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IJC
International Journal of Cancer
Serotype chimeric oncolytic adenovirus coding for GM-CSF for
treatment of sarcoma in rodents and humans
Simona Bramante1, Anniina Koski1, Anja Kipar2,3, Iulia Diaconu1, Ilkka Liikanen1, Otto Hemminki1, Lotta Vassilev4,
€m1,
Suvi Parviainen1, Vincenzo Cerullo1,5, Saila K Pesonen1, Minna Oksanen1, Raita Heiskanen4, Noora Rouvinen-Lagerstro
1
4
6
1,7
1
1,4,6
Maiju Merisalo-Soikkeli , Tiina Hakonen , Timo Joensuu , Anna Kanerva , Sari Pesonen and Akseli Hemminki
1
Cancer Gene Therapy Group, Department of Pathology and Transplantation Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland
Finnish Centre for Laboratory Animal Pathology, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland
3
School of Veterinary Science and Department of Infection Biology, Institute of Global Health,University of Liverpool, Liverpool, United Kingdom
4
Oncos Therapeutics Ltd., Helsinki, Finland
5
Laboratory of Immunovirotherapy, Division of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland
6
Docrates Cancer Center, Helsinki, Finland
7
Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Helsinki, Finland
2
Cancer Therapy
Sarcomas are a relatively rare cancer, but often incurable at the late metastatic stage. Oncolytic immunotherapy has gained
attention over the past years, and a wide range of oncolytic viruses have been delivered via intratumoral injection with positive safety and promising efficacy data. Here, we report preclinical and clinical results from treatment of sarcoma with oncolytic adenovirus Ad5/3-D24-GMCSF (CGTG-102). Ad5/3-D24-GMCSF is a serotype chimeric oncolytic adenovirus coding for
human granulocyte-macrophage colony-stimulating factor (GM-CSF). The efficacy of Ad5/3-D24-GMCSF was evaluated on a
panel of soft-tissue sarcoma (STS) cell lines and in two animal models. Sarcoma specific human data were also collected from
the Advanced Therapy Access Program (ATAP), in preparation for further clinical development. Efficacy was seen in both in
vitro and in vivo STS models. Fifteen patients with treatment-refractory STS (13/15) or primary bone sarcoma (2/15) were
treated in ATAP, and treatments appeared safe and well-tolerated. A total of 12 radiological RECIST response evaluations were
performed, and two cases of minor response, six cases of stable disease and four cases of progressive disease were detected
in patients progressing prior to virus treatment. Overall, the median survival time post treatment was 170 days. One patient
is still alive at 1,459 days post virus treatment. In summary, Ad5/3-D24-GMCSF appears promising for the treatment of
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advanced STS; a clinical trial for treatment of refractory injectable solid tumors including STS is ongoing.V
Sarcomas are malignant tumors of mesenchymal origin. In
humans, they are generally classified as either soft-tissue sarcomas (STS) or primary bone sarcomas. The staging and
treatment approach to both entities differs. Despite being
rare cancer types in general (about 1% of all adult cancers),
sarcomas are among the most frequent tumors of young
adults and constitute 15% of all cancers in children. Moreover, a significant proportion of patients develop metastatic
or inoperable disease that is often incurable with currently
available treatments and ultimately results in death.1,2
Oncolytic viruses are a promising treatment strategy
against cancer. Gene therapy vectors based on serotype 5
adenoviruses have been used most frequently in gene therapy
trials and a multitude of strategies have been pursued to further improve gene delivery and antitumor efficacy. Among
these, the Ad5/3 chimera generated through replacement of
the Ad5 fiber knob with the Ad3 knob has demonstrated
enhanced cancer cell transduction and antitumor efficacy in
preclinical testing.3,4 It has been proposed that this is associated with high expression of the putative receptor for
Key words: oncolytic adenovirus, cancer gene therapy, sarcoma, GM-CSF, animal models of cancer
Additional Supporting Information may be found in the online version of this article.
Conflict of interest: A.H. and O.H. are shareholders in Oncos Therapeutics, Ltd. S.P. is an employee and shareholder in Oncos Therapeutics,
Ltd. A.H. is shareholder and employee in TILT Biotherapeutics Ltd. The other authors declare no competing financial interest.
Grant sponsor: Helsinki Biomedical Graduate Program (HBGP); European Research Council; American Society of Clinical Oncology
(ASCO) Foundation; Helsinki University Central Hospital (HUCH) Research Funds (EVO); Sigrid Juselius Foundation; Academy of Finland;
Biocentrum Helsinki; Biocenter Finland; Cancer Organizations and University of Helsinki
DOI: 10.1002/ijc.28696
History: Received 25 June 2013; Accepted 13 Nov 2013; Online 24 Dec 2013
Correspondence to: A. Hemminki, MD, PhD, or A. Koski, MD, PhD, Cancer Gene Therapy Group, Department of Pathology and
Transplantation Laboratory, Haartman Institute, P.O. Box 21, 00014 University of Helsinki, Haartmaninkatu 3, 00290 Helsinki, Finland,
E-mail: akseli.hemminki@helsinki.fi or anniina.koski@helsinki.fi
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What’s new?
Oncolytic viruses are a promising treatment strategy against cancer. They can also be used as gene-therapy vectors, to further
stimulate the antitumor immune response. In this study, the authors evaluated an adenovirus carrying the gene for GM-CSF,
with positive results in animal models of soft-tissue sarcoma (STS). The study also demonstrated that the virus can spread to
non-injected tumors, suggesting that it might be useful for the treatment of metastatic disease. Furthermore, the treatment
was well-tolerated in human sarcoma patients, with evidence of antitumor efficacy, supporting further clinical trials.
