U.S. patent application number 11/807915 was filed with the patent office on 2008-01-31 for reovirus for the treatment of cellular proliferative disorders.
This patent application is currently assigned to Oncolytics Biotech Inc.. Invention is credited to Matthew C. Coffey, Patrick W.K. Lee, James Strong.
Application Number | 20080026048 11/807915 |
Document ID | / |
Family ID | 22973731 |
Filed Date | 2008-01-31 |
United States Patent
Application |
20080026048 |
Kind Code |
A1 |
Lee; Patrick W.K. ; et
al. |
January 31, 2008 |
Reovirus for the treatment of cellular proliferative disorders
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.
Inventors: |
Lee; Patrick W.K.; (Halifax,
CA) ; Strong; James; (Alberta, CA) ; Coffey;
Matthew C.; (Calgary, CA) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
Oncolytics Biotech Inc.
Calgary
CA
|
Family ID: |
22973731 |
Appl. No.: |
11/807915 |
Filed: |
May 30, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10916777 |
Aug 11, 2004 |
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11807915 |
May 30, 2007 |
|
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10218452 |
Aug 15, 2002 |
6811775 |
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10916777 |
Aug 11, 2004 |
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09594343 |
Jun 15, 2000 |
6455038 |
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10218452 |
Aug 15, 2002 |
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09256824 |
Feb 24, 1999 |
6136307 |
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09594343 |
Jun 15, 2000 |
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Current U.S.
Class: |
424/450 ;
424/93.2 |
Current CPC
Class: |
A61P 35/02 20180101;
A61P 35/04 20180101; A61K 9/0019 20130101; A61P 35/00 20180101;
C12N 2720/12032 20130101; A61P 31/12 20180101; A61K 35/765
20130101; C12N 2720/12232 20130101 |
Class at
Publication: |
424/450 ;
424/093.2 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 48/00 20060101 A61K048/00 |
Claims
1-25. (canceled)
26. A pharmaceutical composition comprising a recombinant reovirus,
a chemotherapeutic agent and a pharmaceutically acceptable
excipient.
27. The pharmaceutical composition of claim 26, wherein the
recombinant reovirus results from reassortment of two or more
strains of reovirus.
28. The pharmaceutical composition of claim 27, wherein the two or
more strains of reovirus are selected from the group consisting of
strain Dearing, strain Abney, strain Jones and strain Lang.
29. The pharmaceutical composition of claim 26, wherein the
recombinant reovirus results from reassortment of two reoviruses
selected from the group consisting of serotype 1 reoviruses,
serotype 2 reoviruses and serotype 3 reoviruses.
30. The pharmaceutical composition of claim 26, wherein the
recombinant reovirus results from co-infection of mammalian cells
with different subtypes of reovirus.
31. The pharmaceutical composition of claim 26, wherein the
recombinant reovirus is naturally-occurring.
32. The pharmaceutical composition of claim 26, wherein the
recombinant reovirus comprises different subtypes of coat proteins
in the resulting virion capsid.
33. The pharmaceutical composition of claim 26, wherein the
recombinant reovirus is immunoprotected.
34. The pharmaceutical composition of claim 26, wherein the
recombinant reovirus is coated in a liposome or micelle.
35. The pharmaceutical composition of claim 26, wherein the
recombinant reovirus comprises mutated coat proteins.
36. The pharmaceutical composition of claim 26, wherein the
chemotherapeutic agent is not BCNU.
37. The pharmaceutical composition of claim 26, wherein the
chemotherapeutic agent is selected from the group consisting of
5-fluorouracil, mitomycin C, methotrexate, hydroxyurea,
cyclophosphamide, dacarbazine, mitoxantrone, doxorubicin,
epirubicin, herceptin, etopside, pregnasome, carboplatin,
cisplatin, taxol, taxotere, tamoxifen, interferon, an aromatase
inhibitor and an LHRH analog.
38. A pharmaceutical composition comprising a modified reovirus, a
chemotherapeutic agent and a pharmaceutically acceptable
excipient.
39. The pharmaceutical composition of claim 38, wherein the
modified reovirus is chemically or biochemically pretreated with a
protease.
40. The pharmaceutical composition of claim 38, wherein the
modified reovirus is coated in a liposome or micelle.
41. The pharmaceutical composition of claim 38, wherein the
modified reovirus comprises mutated coat proteins.
42. The pharmaceutical composition of claim 38, where the modified
reovirus has reduced immunogenecity as compared to wild type
reovirus.
