Canadian Patents Database / Patent 2615684 Summary

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(12) Patent: (11) CA 2615684
(54) English Title: REOVIRUS FOR THE TREATMENT OF CELLULAR PROLIFERATIVE DISORDERS
(54) French Title: REOVIRUS POUR LE TRAITEMENT DE TROUBLES CELLULAIRES PROLIFERANTS
(51) International Patent Classification (IPC):
  • A61K 35/765 (2015.01)
  • A61K 31/664 (2006.01)
  • A61P 35/00 (2006.01)
  • C12N 7/00 (2006.01)
(72) Inventors :
  • LEE, PATRICK W. K. (Canada)
  • STRONG, JAMES (Canada)
  • COFFEY, MATTHEW C. (Canada)
(73) Owners :
  • ONCOLYTICS BIOTECH, INC. (Not Available)
(71) Applicants :
  • ONCOLYTICS BIOTECH INC. (Canada)
(74) Agent: TORYS LLP
(74) Associate agent:
(45) Issued: 2017-02-14
(22) Filed Date: 2000-02-18
(41) Open to Public Inspection: 2000-08-31
Examination requested: 2008-01-22
(30) Availability of licence: N/A
(30) Language of filing: English

(30) Application Priority Data:
Application No. Country/Territory Date
09/256,824 United States of America 1999-02-24

English Abstract

Methods for treating proliferative disorders, by administering reovirus to a Ras-mediated proliferative disorder, are disclosed. The reovirus is administered so that it ultimately directly contacts ras-mediated proliferating cells. Proliferative disorders include but are not limited to neoplasms. Human reovirus, non-human mammalian reovirus, and/or avian reovirus can be used. If the reovirus is human reovirus, serotype 1(e.g., strain Lang), serotype 2 (e.g., strain Jones), serotype 3 (e.g., strain Dearing or strain Abney), as well as other serotypes or strains of reovirus can be used. Combinations of more than one type and/or strain of reovirus can be used, as can reovirus from different species of animal. Either solid neoplasms or hematopoietic neoplasms can be treated.


French Abstract

Des méthodes de traitement de troubles proliférants consistant à administrer un réovirus dans le cas dun trouble proliférant induit par un ras sont décrites. Le réovirus est administré pour quil entre en contact avec les cellules proliférantes induites par un ras. Les troubles proliférants comprennent, entre autres, les néoplasmes. On peut utiliser des réovirus humains, des réovirus de mammifères non humains et/ou des réovirus aviaires. Si le réovirus est un réovirus humain, on peut utiliser le sérotype 1 (p. ex. la souche Lang), le sérotype 2 (p. ex. la souche Jones), le sérotype 3 (p. ex. la souche Dearing ou la souche Abney), ainsi que dautres sérotypes ou souches de réovirus. On peut également utiliser des combinaisons de plus dun type et/ou dune souche de réovirus, ainsi que des réovirus provenant danimaux despèces différentes. Il est possible de traiter soit des néoplasmes solides soit des néoplasmes hématopoïétiques.


Note: Claims are shown in the official language in which they were submitted.

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THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A kit for use in the treatment of a ras-mediated proliferative disorder
comprising a non-
recombinant reovirus and a chemotherapeutic agent, with the proviso that the
chemotherapeutic
agent is not BCNU.
2. The kit of claim 1, wherein the reovirus is an avian reovirus.
3. The kit of claim 1, wherein the reovirus is a mammalian reovirus.
4. The kit of claim 1, wherein the reovirus is a human reovirus.
5. The kit of claim 1, wherein the reovirus is a serotype 1 reovirus, a
serotype 2 reovirus or a
serotype 3 reovirus.
6. The kit of claim 1, wherein the reovirus is a serotype 3 reovirus.
7. The kit of any one of claims 1-6, wherein the chemotherapeutic agent is 5-
fluorouracil,
mitomycin C, methotrexate, hydroxyurea, cyclophosphamide, dacarbazine,
mitoxantrone,
anthracylins, herceptin, etoposide, carboplatin, cisplatin, pregnasome,
taxanes, tamoxifen or
interferon.
8. The kit of any one of claims 6-7, wherein the chemotherapeutic agent is
cyclophosphamide.
9. The kit of any one of claims 1-8, wherein the ras-mediated proliferative
disorder is a
neoplasm.
10. The kit of claim 9, wherein the neoplasm is a solid neoplasm.
11. The kit of claim 9, wherein the neoplasm is lung cancer, prostate
cancer, colorectal
cancer, thyroid cancer, renal cancer, adrenal cancer, liver cancer, pancreatic
cancer, breast
cancer, central nervous system cancer, or peripheral nervous system cancer.
12. The kit of claim 9, wherein the neoplasm is a central nervous system
cancer.

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13. The kit of claim 9, wherein the neoplasm is breast cancer.
14. The kit of claim 9, wherein the neoplasm is a hematopoietic neoplasm.
15. The kit of claim 9, wherein the neoplasm is metastatic.
16. The kit of claim 10, wherein a pharmaceutical composition comprises the
reovirus and
wherein the pharmaceutical composition is formulated for administration by
injection into or
near the solid neoplasm.
17. The kit of any one of claims 1-8, wherein a pharmaceutical composition
comprises the
reovirus and wherein the pharmaceutical composition is formulated for an
administration route
that is intravascular, intrathecal, intravenous, intramuscular, subcutaneous,
intraperitoneal,
topical, oral, rectal, vaginal, nasal, or intratumoral.
18. The kit of any one of claims 1-8, wherein a pharmaceutical composition
comprises the
reovirus and wherein the pharmaceutical composition comprises a dose
sufficient for delivery of
1 to 10 15 plaque forming units of reovirus/kg body weight of a subject.
19. The kit of any one of claims 1-8, wherein the kit comprises a single
dose of a
pharmaceutical composition comprising the reovirus.
20. The kit of any one of claims 1-8, wherein the kit comprises more than
one dose of a
pharmaceutical composition comprising the reovirus.

Note: Descriptions are shown in the official language in which they were submitted.

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FtEOVIRUS FOR THE TREATMENT OF
CELLULAR PROLIFERATIVE DISORDERS '1
CROSS REFERENCE TO RELATED APPLICATION
This application is related to US Patent Nos. 6,136,307 and 6,455,038.
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention pertains to methods for treating ras-mediated
proliferative disorders in a mammal using reovirus.
References
The following publications, patent applications and patents are cited in this
application:
U.S. Patent No. 5,023,252
Armstrong, G.D. et al. (1984), Virology 138:37;
Aronheim, A., et al. ,(1994) Cell, 78:949-961
Barbacid, M., Annu. Rev. Biochem., 56:779-827 (1987);
Berrozpe, G., etal. (1994), Int. Cancer, 58:185-191
Bischoff, J.R. and Samuel, C.E., (1989) Virology, 172:106-115
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Cahill, M.A., et al., Curr. Biol., 6:16-19 (1996);
Chandron and Nibert, "Protease cleavage of reovirus capsid protein mul and
=
mulC is blocked by alkyl sulfate detergents, yielding a new type of infectious

subvirion particle", J. of Virology 72(1):467-75 (1998
Chaubert, P. et al. (1994), Am. J. Path. 144:767; Bos, J. (1989) Cancer Res.
49:4682
Cuff et al., "Enteric reovirus infection as a probe to study inununotoxicity
of the
gastrointestinal tract" Toxicological Sciences 42(2):99-108 (1998)
Der, S.D. etal., Proc. Natl. Acad Sci USA 94:3279-3283 (1997)
Dudley, D.T. etal., Proc. Natl. Acad Sci. USA 92:7686-7689 (1995)
Duncan et al., "Conformational and functional analysis of the C-terminal
globular
head of the reovirus cell attachment protein" Virology 182(2):810-9 (1991)
Fields, B.N. et al. (1996), Fundamental Virology, 3rd Edition, Lippincott-
Raven;
Gentsch, J.R.K. and Pacitti, A.F. (1985), J. Virol. 56:356;
E. Harlow and D. Lane, "Antibodies: A laboratory manual", Cold Spring Harbor
Laboratory (1988)
Helbing, C.C. etal., Cancer Res. 57:1255-1258 (1997)
Hu, Y. and Conway, T.W. (1993), J. Interferon Res., 13:323-328
Laenunli, U.K., (1970) Nature, 227:680-685
Lee. J.M. et al. (1993) PNAS 90:5742-5746;
Lee, P.W.K. etal. (1981) Virology, 108:134-146
Levitzki, A. (1994) Eur. J. Biochem. 226:1; James, P.W., et al. (1994)
Oncogene
9:3601; Bos, J. (1989) Cancer Res. 49:4682
Lowe. S.W. et al. (1994) Science, 266:807-810;
Lyon, H., Cell Biology, A Laboratory Handbook, J.E. Celis, ed., Academic
Press,
1994, p. 232
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Mah et al., "The N-terminal quarter of reovirus cell attachment protein sigma
1
possesses intrinsic virion-anchoring function" Virology 179(1):95-103 (990)
McRae, M.A. and Joklik, W.K., (1978) Virology, 89:578-593
Millis, NE etal. (1995) Cancer Res. 55:14-44;
Mundschau, L.J. and Faller, D.V., (1992) J. Biol. Chem., 267:23092-23098
Nagata, L., et al. ,(1984) Nucleic Acids Res., 12:8699-8710
Paul R.W . et al. (1989) Virology 172:382-385
Raybaud-Diogene. H. et al. (1997) J. Clin. Oncology, /5(3):1030-1038;
Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia
PA 17th ed. (1985)
Robinson, M.J. and Cobb, M.H., Curt-. Opin. Cell. Biol. 9:180-186 (1997);
Rosen, L. (1960) Am. J. Hyg. 71:242;
Sabin, A.B. (1959), Science /30:966
Samuel, C.E. and Brody, M., (1990) Virology, 176:106-113:
Smith, R.E. et at., (1969) Virology, 39:791-800
Stanley, N.F. (1967) Br. Med. Bull. 23:150
Strong, J.E. et al. ,(1993) Virology, /97:405-411;
Strong, J.E. and Lee, P.W.K., (1996) J. Virol., 70:612-616
Trimble, W.S. et al. (1986) Nature, 321:782-784
Turner and Duncan, "Site directed mutagenesis of the C-terminal portion of
reovirus protein sigma1:evidence for a conformation-dependent receptor binding

domain" Virology 186(1):219-27 (1992);
Waters, S.D. etal., J. Biol. Chem. 270:20883-20886 (1995)
Wiessmuller, L. and Wittinghofer, F. (1994), Cellular Signaling 6(3):247-267;
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Wong, H., etal.. (1994) Anal. Biochem., 223:251-258
Yang, Y.L. etal. EMBO J. /4:6095-6106 (1995)
Yu, D. etal. (1996) Oncogene 13:1359
State of the Art
Normal cell proliferation is regulated by a balance between growth-
promoting proto-oncogenes and growth-constraining tumor-suppressor genes.
Tumorigenesis can be caused by genetic alterations to the eenome that result
in the
mutation of those cellular elements that govern the interpretation of cellular

signals. such as potentiation of proto-oncogene activity or inactivation of
tumor
suppression. It is believed that the interpretation of these signals
ultimately
influences the growth and differentiation of a cell, and that
misinterpretation of
these signals can result in neoplastic growth (neoplasia).
Genetic alteration of the proto-oncogene Ras is believed to contribute to
approximately 30% of all human tumors (Wiessmuller, L. and Wittinghofer, F.
(1994), Cellular Signaling 6(3):247-267; Barbacid, M. (1987) A Rev. Biochem.
56, 779-827). The role that Ras plays in the pathogenesis of human tumors is
specific to the type of tumor. Activating mutations in Ras itself are found in
most
types of human malignancies, and are highly represented in pancreatic cancer
(80%), sporadic colorectal carcinomas (40-50%), human lung adenocarcinomas
(15-24%), thyroid tumors (50%) and myeloid leukemia (30%) (Millis, NE etal.
(1995) Cancer Res. 55:1444; Chaubert, P. et al. (1994), Am. J. Path. 144:767;
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Bos, J. (1989) Cancer Res. 49:4682). Ras activation is also demonstrated by
upstream mitogenic signaling elements, notably by tyrosine receptor kiriases
(RTKs). These upstream elements, if amolified or overexpressed, ultimately
result in elevated Ras activity by the signal transduction activity of Ras.
Examples
of this include overexpression of PDGFR in certain forms of glioblastomas, as
well as in c-erbB-2/neu in breast cancer (Levitzki, A. (1994) Eur. J. Biochem.