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oncolytic herpes virus recently met its primary endpoint,
suggesting the approach can work in the context of many
oncolytic platforms.19 We have previously described the use
of a 5/3 capsid chimeric oncolytic adenovirus with a 24-basepair (bp) deletion in the E1A domain and armed with GMCSF (Ad5/3-D24-GMCSF) in preclinical models and cancer
patients.15,20 As demonstrated previously,3,21 the virus replicates selectively in cells with defects in the p16/retinoblastoma (Rb) pathway, which have been reported in most
human cancers.22 Good Laboratory Practices level biodistribution and toxicity studies have been performed in Syrian
hamsters (Oncos Therapeutics, unpublished). The present
study assessed the effect of Ad5/3-D24-GMCSF on sarcoma
cell lines and in animal models, and evaluated the available
sarcoma specific human data from the Advanced Therapy
Access Program.
Material and methods
Adenoviruses
Ad5luc1,6 Ad5/3luc1,6 Ad5lucRGD23 and Ad3CMV-luciferase24 are replication-deficient Ad vectors containing a firefly
luciferase transgene cassette in place of the deleted E1 region.
Ad5luc1 is based on human Ad serotype 5. Ad5/3luc1 contains a chimeric fiber with the tail and shaft domains of Ad
serotype 5 and the knob domain of serotype 3. Ad5lucRGD
contains an Arg-Gly-Asp (RGD) peptide in the HI loop of
the fiber knob domain. Ad3CMV-luciferase is based on
human Ad serotype 3. Ad5/3-D24 is a conditionally replicating adenovirus with a chimeric 5/3 fiber and a 24-bp deletion
in E1A.3 Ad5/3-D24-GMCSF is based on the same backbone,
but it has human GM-CSF inserted in the E3 region.15
Ad5wt is the wild-type Ad5 strain Ad300 from the American
Type Culture Collection (ATCC, Manassas, VA).
Cell lines
SK-LMS-1 human leiomyosarcoma, HT-1080 human fibrosarcoma, RD human rhabdomyosarcoma, and SW872 human
liposarcoma cell lines were obtained from the ATCC. Cells
were grown and maintained under the recommended conditions. The hamster ductus deferens leiomyosarcoma (DDT1MF2) cell line was kindly provided by Professor William S.
M. Wold (St. Louis University, School of Medicine, USA).
This cell line was cultured in 10% DMEM (Dulbecco’s Modified Eagle Medium) with 1% penicillin/streptomycin and 1%
L-glutamine.
Cancer Therapy
serotype 3 adenoviruses, desmoglein-2 (DSG-2),5 on tumor
cells.6,7 In contrast, the coxsackie-adenovirus receptor (CAR),
the proposed primary receptor for serotype 5 adenoviruses, is
expressed in human tumors at variable and often low levels.8
Accordingly, redirecting transduction to DSG-2 via 5/3 knob
chimerism can result in improved treatment efficacy.3,4
Previously, it was thought that the primary efficacy of
oncolytic viruses is based on their selective replication and
lytic effect on cancer cells. Nevertheless, preclinical and clinical evidence suggests that antitumor efficacy is partially mediated by the immune response induced by adenovirus
replication per se.9,10 However, since these immune reactions
are usually not sufficient for clinical responses, immunostimulatory molecules can be inserted into the genome of oncolytic adenoviruses to increase overall antitumor potency and
to stimulate the immune system against tumor cells.11
Granulocyte-macrophage colony-stimulating factor (GMCSF) is one of the most potent immunostimulatory molecules
and can induce antitumor immunity through direct recruitment of natural killer (NK) cells and stimulation of dendritic
cells (DCs), which can eventually result in a CD81 T-cell
attack on both infected and non-infected tumors.12 However,
the systemic use of recombinant GM-CSF is compromised by
toxic side effects caused by off-target activity, and the induction of potentially harmful myeloid-derived suppressor cells.
Further, its efficacy may remain limited by the low local concentration in tumors.13,14 In contrast, local production of
GM-CSF by cancer cells, as directed by an adenovirus, could
ensure both sufficient local target concentration and lower
systemic exposure, resulting in better efficacy and a more tolerable safety profile.15 In this scenario, tumor cell lysis mediated by oncolytic virus replication releases tumor-associated
antigens (TAAs) and increases “danger signals” at the tumor
site, while local production of GM-CSF recruits and activates
DCs. This leads to presentation of antigens to CD41 and
CD81 T-cells by DCs in the local lymph node, resulting in
an adaptive immune response.16 Therefore, the likely mechanism of action of this virus is threefold: (i) oncolysis of cancer cells; (ii) induction of antitumor immunity by the
recruitment and stimulation of DCs and (iii) recruitment of
NK cells to the tumor site.
Indeed, preclinical and clinical reports with GM-CSFencoding adenoviruses have indicated good tolerability and
promising antitumor efficacy.11,15,17,18 Moreover, a global
randomized phase 3 trial featuring a GM-CSF-coding
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In vitro transduction assays
Cells on 24-well plates were infected in triplicates at doses of
1, 10, 100 and 1,000 virus particles (VP)/cell for 2 h at 37 C
in 200 mL of growth medium (GM) supplemented with 2%
fetal bovine serum (FBS). Luciferase assay (E1501 Luciferase
Assay System, Promega, Madison, WI) was performed as
described previously.25
In vitro cytotoxicity assays
Cytotoxicity assays were performed as described earlier.15 For
DDT1-MF2 and SW872 cells, 100 mL of fresh 10% GM was
added to each well 24 h post-infection and plates were incubated at 37 C until cytotoxicity was measured. Cell viability
was measured using CellTiter 96 Aqueous One Solution Cell
Proliferation Assay (Promega) when a cytopathic effect was
observed in cells infected with the highest dilution of any of
the viruses.