43. The pharmaceutical composition of claim 38, wherein the outer
capsid of the modified reovirus has been removed.
44. The pharmaceutical composition of claim 38, wherein the
chemotherapeutic agent is not BCNU.
45. The pharmaceutical composition of claim 38, wherein the
chemotherapeutic agent is selected from the group consisting of
5-fluorouracil, mitomycin C, methotrrexate, hydroxyurea,
cyclophosphamide, dacarbazine, mitoxantrone, doxorubicin,
epirubicin, herceptin, etopside, pregnasome, carboplatin,
cisplatin, taxol, taxotere, tamoxifen, inferferon, an aromatase
inhibitor and an LHRH analog.
46. A kit comprising a pharmaceutical composition comprising a
recombinant reovirus, a chemotherapeutic agent and a
pharmaceutically acceptable excipient.
47. The kit of claim 46, wherein the recombinant reovirus results
from reassortment of two or more strains of reovirus.
48. A kit comprising a pharmaceutical composition comprising a
modified reovirus, a chemotherapeutic agent and a pharmaceutically
acceptable excipient.
49. The kit of claim 48, where the modified reovirus has reduced
immunogenecity as compared to wild type reovirus.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation application of U.S.
application Ser. No. 09/594,343 filed Jun. 15, 2000, which is a
continuation application of U.S. application Ser. No. 09/256,824,
now issued U.S. Pat. No. 6,136,307, filed on Feb. 24, 1999. All
applications to which the instant application claims priority are
herein incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The present invention pertains to methods for treating
ras-mediated proliferative disorders in a mammal using
reovirus.
REFERENCES
[0003] The following publications, patent applications and patents
are cited in this application: [0004] U.S. Pat. No. 5,023,252
[0005] Armstrong, G. D. et al. (1984), Virology 138:37; [0006]
Aronheim, A., et al.,(1994) Cell, 78:949-961 [0007] Barbacid, M.,
Annu. Rev. Biochem., 56:779-827 (1987); [0008] Berrozpe, G., et al.
(1994), Int. J. Cancer, 58:185-191 [0009] Bischoff, J. R. and
Samuel, C. E., (1989) Virology, 172:106-115 [0010] Cahill, M. A.,
et al., Curr. Biol., 6:16-19 (1996); [0011] Chandron and Nibert,
"Protease cleavage of teovirus capsid protein mu1 and mu1C is
blocked by alkyl sulfate detergents, yielding a new type of
infectious subvirion particle", J. of Virology 72(1):467-75 (1998
[0012] Chaubert, P. et al. (1994), Am. J. Path. 144:767; Bos, J.
(1989) Cancer Res. 49:4682 [0013] Cuff et al., "Enteric reovirus
infection as a probe to study immunotoxicity of the
gastrointestinal tract" Toxicological Sciences 42(2):99-108 (1998)
[0014] Der, S. D. et al., Proc. Natl. Acad. Sci. USA 94:3279-3283
(1997) [0015] Dudley, D. T. et al., Proc. Natl. Acad. Sci. USA
92:7686-7689 (1995) [0016] 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) [0017]
Fields, B. N. et al. (1996), Fundamental Virology, 3rd Edition,
Lippincott-Raven; [0018] Gentsch, J. R. K. and Pacitti, A. F.
(1985), J. Virol. 56:356; [0019] E. Harlow and D. Lane,
"Antibodies: A laboratory manual", Cold Spring Harbor Laboratory
(1988) [0020] Helbing, C. C. et al., Cancer Res. 57:1255-1258
(1997) [0021] Hu, Y. and Conway, T. W. (1993), J. Interferon Res.,
13:323-328 [0022] Laemmli, U. K., (1970) Nature, 227:680-685 [0023]
Lee. J. M. et al. (1993) PNAS 90:5742-5746; [0024] Lee, P. W. K.
etal. (1981) Virology, 108:134-146 [0025] Levitzki, A. (1994) Eur.
J. Biochem. 226:1; James, P. W., et al. (1994) Oncogene 9:3601;
Bos, J. (1989) Cancer Res. 49:4682 [0026] Lowe. S. W . et al.
