Download 9 Summary and Discussion

9
Summary and Discussion
Chapter 9
9.1
Summary
The goal of this thesis was to explore novel treatment modalities for high-grade
osteosarcoma. As described in chapter 1, osteosarcoma patients have an overall
5-year survival rate of 50-70% when treated with a combination of surgery and
multi-agent chemotherapy. Osteosaroma patients presenting with metastases
have long-term survival rates of 10-30%. Complete surgical removal of metastases
is essential, because hardly any patient survives without complete surgical
removal of their metastases. In spite of several clinical trials combining different
chemotherapeutics and some new treatment modalities the 5-year survival rate
has not increased over the last two decades. Clearly, new treatment modalities for
osteosarcoma are needed. That virotherapy could be of use for the treatment of
osteosaroma was supported by the finding that the intratumorally injected Ad5∆24RGD, designed to selectively replicate in tumor cells with disrupted pRbpathway, caused significant osteosaroma tumor growth regression in mice (1).
The work presented in this thesis evaluated experimental treatment modalities like
virotherapy and the small-molecule Nutlin for the treatment of osteosarcoma and
its lung metastases.
In chapter 2 we discuss a hurdle for effective virotherapy with conditionally
replicative adenoviruses (CRAds), i.e., adenovirus binding to osteosaroma cells.
This is hampered by low to absent levels of the native adenovirus receptor (CAR)
on osteosaroma cells. Although others have suggested the use of adenovirus with
native binding capacity for osteosaroma treatment, we advocate the use of
retargeted adenoviruses to guarantee efficient tumor cell infection. In this chapter
we show that a CRAd with expanded tropism towards integrins, broadly
expressed on primary osteosaroma tumor cells is more effective than virus with
native tropism.
It is to be expected that new treatment modalities such as adenoviral therapy will
be tested in the clinic in combination with conventional therapies. For
osteosaroma, doxorubicin and cisplatin are widely used chemotherapeutic agents.
Promising results have been obtained in preclinical experiments that
demonstrated synergy between adenoviral therapy and chemotherapeutic agents
like doxorubicin and cisplatin. In chapter 3 we show that combination of the Ad5∆24RGD with doxorubicin or cisplatin indeed resulted in synergistic cell kill on
osteosaroma cell lines. Strikingly, on osteosaroma primary cells such a synergistic
effect was not observed. In contrast, chemotherapeutic agents reduced CRAd
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mediated cell killing. Importantly, the virus did not reduce the effect of
chemotherapy. The adverse combination effect on primary osteosaroma cells was
linked to slow population-doubling time, reduced viral replication and absence of
chemotherapy induced G2 phase cell-cycle arrest. Based on these results we
propose to use chemotherapeutic agents and CRAd therapy in intermittent
administration schemes to obtain maximal anti-tumor effect.
Next to adenoviral cell entry, cell killing potency of the CRAd is of importance.
Clinical trials with adenoviral vectors for the treatment of different cancer types
and its metastases have indicated relative safety but modest clinical effect, urging
the development of more potent adenoviral therapy approaches (2-5). In chapter
4 we evaluated the effect of combining a small-molecule antagonist of MDM2
(Nutlin) with the p53-expressing adenoviral vector Adp53, with the CRAd Ad∆24,
or its p53-expressing derivative Ad∆24-p53 on cell killing properties and
adenoviral replication. Combination of Adp53 and Nutlin increased cell kill on
p53-wild type and p53-deficient tumor cells. Moreover, cell-killing properties of
CRAds increased up to 1,000-fold when combined with Nutlin. Enhanced cell kill
correlated with accelerated viral progeny burst. These findings suggest that
combination of oncolytic adenoviruses and Nutlin is promising to treat
osteosaroma.