Ad5/3-D24-GMCSF replication in hamster sarcoma cells
Hamster leiomyosarcoma DDT1-MF2 cells were plated at 1
3 104 cells/well on two 96-well plates and cultured overnight.
Cells in 10 replicates were infected with Ad5/3-D24-GMCSF
at 0.0001–1,000 VP/cell in 100 mL of GM supplemented with
2% FBS. Cytopathic effect (CPE) was followed for 12 days,
and the amount of infectious particles was calculated daily,
based on the standard TCID50 method.26
Cancer Therapy
Animal experiments
All animal protocols were reviewed and approved by the Experimental Animal Committee of the University of Helsinki and
the Provincial Government of Southern Finland. Three- to fourweek-old female nude/NMRI mice (Harlan, Indianapolis, IN)
were injected with 5 3 106 SK-LMS-1, HT-1080, RD or SW872
cells subcutaneously in both flanks. Viruses diluted in sodium
chloride (NaCl) were injected intratumorally at 7 3 109 VP/
tumor (2 tumors/mouse) on days 1, 4, 8 and 15. Male Syrian
hamsters (Mesocricetus auratus) were obtained from Harlan
(Indianapolis, IN) at 5–6 weeks of age, injected subcutaneously
at four different sites with 5 3 106 DDT1-MF2 cells/site and
randomized into 12 groups, which are described in Supporting
Information. Animals were anesthetized prior to any procedures
and the health status monitored daily. Tumor growth was followed by measuring width and height of the tumors and tumor
volumes were calculated using a formula of 0.5 3 length 3
(width)2. Animals were euthanized according to local animal
care rules when the tumors had exceeded the maximum acceptable size or when any signs of pain or distress were evident.
Histology and immunohistology
Eight hamsters (two animals per group) were treated intratumorally with a single injection of Ad5/3-D24-GMCSF or
Ad5/3-D24 at the high dose, three injections of Ad5/3-D24GMCSF at the low dose, or were mock-treated with NaCl.
Animals were euthanized 9 days after the first treatment and
Oncolytic virus coding for GM-CSF for treatment of sarcoma
necropsied immediately after death. All complete tumors
were collected. From mock-treated hamsters and those
treated with repeated low doses of Ad5/3-D24-GMCSF, samples from heart, lung, liver, spleen, kidney and brain were
also retrieved. All samples were fixed in 10% buffered formalin for 24 h, followed by storage in 70% EtOH and subsequent trimming and routine paraffin wax embedding.
Sections (3–5 mm) were prepared and stained with
hematoxylin-eosin (HE) for histological evaluation or were
used for immunohistology. Immunohistology for the presence
of T and B cells/plasma cells was performed on the tumor
specimens, using cross reacting antibodies against CD3 (rabbit anti-human CD3; Dako, Glostrup, Denmark) and CD79a
(rat anti-human CD79acg; Dako), respectively. The streptavidin peroxidase method with heat pretreatment (citrate buffer
pH 6.0) for antigen retrieval and diaminobenzidin as chromogen was applied, following previously published protocols.27
Viral DNA load in tumors, blood clots and serum of
hamsters
Approximately 25 mg of tumor tissue or blood clot was
digested overnight with proteinase K in tissue lysis buffer
ATL (Qiagen). For serum samples, 6 mL of poly(d)A carrier
DNA (Roche) was added to 200 mL of the diluted serum
sample (1:1 of serum and PBS). Total DNA was extracted
using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA)
and a QIAcube machine, according to the manufacturer’s
instructions. Quantitative PCR targeting the adenoviral E4
gene was performed as described earlier.11,15
Patients
A total of 15 patients with chemotherapy-refractory STS (13/15)
and primary bone sarcomas (2/15) were treated with Ad5/3D24-GMCSF in an Advanced Therapy Access Program, which
is under regulation of the Finnish medicines agency FIMEA as
determined by EC/1394/2007. The patients must have solid
tumors refractory to conventional therapies, progressive disease,
no major organ function deficiencies. The treatment was not
offered if patients had other severe disease or organ malfunction, as described previously.28 To confirm the understanding of
the experimental approach of oncolytical adenovirus treatment
instead of clinical trial, patients signed a written informed consent and treatments were administered according to Good Clinical Practice and the Declaration of Helsinki of World Medical
Association. Some data from these patients has previously been
published elsewhere, focusing on different aspect of the treatments.15,20,28,29 We report here new information regarding clinical responses, viral genomes and neutralizing antibody titers in
patient serum, focusing on sarcoma patients and Ad5/3-D24GMCSF treatments. Analysis of patient samples has been
approved by HUCH operative ethics committee.
Treatment protocol
Treatment protocols have been described previously15,20,28–31
and summarized in Supporting Information.
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Ad5/3luc1 was used to evaluate the effect of neutralizing antibodies in the serum of patients treated with Ad5/3-D24GMCSF, as described previously.11 DNA extraction and realtime PCR for determination of viral titers in patient serum
samples was performed as described previously.11,15
Statistical analysis
Statistics were done with Microsoft Office Excel 2007 and
SPSS v18.0 (SPSS, Chicago, IL). Two-tailed Student’s t tests
and Type III Tests of Fixed Effects on log transformed data
were used. p values of <0.05 were considered significant.
Kaplan–Meier analysis was used to process survival data.
Results
Chimeric 5/3 fiber increases transduction efficacy in
human, but not in hamster STS cell lines
We assessed the transduction of human and hamster STS cell
lines by adenoviruses with different capsid modifications.