(1994) Science, 266:807-810; [0027] Lyon, H., Cell Biology; A
Laboratory Handbook, J. E. Celis, ed., Academic Press. 1994, p. 232
[0028] Mab et al., "The N-terminal quarter of reovirus cell
artachrnent protein sigma 1 possesses intrinsic virion-anchoring
function" Virology 179(1):95-103 (1990) [0029] McRae, M. A. and
Joklik, W. K., (1978) Virology, 89:578-593 [0030] Millis, N E et
al. (1995) Cancer Res. 55:1444; [0031] Mundschau, L. J. and Faller,
D. V., (1992) J. Biol. Chem., 267:23092-23098 [0032] Nagata, L., et
al. ,(1984) Nucleic Acids Res., 12:8699-8710 [0033] Paul R. W. et
al. (1989) Virology 172:382-385 [0034] Raybaud-Diogene. H. et al.
(1997) J. Clin. Oncology, 15(3):1030-1038; [0035] Remington's
Pharmaceutical Sciences, Mace Publishing Company, Philadelphia Pa.
17.sup.th ed. (1985) [0036] Robinson, M. J. and Cobb, M. H., Curr.
Opin. Cell. Biol. 9:180-186 (1997); [0037] Rosen, L. (1960) Am. J.
Hyg. 71:242; [0038] Sabin, A. B. (1959), Science 130:966 [0039]
Samuel, C. E. and Brody, M., (1990) Virology, 176:106-113; [0040]
Smith, R. E. et al., (1969) Virology, 39:791-800 [0041] Stanley, N.
F. (1967) Br. Med. Bull. 23:150 [0042] Strong, J. E. et al.,(1993)
Virology, 197:405-411; [0043] Strong, J. E. and Lee, P. W. K.,
(1996) J. Virol., 70:612-616 [0044] Trimble, W. S. et al. (1986)
Nature, 321:782-784 [0045] 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); [0046] Waters, S. D. et al.,
J. Biol. Chem. 270:20883-20886 (1995) [0047] Wiessmuller, L. and
Wittinghofer, F. (1994), Cellular Signaling 6(3):247-267; [0048]
Wong, H., et al., (1994) Anal. Biochem., 223:251-258 [0049] Yang,
Y. L. et al. EMBO J. 14:6095-6106 (1995) [0050] Yu, D. et al.
(1996) Oncogene 13:1359
[0051] All of the above publications, patent applications and
patents are herein incorporated by reference in their entirety to
the sarne extent as if each individual publication, patent
application or patent was specifically and individually indicated
to be incorporated by reference in its entirety.
State of the Art
[0052] 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 genome that result in the mutation of those
cellular elements that govern the interpretation of cellular
signals, such as potentiation of proto-oncooene activitv 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).
[0053] 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, N E et al. (1995) Cancer Res.
55:1444; Chaubert, P. et al. (1994), Am. J. Path. 144:767; Bos, J.
(1989) Cancer Res. 49:4682). Ras activation is also demonstrated by
upstream mitogenic signaling elements, notably by tyrosine receptor
kinases (RTKs). These upstream elements, if amplified 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).
[0054] 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 tumors
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 antiumor 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.
SUMMARY OF THE INVENTION
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] Also provided is a pharmaceutical composition comprising a
reovirus. a chemotherapeutic agent and a pharmaceutically
acceptable excipient with the proviso that the chemotherapeutic
agent is not BCNU.
[0060] Also provided is a pharmaceutical composition comprising a
modified reovirus and a pharmaceutically acceptable excipient.
[0061] The methods and pharmaceutical compositors 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.
[0062] 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
[0063] FIG. 1 is a depiction of the molecular basis of reovirus
oncolysis, in which the reovirus usurps the host cell Ras signaling
pathway.
[0064] FIG. 2 is a graphic representation of the effects over time
of active (open circles) or inactivated (closed circles) reovirus
serotype 3 (strain Dearing) on tlhe size of murine THC-11 tumors
grown in severe combined immunodeficiency (SCID) mice. The plotted
values represent the mean of the measurements with the standard
error of the mean also shown.
[0065] FIG. 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 measurements with the standard error of the mean also
shown.
[0066] FIG. 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.
[0067] FIG. 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 immunocompetent C3H mice.
[0068] FIG. 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 rumors grown in imnunocompetent C3H mice
previously exposed (open squares) or unexposed (open circles) to
reovirus.
DETAILED DESCRIPTION OF THE INVENTION
[0069] The invention pertains to methods of treating a ras-mediated
proliferative disorder in a mammal, by administering reovirus to
the proliferating cells.
[0070] The name reovirus (Respiratory and enteric orphan 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 130:966). The term "reovirus" refers to all viruses
classified in the reovirus genus.
