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PRODUCER CELLS FOR REPLICATION SELECTIVE VIRUSES
IN THE TREATMENT OF MALIGNANCY
FIELD OF THE INVENTION
The field of the invention is treatment of malignancy using oncolytic
virus agents.
BACKGROUND OF THE INVENTION
Cancer remains one of the leading causes of morbidity and mortality of
humans worldwide. Although certain tumors remain localized at discrete
locations
within the body, at least during certain stages of their growth, other tumors
are
dispersed from their earliest stages or arise in tissues which line body
cavities or
organs. For example, epithelial cancers arise in tissues which line the lungs,
the
ovaries, the exterior of the body, and various body cavities. Epithelial
ovarian cancer
(EOC) is one such epithelial cancer.
Despite the aggressive surgical approaches and combination
chemotherapeutic regimens investigated over the past two decades, EOC remains
a
disease with a grim prognosis. For example, recent statistics indicate that
25,000 new
patients afflicted with EOC are diagnosed yearly in the U.S.; 15,000 deaths
occur there
from this disease yearly. Unfortunately, due to the lack of symptoms, the
majority of
patients afflicted with EOC are diagnosed at a late stage. In addition,
although 70% of
EOC patients initially respond to cisplatin-based chemotherapy, the majority
of these
patients relapse and develop chemotherapy-resistant disease. As a result, the
overall
five-year survival rate is approximately 20% for advanced-stage EOC.
Initially it was believed that anti-cancer gene therapy must involve use
of replication defective viruses to administer the desired transgene in order
to prevent
systemic spread of virus and its associated complications, toxicity, or both.
Therefore,
until recently much of gene therapy for malignant disease has centered on the
delivery
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of a therapeutic gene (i.e. thymidine kinase, cytosine deaminase, p53, and the
like) with
replication defective adenoviruses. This method has several potential
drawbacks.
First, because these vectors are replication defective, it is unclear whether
these viruses
are able to penetrate more than a few tumor cell layers. Second, introduction
of these
viruses into humans induces a strong anti-vectorlviral immune response,
leading to
very transient transgene expression.
HIV-1716 is a replication-competent herpes simplex virus type 1 which
has a 759-by deletion in both copies of the RLI gene which encodes for the
protein
ICP34.5, a major determinant of herpes pathogenicity. Viruses with this
mutation
exhibit drastically reduced neurovirulence. These viruses do not cause
encephalitis
when inoculated either intracerebrally or peripherally into a host. Moreover,
these
mutants replicate as well as their wild-type parental strain (e.g. 17+) in a
variety of
dividing cells lines, but replicate poorly in cells not undergoing mitosis.
These
characteristics make HSV-1716 and other RLI mutants attractive as vectors for
cancer
gene therapy.
Previous studies have demonstrated that RLl mutant herpesviruses like
HSV-1716, replicate well in established dividing human glioma cell lines, as
well as in
primary cell cultures derived from human biopsy material. Infection of these
cultures
result in cell death in the majority of cases. It is also believed that, in
some cell lines,
premature shut-off of host protein synthesis occurs in response to a lack of
expression
of ICP34.5. This has been designated "the double hit phenomenon." In vivo
studies
have also been encouraging. Similar studies have been made with HSV-1 strains
which
lack ribonuclease reductase activity and strains which are multiply-attenuated
(e.g. in
which genes encoding ICP34.5 and ribonuclease reductase are deleted).
Replication
selective adenovirus strains have also been studied. Several groups have shown
efficacy in both immunocompromised and immunocompetent mouse models of
intracranial malignancies. Further, it has been shown that HSV antigen
staining is
restricted to the tumor mass with no spread to adjacent normal tissue.
Similar studies have been performed in animal models of malignant
mesothelioma, a uniformly fatal neoplasia of the lining of the pleural cavity
which does
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not respond well to surgery, chemotherapy or radiation. Kucharczuk et al.
(1997,
Cancer Res. 57:466-471 ) demonstrated that several non-neuronally derived
human cell
lines support HSV-1716 growth in vitro. Further, their in vivo study was based
on a
well characterized intraperitoneal model of human malignant mesothelioma
involving
REN cells injected into SCID mice. Their results indicated reduced tumor
burden and
significantly prolonged survival after intraperitoneal injection of HSV-1716
in tumor-
bearing animals. Although malignant mesothelioma lends itself to study because
of its
location in the lining of the pleural cavity, there is interest in other, more
prevalent,
thoracic malignancies which have poor prognoses unless identified early. Other
malignancies in which morbidity is associated with localized disease include,
for
example, bronchoalveolar cell, bladder, endometrial, cervical, and ovarian
cancers.
Endothelial ovarian cancer (EOC), for example, remains localized
within the peritoneal cavity in a large proportion of patients, ultimately
causing local
morbidity and lethal complications. Because of its localized nature, EOC lends
itself to
intraperitoneal approaches of therapy. One such approach is gene therapy. Gene
therapy comprising either delivering the herpes simplex virus-1 thymidine
kinase
(HSVtk) suicide gene to diseased cells followed by administration of
ganciclovir to the
patient or delivering tumor suppressor genes and/or oncogenes to cells has
been tested
in experimental ovarian cancer models in vitro and in vivo (Tong et al., 1996,
Gynecol.
Oncol. 61:175-179; Behbakht et al., Am. J. Obstet. Gynecol. 175:1260-1265;
Deshane
et al., J. Clin. Invest. 96:2980-2989; Mujoo et al., Oncogene 12:1617-1623).
Sufficiently encouraging preclinical results were attained to justify
initiation of clinical
phase I trials (Link et al., 1996, Human Gene Ther.; Alvarez et al., 1997,
Human Gene
Ther. 8:597-613). However, results from a recent clinical trial using an
adenoviral
vector that comprised an HSVtk gene for treatment of mesothelioma localized in
the
pleural space indicated that adenoviral gene delivery is restricted to a few
superficial
cell layers and that treatment of larger three-dimensional tumors may be
inadequate
(Sterman et al., 1998, Human Gene Ther. 9:1083-1092).
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Replication-competent and replication-restricted viral agents provide a
feasible alternative for cancer therapy. Replication-restricted recombinant
attenuated
forms of herpes simplex virus-1 (HSV-1) represent one family of such agents
(Chambers et al., 1995, Proc. Natl. Acad. Sci. USA 92:1411-1415; Jia et al.,
1994, J.
Natl. Cancer Inst. 86:1209-121 S; Glorioso et al., 1995, Annu. Rev. Microbiol.
49:675-710; Kesari et al., 1995, Lab. Invest. 73:636-648; Kramm et al., 1997,
Hum.
Gene Ther. 8:2057-2068; Nilaver et al., 1995, Proc. Natl. Acad. Sci. USA
92:9829-9833). For example, HSV-1 mutants have been generated that harbor
alterations in genes such as thymidine kinase (tk) or ribonucleotide reductase
(RR) and
exhibit decreased viral replication in non-dividing neuronal cells. The
specificity of the
RR- mutants has been shown to be up to 1,000-fold higher for malignant rodent
cells
than endogenous neural cells (Boviatsis et al., 1994, Human Gene Ther. 5:183-
191).
Another series of HSV-1 mutants has been produced by making
alterations in both copies of the RLI gene, a diploid fragment of the HSV-1
genome
(Chambers et al., 1995, Proc. Natl. Acad. Sci. USA 92:1411-1415; Kramm et al.,
1997,
Hum. Gene Ther. 8:2057-2068; Mineta et al., 1994, Cancer Res. 54:3963-3966;
Pyles
et al., 1997, Human Gene Ther. 8:533-544; Randaao et al., 1995, Virology
211:94-101). Its product, the ICP34.5 protein, has been implicated in
neurovirulence
and is responsible for preventing apoptosis related to premature shut-off of
protein
synthesis in the infected host cells. ICP34.5-null HSV-1 mutants have been
shown to
replicate preferentially in tumor cells, causing a direct oncolytic effect,
but appear to
spare normal differentiated tissues (Randazzo et al., 1996, Virology 223:392-
395;
Brown et al., 1994, J. Gen. Virol. 75:3767-3686). These strains have been
successfully
used to reduce or cure tumors of the central nervous system (CNS) in
experimental
models (Chambers et al., 1995, Proc. Natl. Acad. Sci. USA ; Jia et al., 1994,
J. Natl.
Cancer Inst. 86:1209-1215; Kesari et al., 1995, Lab. Invest. 73:636-648; Kramm
et al.,
1997, Hum. Gene Ther. 8:2057-2068; Nilaver et al., 1995, Proc. Natl. Acad.
Sci. USA
92:9829-9833; Martuza et al., 1991, Science 252:854-856; Mineta et al., 1994,
Cancer
Res. 54:3963-3966; Mineta et al., 1995, Nature Med. 1:938-943; Boviatsis et
al., 1994,
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Human Gene Ther. 5:183-191; Pyles et al., 1997, Human Gene Ther. 8:533-544;
Randazzo et al., 1995, Virology 211:94-101; Andreansky et al., 1997, Cancer
Res.
57:1502-1509; Yazaki et al., 1995, Cancer Res. 55:4752-4756). Thus, the use of
these
vectors alone to kill tumor cells is accepted in the art.
The efficacy of HSV-1716, an ICP34.5 null mutant of HSV-1, for
reducing tumor burden and conferring survival advantage has been demonstrated
in an
intraperitoneal model of malignant mesothelioma in severe combined
immunodeficient
(SCID) mice (Kucharczuk et al., 1997, Cancer Res. 57:466-471). Moreover, these
studies suggested that extra-CNS administration of replication-restricted HSV-
1 is safe.
HSV-1716 administered intraperitoneally to SCID mice appeared to be completely
avirulent. In fact, there was no viral spread outside the tumors, as was
documented in
Kucharczuk et al. (1997, Cancer Res. 57:466-471) by immunohistochemistry and
polymerise chain reaction (PCR) analysis of multiple marine tissues, including
intraperitoneal and retroperitoneal organs as well as distant organs and the
brain. This
was not true for wild-type HSV-1, to which SCID mice were found to be
extremely
sensitive. In fact, intraperitoneal administration of wild-type HSV-1 to SCID
mice led
to rapid systemic spread of the virus and death of the animals within one
week.
Furthermore, administration of HSV-1716 to normal human skin in a marine
xenograft
model was accompanied by no toxicity, while administration of a wild-type HSV-
1 led
to rapid destruction of the xenograft (Randazzo et al., 1996, Virology 223:392-
395).
Use of replication-restricted HSV-1 for extra-CNS malignancies was
recently extended to other tumors. A ribonuclease reductase-deleted mutant was
used
in an experimental animal model of metastatic colorectal carcinoma of the
liver
(Carroll et al., 1996, Ann. Surg. 224:323-329). In separate experiments, a
replication-restricted ICP34.5 mutant was used to treat experimental
metastatic and
subcutaneous melanoma (Randazzo et al., 1995, Virology 211:94-101; Randazzo et
al.,
1997, J. Invest. Dermatol. 108:933-937). In addition, a multi-attenuated
mutant,
HSV-6207, was efficacious for treatment of breast cancer (Toda et al., 1998,
Human
Gene Ther. 9:2173-2185).