Infection with Ad5/3luc1 resulted in the highest transgene
expression efficacy in all studied human STS cell lines (p <
0.05 vs. Ad5luc1 and Ad3CMV-luciferase). Transduction with
Ad5lucRGD virus, which features an integrin binding capsid
modification proposed useful for many tumor types,32 and
thus potentially attractive in the context of sarcoma, was
comparable to Ad5luc1 (Fig. 1a). Transduction of the hamster leiomyosarcoma cell line was most efficient with the
serotype 5 capsid (p < 0.001 vs. Ad5/3luc1 and Ad3CMVluciferase at 10 VP/cell) (Fig. 1b).
Ad5/3-D24-GMCSF exhibits potent oncolytic efficacy in
STS cell lines in vitro
We performed cytotoxicity assays to investigate the oncolytic
potency of Ad5/3-D24-GMCSF in human and hamster STS
cell lines. Keeping in mind that GM-CSF is not expected to
add to efficacy in vitro, it is promising that the cell-killing
efficacy of Ad5/3-D24-GMCSF was superior to that of Ad5wt
and comparable to that of Ad5/3-D24, a similar oncolytic
adenovirus without GM-CSF transgene, in all four human
STS cell lines (Fig. 1c). In the hamster leiomyosarcoma cell
line, the cell-killing efficacies of Ad5/3-D24-GMCSF and
Ad5/3-D24 were comparable, but Ad5wt was slightly more
effective (p 5 0.02 for 100 VP/cell), which is in accord with
the transduction data (Fig. 1d). To assess whether the hamster leiomyosarcoma cells are permissive to Ad5/3-D24GMCSF replication, a progressive infectivity assay was performed. Oncolysis developed over time, suggesting productive
replication (Supporting Information Fig. S1).
Ad5/3-D24-GMCSF exhibits high antitumor efficacy in a
STS xenograft mouse model
To evaluate the antitumor potency of Ad5/3-D24-GMCSF in
vivo, an efficacy study was performed in a STS human xenograft
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model in nude mice (Fig. 2). Because both SK-LMS-1 and
SW872 tumors regressed spontaneously and RD tumors failed
to grow in mice, the experiment was performed with HT-1080
fibrosarcoma cells. Treatment with either Ad5/3-D24 or Ad5/3D24-GMCSF was more effective than the mock injection (p <
0.01), and Ad5/3-D24-GMCSF was more effective than Ad5wt
(p < 0.05). There was no statistically significant difference
between Ad5/3-D24-GMCSF and Ad5/3-D24, which was
expected, as human GM-CSF is not active in mice.33
Multiple dosing with Ad5/3-D24-GMCSF is more effective
than a single injection in an immunocompetent Syrian
hamster STS model
To evaluate the potency of Ad5/3-D24-GMCSF in immunocompetent animals, a dose escalation efficacy study was performed in a hamster STS model. In this model, the potential
effects of the transgene can also be assessed, since human GMCSF has been shown to be functional in Syrian hamsters.11,34
However, a caveat is that the transduction obtained with Ad5/3
was low in semi-permissive hamster cancer cells, as compared
to human cells (Fig. 1b), which results in lower oncolytic
potency than would be expected for human tumors.15 A singleinjection treatment with a medium or high dose of Ad5/3-D24GMCSF resulted in significant tumor growth inhibition, as
compared to mock-treated animals (p < 0.05 and p < 0.01,
respectively) (Supporting Information Fig. S2a). There was no
statistically significant difference in tumor growth after injection of any viruses in a single-low dose (Supporting Information Fig. S2b). However, after injection with the medium and
high viral doses, treatment with oncolytic viruses, but not the
non-replicating control, resulted in slower tumor growth (p <
0.05) (Supporting Information Figs. S2c and S2d). Assessment
of tumor size did not provide evidence of enhanced antitumor
efficacy with the GM-CSF-expressing virus, regardless of the
viral dose (Supporting Information Figs. S2b–S2d). Tumor size
measurements, however, could be impacted by GM-CSFmediated inflammatory swelling.20 In contrast to the single
low-dose injection, serial treatment with a low dose of Ad5/3D24-GMCSF elicited a statistically significant antitumor effect
compared to mock treatment (p < 0.05) (Supporting Information Figs. S2e and S2f).
Intratumoral injection of Ad5/3-D24-GMCSF leads to
systemic viral spread and infection of uninjected tumors
One of the four tumors growing in each hamster was left uninjected. Interestingly, there was no difference in the size of
injected and uninjected tumors when Ad5/3-D24-GMCSF was
used (Fig. 3a). Although statistically non-significant, there was
a trend for more rapid growth of uninjected tumors in the Ad5/
3-D24 group compared to the Ad5/3-D24-GMCSF group,
which is compatible with GM-CSF-mediated systemic effects
(Fig. 3b). While immunological effects would by definition be
predicted to be systemic, we wanted to evaluate if virus dissemination and transduction of metastases also played a role. Quantitative PCR was performed and virus was found in both
Cancer Therapy
Neutralizing antibody titer and detection of virus in blood
samples
Cancer Therapy
Figure 1. Chimeric 5/3 fiber increases transduction of human but not hamster STS cell lines and Ad5/3-D24-GMCSF induces efficient cell
killing in vitro. (a) Human leiomyosarcoma (SK-LMS-1), fibrosarcoma (HT-1080), rhabdomyosarcoma (RD) and liposarcoma (SW872) cell
lines and (b) hamster leiomyosarcoma cells (DDT1-MF2) were analyzed for transduction by luciferase expressing replication-deficient
viruses. Cells were infected and luciferase expression was analyzed from cell lysates 24-h later, expressed as relative light units (RLU).