[0071] Reoviruses are viruses with a double-stranded, segmented RNA
genome. The virions measure 60-80 nm 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.
[0072] 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. el 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).
[0073] 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).
[0074] For mammalian reoviruses, the cell surface recognition
signal is sialic acid (Armstrong, G. D. et al. (1984), Virology
138:37; Gentsch, J. R. K. and Pacitti, A. F. (1985), J. Virol.
56:356; Paul R. W. et al. (1989) Virology 172:382-385) 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.
[0075] 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 rumors were created on
both the right and left hind flanks revealed that reovirus
significantlv 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 directlv
injected with reovirus, indicating that the oncolytic capacity of
the reovirus was systemic as well as local.
[0076] These results indicated that reovirus uses the host cell's
Ras pathway machinery to downregulate PKR and thus reproduce. FIG.
1 depicts the usurpation of the host cell Ras signalling pathway by
reovirus. As shown in FIG. 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-2.alpha.,
and hence the inhibition of viral gene translation. In the case of
EGFR-, Sos-, or ras-transformed cells, the PKR 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.
[0077] The implantation of human tumor cells into SCID mice is
recognized as a well known model system for testing the
effectiveness of various anti-tumor 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.
[0078] 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, pies, non-human primates, and humans. In a
preferred embodiment, the mammal is a human.
[0079] In the methods of the invention, reovirus is administered to
ras-mediated proliferating cells in the individual marnmal.
Representative types of human 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, T3D or
T3A); other strains of reovirus can also be used. In a preferred
embodiment, the reovirus is human reovirus serotype 3, more
preferably 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.
[0080] 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.
[0081] 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 mu1 and mu1C 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 rmay be treated with chymotrypsin
in the presence of micelle forming concentrations of alkyl sulfate
detergents to generate a new infectious subvirion particle.
[0082] 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.
[0083] The reovirus may be modified by incorporation of mutated
coat proteins, such as for example .sigma.1, 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 COS1 cells will result in the
incorporation of the mutated protein into the reovirus virion
particle (Turner and Duncan, "Site directed mutaaenesis of the
C-terminal portion of reovirus protein sigma1: evidence for a
confornation-dependent receptor binding domain" Virology
186(1):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))
[0084] The reovirus is preferably a reovirus modified to reduce or
eliminate an immune reaction to the reovirus. Such modified
reovirus are termed "immunoprotected reovirus". Such modifications
could include packaging of the reovirus in a liposome, a micelle or
other vehicle to mask the reovirus from 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.
[0085] 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 total 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.
[0086] 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 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
proliferative disorder".
[0087] 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 cancerl, 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.
[0088] "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 metastases. The reovirus can also
be administered subcutaneously, intraperitoneally, topically (e.g.,
for melanoma), orally (e.g., for oral or 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).
[0089] 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.
[0090] Immunuocompetent 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
marmnals will result in the lysis of the neoplastic cells.
[0091] 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 immunoprotected, for example, by
protease digestion of the outer capsid or packaging in a
micelle.
[0092] 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 immunotoxicity of the
gastrointestinal tract" Toxicological Sciences 42(2):99-108 (1998))
or alternatively 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 immunoglobulin to the mammal.
[0093] It is contemplated that the reovirus may be administered to
immunocompetent mammals immunized against the reovirus in
conjunction with the administration of anti-antireovirus
antibodies. "Anti-antireovirus 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.
[0094] 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 rumor cells by measuring
the reduction in the size of the tumor in the mammal or the lyvsis
of the tumor cells in vitro.
[0095] A "mammnal 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 been surgically
removed and the mammal is suspected of harboring some residual
tumor cells.
[0096] 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.
[0097] 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.
[0098] 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.
[0099] 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.
[0100] 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.
[0101] 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
compositions are administered by the oral or nasal respiratory
route for local or 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.
[0102] 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. Pat. No. 5,023,252, herein incorporated by
reference. Such patches may be constructed for continuous,
pulsatile, or on demand delivery of pharmaceutical agents.
[0103] Other suitable formulations for use in the present invention
can be found in Remington's Pharmaceutical Sciences.
[0104] 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.
[0105] 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 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 10.sup.15 pfu/kg body
weight, more preferably from about 10.sup.2 pfu/kg body weight to
about 10.sup.13 pfu/kg body weight. For example, for treatment of a
human, approximately 10.sup.2 to 10.sup.17 plaque forming units
(PFU) of reovirus can be used, depending on the type, size and
number of tumors 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.