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Taken together, these studies demonstrate that use of various oncolytic
viruses to kill tumor cells is well accepted, even if prior art uses of such
oncolytic
vectors have been plagued with shortcomings such as low efficacy, low tissue
specificity, rapid clearing of oncolytic viruses, and inability to deliver a
sufficiently
high or prolonged doses of virus to the desired tumor tissue. The present
invention
includes producer cells and methods of using them that overcome the
shortcomings of
the prior art, thereby permitting efficacious delivery of oncolytic viruses to
tumor tissue
and effective treatment of cancers such as EOC.
BRIEF SUMMARY OF THE I1WENTION
The invention relates to a producer cell for administration to a subject
having tumor cells. The producer cell comprises an oncolytic virus which is
capable of
replicating in the producer cell. The producer cell, however, is incapable of
sustained
survival in the body of the subject. In one embodiment, the oncolytic virus is
cytotoxic
with respect to the producer cell in the body of the subject. In another
embodiment, the
producer cell is rendered incapable of sustained survival in the body of the
subject by
exposing the producer cell to a lethal dose of radiation. The lethal dose of
radiation
may be a dose which enhances the burst size of the producer cell (e.g. about 3
Gray).
In yet another embodiment, the producer cell is rendered incapable of
sustained
survival in the body of the subject by incorporating a suicide gene (e.g.
thymidine
kinase or cytosine deaminase) into the producer cell.
In one aspect of the invention, the producer cell exhibits binding affinity
for a tumor cell in the subject, such as an epithelial tumor cell (e.g. an
epithelial ovarian
cancer cell).
In another aspect of the invention, the oncolytic virus is capable of
replicating in a tumor cell of the subject. For example, the oncolytic virus
may be less
capable of replicating in a non-tumor cell of the subject than in the tumor
cell.
In yet another aspect, the oncolytic virus may incapable of replicating in
a non-tumor cell of the subject, or it may be incapable of replicating in any
cell of the
subject. Replication of the oncolytic virus may, for example, be under the
control of a
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tumor-associated transcriptional promoter such as the prostate specific
antigen
promoter or the tumor growth factor-(i promoter.
The producer cell may, for example, be selected from the group
consisting of a PA-1 cell, an REN cell, a PER.C6 cell a 293 cell, a melanoma
cell, a
glioma cell, and a teratocarcinoma cell. Preferably, the producer cell is a PA-
1 cell.
The oncolytic virus may, for example, be selected from the group
consisting of a herpes simplex virus-1, a herpes simplex virus-2, an
adenovirus, a
vesicular stomatitis virus, a Newcastle disease virus, and a vaccinia virus.
When the
oncolytic virus is a herpes simplex virus-1, it preferably does not express
fimctional
ICP34.5. Suitable herpes simplex virus-1 include, but are not limited to, HSV-
1716,
HSV-3410, HSV-3616, HSV-83616, HSV-847, HSV-6207, HSV-7020, HSV-
NVR10, HSV-G92A, HSV-3616-IL-4, and HSV-hrR3. Suitable herpes simplex virus-
2 include, but are not limited to, strain 2701, strain 2616, and strain 2604.
Suitable
adenoviruses include, but are not limited to, ONYX-15, Ad5d1520, Ad5d1312,
CN706,
Add1110, Addll 11, Add1118, and Add1004.
In another aspect, the producer cell further comprises a composition
selected from the group consisting of an immunomodulatory molecule, a
cytokine, a
targeting molecule, a cell growth receptor, an immunoglobulin which is
specific for the
tumor, a nucleic acid encoding an immunomodulatory molecule, a nucleic acid
encoding a cytokine, a nucleic acid encoding a targeting molecule, a nucleic
acid
encoding a cell growth receptor, and a nucleic acid encoding an immunoglobulin
which
is specific for the tumor.
The invention also relates to an anti-tumor agent comprising a
mammalian cell which comprises thymidine lcinase. The mammalian cell exhibits
binding affinity for a tumor cell in a human patient and is incapable of
sustained
survival in the body of the patient. When the mammalian cell is administered
to the
patient, the mammalian cell binds with a tumor cell in the patient. When
gancyclovir is
thereafter administered to the patient, the mammalian cell metabolizes
gancyclovir to
generate a cytotoxic metabolite which is provided to the tumor cell with which
the
mammalian cell has bound.
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The invention further relates to a method of killing tumor cells in a
mammal. This method comprises administering to the mammal a producer cell. The
producer cell comprising a oncolytic virus which is capable of replicating in
the
producer cell. The producer cell is incapable of sustained survival in the
body of the
mammal. The mammal may, for example, be a human afflicted with an epithelial
cancer or a human afflicted with an tumor.
The invention still further relates to use of a producer cell for
manufacture of a medicament for administration to a patient having tumor
cells. The
producer cell comprises a oncolytic virus which is capable of replicating in
the
producer cell. However, the producer cell is incapable of sustained survival
in the body
of the patient.
DETAILED DESCRIPTION
The invention relates to a producer cell for administration to a subject
(e.g. a human patient) having tumor cells. The producer cell comprises any of
a wide
variety of oncolytic viruses (e.g. the herpes simplex virus-1 mutant
designated HSV-
1716). The oncolytic virus is capable of replicating in the producer cell, and
may also
be capable of replicating in tumor cells in the subject. The producer cell is
not capable
of sustained survival in the body of the subject. Because the producer cell
supports
replication of the oncolytic virus, it may contain many (e.g. tens, hundreds,
or
thousands) of copies of the virus. When the producer cell is administered to a
subject,
the copies of the virus escape from the cell and are delivered (e.g. by fluid-
mediated
dispersion or by producer cell-to-tumor cell contact) to tumor cells in the
subject. Once
delivered to tumor cells in the subject, the oncolytic viruses kill the tumor
cells.
Delivery of oncolytic viruses using producer cells has advantages over
prior art direct injection methods of delivering such viruses to tumor cells.
For
example, because the virus is, in some embodiments, capable of replicating in
the
producer cell of the invention, the amount of virus which can be administered
in a
given volume of fluid can be greatly increased, since cells in which a virus
has
replicated may contain tens, hundreds, or even thousands of copies of the
virus. In
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addition, delivery of a virus within a producer cell may enable the virus to
elude the
subject's immune system, increasing the likelihood that the virus will reach
and kill a
tumor cell. Furthermore, if the producer cell used to deliver the oncolytic
virus exhibits
binding affinity for tumor cells in a subject, delivery of the virus using the
producer cell
will increase localization of the virus to the tumor cells in the patient.
This xnay be
particularly important for treatment of tumors that are not discretely
localized. Other
advantages of the producer cells and the methods of using them, as described
herein,
will be apparent to the skilled artisan in view of the present disclosure.
Aefinitions
As used herein, each of the following terms has the meaning associated
with it in this section.
The articles "a" and "an" are used herein to refer to one or to more than
one (i.e. to at least one) of the grammatical object of the article. By way of
example,
"an element" means one element or more than one element.
A "subject" is an animal, preferably a mammal such as a human.
A subject "has tumor cells" if the subject comprises or is suspected to
comprise tumor cells in any form (i.e. in the form of a solid tumor, a
dispersed tumor, a
metastatic tumor cell, or the like).
A tumor cell is "killed" if it is induced to lyse, if it is induced to undergo
apoptosis, or if it is rendered incapable of growing or dividing.
An "oncolytic virus" is any virus which is able to kill a tumor cell by
infecting the tumor cell.
A virus is "cytotoxic with respect to" a cell if the virus is able to kill the
cell after infecting the cell.
An "anti-tumor agent" is a composition of matter which, when applied
to a tumor cell, kills the tumor cell.
A "transcriptional promoter" is a nucleic acid which, when operably
linked with a second nucleic acid encoding a gene product such as an RNA or a
protein,
enables the gene product to be expressed in a cell by virtue of permitting an
RNA
polymerase enzyme to transcribe the second nucleic acid.
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By describing two polynucleotides as "operably linked" as used herein is
meant that a single-stranded or double-stranded nucleic acid moiety comprises
each of
the two polynucleotides and that the two polynucleotides are arranged within
the
nucleic acid moiety in such a manner that at least one of the two nucleic acid
sequences
is able to exert a physiological effect by which it is characterized upon the
other.
A "suicide gene" is a gene which, when expressed in a cell, induces lysis
or apoptosis of the cell or renders the cell incapable of growth or division.
Replication of a virus is "under the control of a promoter" if at least one
gene product required for replication for replication of the virus is operably
linked with
the promoter.
A cell "exhibits binding affinity" for a tumor cell if the cell binds to the
tumor cell with greater affnity than the affinity with which it binds to a non-
tumor cell.
As used herein, a "functional" biological molecule is a biological
molecule in a form in which it exhibits a property by which it is
characterized. A
functional enzyme, for example, is one which exhibits~the characteristic
catalytic
activity by which the enzyme is characterized.
An oncolytic virus is "replication-selective" if it is more capable of
replicating in an tumor cell of a subject than in a non-tumor cell of the
subject.
A goal of viral gene therapy for treatment of malignant disease has been
delivery of a therapeutic/suicide gene (e.g. thymidine kinase, cytosine
deaminase, p53,
etc.) to tumor cells in a subject. In many prior art methods, such genes are
delivered to
tumor cells using a replication defective virus, such as an adenovirus. Use of
replication competent or replication selective viruses for the treatment of
neoplastic
disease provides certain advantages. Such viruses deliver the desired gene to
a larger
percentage of tumor cells. Furthermore, replication of these viruses in vivo
may well be
oncolytic in their own right.
One group of viruses which are useful for gene therapy are replication
competent herpes simplex type 1 viruses (HSV-1) having deletions in the RLl
gene,
which encodes the protein designated ICP34.5. This protein is a major
determinant of
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herpes pathogenicity. Viruses having mutations in this gene exhibit reduced
neurovirulence 105 . For example, they do not cause encephalitis when
inoculated
either intracerebratly or peripherally. Moreover, these mutants replicate as
well as their
wild-type parental strain (e.g. 17+) in a variety of dividing cells lines, but
replicate
poorly in cells not undergoing mitosis. These characteristics make these HSV-1
mutant
viruses attractive for use as oncolytic viruses.
In an effort to increase the efficacy of such oncolytic vectors, the present
inventors have devised a virus delivery method whereby cells that are infected
with the
mutant herpesvirus serve as "producer cells" for an oncolytic virus. Use of
producer
cells to deliver oncolytic viruses in vivo provides several advantages. For
example,
rapidly dividing cell lines have the ability to replicate viruses very
e~ciently,
producing as many as about 6,000 copies of a virus per infected cell. The
producer
cells are, in effect, viral factories which can increase the effective dose of
virus
administered to the patient. Administering producer cells may protect, at
least initially,
the virus from neutralizing host immunity. Producer cell lines may be
engineered to
enhance tumor killing, for example, by selecting or designing producer cells
which
produce cytokines which enhance the oncolytic effects of the virus with which
they are
infected. Oncolytic gene therapy has, until now, focused on localized
malignancies
where tumors can be directly injected with vector (e.g. glioma) or where
vector can be
instilled into a discrete body cavity (e.g. the pleural cavity for malignant
mesothelioma). The producer cell of the invention may be used to treat both
localized
and diffuse or disseminated malignancies. In addition, the presence of
producer cells
may have a positive effect on tumor killing by inducing an immune response
against
the tumor cells.