Each bar represents the mean of triplicates 1 SD. ***p < 0.001; **p < 0.01; *p < 0.05 for Ad5luc1 and Ad3CMV-luciferase (featuring a
fully Ad3 capsid) versus Ad5/3luc1. Transduction with Ad5lucRGD virus was comparable to Ad5luc1. (c) The same human sarcoma cell
lines and (d) hamster sarcoma cells were infected with wild type serotype 5 adenovirus (Ad5wt), a non-replicating chimeric virus (Ad5/
3luc1), Ad5/3-D24-GMCSF, or a chimeric oncolytic virus without GM-CSF (Ad5/3-D24). Cell viability was measured by MTS assays 4 days
after infection for SK-LMS-1 and HT-1080 cells, 14 and 17 days post-infection for RD and SW872 cells, respectively, and 8 days after infection for DDT1-MF2 cells. Cell viability is expressed relative to uninfected control cells as means 1 SD of triplicates. (c) ***p < 0.001; **p <
0.01; *p < 0.05 versus wild type serotype 5 adenovirus. (d) *p < 0.05 versus Ad5/3-D24-GMCSF.
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in a semi-quantitative manner (scores 1–6). Infiltrating Tcells were mainly seen disseminated throughout the tumors,
whereas B-cells and plasma cells were also frequently seen in
the tumor periphery. The differences between groups were
not significant, but there was a trend for more T-cells in
both injected and uninjected tumors of virus-treated animals;
this was highest in those that had received the repeated low
dose of Ad5/3-D24-GMSCF (Supporting Information Figs. S5
and S6).
Figure 2. Ad5/3-D24-GMCSF demonstrates potent antitumor efficacy in a STS xenograft mouse model. 12 nude/NMRI mice were
inoculated s.c. with human fibrosarcoma cells (HT-1080) and
randomized into four groups (six tumors/group). Tumors were
injected with 7 3 109 VP/tumor on days 1, 4, 8, 15 (indicated by
arrows). NaCl injection was used as mock treatment. Tumor sizes
are indicated as percentage respective to day 1, which was set as
100%. Data are presented as mean 1 SD. **p < 0.01; *p < 0.05.
injected and uninjected tumors, suggesting systemic dissemination and consequent delivery to metastases (Fig. 3c, Supporting
Information Fig. S3). Even when all groups were combined to
decrease the impact of biological variation, there was no difference between injected and uninjected tumors, while both
seemed to differ from tumors of mock-treated animals (not significant; Fig. 3d). To corroborate the relevance of the vascular
route, virus was also seen in blood clots and cell-free serum
(Supporting Information Fig. S4). Also, overall there was no
statistically significant difference in the size of uninjected
tumors that contained Ad5/3-D24-GMCSF DNA versus those
that did not contain viral genomes, which suggests that GMCSF produced by the adenovirus can stimulate distal immunological effects also without the presence of virus DNA, and that
overall systemic efficacy may result from both immunity and
dissemination of virus.
Intratumoral Ad5/3-D24-GMCSF injection does not lead to
significant pathological changes in organs or specific
histological features in the tumors
In an attempt to assess potential local or systemic pathological effects of the viruses, the tumors and relevant organs
(heart, lung, liver, spleen, kidney and brain) were subjected
to a histological examination. Apart from the neoplastic processes (described in more detail in Supporting Information),
neither the mock-treated hamsters nor those treated with
multiple injections of Ad5/3-D24-GMSCF exhibited any significant gross or histopathological changes in any of the
examined normal organs.
T-cells in tumors treated with Ad5/3-D24-GMSCF
Tumors were examined for the degree of T-cell (CD31) and
B cell/plasma cell (CD79a1) infiltration and scored blindly
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A total of 24 Ad5/3-D24-GMCSF treatments were given to
15 patients with advanced treatment refractory sarcoma in
the context of an Advanced Therapy Access Program
(ATAP) (Supporting Information Table S1). Treatments were
well-tolerated, with the most common adverse reactions
being grade 1–2 fever, hemoglobin decrease, fatigue, nausea,
pain and cough (Table 1). Grade 3 reactions were reported in
five patients, but none were classified as a SAE (serious
adverse event possibly related to the treatment and leading to
patient hospitalization, malformation or death). The majority
of grade 3 reactions were transient hematological disturbances, and there was one grade 3 fever and one grade 3 neuralgia. One patient (S281) had a grade 4 thrombocytopenia that
was classified as SAE, since she was hospitalized for a thrombocyte infusion. The hospitalization occurred 4 weeks after
the last Ad5/3-D24-GMCSF treatment and the patient had
also received radiotherapy and dexamethasone which may
also have affected the thrombocyte counts.