[0106] 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.
[0107] The compositions are preferably formulated in a unit dosage
form each dosage containing from about 10.sup.2 pfus to about
10.sup.13 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 lto produce the desired therapeutic
effect, in association with a suitable pharmaceutical
excipient.
[0108] It has been found that the reovirus is effective for the
treatment of solid neoplasms in immunocompetent mammals.
Administration of unmodified reovirus directly to the neoplasm
results in oncolysis of the neoplastic cells and, reduction in the
size of the tumor.
[0109] 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.
[0110] It is contemplated that the reovirus may be administered in
conjunction with or in addition to radiation therapy.
[0111] It is further contemplated that the reovirus of the present
invention may be administered 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, milomycin C, methotrexate, hydroxyurea,
cvclophosphamide. dacarbazine, mitoxantrone, 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 tamoxifen and anti-estrogens,
interferons, aromatase inhibitors, progestational agents and LHRH
analogs.
[0112] 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 manmual receives the
reovirus.
[0113] 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 provided for reducing the growth of
metastastic tumors in a mammal comprising administering an
effective amount of a reovirus to the mammal.
Utility
[0114] 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.
[0115] 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
[0116] In the examples below, all temperatures are in degrees
Celsius (unless otherwise indicated) and all percentages are weight
percentages (also unless otherwise indicated).
[0117] In the examples below, the following abbreviations have the
following meanings. If an abbreviation is not defined, it has its
generally accepted meaning:
[0118] .mu.M=micromolar
[0119] mM=millimolar
[0120] M=molar
[0121] ml=milliliter
[0122] .mu.l=microliter
[0123] mg=milligram
[0124] .mu.g=microgram
[0125] PAGE=polyacrylamide gel electrophoresis
[0126] rpm=revolutions per minute
[0127] FBS=fetal bovine serum
[0128] DTT=dithiothrietol
[0129] SDS=sodium dodecyl sulfate
[0130] PBS=phosphate buffered saline
[0131] DMEM=Dulbecco's modified Eagle's medium
[0132] .alpha.-MEM=.alpha.-modified Eagle's medium
[0133] .beta.-ME=.beta.-mercaptoethanol
[0134] MOI=multiplicity of infection
[0135] PFU=plaque forming units
[0136] MAPK=MAP kinase
[0137] phosph-MAPK=phosphorylated-MAP kinase
[0138] HRP=horseradish-peroxidase
[0139] PKR=double-stranded RNA activated protein kinase
[0140] RT-PCR=reverse transcriptase-polymerase chain reaction
[0141] GAPDH=glyceraldehyde-3-phosphate dehydrogenase
[0142] EGFR=epidermal growth. factor receptors
[0143] MEK kinase=mitogen-activated extracellular signal-regulated
kinase
[0144] DMSO=dimethylsulfoxide
[0145] SCID=severe combined immunodeficiency
General Methods
Cells and Virus
[0146] Parental NIH-3T3 and NIH-3T3 cells transfected with the
Harvey-ras (H-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
TNIH#5) 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
.mu.M ZnSO.sub.4. All cell lines were grown in Dulbecco's modified
Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS).
[0147] 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 .mu.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.
[0148] The PKR.sup.+/+ and PKR .degree./.degree. mouse embryo
fibroblasts (MEFs) were obtained from Dr. B. R. G. Williams (the
Cleveland Clinic Foundation) and were grown in .alpha.-MEM
containing fetal bovine serum and antibiotics as previously
described (Yang, Y. L. et al. EMBO J. 14:6095-6106(1995); Der, S.
D. et al., Proc. Nati. Acad. Sci. USA 94:3279-3283 (1997)).
[0149] 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 .beta.-mercaptoethanol
(.beta.-ME) was omitted from the extraction buffer. Reovirus
labelled with [.sup.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.
[0150] Immunofluorescent Analysis of Reovirus Infection
[0151] For the immunofluorescent studies the NIH-3T3, TNIH#5,
H-ras. EJ-ras, 2H1 (+/-ZnSO.sub.4), and THC-11 cells were grown on
coverslips, and infected with reovirus at a multiplicity of
infection (MOI) of .about.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 administration 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
ImmunoChemicals 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, dried and
mounted on slides in 90% glycerol containing 0.1% phenylenediamine,
and viewed with a Zeiss Axiophot microscope on which Carl Zeiss
camera was mounted (the magnification for all pictures was
200.times.).