As mentioned before, osteosaroma metastases (usually confined to the lungs)
predict a poor prognosis. This warrants a systemic approach. Therefore, in the last
4 chapters we focus on systemic delivery of adenoviral vectors and the limitations
encountered. As a proof of principle we injected, in, Ad5-∆24RGD intravenously
into mice bearing human osteosaroma lung metastases (chapter 5). It appeared
that repeated intravenous administration of this CRAd resulted in a significant
decrease in tumor mass and a reduced number of osteosaroma lung metastases.
However, lung metastases were not completely eradicated.
An approach to develop a more potent CRAd is via insertion of toxic transgenes
in the adenoviral genome. A key feature herein is that toxic transgene expression
should be confined to cancer cells, especially relevant for systemic administration
where adenovirus could also infect normal cells. Genes under control of the
major late promoter would theoretically be expressed only during viral replication.
Since CRAds exhibit cancer cell-specific replication properties, replication
dependent expression should be cancer cell-specific. To test this, we constructed
the CRAd Ad∆24.SA-Luc as shown in chapter 6. This CRAd carries a luciferase
gene regulated by the major late promoter through alternative splicing via an
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inserted splice-acceptor (SA) site. We observed high level and replicationdependent transgene expression in cancer cells. However, transgene expression
was also present, albeit at a much lower level, early in the adenoviral replication
cycle, suggesting that this approach would be more suited for transgenes
requiring high expression levels than for highly toxic transgenes.
Another way to protect normal tissues from systemic CRAd delivery is
detargeting, i.e., deleting native binding sites from the viral capsid. Ablation of
CAR and integrin binding sites reduces infection of non-targeted cells, thereby
possibly reducing toxicity and increasing the viral dose available for tumor cell
infection. Therefore, we constructed a CAR- and integrin-binding deficient CRAd
targeted towards the epidermal growth factor receptor (EGFR), which is
abundantly expressed on primary osteosaroma cells (chapter 7). This newly
developed CRAd showed a highly selective targeting profile for EGFR-positive
tumor cells. Ablation of CAR and integrin binding-sites resulted in decreased
infection of human liver as assessed on ex-vivo human liver slices. However,
human liver cells were found to express EGFR. Therefore, EGFR-targeted CRAds
appear not more suitable for systemic administration than a native CRAd.
Importantly, our CRAd design allows relatively simple exchange of binding
specificity. Furthermore, development awaits identification of more stringent
cancer-specific targeting molecules.
The adenovirus type 5 capsid further carries a binding site for heparin sulphate
glycosaminoglycans (HSG). As reported in chapter 8 we constructed a new virus
with ablated tropism for CAR, integrins and HSG. To this end, the adenovirus fiber
was replaced by a chimeric molecule comprising the fiber tail domain and the
reovirus σ1 oligomerization domain. The integrin-binding motif in the penton base
was mutated and a polyhistidine tag was added as a model targeting moiety. In
comparison to the control virus, the new virus showed a 10,000-fold reduced
transduction of mouse liver cells in freshly isolated liver slices. Intravenous
administration of this virus resulted in a significant reduction in liver transduction
and increased blood persistence. This virus thus represents a prototype of a useful
adenovirus design for systemic delivery applications.
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Summary and Discussion
9.2
Discussion
The observation that naturally occurring viruses caused tumor regression
prompted research on virotherapy for cancer. Safety was a major issue and till the
discovery of recombinant DNA technology that allowed genetically engineering
of viruses, little progress was made. Clinial trials for oncolytic viral therapies are
predominantly performed with the DNA viruses: adenovirus, herpes simplex virus
type one and vaccinia virus; and RNA viruses: reovirus and Newcastle disease
virus (6). Advantages of adenovirus are: high achievable virus titers, relatively easy
genetic manipulation and a mild virulence pattern of wild-type adenovirus. That
safety features are a major concern for clinical applicability of adenoviral therapy
is illustrated by the death of a patient treated for ornithine transcarbamylase
deficiency with replication-defective adenovirus. Most likely his death resulted
from adenovirus induced massive cytokine release inducing disseminated
intravascular coagulation and multiorgan failure (7). The immunologic response to
adenoviruses might pose problems, but considerable safety data has accumulated
from several clinical trails.