The invention includes a producer cell for administration to a subject
having tumor cells. In one aspect, the producer cell comprises a suicide gene
(e.g.
thymidine kinase) and exhibits binding affinity for a tumor cell. When
administered to
a patient, the producer cell binds with a tumor cell in the subject, if one is
present.
Expression of the suicide gene, optionally coupled with administration to the
subject of
a substrate of an enzyme encoded by the suicide gene (e.g. ganciclovir when
the suicide
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gene is thymidine kinase) leads to death of the producer cell and, by virtue
of a
bystander effect, to cells located near the producer cell. Thus, if the
producer cell has
bound with a tumor cell in the subject, death of the producer cell induces
death of the
bound tumor cell.
S In an important aspect of the invention , the producer cell comprises an
oncolytic virus. The oncolytic virus is capable of replicating in the producer
cell, but
the producer cell is incapable of sustained survival in the body of the
patient. Thus,
according to this embodiment the producer cell makes many copies of the
oncolytic
virus, and releases them into the subject's body upon the death of the
producer cell. In
another embodiment, the oncolytic virus is not capable of replicating in the
producer
cell, but is nonetheless carried into and released within the body of the
subject by the
producer cell.
The producer cell of the invention is preferably one of many types of
cells which are known to exhibit binding af~mity for tumor cells, but it is
not necessary
that the producer cell exhibit such affinity. For example, the producer cell
may be a
PA-1 cell, an REN cell, a PER C6 cell, a 293 cell, a melanoma cell, a glioma
cell, or a
teratocarcinoma cell. Also preferably, the producer cell is obtained from an
animal of
the same species (and strain, if applicable) as the subject or from a cell
line derived
from such an animal. Preferably, the producer cell is well tolerated by the
subject (i.e.
is not rejected by the immune system of the subject) when the producer cell is
injected
into the patient. By way of example, acceptable producer cells for use in
human
patients include any human cell line which is derived from a human source and
which
does not induce hyperacute rejection when injected into the patient.
Alternatively
acceptable producer cells for use in humans include any cell line which has
been
2S engineered to not induce hyperacute rejection.
The producer cell is not capable of sustained survival in the subject's
body, by which is meant that the producer cell does not endure more than
several
months, several weeks, or several days in the body of the subject following
administration of the producer cell to the subject. Preferably, the producer
cell is not
capable of replicating in the subject's body. Numerous methods are known in
the art
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for rendering cells incapable of sustained survival in a subject, and any of
those
methods may be used to so render the producer cell of the invention. For
example, the
oncolytic virus of the invention may be selected such that it is cytotoxic
with respect to
the producer cell in the body of the subject. Thus, the virus kills not only
tumor cells in
the patient, but also the producer cells which are used to deliver the virus.
The
producer cell may also be rendered incapable of sustained survival in the body
of the
patient by exposing the producer cell to a lethal dose of radiation prior to
providing the
producer cell to the subject. For example, a radiation dose of 20 Gray will
kill nearly
all known cells which might be used as producer cells. Furthermore, it is
understood
that certain lethal doses of radiation enhance the burst size of the oncolytic
virus in the
producer cell. For example, it has been found that when the dose of radiation
is about 3
Gray, the burst size of the HSV-1 variant 6207 in PA-1 cells is enhanced,
relative to
non-irradiated cells of the same type. Furthermore, the producer cell may be
rendered
incapable of sustained survival in the body of the patient by incorporating a
suicide
gene (e.g. thymidine kinase or cytosine deaminase) into the producer cell.
Upon
expression of the suicide gene (optionally coupled with administration to the
subject of
a substrate of an enzyme encoded by the suicide gene, such as ganciclovir when
the
suicide gene is thymidine kinase), the producer cell is killed. Potential
undesirable
immune reactions may be minimized or avoided by using producer cells which are
incapable of sustained survival in the subject.
The producer cell of the invention preferably exhibits binding affinity
for a tumor cell in the patient. Use of such producer cells has a number of
benefits.
For example, binding of a producer cell to a tumor cell necessarily brings the
oncolytic
viruses) in or on the producer cell into close association with the tumor
cell, increasing
the likelihood that the virus will infect and kill the tumor cell.
Furthermore, it is known
that ax least certain viruses may be transmitted by cell-to-cell contact.
Thus, binding of
a producer cell and a tumor cell may enhance targeting of virus to the tumor
cell in this
manner as well. In addition, formation of a producer cell-tumor cell complex
may
generate or expose antigenic regions which can be recognized by the subject's
immune
system, leading to generation of an immune response against the tumor cells.
If the
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producer cells are engineered to contain, display, or express various immune
modulators, growth factors, or suicide genes, binding of the producer cell to
a tumor
cell in the subject localized the biological activity of these molecules to
the tumor site.
For example, the producer cell may comprising an immunomodulatory molecule, a
cytokine, a targeting molecule, a cell growth receptor, an immunoglobulin
which is
specific for the tumor, or a nucleic acid encoding one of these. The cell may
either
naturally comprise one of these molecules or be engineered to comprise the
molecule.
Preferably, the producer cell exhibits binding affinity for an epithelial
tumor cell, such as an epithelial ovarian cancer cell. Because epithelial
tumors (like
dispersed tumors such as various leukemias) are not necessarily present at a
single focal
site in the body of a subject, prior art gene therapy methods have experienced
difficulty
delivering the gene vector to all tumor sites. If the producer cell binds with
one of
these dispersed or widely spread tumor types, then the oncolytic virus of the
invention
will be delivered to the tumor cells wherever they are dispersed or spread.
The oncolytic virus of the invention may be substantially any virus
which is known to exhibit oncolytic activity and which is capable of
replicating in, or at
least being carried by, a producer cell of the invention without ablating the
oncolytic
activity of the virus. In one embodiment, the oncolytic virus of the invention
is also
able to replicate in a tumor cell of the patient, and is preferably less
capable of
replicating in a non-tumor cell of the patient than in a tumor cell of the
patient. For
example, the oncolytic virus may incapable of replicating in a non-tumor cell
of the
patient. Of course, so long as the virus is oncolytic, it may be incapable of
replicating
in any cell of the subject.
An oncolytic virus may be made replication-selective if replication of
the virus is placed under the control of a regulator of gene expression such
as, for
example, a minimal enhancer/promoter region derived from the 5'-flank of the
human
PSA gene (e.g. see Rodriguez et al., 1997, Cancer Res. 57:2559-2563). By way
of
example, the main transcriptional unit of an oncolytic virus may be placed
under
transcriptional control of the tumor growth factor-~3 (TGF-Vii) promoter by
operably
linking virus genes to the TGF-~i promoter. It is known that certain tumor
cells
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overexpress TGF-~3, relative to non tumor cells of the same type. Thus, an
oncolytic
virus wherein replication is subject to transcriptional control of the TGF-(3
promoter is
replication-selective, in that it is more capable of replicating in the
certain tumor cells
than in non-tumor cells of the same type. Similar replication-selective
oncolytic
viruses may be made using any regulator of gene expression which is la~own to
selectively cause overexpression in an affected cell. The replication-
selective oncolytic
virus may, for example, be an HSV strain in which a gene encoding ICP34.5 is
mutated. The oncolytic virus of the invention may also be one which exhibits
binding
affinity for a tumor cell of the subject.
The oncolytic virus of the invention may, for example, be a herpes
simplex virus-1, a herpes simplex virus-2, an adenovirus, a vesicular stout
atitis virus, a
Newcastle disease virus, or a vaccinia virus. Examples of such oncolytic
viruses are
described, for example, in Kirn (1999, In: Gene TheraRv of Cancer, Academic
Press,
San Diego, CA, pp. 235-248).
When the oncolytic virus of the invention is a herpes simplex virus-1, it
is preferably one which does not express functional ICP34.5 protein (e.g. HSV-
1716)
or one of the HSV-1 viruses described in Coukos et al., (1998, Gene Then. Mol.
Biol.
3:79-89). Exemplary HSV-1 viruses include HSV-1716, HSV-3410, HSV-3616, and
HSV-4009. Other replication selective HSV-1 virus strains which may be used as
the
oncolytic virus of the invention include, by way of example and not
limitation, HSV-
83616 (in which the gene encoding ICP34.5 is deleted), HSV-847 (in which genes
encoding proteins 83616 and ICP47 are deleted), HSV-6207 (in which genes
encoding
ICP34.5 and ribonucleotide reductase are deleted}, HSV-7020, HSV-NVR10 (in
which
genes encoding 7020 and ICP47 are deleted), HSV-3616-UB (in which genes
encoding
ICP34.5 and uracil DNA glycosylase are deleted), HSV-G92A (in which the
albumin
promoter is a transcriptional regulated promoter), HSV-3616-IL-4, HSV-hrR3 (in
which the gene encoding ribonucleotide reductase is deleted) and HSV strains
which do
not express functional ICP34.5 and which express a cytokine such as
interleukin-2,
interleukin-4, or GM-CSF.
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When the virus is a herpes simplex virus-2, it may, for example, be
selected from the group consisting of strain 2701, strain 2616, and strain
2604.
When the virus is an adenovirus, it may, for example, be selected from
the group consisting of ONYX-15, Ad5d1520, Ad5d1312, CN706, Add1110, Addll 11,
Addll 18, and Add1004.
The producer cells of the invention may be used in a method of killing
tumor cells in a mammal. The method comprising administering to the mammal a
producer cell comprising a oncolytic virus which is capable of replicating in
the
producer cell. The mammal may be substantially any mammal having tumor cells,
and
is preferably a human patient afflicted with a localized cancer such as
malignant
mesothelioma, EOC, bladder cancer, or the like.
The producer cells of the invention may be selectively engineered to
enhance their organ- or cell-specificity as well as their ability to induce
tumor killing.
Known gene products may be used enhance the oncolytic effect of viruses
delivered to
tumor cells by the producer cells of the invention. For example, increased
levels of
certain cytokines (e.g. interleukins 2, 4, and 6, y-interferon, and tumor
necrosis factor
a) inhibit tumor growth and metastasis. Producer cells which secrete such a
cytokine
in conjunction with virus production will have a synergistic effect on tumor
killing.
Producer cell lines may similarly be modified to enhance immune recruitment to
the
area of administration or decrease the effect of pre-existing immunity to the
vector.
Preliminary experiments have demonstrated an inhibitory effect of
ascites fluid on viral cell killing in vitro. This is thought to be due to the
presence of
IgG in the ascites. It is known that particular viral glycoproteins (gE & gI)
have the
ability to bind the Fc portion of antibodies; thus preventing the antibodies
from binding
antigens on the surface of cells and activating complement. It is also known
that
glycoprotein gC has the ability to inactivate complement directly. A producer
cell
which produces such glycoproteins can absorb and inactivate antibody in
ascitic or
pleural fluid, decreasing inhibition of viral spread.
The invention also includes use as producer cells of genetically-
modified cells which express a suicide gene such as HSV-TK and exhibit binding
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aff pity for tumor cells. These cells are sensitive to ganciclovir because
they convert
ganciclovir into various toxic metabolites. These cells exhibit therapeutic
effect on
tumor cells, based on the "bystander effect," which is death of cells adjacent
to the
genetically-modified cells. The bystander effect is thought to be caused by
transport of
one or more toxic metabolites of ganciclovir from cell to cell through gap
junctions as
well as by induction of an immune response.