Detection of MAP Kinase (ERK) Activity
[0152] The PhosphoPlus 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
[0153] Confluent mon olayers of NIH-3T3, TNIH#5, H-ras, EJ-ras, 2H1
(+/-ZnSO.sub.4), and THC-11 cells were infected with reovirus (MOI
.about.10 PFU/cell). At 12 hours postinfection, the media was
replaced with methionine-free DMEM containing 10% dialyzed FBS and
0.1 mCi/ml [.sup.35S]methionine. After further incubation for 36
hours at 37.degree. C., the cells were washed in phosphate-buffered
saline (PBS) and lysed in the same buffer containing 1% Triton
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.degree. C. until use.
Preparation of Cytoplasmic Extracts for in Vitro Kinase Assays
[0154] Confluent monolayers of the various cell lines were grown on
96 well cell culture plates. At the appropriate time postinfectlion
the media was aspirated off and the cells were lysed with a buffer
containing 20 mM HEPES [pH 7.4], 120 mM KCl, 5 mM MgCl.sub.2, 1 mM
dithiothreitol, 0.5% Nonidet P-40, 2 .mu.g/ml leupeptin, and 50
.mu.g/ml aprotinin. The nuclei were then removed by low-speed
centrifugation and the supernatants were stored at -70.degree. C.
until use.
[0155] Cytoplasmic extracts were normalized for protein
concentrations before use by the Bio-Rad protein microassay method.
Each in vitro kinase reaction contained 20 .mu.l of cell extract,
7.5 .mu.l of reaction buffer (20 mM HEPES [pH 7.4], 120 mM KCl, 5
mM MgCl.sub.2, 1 mM dithiothreitol, and 10% glycerol) and 7.0 .mu.l
of ATP mixture (1.0 .mu.Ci[.gamma.-.sup.32P]ATP in 7 .mu.l of
reaction buffer), and was incubated for 30 minutes at 37.degree. 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 Laemmli SDS-sample buffer or were
either precipitated with agarose-poly(I)poly(C) beads or
immunoprecipitated with an anti-PKR antibody.
Agarose Poly(I)Poly (C) Precipitation
[0156] To each in vitro kinase reaction mixture, 30 .mu.l of a 50%
Ag poly(I)poly(C) Type 6 slurry (Pharmacia LKB Biotechnology) was
added, and the mixture was incubated at 4.degree. 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 KCl, 0.1 mM EDTA, 2 mM dithiothreitol, 10% glycerol) at room
temperature and mixed with 2.times. Laemmli SDS sample buffer. The
beads were then boiled for 5 min, and the released proteins were
analyzed by SDS-PAGE.
Polymerase Chain Reaction
[0157] 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 subjected
to RT-PCR (Wong, H. et al., (1994) Anal. Biochem. 223:251-258)
using random hexanucleotide primers (Pharmacia) and RTase
(GIBCO-BRL) according to the manufacturers' protocol. The cDNAs
from the RT-PCR step were then subjected to selective amplification
of reovirus cDNA using appropriate primers that amplify a predicted
116 bp fragment. These primer sequences were derived from the S1
sequence determined previously (Nagata, L. et al., (1984) Nucleic
Acids Res. 12:8699-8710). The GAPDH primers of Wong, H. et al.,
(1994) Anal. Biochem. 223:251-258 were used to amplify a predicted
306 bp GAPDH fragment which served as a PCR and gel loading
control. Selective amplification of the s1 and GAPDH cDNA's was
performed using Taq DNA polymerase (GIBCO-BRL) according to the
manufacturers' protocol using a Perkin Elmer Gene Amp PCR system
9600. PCR was carried out for 28 cycles with each consisting of a
denaturing step for 30 seconds at 97.degree. C., annealing step for
45 seconds at 55.degree. C., and polymerization step for 60 seconds
at 72.degree. 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
[0158] Immunoprecipitation of .sup.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 .sup.32P-labelled cell lysates
with an anti-PKR antibody (from Dr. Michael Mathews, Cold Spring
Harbor) was similarly carried out. Immunoprecipitates were analyzed
by discontinuous SDS-PAGE according to the protocol of Laemmli
(Laemmli, U. K., (1970) Nature, 227:680-685).
Example 1
Activated Intermediates in the Ras Signalling Pathway Augment
Reovirus Infection Efficiency
[0159] 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 al.,(1993) Virology, 197:405-411; Strong, J. E.
and Lee, P. W. K., (1996) J. Virol., 70:612-616).