The great challenge for treating osteosaroma patients is to cure them from
metastases. Especially for patients with non-operable osteosaroma recurrence (5year survival: 0%) new treatment options are needed. To be successful, the
anticancer agent should reach and kill all metastases and is therefore likely to be
administered systemically. Relatively few adenoviral particles administered this
way will reach the cells of interest because of physical barriers in the body and
nonspecific uptake by other cells. Conditionally replicative adenoviruses, in
contrast to non-replicating adenoviruses, are capable of amplifying the viral input
dose and are therefore promising for systemic treatment. The vast majority of
clinical trials for oncolytic adenoviral therapy tested the utility of ONYX-015 (8).
Although these trials showed relative safety of the adenoviral vector, minimal anticancer response was observed. Efficacy was increased by combination with
chemotherapeutic agents that resulted in objective tumor response in some cases
(9, 10). Failure to prove clinical effect challenged development of more potent
replicating adenoviral vectors. Recent data suggests that the Ad∆24 CRAd is more
potent than ONYX-015 making it appealing to test this CRAd in future clinical
trials (11, 12). It is likely that gene therapy will not be applied as a single
treatment, but will be combined with established therapies. Strikingly, our in vitro
experiments suggested that combination with cisplatin and doxorubicin is best
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not administered simultaneously. This in contrast to combination with Nutlin that
showed increased cell kill when combined. These approaches, sequential
administration of chemotherapy and virus or a combination of Nutlin and virus
should be explored in an in vivo setting to assess effectiveness. Combination
experiments might reveal if adenoviral tumor penetration and subsequent
infectivity is increased and the role of tumor matrix as a possible infection barrier
is decreased.
A concern for adenoviral osteosaroma treatment is the low to absent CAR
expression on osteosaroma cells making these cancer cells refractory to
adenovirus infection (13, 14). To infect osteosaroma cells and minimize infection
of normal cells, CRAds with altered tropism should be engineered. Several
strategies to retarget adenoviral vectors have been employed. Two different
approaches were used in this thesis. One has an integrated RGD-motif in the
adenoviral fiber knob and the other approach uses a bi-specific antibody
produced by the adenovirus itself to achieve tumor cell targeting. The bi-specific
antibody approach has the theoretical advantage of the versatility of this
technique to switch antibodies for different tumor types. However, this two-step
targeting procedure might be less effective in systemic administration
applications. Following injection into the blood stream relatively few viral particles
will infect tumor cells with subsequent low levels of antibody production. It
remains to be investigated if this will be significant to support intratumoral virus
spread. In this regard, adenoviruses with genetically modified capsid proteins
might prove to be more efficient.
Besides targeting viral particles to specific tumor receptors, ablation of native
receptor binding sites seems essential, because of the adenovirus promiscuous
tropism. More than 90% of systemically administered adenovirus transduces the
liver (15-18). Even following local delivery of adenoviral particles to solid tumors a
significant amount of virus is detected in the liver (19). CAR binding site ablation
did not substantially change biodistribution or hepatotoxicity of systemically
delivered adenovirus (18). Ablation of CAR as well as αv-integrin binding sites was
found necessary to reduce liver transduction (more than 700-fold) (15). Based on
these observations we constructed a CAR and integrin binding ablated CRAd with
tropism for the epidermal growth factor receptor (Ad∆24P*F*-425S11). In a pilot
experiment, we injected athymic nude mice intravenously with this CRAd with or
without precomplexation with the bispecific EGFR-specific antibody 425-S11 and
control mice with the parental CRAd Ad∆24-425S11 (that has native plus EGFR-
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Summary and Discussion
specific tropism) with or without 425-S11 precomplexation. Unfortunately, viral
genome copy numbers as analyzed by quantitative PCR analysis in mouse liver
did not differ between the four treatment groups (data not shown). Furthermore,
another study showed that CAR and αvβ3 and αvβ5 integrin expression does not
correlate with vector distribution after systemic adenovirus delivery (20). These
findings put the discussion of the pivotal role of receptor specific uptake via CAR
and integrins and the need for binding site ablation in a different perspective.