P1_?a_ranaceutical_ Compositions
The invention encompasses the preparation and use of medicaments and
pharmaceutical compositions comprising the producer cell of the invention as
an active
ingredient. Such a pharmaceutical composition may consist of the active
ingredient
alone, in a form suitable for administration to a subject, or the
pharmaceutical
composition may comprise the active ingredient and one or more
pharmaceutically
acceptable carriers, one or more additional ingredients, or some combination
of these.
Administration of one of these pharmaceutical compositions to a subject is
useful for
killing tumor cells or arresting tumor growth or spread in the subject, as
described
elsewhere in the present disclosure.
As used herein, the term "pharmaceutically acceptable carrier" means a
chemical composition with which the active ingredient may be combined and
which,
following the combination, can be used to administer the active ingredient to
a subject.
The formulations of the pharmaceutical compositions described herein
may be prepared by any method known or hereafter developed in the art of
pharmacology. In general, such preparatory methods include the step of
bringing the
active ingredient into association with a carrier or one or more other
accessory
ingredients, and then, if necessary or desirable, shaping or packaging the
product into a
desired single- or mufti-dose unit.
Although the descriptions of pharmaceutical compositions provided
herein are principally directed to pharmaceutical compositions which are
suitable for
ethical administration to humans, it will be understood by the skilled artisan
that such
compositions are generally suitable for administration to animals of all
sorts.
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Modification of pharmaceutical compositions suitable for administration to
humans in
order to render the compositions suitable for administration to various
animals is well
understood, and the ordinarily skilled veterinary pharmacologist can design
and
perform such modification with merely ordinary, if any, experimentation.
Subjects to
which administration of the pharmaceutical compositions of the invention is
contemplated include, but are not limited to, humans, primates, and other
mammals.
Pharmaceutical compositions that are useful in the methods of the
invention may be prepared, packaged, or sold in formulations suitable for
intraperitoneal, oral, rectal, vaginal, parenteral, topical, pulmonary,
intranasal, buccal,
ophthalmic, or another route of administration. Other contemplated
formulations
include projected nanoparticles, liposomal preparations, and immunologically-
based
formulations.
A pharmaceutical composition of the invention may be prepared,
packaged, or sold in bulk, as a single unit dose, or as a plurality of single
unit doses.
As used herein, a "unit dose" is discrete amount of the pharmaceutical
composition
comprising a predetermined amount of the active ingredient. The amount of the
active
ingredient is generally equal to the dosage of the active ingredient which
would be
administered to a subject or a convenient fraction of such a dosage such as,
for
example, one-half or one-third of such a dosage.
Controlled- or sustained-release formulations of a pharmaceutical
composition of the invention may be made using conventional technology.
Liquid suspensions, for example, may be prepared using conventional
methods to achieve suspension of the active ingredient in an aqueous or oily
vehicle.
Aqueous vehicles include, for example, water and isotonic saline. Oily
vehicles
ZS include, for example, almond oil, oily esters, ethyl alcohol, vegetable
oils such as
arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and
mineral oils such
as liquid paraffin. Liquid suspensions may further comprise one or more
additional
ingredients including, but not limited to, suspending agents, dispersing or
wetting
agents, emulsifying agents, demulcents, preservatives, buffers, salts,
flavorings,
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coloring agents, and sweetening agents. Oily suspensions may further comprise
a
thickening agent. Known suspending agents include, but are not limited to,
sorbitol
syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum
tragacanth, gum acacia, and cellulose derivatives such as sodium
carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known
dispersing or wetting agents include, but are not limited to, naturally-
occurring
phosphatides such as lecithin, condensation products of an alkylene oxide with
a fatty
acid, with a long chain aliphatic alcohol, with a partial ester derived from a
fatty acid
and a hexitol, or with a partial ester derived from a fatty acid and a hexitol
anhydride
(e.g. polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene
sorbitol
monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known
emulsifying agents include, but are not limited to, lecithin and acacia.
A pharmaceutical composition of the invention may also be prepared,
packaged, or sold in the form of oil-in-water emulsion or a water-in-oil
emulsion. The
oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil
such as
liquid paraffin, or a combination of these. Such compositions may further
comprise
one or more emulsifying agents such as naturally occurring gums such as gum
acacia or
gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin
phosphatide, esters or partial esters derived from combinations of fatty acids
and
hexitol anhydrides such as sorbitan monooleate, and condensation products of
such
partial esters with ethylene oxide such as polyoxyethylene sorbitan
monooleate. These
emulsions may also contain additional ingredients including, for example,
sweetening
or flavoring agents.
A pharmaceutical composition of the invention may be prepared,
packaged, or sold in a formulation suitable for rectal administration. Such a
composition may be in the form of, for example, a suppository, a retention
enema
preparation, and a solution for rectal or colonic irrigation.
Suppository formulations may be made by combining the active
ingredient with a non-irritating pharmaceutically acceptable excipient which
is solid at
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ordinary room temperature (i.e. about 20°C) and which is liquid at the
rectal
temperature of the subject (i.e. about 37°C in a healthy human).
Suitable
pharmaceutically acceptable excipients include, but are not limited to, cocoa
butter,
polyethylene glycols, and various glycerides. Suppository formulations may
further
comprise various additional ingredients including, but not limited to,
antioxidants and
preservatives.
Retention enema preparations or solutions for rectal or colonic irrigation
may be made by combining the active ingredient with a pharmaceutically
acceptable
liquid carrier. As is well known in the art, enema preparations may be
administered
using, and may be packaged within, a delivery device adapted to the rectal
anatomy of
the subject. Enema preparations may further comprise various additional
ingredients
including, but not limited to, antioxidants and preservatives.
A pharmaceutical composition of the invention may be prepared,
packaged, or sold in a formulation suitable for vaginal administration. Such a
composition may be in the form of, for example, a suppository, an impregnated
or
coated vaginally-insertable material such as a tampon, a douche preparation,
or a
solution for vaginal irrigation.
Methods for impregnating or coating a material with a chemical
composition are known in the art, and include, but are not limited to methods
of
depositing or binding a chemical composition onto a surface, methods of
incorporating
a chemical composition into the structure of a material during the synthesis
of the
material (i.e. such as with a physiologically degradable material), and
methods of
absorbing an aqueous or oily solution or suspension into an absorbent
material, with or
without subsequent drying.
Douche preparations or solutions for vaginal irrigation may be made by
combining the active ingredient with a pharmaceutically acceptable liquid
carrier. As
is well known in the art, douche preparations may be administered using, and
may be
packaged within, a delivery device adapted to the vaginal anatomy of the
subject.
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Douche preparations may further comprise various additional ingredients
including, but
not limited to, antioxidants, antibiotics, antifungal agents, and
preservatives.
As used herein, "parenteral administration" of a pharmaceutical
composition includes any route of administration characterized by physical
breaching
of a tissue of a subject and administration of the phannaceutical composition
through
the breach in the tissue. Parenteral administration thus includes, but is not
limited to,
administration of a pharmaceutical composition by injection of the
composition, by
application of the composition through a surgical incision, by application of
the
composition through a tissue-penetrating non-surgical wound, and the like. In
particular, parenteral administration is contemplated to include, but is not
limited to,
subcutaneous, intraperitoneal, intravenous, intraarterial, intramuscular, or
intrasternal
injection and intravenous, intraarterial, or kidney dialytic infusion
techniques.
Formulations of a pharmaceutical composition suitable for parenteral
administration comprise the active ingredient combined with a pharmaceutically
acceptable carrier, such as sterile water or sterile isotonic saline. Such
formulations
may be prepared, packaged, or sold in a form suitable for bolus administration
or for
continuous administration. Injectable formulations may be prepared, packaged,
or sold
in unit dosage form, such as in ampules, in mufti-dose containers containing a
preservative, or in single-use devices for auto-injection or injection by a
medical
practitioner. Formulations for parenteral administration include, but are not
limited to,
suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and
implantable
sustained-release or biodegradable formulations. Such formulations may further
comprise one or more additional ingredients including, but not limited to,
suspending,
stabilizing, or dispersing agents. In one embodiment of a formulation for
parenteral
administration, the active ingredient is provided in dry (i.e. powder or
granular) form
for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water)
prior to
parenteral administration of the reconstituted composition.
The pharmaceutical compositions may be prepared, packaged, or sold in
the form of an injectable aqueous or oily suspension. This suspension or
solution may
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be formulated according to the known art, and may comprise, in addition to the
active
ingredient, additional ingredients such as the dispersing agents, wetting
agents, or
suspending agents described herein. Such injectable formulations may be
prepared
using a non-toxic parenterally-acceptable diluent or solvent, such as water or
1,3-butane diol, for example. Other acceptable diluents and solvents include,
but are
not limited to, Ringer's solution, isotonic sodium chloride solution, and
fixed oils such
as synthetic mono- or di-glycerides. Other parentally-administrable
formulations
which are useful include those which comprise the active ingredient in
microcrystalline
form, in a liposomal preparation, or as a component of a biodegradable polymer
systems. Compositions for sustained release or implantation may comprise
pharmaceutically acceptable polymeric or hydrophobic materials such as an
emulsion,
an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble
salt.
As used herein, "additional ingredients" include, but are not limited to,
one or more of the following: excipients; surface active agents; dispersing
agents; inert
diluents; granulating and disintegrating agents; binding agents; lubricating
agents;
sweetening agents; flavoring agents; coloring agents; preservatives;
physiologically
degradable compositions such as gelatin; aqueous vehicles and solvents; oily
vehicles
and solvents; suspending agents; dispersing or wetting agents; emulsifying
agents,
demulcents; buffers; salts; thickening agents; fillers; emulsifying agents;
antioxidants;
antibiotics; antifungal agents; stabilizing agents; and pharmaceutically
acceptable
polymeric or hydrophobic materials. Other "additional ingredients" which may
be
included in the pharmaceutical compositions of the invention are known in the
art and
described, for example in Genaro, ed., 1985, ReminQton's Pha_rn__,_aceutic-~_1
Sciences,
Mack Publishing Co., Easton, PA, which is incorporated herein by reference.
Another aspect of the invention relates to a kit comprising a
pharmaceutical composition of the invention and an instructional material. As
used
herein, an "instructional material" includes a publication, a recording, a
diagram, or any
other medium of expression which is used to communicate the usefulness of the
pharmaceutical composition of the invention for killing tumor cells in a
subject, for
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preparing producer cells of the invention using one or more components of the
kit, or
for administering the producer cells of the invention to a subject. The
instructional
material of the kit of the invention may, for example, be affixed to a
container which
contains a pharmaceutical composition of the invention or be shipped together
with a
container which contains the pharmaceutical composition. Alternatively, the
instructional material may be shipped separately from the container with the
intention
that the instructional material and the pharmaceutical composition be used
cooperatively by the recipient.
The invention also includes a kit comprising a pharmaceutical
composition of the invention and a delivery device for delivering the
composition to a
subject. By way of example, the delivery device may be a squeezable spray
bottle, a
metered-dose spray bottle, an aerosol spray device, an atomizer, a dry powder
delivery
device, a self propelling solventlpowder-dispensing device, a syringe, a
needle, a
tampon, or a dosage measuring container. The kit may further comprise an
instructional material as described herein.