[0160] 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. ,(l 994) 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.
[0161] Detection of viral proteins was initially carried out using
indirect immunofluorescent microscopy as described above. The
results indicated that whereas the NIH 3T3 cells adopted a
typically flattened, spread-out morphology with marked contact
inhibition, the transformed cells all grew as spindle-shaped cells
with much less contact inhibition. On comparing the uninfected
parental 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
immunofluorescence by 48 hours postinfection (data not shown).
[0162] To demonstrate that viral protein synthesis was more
efficient in the Sos- or Ras-transformed cell lines, cells were
continuously labeled with [.sup.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.
[0163] 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 inimunoprecipitation 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.
[0164] 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).
Example 2
Enhanced Infectibility Conferred by Activated Oncogenes is Not Due
to Long-Term Transformation or the Generalized Transformed State of
the Cell
[0165] 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).
[0166] Cells were either mock-treated or pretreated with 50 .mu.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.
[0167] 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 [.sup.35S]methionine followed
by SDS-PAGE analysis of virus-specific proteins (not shown).
[0168] 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).
[0169] 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-3T3 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 .mu.g/ml)
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.
[0170] A good indicator of activation of the Ras signaling pathway
is the activation of the MAP kinases ERK1 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-Resistant NIH 3T3 Cells
[0171] The step at which reovirus infection is blocked in
nonsusceptible NIH 3T3 cells was also identified. Because virus
binding and virus internalization for nonsusceptible cells were
comparable to those observed for susceptible cells (Strong, J. E.
et al., (1993) Virology, 197:405-411), the transcription of viral
genes was investigated.
[0172] The relative amounts of reovirus S1 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 S1
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
shown). 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.
[0173] 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 kDa Protein is Phosphorylated in Reovirus-Treated NIH 3T3
Cells but not in Reovirus-Infected Transformed Cells
[0174] 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 S1 mRNA
transcripts which have been shown to be potent activators of PKR
(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 a scenario would be that in the case of the
transformed cells, this activation is prevented, allowing viral
protein synthesis to ensue.
[0175] 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.
[0176] 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 wpas 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 .sup.32P-labeled in in
vitro kinase reactions of postinfection lysates of the
Sos-transformed cell lines (data not shown).
[0177] 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 with the enhancement of
viral synthesis. These results therefore eliminated the possibility
that the induction of the 65 kDa phosphoprotein was an NIH
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 Phosphorylation of the 65 kDa Protein Requires Active
Viral Transcription
[0178] 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 NIH 3T3 cells.
For UV-treatment, reovirus was suspended in DMEM to a concentration
of approximately 4.times.10.sup.8 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
[.sup.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 .sup.32P-labeling as before. The results showed that NIH
3T3 cells infected with untreated reovirus produced a prominent 65
kDa .sup.32P-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 requires de novo transcription of the viral genes,
consistent with the identification of the 65 kDa phosphoprotein as
PKR.
Example 6
Identification of the 65 kDa Phosphoprotein as PKR
[0179] To determine whether the 65 kDa phosphoprotein was PKR, a
dsRNA binding experiment was carried out in which poly(I)-poly(c)
agarose beads were added to .sup.32P-labeled lysates, as described
above. After incubation for 30 minutes at 4.degree. C., the beads
were washed, and bound proteins were released and analvzed by
SDS-PAGE. The results showed that the 65 kDa 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 kDa phosphoprotein detected in the
infected H-ras-transformed cell line did not bind to the
Poly(I)-poly(c) agarose. The 65 kDa phosphoprotein was also
immunoprecipitable 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
[0180] 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), J. Interferon Res., 13:323-328). Accordingly, NIH 3T3
cells were exposed to 5 mM 2-aminopurine concurrently with exposure
to reovirus. The cells were labeled with [.sup.35S]methionine from
12 to 48 h postinfection, and lysates were harvested and analyzed
by SDS-PAGE.
[0181] 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 immunoprecipitatino the lysates with an
anti-reoviuus serum. These results demonstrate that PKR
phosphorvlation 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.
[0182] Interferon .beta., known to induce PKR expression, was found
to significantly reduce reovirus replication in Ras-transformed
cells (data not shown).
[0183] 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.sup.+/+ and PKR .degree./.degree. mice (Yang, Y. L. et al. EMBO
J. 14:6095-6106 (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 .degree./.degree. cells than in the PKR.sup.+/+ 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
[0184] Receptor tyrosine Kknases 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. Natt.