226:1; James, P.W., et al. (1994) Oncogene 9:3601; Bos, J. (1989) Cancer Res.
49:4682).
Current methods of treatment for neoplasia include surgery, chemotherapy
and radiation. Surgery is typically used as the primary treatment for early
stages
of cancer; however, many rumors cannot be completely removed by surgical
means. In addition, metastatic growth of neoplasms may prevent complete cure
of
cancer by surgery. Chemotherapy involves administration of compounds having
antitumor activity, such as alkylating agents, antimetabolites, and antitumor
antibiotics. The-efficacy of chemotherapy is often limited by severe side
effects,
including nausea and vomiting, bone marrow depression, renal damage. and
central nervous system depression. Radiation therapy relies on the greater
ability
of normal cells, in contrast with neoplastic cells, to repair themselves after

treatment with radiation. Radiotherapy cannot be used to treat many neoplasms,
however, because of the sensitivity of tissue surrounding the tumor. In
addition,
certain tumors have demonstrated resistance to radiotherapy and such may be
dependent on oncogene or anti-oncogene status of the cell (Lee. J.M. et al.
(1993)
PNAS 90:5742-5746; Lowe. S.W. et al. (1994) Science, 266:807-810; Raybaud-
Diogene. H. et al. (1997) J. Clin. Oncology, 15(3):1030-1038). In view of the
drawbacks associated with the current means for treating neoplastic growth,
the
need still exists for improved methods for the treatment of most types of
cancers.
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SUMMARY OF THE INVENTION
The present invention pertains to a method of treating a ras-mediated
proliferative disorder in a mammal selected from dogs, cats, sheep, goats,
cattle, =
horses, pigs, humans and non-human primates, comprising administering to the
proliferating cells an effective amount of one or more reoviruses in the
absence of
BCNU under conditions which result in substantial lysis of the proliferating
cells.
The reovirus may be a mammalian reovirus or an avian reovirus. The reovirus
may be modified such that the outer capsid is removed, the virion is packaged
in a
liposome or micelle or the proteins of the outer capsid have been mutated. The
reovirus can be administered in a single dose or in multiple doses. The
proliferative disorder may be a neoplasm. Both solid and hematopoietic
neoplasms can be targeted.
Also provided is a method of treating a ras-mediated neoplasm in a human,
comprising administering to the neoplasm a reovirus in an amount sufficient to
result in substantial oncolysis of the neoplastic cells. The reovirus may be
administered by injection into or near a solid neoplasm.
Also provided is a method of inhibiting metastasis of a neoplasm in a
mammal, comprising administering to the mammal a reovirus in an amount
sufficient to result in substantial lysis of the neoplastic cells.
Also provided is a method of treating a suspected ras-mediated neoplasm in
a mammal, comprising surgical removal of the substantially all of the neoplasm

and administration of an effective amount of reovirus at or near to the
surgical site
resulting in oncolysis of any remaining neoplastic cells.
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Also provided is a pharmaceutical composition comprising a reovirus, a
chemotherapeutic agent and a pharmaceutically acceptable excipient wi0 the
proviso that the chemotherapeutic agent is not BCNU.
Also provided is a pharmaceutical composition comprising a modified
reovirus and a pharmaceutically acceptable excipient.
The methods and pharmaceutical compositons of the invention provide an
effective means to treat neoplasia, without the side effects associated with
other
forms of cancer therapy. Furthermore, because reovirus is not known to be
associated with disease, any safety concerns associated with deliberate
administration of a virus are minimized.
The foregoing and other objects, features and advantages of the invention
will be apparent from the following more particular description of preferred
embodiments of the invention, as illustrated in the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a depiction of the molecular basis of reovirus oncolysis, in
which the reovirus usurps the host cell Ras signaling pathway.
Figure 2 is a graphic representation of the effects over time of active (open
circles) or inactivated (closed circles) reovirus serotype 3 (strain Dearing)
on the
size of murine THC-11 tumors grown in severe combined irnmunOdeficiency
(SCID) mice. The plotted values represent the mean of the measurements with
the
standard error of the mean also shown.
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Figure 3 is a graphic representation of the effects over time of active (open
circles) or inactivated (closed circles) reovirus serotype 3 (strain Dearing)
on the
size of human glioblastoma U-87 xenografts grown in SCID mice. The plotted
values represent the mean of the measuremer with the standard error of the
mean also shown.
Figure 4 is a graphic representation of the effects over time of active (open
circles, open squares) or inactivated (closed circles, closed squares)
reovirus
serotype 3 (strain Dearing) on the size of injected/treated (open and closed
circles)
or untreated (open and closed squares) bilateral human glioblastoma U-87
xenografts grown in SCID mice. The plotted values represent the mean of the
measurements with the standard error of the mean also shown.
Figure 5 is a graphic representation of the effects over time of active (open
circles) or inactivated (closed circles) reovirus serotype 3 (strain Dearing)
on the
size of C3H transformed cell mouse tumors in irrununocompetent C3H mice.
Figure 6 is a graphic representation of the effects over time of active (open
circles, open squares) or inactivated (closed circles) reovirus serotype 3
(strain
Dearing) on the size of C3H transformed cell mouse tumors grown in
immunocompetent C3H mice previously exposed (open squares) or unexposed
(open circles) to reovirus.
DETAILED DESCRIPTION OF THE INVENTION
The invention pertains to methods of treating a ras-mediated proliferative
disorder in a mammal, by administering reovirus to the proliferating cells.
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The name reovirus (Respiratory and enteric Qrphan virus) is a descriptive
acronym suggesting that these viruses, although not associated with any known
disease state in humans, can be isolated from both the respiratory and enteric

tracts (Sabin, A.B. (1959), Science /30:966). The term "reovirus" refers to
all
viruses classified in the reovirus genus.
Reoviruses are viruses with a double-stranded, segmented RNA genome.
The virions measure 60-80 rim in diameter and possess two concentric capsid
shells, each of which is icosahedral. The genome consists of double-stranded
RNA in 10-12 discrete segments with a total genome size of 16-27 kbp. The
individual RNA segments vary in size. Three distinct but related types of
reovirus
have been recovered from many species. All three types share a common
complement-fixing antigen.
The human reovirus consists of three serotypes: type 1 (strain Lang or
T1L), type 2 (strain Jones, T2J) and type 3 (strain Dearing or strain Abney,
T3D). The three serotypes are easily identifiable on the basis of
neutralization
and hemagglutinin-inhibition assays (Sabin, A.B. (1959), Science 130:966;
Fields,
B.N. et al. (1996), Fundamental Virology. 3rd Edition, Lippincott-Raven;
Rosen,
L. (1960) Am. J. Hyg. 71:242; Stanley, N.F. (1967) Br. Med. Bull. 23:150).
Although reovirus is not known to be associated with any particular
disease, many people have been exposed to reovirus by the time they reach
adulthood (i.e., fewer than 25% in children < 5 years old, to greater than 50%
in
those 20-30 years old (Jackson G.G. and Muldoon R.L. (1973) J. Infect. Dis.
128:811; Stanley N.F. (1974) In: Comparative Diagnosis of Viral Diseases,
edited
by E. Kurstak and K. Kurstak, 385-421, Academic Press, New York)_
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For mammalian reoviruses, the cell surface recognition signal is sialic acid
(Armstrong, G.D. et al. (1984), Virology /38:37; Gentsch, J.R.K. and Pacitti,
A.F. (1985), J. Virol. 56:356; Paul R.W. et at. (1989) Virology /72:382-385)
Due to the ubiquitous nature of sialic acid, reovirus binds efficiently to a
multitude
of cell lines and as such can potentially target many different tissues;
however,
there are significant differences in susceptibility to reovirus infection
between cell
lines.
As described herein, Applicants have discovered that cells which are
resistant to reovirus infection became susceptible to reovirus infection when
transformed by a gene in the Ras pathway. "Resistance" of cells to reovirus
infection indicates that infection of the cells with the virus did not result
in
significant viral production or yield. Cells that are "susceptible" are those
that
demonstrate induction of cytopathic effects, viral protein synthesis, and/or
virus
production. Resistance to reovirus infection was found to be at the level of
gene
translation, rather than at early transcription: while viral transcripts were
produced, virus proteins were not expressed. Without being limited to a
theory, it
is thought that viral gene transcription in resistant cells correlated with
phosphorylation of an approximately 65 kDa cell protein, determined to be
double-stranded RNA-activated protein kinase (PKR), that was not observed in
transformed cells. Phosphorylation of PKR lead to inhibition of translation.
When phosphorylation was suppressed by 2-aminopurine, a known inhibitor of
PKR, drastic enhancement of reovirus protein synthesis occurred in the
untransformed cells. Furthermore, a severe combined immunodeficiency (SCID)
mouse model in which tumors were created on both the right and left hind
flanks
revealed that reovirus significantly reduced tumor size when injected directly
into
the right-side tumor; in addition, significant reduction in tumor size was
also
noted on the left-side tumor which was not directly injected with reovirus,
indicating that the oncolytic capacity of the reovirus was systemic as well as
local.
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These results indicated that reovirus uses the host cell's Ras pathway
machinery to downregulate PKR and thus reproduce. Figure 1 depicts the
usurpation of the host cell Ras signalling pathway by reovirus. As shown in
Figure 1, for both untransformed (reovirus-resistant) and EGFR-, Sos-, or ras-
transformed (reovirus-susceptible) cells, virus binding, internalization,
uncoating,
and early transcription of viral genes all proceed normally. In the case of
untransformed cells, secondary structures on the early viral transcripts
inevitably
trigger the phosphorylation of PKR, thereby activating it, leading to the
phosphorylation of the translation initiation factor eIF-2a, and hence the
inhibition
of viral gene translation. In the case of EGFR-, Sos-, or ras-transformed
cells, the
NCR phosphorylation step is prevented or reversed by Ras or one of its
downstream elements, thereby allowing viral gene translation to ensue. The
action of Ras (or a downstream element) can be mimicked by the use of 2-
aminopurine (2-AP), which promotes viral gene translation (and hence reovirus
infection) in untransformed cells by blocking PKR phosphorylation.
The implantation of human tumor cells into SCID mice is recognized as a
well known model system for testing the effectiveness of various anti-rumor
agents
in humans. It has previously been shown that pharmaceuticals effective against

human tumors implanted into SCID mice are predictive of their effectiveness
against the same tumors in humans.
Based upon these discoveries, Applicants have developed methods for
treating ras-mediated proliferative disorders in mammals. Representative
mammals include dogs, cats, sheep, goats, cattle, horses, pigs, non-human
primates, and humans. In a preferred embodiment, the mammal is a human.
In the methods of the invention, reovirus is administered to ras-mediated
proliferating cells in the individual mammal. Representative types of human
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reovirus that can be used include type 1 (e.g., strain Lang or T1L): type 2
(e.g.,
strain Jones or T2J); and type 3 (e.g., strain Dearing or strain Abney, TAD or

