, A et al.
(2011). Immunological effects of low-dose cyclophosphamide in cancer patients
treated with oncolytic adenovirus. Mol Ther 19: 17371746.
44. Liikanen, IAL, Kangasniemi, L, Cerullo, V, Nokisalmi, P, Escutenaire, S, Diaconu, I et al.
(2011) Treatment of cancer with oncolytic adenovirus combined with temozolomide,
an autophagy inducer. Mol Ther.
45. Fleischli, C, Sirena, D, Lesage, G, Havenga, MJ, Cattaneo, R, Greber, UF et al. (2007).
Species B adenovirus serotypes 3, 7, 11 and 35 share similar binding sites on the
membrane cofactor protein CD46 receptor. J Gen Virol 88(Pt 11): 29252934.

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