However, vectors with abolished tropism for CAR, integrins and heparin sulphate
glycosaminoglycans (the latter by replacing the fiber shaft domain with that
derived of Ad serotype 35) resulted in a spectacular 15,000-30,000-fold decrease
of liver transduction. Moreover, coagulation factors (factor VII, IX and X), protein
C and complement component C4BP have been shown to play an important role
for hepatocyte transduction with adenovirus serotype 5 (21, 22). These binding
affinities are likely to be located in the fiber knob domain as demonstrated for
coagulation factor IX. Pretreatment with the anticoagulant warfarin resulted in
diminished liver transduction rescued by adding the coagulation factor X (22). In
chapter 8 we report the construction of an adenoviral vector that lacks all known
binding sites. This vector was made by replacing the adenovirus fiber with a
chimeric molecule consisting of the fiber tail domain and the reovirus spike
trimerization domain and by mutating the penton base RGD motif. Removal of
the fiber knob eliminated putative binding sites for blood factors (VII, IX and X),
protein C and complement component C4BP. The new virus indeed exhibited
strongly reduced binding to liver cells and human erythrocytes. In addition, in vivo
biodistribution was improved although liver detargeting was only 6-fold.
Interestingly, transduction of the lungs by the new vector was undetectable.
Effective lung detargeting is an attractive feature for the treatment of osteosaroma
lung metastases. Future experiments should focus on retargeting the new
adenovirus to specific receptors on osteosaroma cells or tumor vasculature. In
general, the effect of abolishing native tropism of the adenovirus seems to hold
great promise but the question if this detargeting effect is maintained when
combined with adequate tumor retargeting is so far unanswered.
A different and complementing approach to improve adenovirus biodistribution
and reduce toxic side effects is based on diminishing non-specific (non-receptor
mediated) adenoviral uptake by liver and other organs. This will extend the halflife of the adenovirus in the bloodstream, resulting in more adenovirus available
for infecting target cells. Several approaches to overcome non-specific uptake of
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the adenoviruses have been tested, including pre-treatment with 6% hestastarch
and GdCl3 to deplete Kupffer cells. This resulted in prolonged bloodstream
persistence and (less than 10-fold) reduced uptake by liver and other organs (23,
24). Even without the above-described improvements, CRAds have achieved
systemic anticancer effects in animal models. For example, the CRAd Ad-OC-E1a,
carrying the essential adenoviral gene E1a under the osteocalcin promoter has
shown encouraging results for the treatment of osteosaroma lung metastases in
vivo (25). We found that infectivity-enhanced, but not native binding site ablated,
Ad5-∆24RGD was capable of inducing regression of osteosaroma primary
subcutaneous tumors and osteosaroma lung metastases in vivo (1) (osteosaroma
lung metastases; chapter 5 of this thesis). This CRAd is designed to selectively
replicate in cancer cells with dysfunctional pRb control. It has been reported that
this mutation still allows some replication in normal cells and tissues like
keratinocytes, normal brain explants and hepatocytes tested in vitro (26-28). A
recent report stated that intravenously delivered Ad5-∆24RGD in cotton rats
caused substantial hepatic toxicity at a dose of 1 x 1011 viral particles but not at 1
x 108 viral particles (29). Safety data in humans are not yet available. Recently, a
clinical trial treating ovarian cancer with this virus was started and a clinical trial
for the treatment of glioblastoma is planned for the near future (30). Safety data
on loco-regional administration from these studies will be important to better
predict feasibility of systemic or loco-regional delivery of Ad5-∆24RGD to treat
metastatic osteosaroma.