The invention is now described with reference to the following
Examples. These Examples are provided for the purpose of illustration only and
the
invention should in no way be construed as being limited to these Examples,
but rather
should be construed to encompass any and all variations which become evident
as a
result of the teaching provided herein.
In order to develop an effective method of vector delivery,
EJ-6-2-Bam-6a cells, an NIH 3T3 fibroblast cell line, were used as producer
cells for
HSV-1716 in a immunocompetent marine model of lung cancer. It was demonstrated
that Lewis Lung Carcinoma (LLC), a spontaneous non-immunogenic marine lung
cancer, is sensitive to treatment using HSV-1716 in vitro as well as in vivo.
LLC supported replication of and was e~ciently lysed by HSV-1716 at
a multiplicity of infection (MOI) of I .0 (<20% of cells were viable four days
following
infection). HSV-1716 replicated in LLC cells with a burst size of 20. In
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EJ-6-2-Bam-6a, the virus replicated with significantly greater efficiency,
exhibiting a
burst size of about 2,200.
It was hypothesized that producer cells would facilitate viral delivery in
an in vivo system. To test this hypothesis, cell mixing studies were performed
using
LLC cells injected into the flanks of C57BL/6 mice. These syngeneic cells, a
fraction
ofwhich were infected with HSV-1716, completely prevented tumor formation by
non-
infected LLC cells in 10 animals~when the cells were mixed at an infected:non-
infected
ratio of 1:10 (P=0.002). Significant growth inhibition was also seen at a
ratio of 1:100.
The mean tumor weight in mice administered non-infected LLC cells was 0.092
gram,
and tumors in the 1:100 treated group averaged 0.026 gram (P~.001).
Analogous experiments using EJ-6-2 cells in place of LLC cells in this
model, demonstrated similar results at an infected:non-infected ratio of 1:10
(P=0.007)
and effective growth retardation at a ratio as low as 1:1000 (tumor weight of
0.224
gram in controls versus 0.072 gram in mice administered a 1:1000 ratio of EJ-6-
2 cells
P=0.02). Without wishing to be bound by any particular theory of operation, it
is
believed that the tumor growth-inhibiting effects was attributable to a dual
effect of the
virus-infected EJ-6-2 cells. First, EJ-6-2 cells have a much greater burst
size than the
LLC, thus drastically increasing the effective dose of virus administered
(MOI).
Second, studies involving irradiated EJ-6-2 cells which were not infected with
HSV-
1716 also demonstrated significant tumor suppression (tumor weight of 0.224
gram in
control mice versus 0.051 gram in mice administered irradiated EJ-6-2 1:10;
p=0.03),
suggesting that EJ-6-2 cells elicited an immune response to this normally
non-immunogenic tumor.
Based on these data, we conclude that HSV-1716 is useful as a new
gene therapy vector for, at least, lung cancer and that the use of allogeneic
producer
cells such as EJ-6-2 can enhance the oncolytic efficacy of virus both by
increasing the
effective MOI of the virus and by inducing an inflammatory response within the
tumor
milieu.
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Epithelial ovarian cancer (EOC) remains localized within the peritoneal
cavity in many patients, lending itself to intraperitoneal therapeutic
approaches. In the
experiments presented in this Example, the effect of intraperitoneally
administering a
replication-selective herpes simplex virus-1 (rsHSV-I) on EOC cells was
assessed.
The rsHSV-1 was administered by direct injection of virus particles and by
injection of
producer cells which had been infected using the rsHSV-1. Irradiated human
teratocarcinoma PA-1 cells were used as the producer cells.
The rsHSV-1 used in the experiments in this Example, HSV-1716, is a
replication-competent attenuated strain of HSV-1 which does not express
ICP34.5.
Contacting EOC cells with HSV-1716 in vitro induced dose-dependent oncolysis.
A
single intraperitoneal administration of S x 106 plaque-forming units (pfu) of
HSV-1716
resulted in significant reduction of tumor volume and tumor spread and
increase in
survival in a mouse xenograft model of EOC. PA-1 cells support HSV-1716
replication in vitro and bind preferentially to human ovarian carcinoma
surfaces,
relative to mesothelial surfaces, both in vitro and in vivo.
In comparison to intraperitoneal administration of HSV-1716 alone,
intraperitoneal administration of irradiated PA-1 cells infected with HSV-1716
induced
significantly greater tumor reduction in the two xenograft models tested.
Significant
prolongation of mean survival associated with injection of HSV-1716-infected
cells,
but not associated with injection of HSV-1716 alone, was also observed in one
model.
Histologic evaluation indicated the presence of extensive necrosis at tumor
sites
infected with HSV-1716. Immunohistochemistry to detect HSV-1716 indicated that
areas of viral infection were present within tumor nodules, and that such
infection
persisted for several weeks following treatment. Administration of HSV-1716-
infected
producer cells induced more widespread infection of tumor tissue by the virus.
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The experiments presented in this Example indicate that
replication-competent attenuated HSV-1 exerts a potent oncolytic effect on
EOC, and
that this oncolytic effect may be enhanced by administering the virus in the
form of
virus-infected producer cells. Without wishing to be bound by any particular
theory of
operation, it is believed that the enhanced oncolytic effect of producer cell-
delivered
virus, relative to virus alone, is attributable to amplification of the number
of virus
particles delivered to tumor tissue and to preferential binding of producer
cells to tumor
surfaces.
The materials and method used in the experiments presented in this
Example are now described.
Virus
Isolation of HSV-1716 has been described previously (MacLean et al.,
1991, J. Gen. Virol. 72:631-639). The genome of this virus contains a 759-base-
pair
deletion located within each copy of the BamHl fragment of the long repeat
region of
the genome. These deletions encompass most of the gene encoding ICP34.5. The
mutant therefore does not express this protein. Passage of the virus has also
been
described (MacLean et al., 1991, J. Gen. Virol. 72:631-639; Kucharczuk et al.,
1997,
Cancer Res. 57:466-471 ).
Epithelial ovarian cancer cell lines SKOV3, IVIH:OVCAR3, CaOV3,
and human ovarian teratocarcinoma line PA-1 have been previously described,
and
were obtained from the American Tissue Culture Collection (Plainview, MD;
Tainsky
et al., 1988, Anticancer Res. 8:899-913). The A2780 EOC cell line was obtained
from
the Fox Chase Cancer Center (Philadelphia, PA).
Primary ovarian cultures were obtained from patients afflicted with
advanced EOC, at stages III or IV, according to the criteria set by the
International
Federation of Gynecologists and Obstetricians (DiSaia et al., 1993, In:
Clinical
Gynecologic Oncology, Mosby-Year Book, Inc., St. Louis, MO). Malignant
effusions,
obtained at the time of exploratory laparotomy or diagnostic/therapeutic
paracentesis,
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were centrifuged at 300xg for 10 minutes at room temperature, and the cell
pellets were
collected and seeded in standard tissue culture media, as described herein.
These cells
assumed an epithelial phenotype and their malignaat nature was confirmed by a
rapid
doubling time (average 18 hours) and their immortalized behavior in vitro. EOC
cells
were passaged 4-5 times prior to using them in experiments.
Normal peritoneal mesothelial cells were obtained from intraoperative
pelvic peritoneal lavages carried out using normal saline in patients
undergoing
laparotomy for benign pelvic pathology (e.g. pelvic relaxation, uterine
myomata).
Lavage fluids were centrifuged at 300xg for 10 minutes at room temperature and
the
cell pellets were collected and seeded in standard media (see below). These
cells
assumed an epithelial-like phenotype and grew in a cobblestone pattern. Their
doubling time was longer than that of primary EOC cultures (average 36 hours),
and
their non-malignant nature was confirmed by the fact that they propagated for
only a
few (4-5) passages even in the presence of growth factors.
To eliminate macrophages from primary cultures, culture media were
aspirated 30 min following plating, and suspended cells were re-seeded in new
culture
flasks. All cell lines and EOC primary isolates were cultured under standard
conditions
(37°C in a 5% C02 atmosphere) in RPMI media comprising 10% (v/v)
heat-inactivated fetal calf serum (FCS) and antibiotics. For normal
mesothelial cells,
media were supplemented with a mixture of growth factors (SerXtendTM, Irvine
Scientific, Santa Anna, CA) at 0.1 % dilution.
Assessment of C, otoxicil in vitro
Cells were incubated in 96-well plates at a density of 3 X 103 cells per
well. The cells were incubated in the presence of HSV-1716 at multiplicities
of
infection (MOI) of 0.1 and 1 in serum-free media for one hour. Serum-enriched
media
was subsequently added and cultures were observed for four days. Cell
proliferation
assays were performed using a chromogenic kit (CellTiter AQueous96TM, Promega,
Madison, WI) and colorimetric assays were performed using a microplate ELISA
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reader (Bio-Tek Inshuments, Winooski, VT). Cytopathic effects (CPE) were
documented by phase microscopy.
Flow Cytome ~3rsis of HSV nt ~enc
PA-1 cells were cultured in T25 flasks until they reached 60%
confluence. The cells were then infected with 0.5-2.5 MOI of HSV-1716, as
described
above. Cells were harvested at 16 hours using a 0.05% trypsin solution, washed
once
with phosphate buffered saline (PBS) and fixed and permeabilized using 70%
methanol
at -20°C for 20 minutes. Cells were labeled using a polyclonal antibody
(obtained
from American Qualex, La Mirada, CA) which specifically binds with HSV-1
proteins,
which was used at a dilution of 1:250, and a secondary anti-rabbit fluorescein
isothiocyanate (FITC)-conjugated antibody (obtained from Jackson
Immunoresearch
Laboratories Inc., West Groove, PA), which was used at a dilution of 1:250.
Flow
cytometric analysis was performed using an EPICSTM XL flow cytometer (Coulter
Corporation, Hialeah, FL).
One-Step Growth ('_~rvec
PA-1 teratocarcinoma cells were incubated overnight in 6-well plates at
a density of 4x 105 cells per well under standard culture conditions and were
then
infected with HSV-1716 at 0.3 MOI. In parallel experiments, PA-1 cells were
subjected to a single 20 Gray dose of ionizing radiation one hour prior to
infection with
HSV-1716. Cells were harvested in the accompanying medium by mechanical
scraping one hour after infection with the virus (designated'0 hours') as well
as 6, 20,
24, and 48 hours later and stored at -80°C. One-step growth curves were
generated as
described (Kucharczuk et al., 1997, Cancer Res. 57:466-471).
In vivo Adhesion Assays
PA-1 teratocarcinoma cells were labeled using a rhodamine fluorescent
dye (PKH26 Red Fluorescent Cell Linker Kit, Sigma Chemical Co., St. Louis, MO)
as
recommended by the manufacturer. Briefly, cells were harvested using a 0.05%
(w/v)
trypsin solution, re-suspended in PBS, and incubated with a 1:250 dilution of
the dye at
for 8 minutes. The labeling reaction was termination by addition of 100% FCS.
Cells
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were washed, suspended in RPMI media containing 10% FCS, and injected
intraperitoneally (at 5 x 106 cells per animal) into SCID mice having
intraperitoneal
SKOV3 tumors, as described below.