T3A); other strains of reovirus can also be used. In a preferred embodiment,
the
reovirus is human reovirus serotype 3, more; .eferably the reovirus is human
reovirus serotype 3, strain Dearing. Alternatively, the reovirus can be a non-
human mammalian reovirus (e.g., non-human primate reovirus, such as baboon
reovirus; equine; or canine reovirus), or a non-mammalian reovirus (e.g.,
avian
reovirus). A combination of different serotypes and/or different strains of
reovirus, such as reovirus from different species of animal, can be used.
The reovirus may be naturally occurring or modified. The reovirus is
"naturally-occurring": when it can be isolated from a source in nature and has
not
been intentionally modified by humans in the laboratory. For example, the
reovirus can be from a "field source": that is, from a human patient.
The reovirus may be modified but still capable of lytically infecting a
mammalian cell having an active ras pathway. The reovirus may be chemically or
biochemically pretreated (e.g., by treatment with a protease, such as
chymotrypsin
or trypsin) prior to administration to the proliferating cells. Pretreatment
with a
protease removes the outer coat or capsid of the virus and may increase the
infectivity of the virus. The reovirus may be coated in a liposome or micelle
(Chandron and Nibert, "Protease cleavage of reovirus capsid protein mul and
muIC is blocked by alkyl sulfate detergents, yielding a new type of infectious

subvirion particle", J. of Virology 72(1):467-75 (1998)) to reduce or prevent
an
immune response from a mammal which has developed immunity to the reovirus.
For example, the virion may be treated with chymotrypsin in the presence of
micelle forming concentrations of alkyl sulfate detergents to generate a new
infectious subvirion particle.
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The reovirus may be a recombinant reovirus from two or more types of
reoviruses with differing pathogenic phenotypes such that it contains
different
antigenic determinants thereby reducing or preventing an immune response by a
mammal previously exposed to a reovirus subtype. Such recombinant virions can
be generated by co-infection of mammalian cells with different subtypes of
reovirus with the resulting resorting and incorporation of different subtype
coat
proteins into the resulting virion capsids.
The reovirus may be modified by incorporation of mutated coat proteins,
such as for example o I, into the virion outer capsid. The proteins may be
mutated by replacement, insertion or deletion. Replacement includes the
insertion
of different amino acids in place of the native amino acids. Insertions
include the
insertion of additional amino acid residues into the protein at one or more
locations. Deletions include deletions of one or more amino acid residues in
the
protein. Such mutations may be generated by methods known in the art. For
example, oligonucleotide site directed mutagenesis of the gene encoding for
one of
the coat proteins could result in the generation of the desired mutant coat
protein.
Expression of the mutated protein in reovirus infected mammalian cells in
vitro
such as COSI cells will result in the incorporation of the mutated protein
into the
reovirus virion particle (Turner and Duncan, "Site directed mutagenesis of the
C-
terminal portion of reovirus protein sigma!: evidence for a conformation-
dependent receptor binding domain" Virology 186(I):219-27 (1992); Duncan et
al., "Conformational and functional analysis of the C-terminal globular head
of the
reovirus cell attachment protein" Virology 182(2):810-9 (1991); Mah et al.,
"The
N-terminal quarter of reovirus cell attachment protein sigma 1 possesses
intrinsic
virion-anchoring function" Virology 179(1):95-103 (1990))
The reovirus is preferably a reovirus modified to reduce or eliminate an
immune reaction to the reovirus. Such modified reovirus are termed
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"immunoprotected reovirus". Such modifications could include packaging of the
reovirus in a liposome, a micelle or other vehicle to mask the reovirus frolm
the
mammals immune system. Alternatively, the outer capsid of the reovirus virion
particle may be removed since the proteins present in the outer capsid are the
major determinant of the host humoral and cellular responses.
A "proliferative disorder" is any cellular disorder in which the cells
proliferate more rapidly than normal tissue growth. Thus a "proliferating
cell" is
a cell that is proliferating more rapidly than normal cells. The proliferative

disorder, includes but is not limited to neoplasms. A neoplasm is an abnormal
tissue growth, generally forming a distinct mass, that grows by cellular
proliferation more rapidly than normal tissue growth. Neoplasms show partial
or
tot& lack of structural organization and functional coordination with normal
tissue.
These can be broadly classified into three major types. Malignant neoplasms
arising from epithelial structures are called carcinomas, malignant neoplasms
that
originate from connective tissues such as muscle, cartilage, fat or bone are
called
sarcomas and malignant tumors affecting hematopoetic structures (structures
pertaining to the formation of blood cells) including components of the immune

system, are called leukemias and lymphomas. A tumor is the neoplastic growth
of
the disease cancer. As used herein, a "neoplasm", also referred to as a
"tumor",
is intended to encompass hematopoietic neoplasms as well as solid neoplasms.
Other proliferative disorders include, but are not limited to
neurofibromatosis.
At least some of the cells of the proliferative disorder have a mutation in
which the Ras gene (or an element of the Ras signaling pathway) is activated,
either directly (e.g., by an activating mutation in Ras) or indirectly (e.g.,
by
activation of an upstream element in the Ras pathway). Activation of an
upstream
element in the Ras pathway includes, for example, transformation with
epidermal
growth factor receptor (EGFR) or Sos. A proliferative disorder that results,
at
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least in part, by the activation of Ras, an upstream element of Ras, or an
element
in the Ras signalling pathway is referred to herein as a "Ras-mediated
prpliferative
disorder".
One neoplasm that is particularly susceptible to treatment by the methods
of the invention is pancreatic cancer, because of the prevalence of Ras-
mediated
neoplasms associated with pancreatic cancer. Other neoplasms that are
particularly susceptible to treatment by the methods of the invention include
breast
cancer, central nervous system cancer (e.g., neuroblastoma and glioblastoma),
peripheral nervous system cancer, lung cancer, prostate cancer, colorectal
cancer,
thyroid cancer, renal cancer, adrenal cancer, liver cancer, lymphoma and
leukemia. One proliferative disorder that is particularly susceptible to
treatment
by the methods of this invention include neurofibromatosis 1 because of the
activation of the ras pathway.
"Administration to a proliferating cell or neoplasm" indicates that the
reovirus is administered in a manner so that it contacts the proliferating
cells or
cells of the neoplasm (also referred to herein as "neoplastic cells"). The
route by
which the reovirus is administered, as well as the formulation, carrier or
vehicle,
will depend on the location as well as the type of the neoplasm. A wide
variety of
administration routes can be employed. For example, for a solid neoplasm that
is
accessible, the reovirus can be administered by injection directly to the
neoplasm.
For a hematopoietic neoplasm, for example, the reovirus can be administered
intravenously or intravascularly. For neoplasms that are not easily accessible

within the body, such as metastases or brain tumors, the reovirus is
administered
in a manner such that it can be transported systemically through the body of
the
mammal and thereby reach the neoplasm (e.g., intrathecally, intravenously or
intramuscularly). Alternatively, the reovirus can be administered directly to
a
single solid neoplasm, where it then is carried systemically through the body
to
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metastases. The reovirus can also be administered subcutaneously,
intraperitoneally, topically (e.g., for melanoma), orally (e.g., for oral
.tisr
esophageal neoplasm), rectally (e.g., for colorectal neoplasm), vaginally
(e.g., for -
cervical or vaginal neoplasm), nasally or by inhalation spray (e.g., for lung
neoplasm).
Reovirus can be administered systemically to mammals which are immune
compromised or which have not developed immunity to the reovirus epitopes. In
such cases, reovirus administered systemically, i.e. by intraveneous
injection, will
contact the proliferating cells resulting in lysis of the cells.
Irrununocompetent mammals previously exposed to a reovirus subtype may
have developed humoral and/or cellular immunity to that reovirus subtype.
Nevertheless, it has been found that direct injection of the reovirus into a
solid
tumor in immunocompetent mammals will result in the lysis of the neoplastic
cells.
On the other hand, when the reovirus is administered systemically to
immunocompetent mammals, the mammals may produce an immune response to
the reovirus. Such an immune response may be avoided if the reovirus is of a
subtype to which the mammal has not developed immunity, or the reovirus has
been modified as previously described herein such that it is irnmunoprotected.
for
example, by protease digestion of the outer capsid or packaging in a micelle.
Alternatively, it is contemplated that the immunocompetency of the
mammal against the reovirus may be suppressed either by the co-administration
of
pharmaceuticals known in the art to suppress the immune system in general
(Cuff
et al., "Enteric reovirus infection as a probe to study ineununotoxicity of
the
gastrointestinal tract" Toxicological Sciences 42(2):99-108 (1998)) or
alternatively
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the administration of anti-antireovirus antibodies. The humoral immunity of
the
mammal against reovirus may also be temporarily reduced or suppressed by
plasmaphoresis of the mammals blood to remove the anti-reovirus antibodies.
The
humoral immunity of the mammal against reovirus may additionally be
temporarily reduced or suppressed by the intraveneous administration of non-
specific inununoglobulin to the mammal.
It is contemplated that the reovirus may be administered to
inununocompetent mammals immunized against the reovirus in conjunction with
the administration of anti-antireovirus antibodies. "Anti-amireovirus
antibodies"
are antibodies directed against anti-reovirus antibodies. Such antibodies can
be
made by methods known in the art. See for example "Antibodies: A laboratory
manual" E. Harlow and D. Lane, Cold Spring Harbor Laboratory (1988). Such
anti-antireovirus antibodies may be administered prior to, at the same time or

shortly after the administration of the reovirus. Preferably an effective
amount of
the anti-antireovirus antibodies are administered in sufficient time to reduce
or
eliminate an immune response by the mammal to the administered reovirus.
The term "substantial lysis" means at least 10% of the proliferating cells
are lysed, more preferably of at least 50% and most preferably of at least 75%
of
the cells are lysed. The percentage of lysis can be determined for tumor cells
by
measuring the reduction in the size of the rumor in the mammal or the lysis of
the
tumor cells in vitro.
A "mammal suspected of having a proliferative disorder" means that the
mammal may have a proliferative disorder or tumor or has been diagnosed with a

proliferative disorder or tumor or has been previously diagnosed with a
proliferative disorder or tumor, the tumor or substantially all of the tumor
has
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been surgically removed and the mammal is suspected of harboring some residual

tumor cells.
This invention also includes pharmaceutical compositions which contain, as
the active ingredient, one or more of the reoviruses associated with
"pharmaceutically acceptable carriers or excipients". In making the
compositions
of this invention, the active ingredient/reovirus is usually mixed with an
excipient,
diluted by an excipient or enclosed within such a carrier which can be in the
form
of a capsule, sachet, paper or other container. When the pharmaceutically
acceptable excipient serves as a diluent, it can be a solid, semi-solid, or
liquid
material, which acts as a vehicle, carrier or medium for the active
ingredient.
Thus, the compositions can be in the form of tablets, pills, powders,
lozenges,
sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols
(as a
solid or in a liquid medium), ointments containing, for example, up to 10% by
weight of the active compound, soft and hard gelatin capsules, suppositories,
sterile injectable solutions, and sterile packaged powders.
Some examples of suitable excipients include lactose, dextrose, sucrose,
sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,
tragacanth,
gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, sterile water, syrup, and methyl cellulose. The formulations can
additionally include: lubricating agents such as talc, magnesium stearate, and
mineral oil; wetting agents; emulsifying and suspending agents; preserving
agents
such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring
agents. The compositions of the invention can be formulated so as to provide
quick, sustained or delayed release of the active ingredient after
administration to
the patient by employing procedures known in the art.
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For preparing solid compositions such as tablets, the principal active
ingredient/reovirus is mixed with a pharmaceutical excipient to form a solid
preformulation composition containing a homogeneous mixture of a compound of
the present invention. When referring to these preformulation compositions as
homogeneous, it is meant that the active ingredient is dispersed evenly
throughout
the composition so that the composition may be readily subdivided into equally

effective unit dosage forms such as tablets, pills and capsules.
=
The tablets or pills of the present invention may be coated or otherwise
compounded to provide a dosage form affording the advantage of prolonged
action. For example, the tablet or pill can comprise an inner dosage and an
outer
dosage component, the latter being in the form of an envelope over the former.