A transient decrease in lymphocyte numbers in the
peripheral blood was frequently observed: two patients had
lymphocytes counts between 1.3 and 0.8 3 109/L, five
patients between 0.8 and 0.5 3 109/L, six patients between
0.5 and 0.2 3 109/L and three patients below 0.2 3 109/L
following virus treatments. We speculate that lymphocyte
redistribution from blood to tissues may actually reflect
mechanistic issues instead of being classical “lymphopenia”
caused by bone marrow depression,35,36 but this remains to
be further evaluated. Furthermore, lymphocyte counts were
measured by automated machine counting, rather than manual microscopy, which may have affected the reliability of
the values. For these two reasons, the self-limiting and spontaneously recovering (in a few days) lymphocyte decrease in
the peripheral blood was not considered an adverse
reaction.20,29
Neutralizing antibody titer and presence of viral genomes
in the serum of patients after virus treatment
Prior to the treatment with Ad5/3-D24-GMCSF, 9/12 evaluable patients had detectable neutralizing antibody titers
against Ad5/3, often in the low to intermediate range. After
the treatment, the titer increased in 10/12 analyzed patients
(Table 2), and remained stable in the other two. Prolonged
presence of virus genome in the serum, and especially
increasing virus titers, have been suggested as a sign of virus
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Safety of Ad5/3-D24-GMCSF in sarcoma patients
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Oncolytic virus coding for GM-CSF for treatment of sarcoma
Figure 3. In Syrian hamsters, virus is able to transduce uninjected tumors via blood. Syrian hamsters were inoculated s.c. with hamster
leiomyosarcoma cells (DDT1-MF2) and treated intratumorally with a single low (2.1 3 108 VP/tumor), medium (2.1 3 109 VP/tumor) or
high dose (2.1 3 1010 VP/tumor) injection of (a) Ad5/3-D24-GMCSF or (b) Ad5/3-D24. One out of the four tumors per animal was left without any injection. Black columns represent injected tumors (low 1 medium 1 high dose grouped together, 45 tumors) 1 SE. White columns represent uninjected tumors (15 tumors) 1 SE. Tumor size (8 days after virus injection) of injected tumors was set to 100% and the
size of the uninjected tumors was calculated relative to this. (c,d) Tumors of four animals per group were collected and stored at 280 C.
Total DNA was extracted and viral DNA load was studied with quantitative PCR. Viral E4 copy numbers were normalized to genomic DNA
with GAPDH primers. Each icon represents one tumor; horizontal line indicates the median of the group. (c) n 5 12 uninjected and n 5 36
injected tumors/virus group, white icons and black icons respectively. Low, medium and high dose of each virus are grouped together. n 5
4 uninjected and n 5 12 injected tumors in case of multiple injections with Ad5/3-D24-GMCSF. (d) All treatment groups are combined. n 5
40 uninjected and n 5 120 injected tumors.
replication, as injected virus is rapidly cleared from the
bloodstream.37–40 Measurable viral DNA levels in the serum
were found in 10/12 patients at one day after Ad5/3-D24GMCSF treatment, and in 8/12 patients beyond day 2. In
two patients, the viral DNA load increased between day 1
and day 4. As observed also previously,15 there was no clear
correlation between neutralizing antibody titers and virus levels in the blood.
Efficacy of Ad5/3-D24-GMCSF in sarcoma patients
All patients had progressing disease refractory to conventional therapies prior to entering ATAP. The tumors of nine
patients were evaluable for treatment responses according to
RECIST 1.1.41
Three out of nine patients received two Ad5/3-D24GMCSF treatments and were evaluated after each treatment.
Thus, 12 evaluations were performed overall, and 2 minor
responses (MR), 6 stable diseases (SD) and 4 progressive
diseases (PD) were observed (Table 2). Endometrial sarcoma patient S149 showed a 27% decrease of one injected
lesion and a total of 15% reduction of both injected and
uninjected lesions, after a single treatment with Ad5/3-D24GMCSF.29 A subsequent serial treatment including Ad5/3D24-GMCSF resulted in SD, and the patient’s overall survival was 923 days from the first Ad5/3-D24-GMCSF
treatment.
Median survival was 170 days after the first Ad5/3-D24GMCSF treatment (Fig. 4) and there was one alveolar sarcoma patient still alive at the end of follow-up, 1,459 days
after receiving Ad5/3-D24-GMCSF. This is unusual in
patients with high grade soft-tissue sarcoma metastatic to the
lungs.
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Int. J. Cancer: 135, 720–730 (2014) V
727
Bramante et al.
Grade
1–2–3–4
Hematological
Anemia
5–3–1–0
Leukocytopenia
2–3–1–0
Thrombocytopenia
1–0–1–1
Constitutional symptoms
Fever
8–8–1–0
Chills
1–0–0–0
Fatigue
7–7-0–0
Edema
2–1–0–0
Dizziness, vertigo
1–1–0–0
Rigors
4–1–0–0
Flu like symptoms
1–0–0–0
Pain
Injection site
3–1–0–0
Abdominal
2–1–0–0
Tumor
0–1–0–0
Pleural
2–0–0–0
Hip
0–1–0–0
Head
2–1–0–0
Neuralgia
0–0–1–0
Others
2–7–0–0
Gastrointestinal system
Nausea
5–3–0–0
Vomiting
2–0–0–0
Anorexia
0–1–0–0
Loss of appetite
1–0–0–0
Constipation, obstruction
1–0–0–0
Respiratory system
Dyspnea
1–3–0–0
Cough
4–3–0–0
Sore throat
0–1–0–0
Dry mouth
1–0–0–0
Infection (bronchus)
0–1–0–0
Sinusitis
0–1–0–0
Hyperventilation
1–0–0–0
Metabolic/laboratory
ALT increased
2–0–0–0
AST increased
2–0–0–0
Hyperbilirubinemia
1–0–0–0
Hyponatremia
3–0–0–0
Other
Depression
0–1–0–0
Muscle weakness
0–1–0–0
Hypotension
1–0–0–0
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase. Numbers indicate the number of patient cases out of 15.
C 2013 UICC
Int. J. Cancer: 135, 720–730 (2014) V
We assessed the efficacy and safety of an Rb-p16 selective 5/
3 capsid chimeric oncolytic adenovirus armed with GM-CSF
in the treatment of sarcomas. Our in vitro experiments
demonstrate that human sarcoma cell lines are permissive
and susceptible to human adenoviruses and 5/3 chimerism
emerged as a favoured capsid configuration. In contrast, 5/3
chimerism did not enhance the transduction of hamster
leiomyosarcoma cells, which suggests that Syrian hamster
sarcoma cells express lower levels of the receptor(s) relevant
for Ad5/3 than human cells. At this point, it is not known
if hamster DSG-2 allows binding to the human Ad3 fiber.