Systemic administration will be the most suited for diffuse metastasized disease.
However, the majority of osteosaroma metastases are located in the lungs. This
allows loco-regional application methods such as isolated lung perfusion and
aerosol inhalation. These routes of administration could allow high doses of
adenoviral vectors with presumed fewer side effects, because liver and other
organs are bypassed. If these administration routes are safe for lung and upper
airway tissues, in case of inhalation therapy, and show significant anti-tumor
effects awaits experimental verification. A potential disadvantage of inhalation
therapy is spread of the adenoviral vector to the environment via exhalation.
Furthermore it is speculated that via this inhalation technique genetically modified
adenoviral vectors might encounter wild-type adenoviruses. It is hypothesized that
this might lead to recombination of these modified adenoviruses with wild-type
adenoviruses.
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Summary and Discussion
All adjustments to the procedures and modifications of the virus to increase safety
should outweigh the potentially associated reduction in treatment efficacy.
Careful experimental evaluation is needed. However, these efforts should not
slow down the process of developing the current generation of promising CRAds
like Ad5-∆24RGD for clinical applicability.
Perspectives
We have shown that Ad5-∆24RGD is capable of inducing tumor regression of
primary subcutaneous OS tumors and pulmonary osteosaroma metastases in
preclinical models. The next step is to optimize the treatment of lung metastases
bearing mice with Ad5-∆24RGD with or without chemotherapy (in intermittant
administration schemes). Prolonged administration of intravenous Ad5-∆24RGD
with or without chemotherapy in a survival experiment will elucidate if this
treatment results in prolonged survival. If prolonged survival is observed one
might proceed by evaluating the treatment in a clinical trial. An advantage is that
by that time safety data of this particular virus will have been collected by two
clinical trials planned for the near future (30). One of these trials in planned in the
Netherlands. Legislation demands strict application rules to prevent genetically
modified agents to be released into the environment. When this hurdle has been
taken and application is regarded safe, other trials with replicating agents are
more likely to follow. A major requirement to succesfully initiate a clinical trial
with Ad5-∆24RGD for osteosaroma is collaboration with other institutes to reach
adequate numbers of osteosaroma patients. Most likely patients with (nonoperable) recurrent osteosarcoma, given their poor prognosis, are included first.
Besides trying to reach the clinic with Ad5-∆24RGD for osteosaroma metastases
preclinical testing should continue in order to develop more potent adenoviral
therapeutic regimens. So far, systemically administered CRAds to combat
metastastic cancer have shown modest local efficacy in recent clinical trials (3, 5,
10).
Enhanced delivery to tumor cells, in our case predominantly lung metastases, can
be achieved via detargeting strategies or different administration routes by simply
bypassing scavenger sites. Therefore isolated lung perfusion to increase
therapeutic efficacy is attractive and certainly to be tested in the context of
oncolytic viral therapy. Preclinical studies should reveal if lung tumor nodules are
efficiently killed alongside with limited toxicity to normal lung tissue. Ad5∆24RGD is the vector of choice because native ablated adenoviral vectors as
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shown in chapter 7 exhibited reduced cell killing properties without guaranteed
reduced toxicity. In case of diffuse metastatic disease where isolated lung
perfusion is not feasible, detargeting strategies could reveal their potential.
An exciting avenue that is worth exploring is to use certain cells types with
intrinsic tumor targeting properties as carriers for the virus. In elegant
experiments, antigen-specific T-cells have been used to deliver viruses encoding
cytokines or suicide genes to sites of metastasis (31). Recently, naïve T-cells have
been succeffully employed to target oncolytic vesicular stomatitis virus to lymph
nodes harboring metastatic cells (32). These methods are well suited for
metastatic disease and obviate the need for high titer injection of the virus in the
bloodstream. This warrants exploring whether T-cells can be used as “homing
devices” in the context of the treatment of osteosarcoma metastatic disease using
Ad5-∆24RGD.
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