The two components can be separated by an enteric layer which serves to resist

disintegration in the stomach and permit the inner component to pass intact
into
the duodenum or to be delayed in release. A variety of materials can be used
for
such enteric layers or coatings, such materials including a number of
polymeric
acids and mixtures of polymeric acids with such materials as shellac. cetyl
alcohol. and cellulose acetate.
The liquid forms in which the novel compositions of the present invention
may be incorporated for administration orally or by injection include aqueous
solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored
emulsions with edible oils such as corn oil, cottonseed oil, sesame oil,
coconut oil,
or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
Compositions for inhalation or insufflation include solutions and
suspensions in pharmaceutically acceptable, aqueous or organic solvents, or
mixtures thereof, and powders. The liquid or solid compositions may contain
suitable pharmaceutically acceptable excipients as described herein.
Preferably the
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compositions are administered by the oral or nasal respiratory route for local
or
1
systemic effect. Compositions in preferably pharmaceutically acceptable
solvents
may be nebulized by use of inert gases. Nebulized solutions may be inhaled
directly from the nebulizing device or the nebulizing device may be attached
to a
face mask tent, or intermittent positive pressure breathing machine. Solution,
suspension, or powder compositions may be administered, preferably orally or
nasally, from devices which deliver the formulation in an appropriate manner.
Another preferred formulation employed in the methods of the present
invention employs transdermal delivery devices ("patches"). Such transdermal
patches may be used to provide continuous or discontinuous infusion of the
reovirus of the present invention in controlled amounts. The construction and
use
of transdermal patches for the delivery of pharmaceutical agents is well known
in
the art. See, for example, U.S. Patent 5,023,252,
Such patches may be constructed for continuous, pulsatile, or on
demand delivery of pharmaceutical agents.
Other suitable formulations for use in the present invention can be found in
Remington 's Pharmaceutical Sciences.
The reovirus or the pharmaceutical composition comprising the reovirus
may be packaged into convenient kits providing the necessary materials
packaged
into suitable containers. It is contemplated the kits may also include
chemotherapeutic agents and/or anti-antireovirus antibody.
The reovirus is administered in an amount that is sufficient to treat the
proliferative disorder (e.g., an "effective amount"). A proliferative disorder
is
"treated" when administration of reovirus to the proliferating cells effects
lysis of
the proliferating cells. This may result in a reduction in size of the
neoplasm, or
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in a complete elimination of the neoplasm. The reduction in size of the
neoplasm,
or elimination of the neoplasm, is generally caused by lysis of neoplastic
cells
("oncolysis") by the reovirus. Preferably the effective amount is that amount
able
to inhibit tumor cell growth. Preferably the effective amount is from about
1.0
pfu/kg body weight to about le phi/kg body weight, more preferably from
about 102 pfu/kg body weight to about 10' pfu/kg body weight. For example,
for treatment of a human, approximately 102 to 101 plaque forming units (PFU)
of
reovirus can be used, depending on the type, size and number of rumors
present.
The effective amount will be determined on an individual basis and may be
based.
at least in part, on consideration of the type of reovirus; the chosen route
of
administration; the individual's size, age, gender; the severity of the
patient's
symptoms; the size and other characteristics of the neoplasm; and the like.
The
course of therapy may last from several days to several months or until
diminution
of the disease is achieved.
The reovirus can be administered in a single dose, or multiple doses (i.e.,
more than one dose). The multiple doses can be administered concurrently, or
consecutively (e.g., over a period of days or weeks). The reovirus can also be

administered to more than one neoplasm in the same individual.
The compositions are preferably formulated in a unit dosage form, each
dosage containing from about 102 pfus to about 10'3 pfus of the reovirus. The
term "unit dosage forms" refers to physically discrete units suitable as
unitary
dosages for human subjects and other mammals, each unit containing a
predetermined quantity of reovirus calculated to produce the desired
therapeutic
effect, in association with a suitable pharmaceutical excipient.
It has been found that the reovirus is effective for the treatment of solid
neoplasms in iznmunocompetent mammals. Administration of unmodified reovirus
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directly to the neoplasm results in oncolysis of the neoplastic cells and
reduction in
the size of the tumor.
It is contemplated that the reovirus may be administered in conjunction
with surgery or removal of the neoplasm. Therefore, provided herewith are
methods for the treatment of a solid neoplasm comprising surgical removal of
the
neoplasm and administration of a reovirus at or near to the site of the
neoplasm.
It is contemplated that the reovirus may be administered in conjunction
with or in addition to radiation therapy.
It is further contemplated that the reovirus of the present invention may be
admMistered in conjunction with or in addition to known anticancer compounds
or
chemotherapeutic agents. Chemotherapeutic agents are compounds which may
inhibit the growth of tumors. Such agents, include, but are not limited to, 5-
fluorouracil, mitomycin C, methotrexate, hydroxyurea, cyclophosphamide,
dacarbazine, miroxantrone, anthracyclins (Epirubicin and Doxurubicin),
antibodies
to receptors, such as herceptin, etopside, pregnasome, platinum compounds such
as carboplatin and cisplatin, taxanes such as taxol and taxotere, hormone
therapies
such as tarnoxifen and anti-estrogens, interferons, aromatase inhibitors,
progestational agents and LHRH analogs.
Preferably the reovirus is administered in the absence of 1,3-bis(2-
chloroethyl)-1-nitrosourea (BCNU). For example, the 1,3-bis(2-chloroethyl)-1-
nitrosourea (BCNU) is not administered to the mammal either before, during or
after the mammal receives the reovirus.
The reovirus of the present invention have been found to reduce the growth
of tumors that are metastatic. In an embodiment of the invention, a method is
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provided for reducing the growth of metastastic tumors in a mammal comprising
administering an effective amount of a reovirus to the mammal.
Utility
The reoviruses of the present invention may be used for a variety of
purposes. They may be used in methods for treating ras-mediated proliferative
disorders in a mammal. The reovirus may be used to reduce or eliminate
neoplasms. They may be used in methods for treating metastases. They may be
used in conjunction with known treatments for cancer including surgery,
chemotherapy and radiation.
In order to further illustrate the present invention and advantages thereof,
the following specific examples are given but are not meant to limit the scope
of
the claims in any way.
EXAMPLES
In the examples below, all temperatures are in degrees Celsius (unless
otherwise indicated) and all percentages are weight percentages (also unless
otherwise indicated).
In the examples below, the following abbreviations have the following
.meanings. If an abbreviation is not defined, it has its generally accepted
meaning:
AM= micromolar
rnM = millimolar
molar
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ml = milliliter
microliter
mg = milligram
microgram
PAGE polyacrylamide gel electrophoresis
rpm = revolutions per minute
FBS = fetal bovine serum
DTF = dithiothrietol
SDS = sodium dodecyl sulfate
PBS = phosphate buffered saline
DMEM = Dulbecco's modified Eagle's medium
a-MEM a-modified Eagle's medium
p-mercaptoethanol
MOI = multiplicity of infection
PFU = plaque forming units
MAPK = MAP kinase
phosph-MAPK = phosphorylated-MAP kinase
HRP = horseradish-peroxidase
PKR = double-stranded RNA activated protein kinase
RT-PCR = reverse transcriptase-polymerase chain reaction
GAPDH = glyceraldehyde-3-phosphate dehydrogenase
EGFR = epidermal growth factor receptors
MEK kinase = mitogen-activated extracellular signal-regulated
kinase
DMSO = dimethylsulfoxide
SCID = severe combined immunodeficiency
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General Methods
Cells and Virus
Parental NIH-3T3 and NIH-3T3 cells transfected with the Harvey-ras
ras) and EJ-ras oncogenes were a generous gift of Dr. Douglas Faller (Boston
University School of Medicine). NIH-3T3 cells along with their Sos-transformed
counterparts (designated TNIH115) were a generous gift of Dr. Michael Karin
(University of California, San Diego). Dr. H.-J. Kung (Case Western Reserve
University) kindly donated parental NIH-3T3 cells along with NIH-3T3 cells
transfected with the v-erbB oncogene (designated THC-11). 2H1 cells, a
derivative of the C3H 10T1/2 murine fibroblast line, containing the Harvey-ras
gene under the transcriptional control of the mouse metallothionein-I promoter

were obtained from Dr. Nobumichi Hozumi (Mount Sinai Hospital Research
Institute). These 2H1 cells are conditional ras transformant that express the
H-ras
oncogene in the presence of 50 AM ZnSO4. All cell lines were grown in
Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine
serum (FBS).
The NIH-3T3 tet-myc cells were obtained from Dr. R.N. Johnston
(University of Calgary) and were grown in DMEM containing 10% heat-inactivated
FBS and antibiotics in the presence or absence of 2 g/ml tetracycline
(Helbing,
C.C. et al., Cancer Res. 57:1255-1258 (1997)). In the presence of
tetracycline,
expression of the human c-myc gene is repressed. Removal of tetracycline
results in
the elevation of expression of c-myc by up to 100-fold in these cells, which
also
display a transformed phenotype.
The PKR */ and PKR 'V mouse embryo fibroblasts (MEFs) were obtained
from Dr. B.R.G. Williams (the Cleveland Clinic Foundation) and were grown in a-

MEM containing fetal bovine serum and antibiotics as previously described
(Yang,
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Y.L. etal. EMBO /4:6095-6106 (1995); Der, S.D. et al., Proc. Natl. Acad. Sci.
USA 94:3279-3283 (1997)).
The Dearing strain of reovirus serotype 3 used in these studies was
propagated in suspension cultures of L cells and purified according to Smith
(Smith, R.E. et al., (1969) Virology, 39:791-800) with the exception that (3-
mercaptoethanol (n-ME) was omitted from the extraction buffer. Reovirus
labelled with [35S]methionine was grown and purified as described by McRae and