As suggested by Hemmi et al.,42 rodent cells, including Chinese hamster ovarian (CHO) cells, do not support Ad3
binding and they do not stain with a DSG-2 antibody. However, it should be noted that Syrian and Chinese hamsters
are two different strains, and Ad5/3 only shares the fiber
knob with Ad3, and may be able to enter cells also through
other mechanisms, at least in vitro. It would be interesting
to study this further, but Syrian hamster reagents for these
assays are currently not available. Nevertheless, our data
suggests that Ad5/3-D24-GMCSF is able to enter both
human and hamster sarcoma cells, resulting in productive
replication and oncolysis (Fig. 1, Supporting Information
Fig. S1).
Xenograft models are widely employed in cancer research
since they allow the use of human cells. Therefore, we
employed a STS xenograft mouse model and confirmed that
the antitumor efficacy of Ad5/3-D24-GMCSF was equivalent
to the corresponding virus without an immunostimulatory
transgene. To extend these studies into an immunocompetent
model, where the GM-CSF-transgene product is also active,
also studies in Syrian hamsters were performed, despite the
concern that this animals may not be fully permissive to 5/3
chimeric viruses.
We have previously shown that human GM-CSF produced by oncolytic adenovirus is functional in hamsters and
that virus-produced GM-CSF induces tumor-specific immunity.11 In our current experiment, assessment of tumor size
did not indicate improved antitumor efficacy with GM-CSFexpressing virus compared to the control without GM-CSF.
In addition to tumor size measurements, which could be
impacted by GM-CSF-mediated inflammatory swelling
(“pseudo-progression”), survival data was also collected.
However, due to rapid tumor progression and frequent
necrosis, all hamsters had to be killed 9 days after the first
virus injection as required by the animal permit. Thus, the
data does not reflect the actual survival of the animals. Taken
together, our data indicate that this hamster model—the only
hamster sarcoma model currently available—is not optimal
for the evaluation of the overall oncolytic potency of Ad5/3D24-GMCSF, since the transduction was suboptimal, the
rapid tumor growth might not have allowed the oncolytic
potency of the virus to be fully effective. In particular, 9 days
is probably not enough for adaptive immunity to be induced
Cancer Therapy
Discussion
Table 1. Adverse reactions
728
Oncolytic virus coding for GM-CSF for treatment of sarcoma
Table 2. Clinical responses, viral genomes and neutralizing antibody titers in patient serum
Treatment
number
Patient
code
RECIST (or other sign
of treatment outcome)
Virus in blood (VP/mL), days
post-treatment
0
1–2
3–7
8–35
Neutralizing
antibody titer
0
1–2
weeks
Survival
(days)1
3–5
weeks
1
S67
NA, tumor softer
0
<500
<500
0
0
1,024
1,024
221
2
S70
SD (15.9%)
0
571
336,166
<500
0
0
4,096
77
3
S100
NA
0
0
<500
1,024
1,024
69 (118)
4
S119
SD (16.6%),
tumor softer
0
0
4,096
14592
SD (112%),
tumor softer
0
0
0
PD (137.6%)
0
<500
0
MR (215%)
0
<500
<500
5
6
S135
7
S149
SD (10.9%)3
8
9
S153
NA
10
S188
NA
0
256
0
64
0
1024
1,024
4,096
256
1,024
4,096
212 (275)
0
4,096
4,096
16,384
923 (951)
<500
1,024
1,024
51 (121)
4
4,096
56
0
500
<500
<500
3
11
S202
MR (210.3%)
12
S210
PD (137%)3,
symptoms: improv.
13
S212
PMD3
87
14–16
S216
NA3
108
17–19
S281
SMD (18.1%)3,
symptoms: improv.
20
NA
21
NA5, Symptoms: worse
0
21,621
16
1,0244
<500
256
4,096
277
469
16
16
170
4
1,024
645
1,0244
4,0966
<500
22
S282
NA
23
S171
PD (120.6%)3
0
<500
SD (20.4%)
0
0
24
3,723
<500
10
<500
0
1
Numbers in parenthesis indicate the survival “from the 1st viral treatment”. Otherwise “from the 1st Ad5/3-D24-GMCSF treatment.”
Patient alive at manuscript submission.
3
Serial treatment, response evaluation done after the third treatment.
4
Post-treatment day 1.
5
Magnetic Resonance Imaging: brain metastasis (brain had not been imaged before).
6
>5 weeks.
Abbreviations: PD 5 progressive disease, SD 5 stable disease, MR 5 minor response, PMD 5 progressive metabolic disease (in PET31), SMD 5 stable metabolic disease, NA 5 not available, improv 5 improvement.
Cancer Therapy
2
by GM-CSF. Nevertheless, as Syrian hamsters are semipermissive for productive replication of human adenovirus
serotype 5—although perhaps not true to the same degree for
the 5/3 chimera—they have been considered the best available immunocompetent animal model for oncolytic
adenoviruses.43
Productive replication of human adenoviruses is a complex process with multiple post-entry steps and perhaps some
of these steps are compromised in hamsters, as reported for
mice,44 despite our evidence suggesting in vitro transduction,
replication over time and killing of the hamster leiomyosarcoma cells with Ad5/3 viruses (Figs. 1b and 1d, Supporting
Information Fig. S1).