Eight hours after PA-1 cell injection, the animals were sacrificed, and
the parietal peritoneum was entirely dissected into four specimens. Each of
the
specimens contained alinost entirely one of the four different segments of the
parietal
peritoneum (i.e. the diaphragmatic segment, the ventral segment, the lateral
left
segment, or the lateral right segment). In addition, random biopsies were
obtained
from the mesentery and the visceral peritoneum. Six micrometer sections were
prepared, mounted in FluoromountTM medium (Fisher, Pittsburgh, PA) containing
2.5% 1,4-diazabicyclo-(2,2,2)octane (DABCO, Polyscience, Warnngton, PA) to
prevent fluorescent quenching, and examined using a Zeiss microscope.
For quantitative analysis of in vivo adhesion, 20 random fields per slide
(at 40x magnification) were inspected on each of five slides cut 18
micrometers apart
from each other from each of the four different segments of the parietal
peritoneum and
from each of two sections of mesenteric areas. Fields were selected in such a
way that
they contained only tumor surface or tumor-free normal peritoneum. The number
of
fields containing SKOV3 tumor with or without adhering fluorescent PA-1 cells
and
those containing normal peritoneum with or without adhering fluorescent PA-1
cells
were counted.
In vitro Adhesion As~vs
Normal peritoneal mesothelial cells, PA-1 teratocarcinoma cells, and
EOC cells of type SKOV3, CaOV3, NIH:OVCAR3, or A2780 were incubated in
48-well plates at a density of 3 x 1,04 to 4x 104 cells per well and allowed
to form 100%
confluent monolayers. Teratocarcinoma PA-1 cells cultured in T25 flasks were
starved
with methionine-free media for 2 hours and then incubated overnight with
35S_methionine-containing media (50 microcuries per milliliter) containing 1%
dialyzed FCS. Radio-labeled PA-1 cells were harvested by short exposure to
0.05%
trypsin solution, washing once with serum-free media, centrifugation at 300xg
for S
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minutes at room temperature, and re-suspension in media containing 1% FCS. PA-
1
cells were then seeded at 5 X 103 per well on cell monolayers and allowed to
interact
with adherent mesothelial or ovarian cancer cells for 30 minutes.
Following co-incubation of PA-1 and mesothelial or cancer cells, the
wells were washed three times with PBS to remove non-adherent cells. Adherent
cells
were harvested using 0.1 % trypsin solution, transferred to scintillation
vials, and
enumerated using a scintillation counter (model LS 6500, Beckman Instruments,
Fullerton, CA).
In separate experiments, PA-1 teratocarcinoma cells were radio-labeled
using 35S-methionine, as described above, and subjected to ionizing radiation
(20
Gray) and/or infection with HSV-1716 (at 2 MOI). Cells subjected to these
treatments
were permitted to interact with the different monolayer substrates, and
adhesion was
measured, as described above.
In vivo Xenoeraft Model of F~ithelial Ovarian Cancer
Six- to eight-week-old female CB 17 SCID mice (Charles River,
Wilmington, MA) were housed in an isolation unit. A2780 and SKOV3 cells were
produced using standard culture conditions until they reached 70% confluence.
The
cells were harvested using 0.05% (w/v) trypsin solution, washed with serum-
enriched
media, and centrifuged at 300xg for 5 minutes at 4°C. About 5x106 SKOV3
cells or
1 x 106 A2780 cells per animal were injected intraperitoneally in 0.5
milliliter of RPMI
medium containing 10% FCS and 1 % SerXtendTM (Irvine Scientific). Five mice
were
sacrificed at selected times to confu~n the presence of intraperitoneal tumors
prior to
administering treatment in each experiment. At the end of the experiments,
animals
were sacrificed and intraperitoneal twnors were assessed for spread and
volume.
A semi-quantitative scoring system was devised to assess tumor spread.
Five areas were screened for the presence of tumor:
1 ) the inj ection site;
2) the parietal peritoneum and diaphragm;
3) the small bowel mesentery and omentum;
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4) the lesser omentum and hepatic hilum; and
5) the retroperitoneum.
Each site was assigned a score of 0-3 as following: "0" if no microscopic
tumor was
seen; "1" if one or more microscopic tumors were visible with the aid of a
dissecting
microscope at 2.5 x magnification; "2" if tumor nodules less than 5
millimeters in
diameter were visible; and "3" if tumor nodules equal to or more than 5
millimeters in
diameter were present. The presence of ascites resulted in addition of one
point in the
scoring system. The maximum score accumulated in this scale was therefore 16.
All
visible tumor nodules were dissected from surrounding normal peritoneum and
viscera,
and the total weight of infra-abdominal tumor was determined for each animal.
PA-1 human teratocarcinoma cells were exposed to ionizing radiation at
a single dose of 20 Gray and were then infected with HSV-1716 at an MOI of 2.
Two
hours after infection, cells were washed with PBS and harvested using a 0.05%
(w/v)
trypsin solution. Cells were washed twice in media comprising 10% heat-
inactivated
FCS, centrifuged at 300xg for S minutes, and re-suspended in RPMI media
comprising
1 % heat-inactivated FCS. About 5 X 106 cells were injected intraperitoneally
into
tumor-bearing animals, as described below. Control tumor-bearing animals
received
PA-1 teratocarcinoma cells that had been exposed to the same amount of
radiation but
that were not infected with HSV-1716. To confirm that irradiated PA-1 cells
did not
cause tumorigenicity, non tumor-bearing mice received a flank (n=10) or an
intraperitoneal (n=10) administration of 5 x 106 irradiated (but not infected)
PA-1 cells
and were observed for 16 weeks.
At selected times, animals received a single intraperitoneal dose of
S X 106 plaque-forming units (pfu) HSV-1716 at in 500 milliliters of serum-
free RPMI
medium. This dose was previously determined to be effective to reduce
mesothelioma
tumor load (Kucharczuk et aL, 1997, Cancer Res. 57:466-471). Control animals
received a similar volume of virus-free medium. A separate group of animals
received
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an iatraperitoneal injection of SX 106 pfu of irradiated PA-1 teratocarcinoma
cells
which had been infected with HSV-1716 at an MOI of 2, as described above. A
second
control group received irradiated, but non-infected, PA-1 cells.
Animals were sacrificed at selected times and intraperitoneal tumors
were assessed in the animals. For survival experiments, animals were injected
with
tumor and treated as above. Animals were observed daily and removed from the
cages,
if found dead, or sacrificed, if they appeared severely ill. Tumor weight was
estimated
in sacrificed animals as described above. Survival experiments were repeated
twice for
each EOC cell line. Kaplan-Meier survival curves were computed utilizing the
StatViewTM computer program.
Histology and Immunohistochemistrv
Tumors obtained from treated and control animals were immediately
fixed in formalin (3.7% (v/v) formaldehyde in PBS) and embedded in paraffin.
Six-
micrometer-thick sections were de-paraffinized and stained using hematoxylin-
eosine
(H&E). For immunohistochemical analysis, the slides were subjected to antigen
retrieval at 105 °C for 10 minutes in 0.1 normal citric acid and
incubated with a
monoclonal antibody which binds specifically with HSV-1 (DAKO, Carpenteria,
California) at a dilution of 1:4 for 30 minutes at 47°C. A
horseradish
peroxidase-conjugated anti-mouse antibody (Vectastain, Vector Laboratories,
Burlingame, CA) was used at a dilution of 1:400. Slides were processed using
the
ABCTM Kit (Vector Laboratories) according to the manufacturer's instructions.
Mouse
pre-immune serum was used at a dilution of 1:4 as a negative control (DAKO,
Carpenteria, California).
Statistical Analysis
Differences in values obtained during in vitro adhesion assays and
differences in tumor weights in animal experiments were determined using one-
way
ANOVA. Post-hoc comparisons of specific paired groups were performed using the
t-test. Differences in in vivo adhesion assays were computed using the Chi-
square test
for a 2X2 contingency table. Odds ratios (OR) were computed using the formula
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OR--axd/bXc from the 2x2 contingency tables. Survival curies were analyzed
using
the Mantel-Cox Logrank test. Statistical significance was set at p <0.05.
Results are
expressed as the mean t standard error.
The results of the experiments presented in this Example are now
described.
HSV-1716 Exerts an Oncolvtic Effect on Epithelial Ovarian Cancer in vitro
In order to assess the oncolytic effect of HSV-1716 on EOC cells in
vitro, primary EOC cultures and established EOC cell lines were exposed to HSV-
1716
at 0.1 and 1 MOI. Cell survival was assessed by proliferation colorimetric
assays and
evaluation of cytopathic effect (CPE), as assessed by phase-contrast
microscopy.
HSV-1716 exerted a direct dose-dependent cytolytic effect on all EOC cell
lines and on
human teratocarcinoma PA-1 cell fine.
Primary EOC cultures obtained from patients afflicted with advanced
disease displayed 10- to 20-fold higher sensitivity to cytolysis by HSV-1716
than
established EOC cell lines. EOC cell line A2780 was the most sensitive of the
cell
lines tested, and SKOV3 was the least sensitive of those tested.
Teratocarcinoma PA-1
cells were highly sensitive to HSV-1716-induced cytolysis. The sensitivity of
PA-1
cells was comparable to that of primary EOC cultures.
These sensitivity observations were confirmed by phase microscopy.
100% of primary EOC cells, A2780 cells, and teratocarcinoma PA-1 cells
exhibited
CPE within 24-48 hours of exposure at HSV-1716 at 1 MOI. About 95-99% of
1VIH:OVCAR3, SKOV3, and CaOV3 cells exhibited CPE within four days of
exposure.
HSV-1716 Exerts an Oncolsrtic Effect on Epithelial Ovarian Cancer in vivo
In order to assess the efficacy of HSV-1716 for treating established
intraperitoneal EOC, a marine xenograft model of EOC was used together with
two
well characterized EOC cell lines, namely cell lines SKOV3 and A2780. These
two
cell lines were selected because, in addition to being well characterized,
these two cell
lines exhibited the least and greatest, respectively, sensitivity to cytolysis
by HSV-1716
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in vitro. The oncolytic effect of HSV-1716 was evaluated by examining
xenografted
mice having different tumor burdens, by varying the duration of treatment, and
by
using the two aforementioned cell lines to generate the tumors, thereby
yielding
intraperitoneal tumors having different volumes.
To assess the effect of HSV-1716 in minimally-spread EOC, the virus
was administered one week following the injection of SKOV3 ovarian cancer
cells into
mice. Intraperitoneal injection of 5 x 106 SKOV3 cells resulted in formation
of
microscopic tumors) at the diaphragm and occasionally at the omentum,
mesentery, or
lesser omentum within one week. The tumor score (as described above) for these
mice
was 2/16. The tumor weight in these mice (n=5) was 202 ~ 7.1 milligrams.
One week after injecting SKOV3 cells into the mice, a group (n=10) of
animals received a single dose of HSV-1716, while a group of control animals
(n=10)
received medium only. Animals were S weeks later to evaluate tumors. Animals
that
received virus displayed significantly smaller tumors compared to control
animals
(p<0.001), as indicated in Table 1. Moreover, HSV-treated animals displayed
tumors
similar to their pre-treatment counterparts (p--0.78). Tumor spread was
advanced in
untreated animals (tumor score 15.5/16), while it remained similar to pre-
treatment
tumor spread in the HSV-treated animals (tumor score 2.2/16).