Joklik (McRae, M.A. and Joklik, W.K., (1978) Virology, 89:578-593). The
particle/PFU ratio for purified reovirus was typically 100/1.
Immunofluorescent analysis of reovirus infection
For the immunaluorescent studies the NIH-3T3, TNIH#5, H-ras, EJ-ras,
2111 (+/- ZnSO4), and THC-11 cells were grown on coverslips, and infected with

reovirus at a multiplicity of infection (MOD of ¨10 PFU cell or mock-infected
by
application of the carrier agent (phosphate-buffered saline, PBS) to the cells
in an
identical fashion as the adminisiration of virus to the cells. At 48 hours
postinfection, cells were fixed in an ethanol/acetic acid (20/1) mixture for 5

minutes, then rehydrated by sequential washes in 75%, 50% and 25% ethanol,
followed by four washes with phosphate-buffered saline (PBS). The fixed and
rehydrated cells were then exposed to the primary antibody (rabbit polyclonal
anti-
reovirus type 3 serum diluted 1/100 in PBS) [antiserum prepared by injection
of
rabbits with reovirus serotype 3 in Freund's complete adjuvant, and subsequent

bleedings] for 2 hours at room temperature. Following three washes with PBS,
the cells were exposed to the secondary antibody [goat anti-rabbit IgG (whole
molecule)-fluorescein isothiocyanate conjugate (FITC) [Sigma IrrununoChemicals
F-0382] diluted 1/100 in PBS containing 10% goat serum and 0.005% Evan's
Blue] for 1 hour at room temperature. Finally, the fixed and treated cells
were
washed three more times with PBS and then once with double-distilled water,
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dried and mounted on slides in 90% glycerol containing 0.1% phenylenediamine,
and viewed
with a Zeiss AxiophotTM microscope on which Carl Zeiss camera was mounted (the

magnification for all pictures was 200X).
Detection of MAP Kinase (ERK) Activity
The PhosphoPlusTM p44/42 MAP kinase (Thr202/Tyr204) Antibody kit (New
England Biolabs) was used for the detection of MAP kinase in cell lysates
according to the
manufacturer's instructions. Briefly, subconfluent monolayer cultures were
lysed with the
recommended SDS-containing sample buffer, and subjected to SDS-PAGE, followed
by
electroblotting onto nitrocellulose paper. The membrane was then probed with
the primary
antibody (anti-total MAPK or anti-phospho-MAPK), followed by the horseradish
peroxidase
(HRP)-conjugated secondary antibody as described in the manufacturer's
instruction manual.
Radiolabelling of reovirus-infected cells and preparation of lysates
Confluent monolayers of NIH-3T3, TNIH#5, H-ras, EJ-ras, 2H1 (+/- ZnSO4), and
THC-11 cells were infected with reovirus (MOI ¨ 10 PFU/cell). At 12 hours
postinfection,
the media was replaced with methionine-free DMEM containing 10% dialyzed FBS
and 0.1
mCi/m1 [35S]methionine. After further incubation for 36 hours at 37 C, the
cells were washed
in phosphate-buffered saline (PBS) and lysed in the same buffer containing 1%
TritonTm X-
100, 0.5% sodium deoxycholate and 1 mM EDTA. The nuclei were then removed by
low
speed centrifugation and the supernatants were stored at -70 C until use.
Preparation of cytoplasmic extracts for in vitro kinase assays
Confluent mono layers of the various cell lines were grown on 96 well cell
culture
plates. At the appropriate time postinfection the media was aspirated off and
the cells were
lysed with a buffer containing 20 mM HEPES [pH 7.4], 120 mM KC1, 5 mM MgC12, 1
mM
dithiothreitol, 0.5% Nonidet P-40, 2 j.ig/m1

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leupeptin, and 50 pg/m1 aprotinin. The nuclei were then removed by low-speed
1
centrifugation and the supernatants were stored at -70 C until use.
Cytoplasmic extracts were normalized for protein concentrations before use
by the Bio-Rad protein microassay method. Each in vitro kinase reaction
contained 20 /.11 of cell extract, 7.5 Al of reaction buffer (20 mM HEPES [pH
7.41,
120 mM KC1, 5 mM MgC12, 1 mM dithiothreitol, and 10% glycerol) and 7.0 1
of ATP mixture (1.0 Ci[y-3213]ATP in 7 p.1 of reaction buffer), and was
incubated
for 30 minutes at 37 C (Mundschau, L.J.,and Faller, D.V., J. Biol. Chem.,
267:23092-23098 (1992)). Immediately after incubation the labelled extracts
were
either boiled in Laerrunii SDS-sample buffer or were either precipitated with
agarose-poly(I)poly(C) beads or immunoprecipitated with an anti-PICR antibody.
Agarose poly(I)poly(C) precipitation
To each in vitro kinase reaction mixture, 30 pi of a 50% Ag poly(Dpoly(C)
Type 6 slurry (Pharmacia LKB Biotechnology) was added, and the mixture was
incubated at 4 C for 1 h. The Ag. poly(I)poly(C) beads with the absorbed,
labelled proteins were then washed four times with wash buffer (20 mM HEPES
[7.5 pH], 90 mM KC1, 0.1 mM EDTA, 2 mM dithiothreitol, 10% glycerol) at
room temperature and mixed with 2X Laemmli SDS sample buffer. The beads
were then boiled for 5 min, and the released proteins were analyzed by SDS-
PAGE.
Polymerase chain reaction
Cells at various times postinfection were harvested and resuspended in ice
cold TNE (10 mM Tris [pH 7.8], 150 mM NaCl, 1 mM EDTA) to which NP-40
was then added to a final concentration of 1%. After 5 minutes, the nuclei
were
pelleted and RNA was extracted from the supernatant using the
phenol:chloroform
procedure. Equal amounts of total cellular RNA from each sample were then
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subjected to RT-PCR (Wong, H. et al., (1994) Anal. Biochem. 223:251-258) using
random
hexanucleotide primers (Pharmacia) and reverse transcriptase (GIBCO-BRL)
according to
the manufacturers' protocol. The cDNA's from the RT-PCR step was then
subjected to
selective amplification of reovirus cDNA sl cDNA using the primer 5'-
AATTCGATTTAGG
TGACACTATAGCTATTGGTCGGATG-3' (SEQ ID NO:1) and 5'-CCCTITTGACAGTGA
TGCTCCGTTATCACTCG-3' (SEQ ID NO:2) that amplify a predicted 116 bp fragment.
These primer sequences were derived from the Si sequence determined previously
(Nagata,
L. et al., (1984) Nucleic Acids Res. 12:8699-8710). The GAPDH primers (Wong,
H. et al.,
(1994) Anal. Biochem. 223:251-258), 5'-CGGAGTCAACGGATTTGGTCGTAT-3' (SEQ ID
NO:3) and 5'-AGCCTTCTCCATGGTGGTGAAGAC-3' (SEQ ID NO:4) were used to
amplify a predicted 306 bp GAPDH fragment which served as a PCR and gel
loading control.
Selective amplification of the sl and GAPDH cDNA's was performed using Taq DNA

polymerase (GIBCO-BRL) according to the manufacturers' protocol using a Perkin
Elmer
Gene AmpTM PCR system 9600. PCR was carried out for 28 cycles with each
consisting of a
denaturing step for 30 seconds at 97 C, annealing step for 45 seconds at 55 C,
and
polymerization step for 60 seconds at 72 C. PCR products were analyzed by
electrophoresis
through an ethidium bromide-impregnated TAE-2% agarose gel and photographed
under
ultra-violet illumination with Polaroid 57 film.
Immunoprecipitation and SDS-PAGE Analysis
Immunoprecipitation of 35S-labelled reovirus-infected cell lysates with anti-
reovirus
serotype 3 serum was carried out as previously described (Lee, P. W. K. et al.
(1981)
Virology, 108:134-146). Immunoprecipitation of 32P-labelled cell lysates with
an anti-PKR
antibody (from Dr. Michael Mathews, Cold Spring Harbor) was similarly carried
out.
Immunoprecipitates were analyzed by

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discontinuous SDS-PAGE according to the protocol of Laeinmli (Laem1ili, U.K.,
(1970) Nature, 227:680-685).
EXAMPLE I. Agivated Intermediates in the Ras Signalling Pathway
Augment Reovirus Infection Efficiency
It was previously shown that 3T3 cells and their derivatives lacking
epidermal growth factor receptors (EGFR) are poorly infectible by reovirus,
whereas the same cells transformed with either EGFR or v-erb B are highly
susceptible as determined by cytopathic effects, viral protein synthesis, and
virus
output (Strong, J.E. et at. ,(1993) Virology, 197:405-411; Strong, J.E. and
Lee,
P.W.K., (1996) J. Virol., 70:612-616).
To determine whether downstream mediators of the EGFR signal
transduction pathway may be involved, a number of different NIH 3T3-derived,
transformed with constitutively activated oncogenes downstream of the EGFR,
were assayed for relative susceptibility to reovirus infection. Of particular
interest
were intermediates in the ras signalling pathway (reviewed by Barbacid, M.,
Annu. Rev. Biochem., 56:779-827 (1987); Cahill, M.A., et al., Curr. Biol.,
6:16-
19 (1996). To investigate the Ras signalling pathway, NIH 3T3 parental cell
lines
and NIH 3T3 lines transfected with activated versions of Sos (Aronheim, A., et

al. ,(1994) Cell, 78:949-961) or ras (Mundschau, L.J. and Faller, D.V., (1992)
J.
Biol. Chem., 267:23092-23098) oncogenes were exposed to reovirus, and their
capacity to promote viral protein synthesis was compared.
Detection of viral proteins was initially carried out using indirect
immunofluorescent microscopy as described above. The results indicated that
whereas the NM 3T3 cells adopted a typically flattened, spread-out morphology
with marked contact inhibition, the transformed cells all grew as spindle-
shaped
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cells with much less contact inhibition. ,On comparing the uninfected p4rental
cell
lines with the various transformed cell lines, it was apparent that the
morphology
of the cells was quite distinct upon transformation. Upon challenge with
reovirus,
it became clear that parental NIH 3T3 line was poorly infectible (<5%),
regardless of the source of the parental NIH 3T3 line. In contrast, the
transfected
cell lines each demonstrated relatively pronounced imrnunofluorescence by 48
hours postinfection (data not shown).
To demonstrate that viral protein synthesis was more efficient in the Sos-
or Ras-transformed cell lines, cells were continuously labeled with [35S]-
methionine from 12 to 48 hr postinfection and the proteins were analyzed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as
described above.
The results showed clearly that the levels of viral protein synthesis were
significantly higher in the Sos- or Ras-transformed cells than in parental NIH
3T3
cells. The identities of the viral bands were confirmed by
itrununoprecipitation of
the labeled proteins with polyclonal anti-reovirus antibodies. Since the
uninfected
NIH 3T3 cells and their transformed counterparts displayed comparable levels
of
cellular protein synthesis and doubling times (data not shown), the observed
difference in the level of viral protein synthesis could not be due to
intrinsic
differences in growth rates or translation efficiencies for these cell lines.
The long-term fate of infected NIH-3T3 cells was followed by passaging
these cells for at least 4 weeks. They grew normally and appeared healthy,
with no
sign of lytic or persistent infection; no virus could be detected in the
medium after
this time (data not shown).
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EXAMPLE 2. Enhanced Infectibility Conferred by Activated OrtFogenes
is
Not Due to Long-term Transformation or the Generalized
Transformed State of the Cell
To determine whether the differences in susceptibility may be the result of
long-term effects of transformation, or the result of the activated oncogene
itself, a
cell line expressing a zinc-inducible cellular Harvey-ras (c-H-ras) gene was
tested
for susceptibility to reovirus infectibility, as described above. These cells,
called
2H1, were derived from the C3H 10T1/2 cell line which is poorly infectible by
reovirus (data not shown), and carry the c-H-ras gene under the control of the
mouse metallothionine-I promoter (Trimble, W.S. et al. (1986) Nature, 321:782-
784).
Cells were either mock-treated or pretreated with 50 M ZnSO, 18 hours
prior to infection or mock-infection (administration of carrier agent),
followed by
indirect immunofluorescent analysis of these cells at 48 hours postinfection
or
mock-infection.
The results (not shown) demonstrated that uninduced cells were poorly
infectible (<5%) whereas those induced for only 18 hours were much more
susceptible (>40%). Enhanced viral protein synthesis in the Zn-induced 2H1
cells was further confirmed by metabolic labeling of the cells with
rSlmethionine
followed by SDS-PAGE analysis of virus-specific proteins (not shown).
Based on these observations, the augmentation of reovirus infection
efficiency in the transformed cells is a direct result of the activated
oncogene
product(s), and not due to other factors such as aneuploidy often associated
with
long-term transformation, or other accumulated mutations that may be acquired
under a chronically transformed state (e.g., p53 or myc activation).
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To show further that susceptibility to reovirus infection is not a result of
transformation per se (i.e., a result of the transformed state of the host
cell), NIH-
313 cells containing a tetracycline-controlled human c-myc gene (tet-myc
cells)
were examined (Helbing, C.C. et aL, Cancer Res. 57:1255-1258 (1997)). These
cells normally are maintained in tetracycline (2 p.g/m1) which represses the
expression of c-myc. Removal of tetracycline under normal growth conditions
(10%
fetal bovine serum) leads to accumulation of the c-Myc protein and the cells
display
a transformed phenotype. We found that these cells were unable to support
virus
growth either in the presence or in the absence of tetracycline (data not
shown),
suggesting that susceptibility to reovirus infection is not due to the general
transformed state of the host cell, but rather requires specific
transformation by
elements of the Ras signaling pathway.
A good indicator of activation of the Ras signaling pathway is the activation
of the MAP kinases ERK I and ERK2 (for a review, see Robinson, M.J. and Cobb,
M.H., Curr. Opin. Cell. Biol. 9:180-186 (1997)). In this regard, it was found
that,
compared with untransformed cells, Ras-transformed cells have a significantly
higher ERK1/2 activity (data not shown). Furthermore, an examination of a
number
of human cancer cell lines has revealed an excellent correlation between the
level of
ERK1/2 activity and susceptibility to reovirus infection (data not shown),
although
ERK1/2 itself does not appear to play any role in it. Mouse L cells and human
HeLa
cells, in which reovirus grows very well, both manifest high ERK1/2 activity
(data
not shown).
EXAMPLE 3. Viral Transcripts are Generated but Not Translated in
,
Reovirus-R.esistant NIH 3T3 Cells
The step at which reovirus infection is blocked in nonsusceptible NIH 3T3
cells was also identified. Because virus binding and virus internalization for
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nonsusceptible cells were comparable to those observed for susceptible cells
(Strong, J.E. et al., (1993) Virology, /97:405-411), the transcription Of
viral
genes was investigated.
The relative amounts of reovirus Si transcripts generated in NIH 3T3 cells
and the Ras-transformed cells during the first 12 hours of infection were
compared
after amplification of these transcripts by polymerase chain reaction (PCR),
as
described above. The results demonstrated that the rates of accumulation of Si