The use of GM-CSF for immunotherapy has been extensively discussed, given its dual effects on the immune sys-
tem. In mice, local administration of GM-CSF results in
local recruitment of activated DCs and stimulation of the
immune response.45 However, it has been proposed that
there is a “safety level” above which systemic GM-CSF
recruits myeloid suppressor cells that impair the generation
of tumor-specific immune responses.13 Importantly, these
unwanted effects of GM-CSF are mediated by the systemic
and not local concentration of this cytokine.13 The beneficial
effects of GM-CSF on antigen presenting cells might be
achieved with local production levels without increasing systemic concentrations to the threshold above which GM-CSF
results in immune suppression. To support local replicationlinked production of GM-CSF by Ad5/3-D24-GMCSF, we
have previously observed the absence of significant changes
in systemic levels of GM-CSF, following its intratumoral
C 2013 UICC
Int. J. Cancer: 135, 720–730 (2014) V
Figure 4. Overall survival of the patients after Ad5/3-D24-GMCSF
treatments. Kaplan–Meier analysis of the survival of patients after
first treatment with Ad5/3-D24-GMCSF. One patient was alive at
the end of follow-up (7th May 2013), with a survival of 1,459
days.
production in immunocompetent Syrian hamsters and in
cancer patients.15
Systemic effects would be useful for the treatment of
metastatic cancer. With regard to oncolytic viruses, these
could be achieved in two ways: through release of virus
progeny from injected tumors into the blood with subsequent transduction of metastases and/or through induction
of a systemic antitumor immune response. We observed
that the mean size of the uninjected hamster tumors was
not different from that of those injected with Ad5/3-D24GMCSF (in the same animal), indicating that the virus
treatments had systemic effects. Both Ad5/3-D24-GMCSF
and Ad5/3-D24 virus particles were also shown to spread
systemically and infect distant uninjected tumors, in the
presence of an intact immune system. Viral DNA was not
detected in all injected tumors and in some cases the
presence of virus in uninjected tumors was higher than in
injected tumors. This may be a consequence of fast elimination of virus-infected cells in injected tumors and/or a
less prominent antiviral response in uninjected tumors,
due to a smaller initial virus load. Nevertheless, we conclude that our data is compatible with dissemination of
intratumorally injected virus to distant tumors through
the blood stream. We did not observe differences in viral
copy numbers present in tumors between replicationcompetent viruses (Ad5/3-D24-GMCSF and Ad5/3-D24)
compared to the replication-deficient control virus (Ad5/
3luc1). However, virus copy number at a given time point
does not reflect past replication of virus, as cells allowing
replication die in the process. As Ad5/3luc1 is not
expected to cause oncolysis, it may be found in tissues
for longer periods than a replicating virus. Thus,
virus genomes found at 8 days may represent
daughter virions of virus that has replicated in the tumor
(oncolytic viruses) or inactive input viruses (non-replicating viruses).
C 2013 UICC
Int. J. Cancer: 135, 720–730 (2014) V
The presence of T-cells also in the tumors of untreated
animals might be a result of the non-syngeneic nature of the
model. Syrian hamsters are not inbred in the laboratory, and
thus growth of non-syngeneic tumors would logically be
expected to attract both effector and suppressive T-cells. The
fact that non-syngeneic xenografts can even be grown results
from an evolutionary bottle-neck effect, resulting in all Syrian hamsters being genetically similar.46,47 Moreover, it
should be noted that CD3 is pan-T-cell marker and therefore does not separate between effector and regulatory Tcells. Unfortunately, there are currently no reagents available
that would allow the evaluation of T-cell subsets in
hamsters.
Despite the aforementioned limitations inherent to the
animal model, the short duration of the experiment, and the
lack of significant difference due to biological variation, it is
noteworthy that there was evidence of pronounced T-cell
infiltration in treated tumors. Some tumors, and in particular
the low dose Ad5/3-D24-GMCSF-treated ones, exhibited heterophil (the hamster equivalent of neutrophils of other species) infiltration, mainly in association with granulation tissue
formation. It remains to be evaluated whether this could be a
local effect of GM-CSF which is known to recruit
neutrophils.48
A total of 15 patients with sarcoma refractory to other
forms of therapy were treated with Ad5/3-D24-GMCSF.
Treatments resulted in disease stabilization or tumor shrinkage in 8 out of 12 available radiological evaluations and
were generally well-tolerated. Although the heterogeneity of
the treatment regimen can be seen as an issue in ATAP,
which aims at patient benefit instead of rigorous scientific
data, it can also be seen as a strength in the sense that these
are real-life patients instead of a highly selected trial
population.49,50
Here, we reported preclinical in vitro and in vivo data
(from two different animals) and some preliminary human
experience. In summary, our results indicate that Ad5/3D24-GMCSF mediates efficacy in vitro and in animal models of STS. Moreover, the virus appears to be safe in sarcoma patients, with promising signs of possible antitumor
activity. These preliminary findings should be followed up
with clinical trials. Moreover, a randomized trial would be
required for demonstration of survival over controls. Thus,
it is exciting that a clinical trial featuring Ad5/3-D24GMCSF for treatment of refractory injectable solid tumors
including STS is ongoing, and randomized trials are in
planning.
Acknowledgements
The authors thank all current and previous technicians in the
Cancer Gene Therapy Group and the Finnish Centre of Laboratory Animal Pathology, Faculty of Veterinary Medicine,
University of Helsinki, for excellent technical support. They
thank Oncos Therapeutics Ltd. for expert assistance, and the
Docrates and Eira hospital personnel for help.
Cancer Therapy
729
Bramante et al.
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Oncolytic virus coding for GM-CSF for treatment of sarcoma
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