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Table 1
Tumor Weight,
milligrams
Week during Pre- Non-Treated HSV-1716
which Treatment (at week (at week 6)
Treatment Began 6)
SKOV3 1 202 ~ 7 1,147 ~ 11 191 t lA
Tumors 3 837 ~9 1,249 ~ 13.2732 ~ 3A
A2780 1 1,800 X51118,705 ~ 2,870 t 923A
Tumors 3 8,900 ~ 2,120 10,032 t 1,812B
92I 19,100 ~
1,115
Notes:
A p<0.001
B p<0.05
In order to assess the role of HSV-1716 in more advanced EOC,
xenografted SKOV3 tumors were allowed to grow for three weeks before the mice
were injected with virus. At three weeks and without virus treatment, larger
intraperitoneal tumors were observed (tumor score: 3.5/16; tumor weight: 837 t
9.1
milligrams; n=5) than the tumors observed following only one week of growth.
Virus-
treated animals received a single dose of HSV-1716 intraperitoneally, and
control
animals received only medium. Animals were sacrificed at 6 weeks (i.e. three
weeks
after treatment) to evaluate tumors. Animals that received HSV-1716 (n=I O)
displayed
significantly smaller tumors compared to control animals (n--10; p<0.001).
Moreover,
HSV-treated animals displayed similar tumors to their pre-treatment
counterparts
(p=0.86). Tumor spread was again advanced in untreated animals (tumor score
16/16),
while it remained similar to pre-treatment in the HSV-treated animals (tumor
score
3.7/16).
In order to assess the role of HSV-1716 in bulky EOC, the A2780 cell
Line was injected into mice. Administration of 1 X 106 A2780 cells resulted in
growth of
bulky intraperitoneal tumors characterized by the presence of tumor nodules
having
diameters of from 0.5 to 2 millimeters in diameter throughout the abdominal
cavity
within one week (tumor score 16/16; tumor weight: 1860 t 532 milligrams; n--
5). One
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week after injection of A2780 cells, one group of animals (n=10) received a
single dose
of HSV-1716, and a group of control animals (n=10) received medium only.
Animals
were sacrificed 5 weeks later in order to evaluate tumors. HSV-treated animals
displayed significantly smaller tumors than control animals that received
saline at week
one (p<0.001; n=10). No difference in tumor weight was detected between the
HSV-treated animals and their pre-treatment counterparts (p~.86; n=10).
In order to assess the role of HSV-1716 in more widespread bulky
intraperitoneal EOC, A2780 tumors were allowed to grow for three weeks
following
cell injection. At that time, tumor nodules having diameters of from 4 to
about 14
millimeters were observed throughout the abdominal cavity of each mouse (tumor
score 16/16; tumor weight: 8150 ~ 912 mg; n=5). Administration of a single
dose of
HSV-1716 three weeks following cell injection resulted in arrest of tumor
growth at 6
weeks (three weeks later), relative to the pre-treatment counterpart mice. HSV-
treated
animals displayed significantly smaller tumors at 6 weeks, compared to control
animals
that received media (p<0.05; n=10). In addition, no significant differences in
tumor
weight were observed between the animals treated with HSV-1716 at week three
and
their pre-treatment counterparts (p=0.66; n=10).
HSV-1716 Efficiently Infects and R~licates in Irradiated PA-1 Cells
A difficulty of comparing the effect of virus administered alone and
virus administered by way of producer cells relates to controlling the number
of
particles inoculated. To minimize the difference in the virus load initially
administered
to xenografted mice, the lowest MOI at which 100% of PA-1 cells were infected
with
HSV-1716 was determined. PA-1 cells were incubated with increasing doses of
the
virus and the rate of infection of the cells was determined by flow cytometry.
These
experiments indicated that approximately 100% of PA-1 cells were infected at
an MOI
of 2. At this MOI, each PA-1 cell presumably was initially infected by one or
at most
two viral particles.
It was hypothesized that producer cells would lead to significant
amplification of the viral load delivered intraperitoneally to xenografted
mice in vivo.
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To assess the magnitude of viral replication in PA-1 cells in vitro, the viral
burst size
(i.e. the number of infectious virus particles released from a PA-1 cell upon
cytolysis
following infection of the cell with the virus and incubation) was determined.
The
burst size of HSV-1716 in PA-1 teratocarcinoma cells was 200 in the absence of
ionizing radiation. Because administration of HSV-1 infected producer cells
into
humans will most likely be performed after eliminate the risk of administering
potentially uninfected producer cells (i.e. by irradiating the cells), PA-1
cells were
subjected to a single (lethal) dose of 20 Gray one hour prior to infection,
and viral
replication in the irradiated cells was assessed. Burst size experiments
indicated that
irradiated PA-1 cells infected with HSV-1716 supported viral replication with
a burst
size of 70 following irradiation at 20 Gray. It was furthermore found that
irradiation of
the cells infected with HSV-6207 using a radiation dose of 3 Gray
significantly
increased the burst size, relative to non-irradiated cells.
PA-1 Producer Cells Preferentially Bind to Epithelial Ovarian Cancer Surface
Compared to Normal Peritoneum in vivo
In order to assess the behavior of the producer cells in the marine
xenograft model, fluorescently labeled PA-1 cells were injected
intraperitoneally to
SKOV3 tumor-bearing mice. Overall, a large number of fluorescent PA-1 cells
adhered to areas of the diaphragm or other peritoneal surfaces that were
covered by
tumor. In some areas, PA-1 cells almost formed a monolayer covering the SKOV3
preexisting tumors. In the absence of tumor nodules, there were few and
isolated
fluorescent PA-1 cells attaching to normal peritoneal surfaces.
When the number of tumor fields containing adherent PA-1 cells were
compared to the number of non-tumor peritoneum fields containing adherent PA-1
cells, it was observed that there was a significantly higher frequency of
binding of
PA-1 cells to tumor surfaces than to nornnal peritoneum. Specifically, 90
random
diaphragmatic areas harboring SKOV3 tumor cells were examined and 91.1%
displayed adherent PA-1 cells, while among 110 diaphragmatic areas with no
tumor
cells, only 18.1% displayed PA-1 adherent cells (p<0.0001, OR=49.9), as
indicated in
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Table 2A. If only the areas displaying more than 3 PA-1 cells were considered,
91.1%
of tumor-positive and 6.3% of tumor-negative microscopic fields had PA-1 cells
bound
thereto (p<0.0001, OR--158.4). Similar results were obtained analyzing lateral
or
ventral parietal peritoneum and mesenteric peritoneum. Among 89 areas euamined
that
harbored SKOV3 tumor, 75.3% had adherent PA-1 cells, while among 241 areas
with
no tumor inspected, only 14.1% had adherent PA-1 cells (p<0.0001, OR--18.6).
In
these tissues, if only the microscopic fields displaying more than 3 PA-1
cells were
considered, 79.8% of tumor-positive and 9.9% of tumor-negative microscopic
fields
displayed PA-1 cells (p<0.0001, OR=36.1). These results are tabulated in Table
2B.
Table 2A
Tumor Cells PresentTumor Cells Not Present
in the Field in the Field
PA-1 Cells Bound82 (91.1%) 20 (18.2%)
to the Field
PA-1 Cells Not 8 (8.9%) 90 (81.8%)
Bound to the
Field
Table
2B
Tumor Cells PresentTumor Cells Not Present
in the Field in the Field
PA-1 Cells Bound67 (75.3%) 34 (14.1%)
to the Field
PA-1 Cells Not 22 (24.7%) 207 (85.9%)
Bound to the
Field
PA-1 Producer Cells Preferential_~v Bind to F,pithelial_ Ova_ria_n Ca'nc"~
SL~~P
The observed differences between PA-1 cell binding to tumor and non-
tumor cells in vivo could have been related to molecular factors controlling
cell-cell
interactions between PA-1 cells and marine mesothelial cells. Alternatively,
these
differences have been related to physical forces governing peritoneal fluid
circulation,
which might direct both SKOV3 tumor cells and PA-1 cells towards the same
peritoneal sites. In addition, these differences have been caused by decreased
affinity
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of PA-1 teratocarcinoma cells to marine tissues as compared to human tissues.
In order
to analyze the interaction of PA-1 cells with human EOC and normal peritoneal
cells,
in vitro adhesion assays were performed to compare adhesion of PA-1
teratocarcinoma
cells to normal human peritoneum mesothelial cells, several EOC cell lines,
and
primary EOC cultures. These experiments indicated that PA-1 teratocarcinoma
cells
displayed significantly higher binding to ovarian cancer cells than to normal
human
mesothelium (p<0.001 for each mesothelial culture versus each ovarian cancer
primary
culture or cell line except A2780). To assess the effect of radiation or HSV
infection
on PA-1 adhesion, adhesion of PA-1 teratocarcinoma cells to different ovarian
cancer
cell lines following radiation was compared with adhesion following infection
with
HSV-1716 and adhesion after both radiation treatment and infection with virus.
These
treatments did not significantly affect PA-1 adhesion to cancer cells or
normal
mesothelial cells.
PA-1 Producer Cells Effectively Deliver HSV-1716 in vivo
In order to assess the suitability of producer cells for delivering
HSV-1716 particles to tumor cells in vivo, direct administration of HSV-1716
(i.e. via
injection of a suspension of virus particles) was compared with administration
of PA-1
cells infected with HSV-1716. One week following the administration of A2780
tumor
cells, SCID mice received a single intraperitoneal administration of either
virus alone
or HSV-infected PA-1 cells. Control animals received medium or non-infected,
irradiated PA-1 cells, respectively. Four weeks later, a significant increase
in tumor
weight was noted in control animals (20 f 2.5 grams; n=20) and in animals
which
received non-infected, irradiated PA-1 cells (22.3 t 3 grams; n=20), relative
to
pre-treatment animals (1.9 ~ 0.1 grams; n=5; p<0.001 for both). Administration
of
HSV-1716 alone (n=20) allowed tumors to grow slightly, as evidenced by a small
increase in tumor weight (3.4 t 0.2 grams), relative to pre-treatment animals
(p<0.05).
Injection of PA-1 cells infected with HSV-1716 (n=20) inhibited tumor growth (
tumor
weight = 2 t 0.2 grams), relative to pre-treatment animals (p=0.198). Tumors
in
HSV-treated animals and in those treated with HSV-infected producer cells were
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significantly smaller than those of their control (p<0.01 and p<0.001,
respectively).
Tumors in animals treated with HSV-infected producer cells were significantly
smaller
than those in animals which were treated with HSV-1716 alone (p<0.05).
Similar results were obtained in experiments performed using SKOV3
cells injected intraperitoneally (5x106 cells per animal) into SCID mice, as
indicated in
Table 3. One week following injection of tumor cells, control animals received
medium (group 1, n=20) or irradiated, non-infected PA-1 cells (group 2, n=20).
Virus-
treated animals received a single int<aperitoneal injection of virus (group 3,
n=20) or
HSV-infected PA-1 producer cells (group 4, n=20), respectively (Table 3).
Animats
from each group were sacrificed at 4 and 7 weeks following treatment. Control
animals from group 1 (n=10) exhibited a 6-fold increase in tumor weight
(p<0.01 ) and
extensive tumor spread at 4 weeks (tumor score 15.5/16), relative to pre-
treaxcnent
animals sacrificed at 1 week (n=5; tumor score 2.5/16). A 10-fold increase in
tumor
weight (p<0.001 versus pre-treatment) and further tumor spread (tumor score
16/16)
was noted at 7 weeks (n=10). Control animals from group 2 (n--10) exhibited a
6.5-fold increase in tumor weight (p<0.001 versus pre-treatment) and extensive
tumor
spread (tumor score 15.9/16) at 4 weeks. A 12-fold increase in tumor weight
(p<0.0p1
versus pre-treatment) and diffuse tumor spread (score 16/16) were noted at 7
weeks
(n=10) in these animals. There was no significant difference in tumor growth
or tumor
spread between the control groups receiving media or irradiated PA-1 cells.