transcripts in the two cell lines were similar, at least up to 12 hours
postinfection.
Similar data were obtained when rates of accumulation of other reovirus
transcripts were compared (data not shov.-n). These results demonstrate that
infection block in nonsusceptible cells is not at the level of transcription
of viral
genes, but rather, at the level of translation of the transcripts.
At later times, the level of viral transcripts present in untransformed
NIH-
3T3 cells decreased significantly whereas transcripts in transformed cells
continued
to accumulate (data not shown). The inability of these transcripts to be
translated in
NIH-3T3 cells probably contributed to their degradation. As expected, the
level of
viral transcripts in infected L cells was at least comparable with that in
infected Ras-
transformed cells (data not shown).
EXAMPLE 4. A 65 IdDa Protein is Phosphorylated in Reovirus-treated
NIH 3T3 Cells but Not in Reovirus-infected Transformed
cf.115
Because viral transcripts were generated, but not translated, in NIH 3T3
cells, it was investigated whether the double-stranded RNA (dsRNA)- activated
protein kinase, PKR, is activated (phosphorylated) in these cells (for
example, by
Si mRNA transcripts which have been shown to be potent activators of PKR
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(Bischoff, J.R. and Samuel, C.E., (1989) Virology, 172:106-115), which in turn

leads to inhibition of translation of viral genes. The corollary of such d
scenario
would be that in the case of the transformed cells, this activation is
prevented,
allowing viral protein synthesis to ensue.
NIH 3T3 cells and v-erbB- or Ras- transformed cells (designated THC-11
and H-ras, respectively) were treated with reovirus (i.e., infected) or mock-
infected (as above), and at 48 hours post treatment, subjected to in vitro
kinase
reactions, followed by autoradiographic analysis as described above.
The results clearly demonstrated that there was a distinct phosphoprotein
migrating at approximately 65 kDa, the expected size of PKR, only in the NIH
3T3 cells and only after exposure to reovirus. This protein was not labeled in
the
lysates of either the uninfected transformed cell lines or the infected
transformed
cell lines. Instead, a protein migrating at approximately 100 kDa was found to
be
labeled in the transformed cell lines after viral infection.' This protein was
absent
in either the preinfection or the postinfection lysates of the NIH 3T3 cell
line, and
was not a reovirus protein because it did not react with an anti-reovirus
serum that
precipitated all reovirus proteins (data not shown). A similar 100 kDa protein
was
also found to be 'P-labeled in in vitro kinase reactions of postinfection
lysates of.
the Sos-transformed cell lines (data not shown).
That intermediates in the Ras signalling pathway were responsible for the
lack of phosphorylation of the 65 kDa protein was further confirmed by the use
of
the 2H1 cells which contain a Zn-inducible Ras oncogene. Uninduced 2H1
cells(relatively resistant to reovirus infection, as shown above), were
capable of
producing the 65 kDa phosphoprotein only after exposure to virus. However,
2H1 cells subjected to Zn-induction of the H-Ras oncogene showed significant
impairment of the production of this phosphoprotein. This impairment coincided
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with the enhancement of viral synthesis. These results therefore eliminated
the
possibility that the induction of the 65 kDa phosphoprotein was an Nth 3T3-
specific event, and clearly established the role of Ras in preventing (or
reversing)
induction of the production of this phosphoprotein. The Zn-induced 2H1 cells
did
not produce the 100 kDa phosphoprotein seen in the infected, chronically
transformed H-Ras cells.
EXAMPLE 5. Induction of Phosphorvlation of the 65 kDa Protein
Requires
Active Viral Transcription
Since production of the 65 kDa phosphoprotein occurred only in cells that
were resistant to reovirus infection, and only after the cells were exposed to
reovirus, it was investigated whether active viral transcription was required
for
production of the 68 kDa phosphoprotein. Reovirus was UV-treated to inactivate

its genome prior to administration of the reovirus to Nil-I 3T3 cells. For UV-
treatment, reovirus was suspended in DMEM to a concentration of approximately
4 x 108 PFU/mL and exposed to short-wave (254 nm) UV light for 20 minutes.
UV-inactivated virus were non-infectious as determined by lack of cytopathic
effects on mouse L-929 fibroblasts and lack of viral protein synthesis by
methods
of [35S]-methionine labelling as previously described. Such UV treatment
abolished viral gene transcription, as analyzed by PCR, and hence viral
infectivity
(data not shown). The cells were then incubated for 48 hours, and lysates were
prepared and subjected to in vitro 3213-labeling as before. The results showed
that
NIH 3T3 cells infected with untreated reovirus produced a prominent 65 kDa
3213-
labelled band not found in uninfected cells. Cells exposed to UV-treated
reovirus
behaved similarly to the uninfected control cells, manifesting little
phosphorylation
of the 65 kDa protein. Thus, induction of the phosphorylation of the 65 kDa
phosphoprotein is not due to dsRNA present in the input reovirus; rather, it
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requires de novo transcription of the viral genes, consistent with the
identification
of the 65 IcDa phosphoprotein as PKR.
EXAMPLE 6. Identification of the 65 kDa Phosphoprotein as PKR
To determine whether the 65 IcDa phosphoprotein was PKR, a dsRNA
binding experiment was carried out in which poly(I)-poly(c) agarose beads were
added to 'P-labeled lysates , as described above. After incubation for 30
minutes
at 4 C, the beads were washed, and bound proteins were released and analyzed
by
SDS-PAGE. The results showed that the 65 IcDa phosphoprotein produced in the
postinfection NIH 3T3 cell lysates was capable of binding to dsRNA: such
binding
is a well-recognized characteristic of PKR. In contrast, the 100 IcDa
phosphoprotein detected in the infected H-ras-transformed cell line did not
bind to
the Poly(I)-poly(c) agarose. The 65 IcDa phosphoprotein was also
itrununoprecipitable with a PKR-specific antibody (provided by Dr. Mike
Mathews, Cold Spring Harbor Laboratory), confirming that it was indeed PKR.
EXAMPLE 7. PKR Inactivation or Deletion Results in enhanced
Infectibility of Untransformed Cells
If PKR phosphorylation is responsible for the shut-off of viral gene
translation in NIH-3T3 cells, and one of the functions of the activated
oncogene
product(s) in the transformed cells is the prevention of this phosphorylation
event,
then inhibition of PKR phosphorylation in NIH-3T3 cells by other means (e.g.
drugs) should result in the enhancement of viral protein synthesis, and hence
infection, in these cells. To test this idea, 2-aminopurine was used: This
drug has
been shown to possess relatively specific inhibitory activity towards PKR
autophosphorylation (Samuel, C.E. and Brody, M., (1990) Virology, 176:106-
113; Hu, Y. and Conway, T.W. (1993),]. Interferon Res., /3:323-328).
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Accordingly, NIH 3T3 cells were exposed to 5 mM 2-aminopurine concurrently
with exposure to reovirus. The cells were labeled with [35S]methioninel from
12 to
48 h postinfection, and lysates were harvested and analyzed by SDS-PAGE.
The results demonstrated that exposure to 2-aminopurine resulted in a
significantly higher level of viral protein synthesis in NIH 3T3 cells (not
shown).
The enhancement was particularly pronounced after immunoprecipitating the
lysates with an anti-reovirus serum. These results demonstrate that PKR
phosphorylation leads to inhibition of viral gene translation, and that
inhibition of
this phosphorylation event releases the translation block. Therefore,
intermediates
in the Ras signalling pathway negatively regulate PKR, leading to enhanced
infectibility of Ras-transformed cells.
Interferon f3, known to induce PKR expression, was found to significantly
reduce reovirus replication in Ras-transformed cells (data not shown).
A more direct approach to defining the role of PKR in reovirus infection is
through the use of cells that are devoid of PKR. Accordingly, primary embryo
fibroblasts from wild-type PKR -r and PKR 0/0 mice (Yang, Y.L. et al. EMBO J.
/4:6095-6106 (1995)) were compared in terms of susceptibility to reovirus
infection.
The results clearly showed that reovirus proteins were synthesized at a
significantly
higher level in the PKR 0/0 cells than in the PKR 'V* cells. These experiments
demonstrated that PKR inactivation or deletion enhanced host cell
susceptibility to
reovirus infection in the same way as does transformation by Ras or elements
of the
Ras signaling pathway, thereby providing strong support of the role of
elements of
the Ras signaling pathway in negatively regulating PKR.
EXAMPLE 8 Inactivation of PKR in Transformed Cells Does Not Involve MEK
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Receptor tyrosine kinases such as EGFRs are known to stimulate the
mitogen-activated or extracellular signal-regulated kinases (ERK1/2) via Ras
(see
Robinson, M.J. and Cobb, M.H., Curr. Opin. Cell. Biol. 9:180-186 (1997)). This

stimulation requires the phosphorylation of ERK1/2 by the mitogen-activated
extracellular signal-regulated kinase, kinase MEK, which itself is activated
(phosphorylated) by Raf, a serine-threonine kinase downstream of Ras. To
determine if MEK activity was required for PKR inactivation in transformed
cells,
the effect of the recently identified MEK inhibitor PD98059 (Dudley, D.T. et
al.,
Proc. Natl. Acad. Sci. USA 92:7686-7689 (1995); Waters, S.D. et al., J. Biol.
Chem.
270:20883-20886 (1995)) on infected Ras-transformed cells was studied.
H-Ras-transformed cells were grown to 80% confluency and infected with
reovirus at an MO1 of approximately 10 p.f.u./cell. PD98059 (Calbiochem),
dissolved in dimethylsulfoxide (DMSO), was applied to the cells at the same
time as
the virus (final concentration of PD98059 was 50 1.1M). The control cells
received
an equivalent volume of DMSO. The cells were labeled with 'S-methionine from
12 to 48 hours post-infection. Lysates were then prepared, immunoprecipitated
with
the polyclonal anti-reovirus serotype 3 serum and analyzed by SDS-PAGE.
The results (data not shown) showed that PD98059, at a concentration that
effectively inhibited ERK1/2 phosphorylation, did not inhibit reovirus protein
synthesis in the transformed cells. On the contrary, PD98059 treatment
consistently
caused a slight enhancement of viral protein synthesis in these cells; the
reason for
this is under investigation. Consistent with the lack of inhibition of viral
protein
synthesis in the presence of PD98059, the PKR in these cells remained
unphosphorylated (data not shown). As expected, PD98059 had no effect on
reovirus-induced PKR phosphorylation in untransformed NIH-3T3 cells (data not
shown). These results indicated that MEK and ERK1/2 are not involved in PKR
activation.
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EXAMPLE 9. In Vivo Oncolytic Capability of Reovirus
A seve-e combined immunodeficiency (SCID) host tumor model was used
to assess the efficacy of utilizing reovirus for tumor reduction. Male and
female
SCID mice (Charles River, Canada) were injected with v-erbB-transformed NIH
3T3 mouse fibroblasts (designated THC-11 cells) in two subcutaneous sites
overlying the hind flanks. In a first trial, an injection bolus of 2.3 X 105
cells in
100 Al of sterile PBS was used. In a second trial, an injection bolus of 4.8 X
106
cells in 100 pl PBS was used. Palpable tumors were evident approximately two
to three weeks post injection.
Reovirus serotype three (strain Dearing) was injected into the right-side
tumor mass (the "treated tumor mass") in a volume of 20 Al at a concentration
of
1.0 X 10 plaque forming units (PFU)/ml. The left-side tumor mass (the
"untreated tumor mass") was left untreated. The mice were observed for a
period
of seven days following injection with reovirus, measurements of tumor size
were
taken every two days using calipers, and weight of tumors was measured after
sacrifice of the animals. All mice were sacrificed on the seventh day. Results
are
shown in Table 1.
Table 1 Tumor Mass after Treatment with Reovirus
Trial 1 (n=8) mean untreated tumor mass 602 mg
mean treated tumor mass 284 mg
Trial 2 (n = 12) mean control tumor mass 1523.5
mg
mean untreated tumor mass 720.9 mg
mean treated tumor mass 228.0 mg
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The treated tumor mass was 47% of that of the untreated tumor mass in trial 1,

and 31.6% of the untreated tumor mass in trial 2. These results indicated that
the
virus-treated tumors were substantially smaller than the untreated tumors, and
that
there may be an additional systemic effect of the virus on the untreated tumor
mass.
Similar experiments were also conducted using unilateral introduction of
tumor cells. SCID mice were injected subcutaneously and unilaterally in the
hind
flank with v-erbB-transformed NIH 3T3 mouse fibroblasts (THC-11 cells).
Palpable
tumors (mean area 0.31 cm2) were established after two weeks. Eight animals
were
then given a single intratumoral injection of 1.0 x 10 PFUs of reovirus
serotype 3
(strain Dearing) in phosphate-buffered saline (PBD). Control tumors (n=10)
were
injected with equivalent amounts of UV-inactivated virus. Tumor growth was
followed for 12 days, during which time no additional reovirus treatment was
administered.
Results, shown in Figure 2; demonstrated that treatment of these tumors with
a single dose of active reovirus (open circles) resulted in dramatic
repression of
tumor growth by the thirteenth day (endpoint), when tumors in the control
animals
injected with a single dose of inactivated reovirus (closed circles) exceeded
the
acceptable tumor burden. This experiment was repeated several times and found
to
be highly reproducible, thus further demonstrating the efficacy of reovirus in
repressing tumor growth.
EXAMPLE 10: In Vivo
Oncolytic Capability of Reovirus Against Human
Breast Cancer-Derived Cell Lines
In vivo studies were also carried out using human breast carcinoma cells in
a SCID mouse model. Female SCID mice were injected with 1 x 106 human
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breast carcinoma MDA-MB468 cells in two subcutaneous sites, overlying both
hind flanks. Palpable rumors were evident approximately two to four 1.veeks
post
injection. Undiluted reovirus serotype three (strain Dearing) was injected
into the
right side tumor mass in a volume of 20 Al at a concentration of 1.0 x 10'
PFU/ml. The following results were obtained:
Table 2 Tumor Mass After Treatment with Reovirus
TREATMENT mean untreated tumor mean treated tumor mass
mass (left side) (right side)
Reovirus (N=8) 29.02 g 38.33 g
Control (N=8) 171.8 g 128.54 g
*Note: One of the control mice died early during the treatment phase. None of
the reovirus-treated mice died.
Although these studies were preliminary, it was clear that the size of the
tumors in the reovirus-treated animals was substantially lower than that in
the
untreated animals. However, the=size of the tumors on the right (treated) side
of
the reovirus-treated animals was slightly larger on average than the left
(untreated)
side. This was unexpected but may be explained by the composition of the mass
being taken up by inflammatory cells with subsequent fibrosis, as well as by
the
fact that these tumors were originally larger on the right side on average
than the
left. The histologic composition of the tumor masses is being investigated.
These
results also support the systemic effect the reovirus has on the size of the
untreated
tumor on the contralateral slide of reovirus injection.
EXAMPLE 11. Susceptibility of Additional Human Tumors to Reovirus
Oncolysis
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In view of the in vivo results presented above, the oncolytic capability
observed in murine cells was investigated in cell lines derived from
addlitional
human tumors.
Cells and Virus
All cell lines were grown in Dulbecco's modified Eagle's medium (DMEM)
containing 10% fetal bovine serum (FBS).
The Dearing strain of reovirus serotype 3 used in these studies was
propagated in suspension cultures of L cells and purified according to Smith
(Smith_
R.E. et al., (1969) Virology, 39:791-800) with the exception that P-
mercaptoethanol
(P-ME) was omitted from the extraction buffer. Reovirus labelled with
[35S]methionine was grown and purified as described by McRae and Joklik
(McRae,
M.A. and Joklik, W.K., (1978) Virology, 89:578-593). The particle/PFU ration
for
purified reovirus was typically 100/1.
Cytopathic effects of reovirus on cells
Confluent monolayers of cells were infected with reovirus serotype 3 (strain
Dearing) at a multiplicity of infection (M01) of approximately 40 plaque
forming
units (PFU) per cell. Pictures were taken at 36 hour postinfection for both
reovirus-
infected and mock-infected cells.
Immunolluorescent analysis of reovirus infection
For the immunofluorescent studies the cells were grown on coverslips, and
infected with reovirus at a multiplicity of infection (M01) of ¨10 PFU/cell or
mock-
infected as described above. At various times postinfection, cells were fixed
in an
ethanol/acetic acid (20/1) mixture for 5 minutes, then rehydrated by
subsequential
washes in 75%, 50% and 25% ethanol, followed by 4 washes with phosphate-
buffered saline (PBS). The fixed and rehydrated cells were then exposed to the
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primary antibody (rabbit polyclonal anti-reovirus type 3 serum diluted 1/100
in PBS) for 2 hr
at room temperature. Following 3 washes with PBS, the cells were exposed to
the secondary
antibody [goat anti-rabbit IgG (whole molecule) fluorescein isothiocyanate
(FITC) conjugate
diluted 1/100 in PBS containing 10% goat serum and 0.005% Evan's Blue
counterstain] for 1
hour at room temperature. Finally, the fixed and treated cells were washed 3
more times with
PBS, followed by 1 wash with double-distilled water, dried and mounted on
slides in 90%
glycerol containing 0.1% phenylenediamine, and viewed with a Zeiss AxiophotTM
microscope mounted with a Carl Zeiss camera (magnification for all pictures
was 200X).
Infection of cells and quantitation of virus
Confluent monolayers of cells grown in 24-well plates were infected with
reovirus at
an estimated multiplicity of 10 PFU/cell. After 1 hour incubation at 37 C, the
monolayers
were washed with warm DMEM-10% FBS, and then incubated in the same medium. At
various times postinfection, a mixture of NP-40 and sodium deoxycholate was
added directly
to the medium on the infected monolayers to final concentrations of 1% and
0.5%,
respectively. The lysates were then harvested and virus yields were determined
by plaque
titration on L-929 cells.
Radiolabelling of Reovirus-Infected Cells and Preparation of Lysates
Confluent monolayers of cells were infected with reovirus (MOI ¨10 PFU/cell).
At
various times postinfection, the media was replaced with methionine-free DMEM
containing
10% dialyzed PBS and 0.1 mCi/m1 [35S]methionine. After further incubation for
1 hour at
37 C, the cells were washed in phosphate-buffered saline (PBS) and lysed in
the same buffer
containing 1% TritonTm X-100, 0.5% sodium deoxycholate and 1 mM EDTA. The
nuclei
were then removed by low speed centrifugation and the supernatants was stored
at 70 C until
use.
Immunoprecipitation and SDS-PAGE analysis

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Immunoprecipitation of [35S]-labelled reovirus-infected cell lysates with anti-