Table 3
Tumor Weight, Milligrams
Pre- Treatment
TreatmentCrroup Number4 Weeks Post Treatment7 Weeks Post-Treatment
1 1.225 t 0.09 2.256 ~ 0.06
0.214 2 1.412 f 0.1 2.49410.16
f
0.032 3 0.225 t 0.003 0.380 ~ 0:06
4 0.185 ~ 0.04 0.13110.09
_ ,4p _
S~ST'ITUTE SHEET (RULE 26)
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Animals treated with HSV-1716 (group 3, n=10) exhibited significantly
less tumor growth (p<0.001 ) and spread (tumor score 2.5/ 16) at 4 or 7 weeks
than
control group 1. The mice of group 3 exhibited no difference in tumor weight
at 7
weeks, relative to pre-treahnent animals (p=0.61), suggesting stabilization of
disease.
Animals receiving HSV-infected producer cells (group 4) also had significantly
smaller
tumors (p<0.001) and less tumor spread (score 2/16) at 4 and 7 weeks than
their
corresponding controls (group 2). The mice of group 4 exhibited no tumor
progression
at 4 weeks (p=0.43), and significant tumor regression by week 7 (p<0.05 versus
pre-treatment). In addition, the mice of group 4 exhibited a significantly
smaller tumor
burden at 7 weeks than HSV-1716-treated animals (group 3, p<0.05). Remarkably,
in
the group receiving HSV-infected PA-1 producer cells, 20% of the animals
displayed
no detectable EOC, either grossly or under the dissecting microscope at 4
weeks
(n=4/20) or at 7 weeks (n=4/20).
In order to assess the carcinogenic potential of PA-1 producer cells, 6-
to 8-week-old SCID mice (n=10) were injected intraperitoneally with 5 x 106
irradiated
PA-1 cells infected with HSV-1716 at 2 MOI. The mice were observed for 16
weeks
and then sacrificed in order to evaluate the presence of intraperitoneal
tumors. No
microscopic or gross tumor could be detected. In addition, 10 animals were
injected in
the flank with 5 X 106 PA-1 cells, which were prepared in a similar manner.
Similarly,
no tumor was detected over 16 weeks.
Mice
Survival experiments performed using animals bearing SKOV3 tumors
confirmed the oncolytic activity of HSV-1716 against EOC cells. A single dose
of
HSV-1716 was administered four weeks following the injection of tumor cells, a
time
at which there was a sizable intraperitoneal tumor burden. Administration of
HSV-
1716 resulted in approximate doubling of the mean survival time (17 weeks;
n=20),
relative to control animals, which received virus-free medium (9 weeks; n=20;
p<0.001). Moreover, administration of HSV-infected PA-1 cells (n=20), as
described
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herein, slightly extended the mean survival time, relative to the survival
time
corresponding to administration of HSV-1716 (20 weeks; p<0.01 versus HSV-1716
alone).
Similar experiments were performed in xenografted mice bearing A2780
tumors. Injection of a single dose of HSV-1716 four weeks following injection
of
tumor cells, a time at which there was extensive intraperitoneal tumor, led to
doubling
of animal survival (13 weeks, n=20), relative to controls (6 weeks, n=20}.
Administration of PA-1 cells infected with HSV-1716 to such animals did not
further
extend the mean animal survival time ( 13.5 weeks, n=20), relative to
administration of
HSV-1716 alone (p=0.987). However, when moribund animals were sacrificed in
the
treated groups, the amount of intraperitoneal tumor encountered was minimal in
HSV-treated animals (tumor weight 1.9 ~ 0.4 grams; n=4), but even smaller in
animals
which were administered PA-1 cells infected with HSV-1716 (tumor weight 0.9 t
0.4
grams, n=3). These (PA-1/HSV) animals exhibited large tumors at the abdominal
injection site, which were ulcerated and necrotic, suggesting that death might
have
ensued due to infectious or tumor-related complications stemming from the
injection
.site tumors. It is possible that survival would have been improved in the
absence of
these subcutaneous tumors. This was in sharp contrast to the untreated
animals, which
exhibited very bulky intraperitoneal tumors (tumor weight 22.7 t 3.1 grams,
n=4),
most of which were associated with hemorrhagic ascites.
HSV-1716 Penetrates Deeg,~y in Tumor Nodules and Causes Extensive Necrosis in
a
Dose-Dependent Manner
Microscopic examination of control non-treated tumors revealed solid
sheets of tumor tissue having well-established vascularization. Examination of
this
tissue under higher magnification revealed numerous mitoses, indicating rapid
tumor
growth, many capillaries, and no inflammatory infiltrate. On the contrary,
tumors
obtained from animals treated with HSV-1716 or with HSV-infected producer
cells
exhibited extensive necrosis. Examination of these tumor tissues at high power
revealed extensive cell vacuolization, suggesting HSV-induced CPE. Occasional
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polymorphonucleate leukocytes were also visible within the necrotic areas of
the
tumor. Immunohistochemical detection of HSV revealed the presence of virus
within
the necrotic areas, wherein many vacuolized cells stained strongly for HSV.
Cells
located near, but not within, necrotic areas exhibited characteristic signs of
cell death
did not stain positive for the presence of HSV, suggesting operation of a
bystander
killing effect associated with HSV in these cells.
In HSV-treated animals (i.e. those treated with virus alone and those
treated with virus-infected producer cells), the virus was present deep within
solid
tumor nodules. In distinct areas of the tumor nodules, viral spread was
detectable
several weeks following a single intraperitoneal injection.
Immunohistochemical
evaluation of tumors obtained from animals treated with HSV-infected PA-1
cells
indicated that even larger areas of the tumor nodules were subject to viral
infection,
relative to animals treated with HSV-1716 alone. Areas of viral infection were
primarily found in the interface between uninfected tumor and areas of
necrosis,
suggesting that, by replicating, the virus advanced into the tumor leaving
behind an
area of necrosis. In HSV-treated mice, the virus could not be detected in
normal
marine tissues such as liver, pancreas, kidney, adrenal, spleen, small bowel
myenteric
plexus, or brain.
The results of the experiments presented in this Example demonstrate
that intraperitoneal administration of HSV-1716 to tumor-bearing animals
results in
significant reduction or arrest of EOC tumor growth. The effect of a single
intraperitoneal dose to stabilize intraperitoneal disease was noted even with
bulky
tumors and with different cell lines. Those results merely confirm what was
hypothesized in the prior art. However, the results of the experiments
presented in this
Example also demonstrate that administration of producer cells infected with
an
oncolytic virus such as HSV-1716 is at least as, if not more, effective for
killing tumor
cells in an animal and preventing tumor growth and spread. These experiments
therefore represent a significant advance over prior art methods of delivering
oncolytic
viruses to subjects.
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These results also demonstrate that delivery of oncolytic virus using
producer cells resulted in infection of larger areas of intraperitoneal tumors
by the virus
and in deeper penetration of the virus within tumor nodules, relative to virus
administered by direct injection. Without wishing to be bound by any
particular theory
of operation, it is understood that two possible mechanisms may account for
the
improved virus delivery effected by use of producer cells. First, based on the
in vitro
observations described herein, lysis of infected producer cells within the
peritoneal
cavity may have released large amounts of viral particles within the
peritoneal fluid.
Second, since HSV is known to propagate by direct cell-to-cell spread
(Dingwell et al.,
1994, J. Virol. 68:834-845; Weeks et al., 1997, Biochem. Biophys. Res. Common.
235:31-35), producer cells may have promoted direct cell-to-cell viral
infection of
tumor cells by binding to EOC cells. PA-1 cells appeared to adhere
predominantly to
areas of peritoneum that were covered by established tumor and not to close-by
areas of
normal peritoneum. There was a close correlation between the presence of tumor
and
PA-1 cells, as calculated by odds ratios. It is possible that the physical
forces
governing fluid migration within the peritoneal cavity were partially
responsible for
this effect or that species-related differences in the affinity of interaction
might have
influenced these findings. However, the in vitro studies performed using human
normal mesothelial cells demonstrated that PA-1 cells bound predominantly to
EOC
surfaces compared to normal human mesothelium. This suggests that cell-cell
interactions promote adhesion of PA-1 producer cells to tumor cells, and that
such
adhesion promotes transfer of virus from producer cells to tumor cells.
It may be that, by virtue of viral amplification and through direct
adhesion of producer cells to tumor cells, a significantly larger amount of
virus infected
the intraperitoneal tumor cells. Although immune neutralization of HSV was not
an
issue with the SCID model, this may occur in the peritoneal cavity of an
immunocompetent host by complement or neutralizing antibodies, which may be
present in the peritoneal fluid. Indeed, anti-herpes antibodies are highly
prevalent in
the adult population. Use of producer cells to deliver oncolytic virus might
partly
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circumvent this immune barner because cell-to-cell spread is less affected by
neutralizing antibodies than infection by free virus.
~urvivai_ in a Mu_rine ntraneritoneai_ Tumor Model
HSV-1716, a replicating herpes simplex type 1 virus with a mutation in
the gamma 34.5 gene, is non-neurovintlent and has shown efficacy as an
oncolytic
treatment of mesothelioma in immunodeficient mice. To further evaluate its
possible
role in cancer gene therapy, an immunocompetent animal model was investigated.
EJ-62, a ras-mutated marine fibroblast, grows well in the peritoneum of
immunocompetent BALB/c mice and, unlike most marine cells, is sensitive to
herpesvirus infection. The effects of HSV-1716 in this syngeneic
intraperitoneal
model, which resembles mesothelioma and ovarian cancer, were studied, and the
efficacy of both single and multiple virus injections of virus were evaluated.
Use of
producer cells in this model was also investigated.
Producer cells were infected ex vivo with the virus, irradiated, and then
injected intraperitoneally. CeII viability studies were performed on EJ-62 at
varying
MOI values. At an MOI of 1 or greater, greater than 90% cell death, relative
to control
cells, was observed. MOI values as low as 0.01 resulted in approximately 75%
cell
death, relative to control cells.
Established intraperitoneal tumors were treated with virus injections in a
single (4x 106 particles) dose, multiple doses (three doses given every fourth
day), or a
single comparable dose of producer cells. Prolonged survival was observed in
all
treated groups, relative to control cells. The median survival of control
cells was about
30 days. The median survival of cells treated with a single viral injection
was about 62
days. Cells treated with multiple virus injections exhibited >60% survival at
70 days.
Cells treated with a single dose of producer cells exhibited >65% survival at
70 days.
The disclosures of each and every patent, patent application, and
publication cited herein are hereby incorporated herein by reference in their
entirety.
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While this invention has been disclosed with reference to specific
embodiments, it is apparent that other embodiments and variations of this
invention
may be devised by others skilled in the art without departing from the true
spirit and
scope of the invention. The appended claims are intended to be construed to
include all
such embodiments and equivalent variations.
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