reovirus serotype 3 serum was carried out as previously described (Lee, P.W.K.
et
al. (1981) Virology, 108:134-146). lmmunoprecipitates were analyzed by
discontinuous SDS-PAGE according to the protocol of Laemmli (Laemmli, U.K.,
(1970) Nature, 227:680-685).
Breast Cancer
The c-erbB-2/neu gene encodes a transmembrane protein with extensive
homology to the EGFR that is overexpressed in 20-30% of patients with breast
cancer (Yu, D. et al. (1996) Oncogene 13:1359). Since it has been established
herein that Ras activation, either through point mutations or through
augmented
signaling cascade elements upstream of Ras (including the c-erbB-2/neu
homologue
EGFR) ultimately creates a hospitable environment for reovirus replication, an
array
of cell lines derived from human breast cancers were assayed for reovirus
susceptibility. The cell lines included MDA-MD-435SD (ATCC deposit HTB-I29),
MCF-7 (ATCC deposit HTB-22), T-27-D (ATCC deposit HTB-133), BT-20 (ATCC
deposit HTB-19). HBL-100 (ATCC deposit HTB-124), MDA-MB-468 (ATCC
deposit HTB-I32), and SKBR-3 (ATCC deposit HTB-30).
Based upon induction of cytopathic effects, and viral protein synthesis as
measured by radioactive metabolic labeling and immunofluorescence as described
above, it was found that five out of seven of the tested breast cancers were
susceptible to reovirus infection: MDA-MB-435S, MCF-7, T-27-D, MDA MB-468,
and SKBR-3 were exquisitely sensitive to infection, while BT-20 and HBL-100
demonstrated no infectibility.
Brain Glioblastoma
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Next a variety of cell lines derived from human brain glioblastothas was
investigated. The cell lines included A-172, U-118, U-178, U-563, U-51, U-87
and U-373 (cells were a generous gift from Dr. Wee Yong, University of
Calgary).
Six out of seven glioblastoma cell lines demonstrated susceptibility to
reovirus infection, including U-118, U-178, U-563, U-251. U-87 and U-373,
while
A-172 did not demonstrate any infectibility, as measured by cytopathic
effects,
immunofluorescence and [35S]-methionine labeling of reovirus proteins.
The U-87 glioblastoma cell line was investigated further. To assess the
sensitivity of U-87 cells to reovirus, U-87 cells (obtained from Dr. Wee Yong.
University of Calgary) were grown to 80% confluency and were then challenged
with reovirus at a multiplicity of infection (MOI) of 10. Within a period of
48 hours
there was a dramatic, widespread cytopathic effect (data not shown). To
demonstrate further that the lysis of these cells was due to replication of
reovirus, the
cells were then pulse-labeled with [35S]methionine for three hour periods at
various
times postinfection and proteins were analyzed by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) as described above. The results
(not shown) clearly demonstrated effective reovirus replication within these
cells
with resultant shutoff of host protein synthesis by 24 hours postinfection.
The U-87 cells were also introduced as human tumor xenografts into the hind
flank of 10 SCID mice. U-87 cells were grown in Dulbecco's modified Eagle's
medium containing 10% fetal bovine serum, as described above. Cells were
harvested, washed, and resuspended in sterile PBS; 2.0 x 106 cells in 100 ul
were
injected subcutaneously at a site overlying the hind flank in five- to eight-
week old
male SCID mice (Charles River, Canada). Tumor growth was measured twice
weekly for a period of four weeks. Results, shown in Figure 3, demonstrated
that
treatment of U-87 tumors with a single intratumoral injection of 1.0 x 10 PFUs
of
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live reovirus (open circles, n=5) resulted in drastic repression of tumor
growth,
including tumor regression by the fourth week post-treatment (P=0.008), ill
comparison with treatment of tumors with a single intratumoral injection of
the same
amount of UV-inactivated reovirus (closed n=5).
Hematoxylin/eosin (HE)-staining of the remaining microfoci of the tumors
treated with active virus, performed as described (H. Lyon, Cell Biology A
Laboratory Handbook, J.E. Celis, ed., Academic Press, 1994, p. 232) revealed
that
the remaining tumor mass consisted largely of normal stroma without
appreciable
numbers of viable tumor cells, nor was there any evidence of infiltration of
tumor
cells into the underlying skeletal muscle (data not shown). Necrosis of tumor
cells
was due to direct lysis by the virus, the same mechanism of cell killing as by

reovirus in vitro.
To determine if there was viral spread beyond the tumor mass,
immunofluorescent microscopy using antibodies directed against total reovirus
proteins was conducted as described above, on paraffin sections of the tumor
and
adjoining tissue. It was found that reovirus-specific proteins were confined
to the
tumor mass; no viral staining was detected in the underlying skeletal muscle
(data
not shown). As expected, viral proteins were not present in tumors injected
with the
UV-inactivated virus (data not shown). These results demonstrated that
reovirus
replication in these animals was highly tumor specific with viral
amplification only
in the target U-87 cells.
Since most tumors are highly vascularized, it was likely that some virus
could enter the blood stream following the lysis of the infected tumor cells.
To
determine if there was systemic spread of the virus, blood was harvested from
the
treated and control animals, and serially diluted for subsequent plaque
titration.
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Infectious virus was found to be present in the blood at a concentration of 1
x 105
PFUs/m1 (data not shown).
The high degree of tumor specificity of the virus, combined with systemic
spread, suggested that reovirus could be able to replicate in glioblastoma
tumors
remote from the initially infected tumor, as demonstrated above with regard to
breast
cancer cells_ To verify this hypothesis, SCID mice were implanted bilaterally
with
U-87 human tumor xenografts on sites overlying each hind flank of the animals.

These tumors were allowed to grow until they measured 0.5 x 0.5 cm. The left-
side
tumors were then injected with a single dose (1 x 10 pfu) of reovirus in
treated
animals (n=5); control animals (n=7) were mock-treated with UV-inactivated
virus.
Tumors were again measured twice weekly for a period of four weeks.
Results, shown in Figure 4, demonstrated that inhibition and eventual
regression of both the treated/injected (circles) and untreated tumor masses
(squares)
occurred only in the live reovirus-treated animals (open circles and squares),
in
contrast with the inactivated reovinis-treated animals (closed circles and
squares).
Subsequent immunofluorescent analysis revealed that reovirus proteins were
present
in both the ipsilateral (treated) as well as the contralateral (untreated)
tumor,
indicating that regression on the untreated side was a result of reovirus
oncolysis
(data not shown).
Pancreatic Carcinoma
Cell lines derived from pancreatic cancer were investigated for their
susceptibility to reovirus infection. The cell lines included Capan-1 (ATCC
deposit
HTB-79), BxPC3 (ATCC deposit CRL-I687), MIAPACA-2 (ATCC deposit CRL-
1420), PANC-1 (ATCC deposit CRL-I469), AsPC-I (ATCC deposit CRL-1682)
and Hs766T (ATCC deposit HTB-134).
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Five of these six cell lines demonstrated susceptibility to reovirus infection

including Capan-1, MIAPACA-2, PANC-1, AsPC-I and Hs766T, whereas BxPC3
demonstrated little infectability as assayed by virus-induced cytopathological

effects, inununofluorescence and [35S]-labelling. Interestingly, four of the
five cell
lines demonstrating susceptibility to reovirus oncolysis have been shown to
possess
transforming mutations in codon 12 of the K-ras gene (Capan-I, MIAPACA-2,
PANC-1 and A5PC-1) whereas the one lacking such susceptibility (BxPC3) has
been shown to lack such a mutation (Berrozpe, G., etal. (1994), Int. J.
Cancer,
58:185-191). The status of the other K-ras codons is currently unknown for the
Hs766T cell line.
EXAMPLE 12. Use of Reovirus as an Oncolytic Agent in Immune-
Competent
Animals
A syngeneic mouse model was developed to investigate use of reovirus in
immune-competent animals rather than in SCID mice as described above. C3H
mice (Charles River) were implanted subcutaneously with 1.0 x 106 PFUs ras-
transformed C3H cells (a gift of D: Edwards, University of Calgary). Following

tumor establishment, mice were treated with a series of intratumoral
injections of
either live reovirus (1.0 x 108 PFUs) or UV-inactivated reovirus. Following an

initial series (six injections over a nine-day course), test animals received
a treatment
of dilute reovirus (1.0 x I 0 PFUs) every second day. Mock-treated animals
received an equivalent amount of UV-inactivated virus.
Figure 5 demonstrates that reovirus was an effective oncolytic agent in these
immune competent animals. All of the test animals showed regression of tumors;
5
of the 9 test animals exhibited complete tumor regression after 22 days; a
point at
which the control animals exceeded acceptable tumor burden. Furthermore, there
were no identifiable side effects in the animals treated with reovirus.
SUBSTITUTE SHEET (RULE 26)

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To assess the effects of previous reovirus exposure on tumor repression and
regression, one-half of a test group was challenged with reovirus
(intramtiscular
injection of 1.0 x 108 PFUs, type 3 Dearing) prior to tumor establishment. Two

weeks after challenge, neutralizing antibodies could be detected in all
exposed
animals. Following tumor establishment, animals were treated with a series of
intratumoral injections of either live or UV-inactivated reovirus, as
described above. =
Figure 6 demonstrates that animals with circulating neutralizing antibodies to
reovirus (i.e., those challenged with reovirus prior to tumor establishment)
exhibited
tumor repression and regression similar to those animals in which there was no
prior
exposure to reovirus. Thus, reovirus can serve as an effective oncolytic agent
even
in immune-competent animals with previous exposure to reovirus.
The scope of the claims should not be limited by the preferred embodiments
set forth in the examples, but should be given the broadest interpretation
consistent
with the description as a whole.

A single figure which represents the drawing illustrating the invention.

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Title Date
Forecasted Issue Date 2017-02-14
(22) Filed 2000-02-18
(41) Open to Public Inspection 2000-08-31
Examination Requested 2008-01-22
(45) Issued 2017-02-14
Expired 2020-02-18

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Filing $400.00 2008-01-22
Maintenance Fee - Application - New Act 2 2002-02-18 $100.00 2008-01-22
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Maintenance Fee - Application - New Act 4 2004-02-18 $100.00 2008-01-22
Maintenance Fee - Application - New Act 5 2005-02-18 $200.00 2008-01-22
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Maintenance Fee - Application - New Act 8 2008-02-18 $200.00 2008-01-22
Maintenance Fee - Application - New Act 9 2009-02-18 $200.00 2009-02-09
Maintenance Fee - Application - New Act 10 2010-02-18 $250.00 2010-02-09
Maintenance Fee - Application - New Act 11 2011-02-18 $250.00 2011-01-20
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Maintenance Fee - Application - New Act 13 2013-02-18 $250.00 2013-02-07
Maintenance Fee - Application - New Act 14 2014-02-18 $250.00 2014-01-28
Maintenance Fee - Application - New Act 15 2015-02-18 $450.00 2015-01-22
Maintenance Fee - Application - New Act 16 2016-02-18 $450.00 2016-02-17
Final Fee $300.00 2016-12-30
Maintenance Fee - Application - New Act 17 2017-02-20 $450.00 2017-01-23
Maintenance Fee - Patent - New Act 18 2018-02-19 $450.00 2018-01-24
Maintenance Fee - Patent - New Act 19 2019-02-18 $450.00 2019-02-07
Current owners on record shown in alphabetical order.
Current Owners on Record
ONCOLYTICS BIOTECH, INC.
Past owners on record shown in alphabetical order.
Past Owners on Record
COFFEY, MATTHEW C.
LEE, PATRICK W. K.
STRONG, JAMES
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.

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Abstract 2008-01-22 1 56
Description 2008-01-22 50 2,092
Claims 2008-01-22 3 111
Drawings 2008-01-22 6 89
Representative Drawing 2008-03-10 1 12
Cover Page 2008-04-03 1 45
Description 2010-08-06 50 2,098
Claims 2010-08-06 3 103
Claims 2011-09-22 3 87
Description 2012-10-19 50 2,095
Claims 2013-10-02 3 87
Claims 2014-09-30 3 89
Claims 2015-11-05 2 67
Representative Drawing 2017-01-12 1 10
Cover Page 2017-01-12 1 43
Correspondence 2008-03-27 2 67
Prosecution-Amendment 2011-09-22 7 292
Assignment 2008-01-22 5 159
Correspondence 2008-02-06 1 38
Correspondence 2008-04-03 1 15
Prosecution-Amendment 2008-11-03 2 56
Correspondence 2009-01-13 1 15
Prosecution-Amendment 2009-09-01 1 31
Prosecution-Amendment 2010-02-26 3 120
Prosecution-Amendment 2010-08-06 9 389
Prosecution-Amendment 2011-03-22 3 130
Prosecution-Amendment 2012-10-19 5 259
Prosecution-Amendment 2012-04-19 3 131
Prosecution-Amendment 2013-04-02 3 154
Prosecution-Amendment 2014-03-31 3 123
Prosecution-Amendment 2013-10-02 8 390
Prosecution-Amendment 2014-09-30 6 236
Prosecution-Amendment 2015-05-05 6 384
Prosecution-Amendment 2015-11-05 7 409
Correspondence 2016-12-30 1 38
Assignment 2002-02-08 5 215
Assignment 2002-02-08 5 212
Assignment 2002-02-08 5 212