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.
[0185] H-Ras-transformed cells were grown to 80% confluency and
infected with reovirus at an MOI 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 .mu.M). The control cells received
an equivalent volume of DMSO. The cells were labeled with
.sup.35S-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.
[0186] 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.
Example 9
In Vivo Oncolytic Capability of Reovirus
[0187] A severe combined immunodeficiency (SCID) host rumor 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 NTH 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.times.10.sup.5 cells in 100 .mu.l of sterile PBS was used. In a
second trial, an injection bolus of 4.8.times.10.sup.6 cells in 100
.mu.l PBS was used. Palpable tumors were evident approximately two
to three weeks post injection.
[0188] Reovirus serotype three (strain Dearing) was injected into
the right-side tumor mass (the "treated tumor mass") in a volume of
20 .mu.l at a concentration of 1.0.times.10.sup.7 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-US-00001 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
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.
[0189] 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
cm.sup.2) were established after twvo weeks. Eight animals were
then given a single intratumoral injection of 1.0.times.10.sup.7
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.
[0190] Results, shown in FIG. 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
[0191] In vivo studies were also carried out using human breast
carcinoma cells in a SCID mouse model. Female SCID mice were
injected with 1.times.10.sup.6 human breast carcinoma MDA-MB468
cells in two subcutaneous sites, overlying both hind flanks.
Palpable tumors were evident approximately two to four weeks post
injection. Undiluted reovirus serotype three (strain Dearing) was
injected into the right side tumor mass in a volume of 20 .mu.l at
a concentration of 1.0.times.10.sup.7 PFU/ml. The following results
were obtained: TABLE-US-00002 TABLE 2 Tumor Mass After Treatment
with Reovirus TREATMENT mean untreated tumor mean treated tumor
mass (left side) mass (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.
[0192] 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
[0193] In view of the in vivo results presented above, the
oncolytic capability observed in murine cells was investigated in
cell lines derived from additional human tumors.
Cells and Virus
[0194] All cell lines were grown in Dulbecco's modified Eagle's
medium (DMEM) containing 10% fetal bovine serum (FBS).
[0195] 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 .beta.-mercaptoethanol
(.beta.-ME) was omitted from the extraction buffer. Reovirus
labelled with [.sup.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
[0196] Confluent monolayers of cells were infected with reovirus
serotype 3 (strain Dearing) at a multiplicity of infection (MOI) of
approximately 40 plaque forming units (PFU) per cell. Pictures were
taken at 36 hour postinfection for both reovirus-infected and
mock-infected cells.
Immunofluorescent Analysis of Reovirus Infection
[0197] For the immunofluorescent studies the cells were grown on
coverslips, and infected with reovirus at a multiplicity of
infection (MOI) of .about.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 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 Axiophot microscope mounted with a Carl Zeiss camera
(magnification for all pictures was 200.times.).
Infection of Cells and Quantitation of Virus
[0198] 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.degree. 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
[0199] Confluent monolayers of cells were infected with reovirus
(MOI .about.10 PFU/cell). At various times postinfection, the media
was replaced with methionine-free DMEM containing 10% dialyzed PBS
and 0.1 mCi/mi [.sup.35S]methionine. After further incubation for 1
hour at 37.degree. C., the cells were washed in phosphate-buffered
saline (PBS) and lysed in the same buffer containing 1% Triton
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.degree. C. until use.
Immunoprecipitation and SDS-PAGE Analysis
[0200] Immunoprecipitation of [.sup.35S]-label led
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). Immunoprecipitates were analyzed by
discontinuous SDS-PAGE according to the protocol of Laemmli
(Laemmli, U. K., (1970) Nature, 227:680-685).
Breast Cancer
[0201] 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-129), 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-MB468 (ATCC deposit
HTB-132), and SK-BR-3 (ATCC deposit HTB-30).
[0202] 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 infectibiiity.
Brain Glioblastoma
[0203] Next a variety of cell lines derived from human brain
glioblastomas was investigated. The cell lines included A-172,
U-118, U-178, U-563, U-251, U-87 and U-373 (cells were a generous
gift from Dr. Wee Yong, University of Calgary).
[0204] 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 [.sup.35S]-methionine labeling of reovirus
proteins.
[0205] 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 [.sup.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.
[0206] 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.times.10.sup.6 cells in 100 .mu.l
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 FIG. 3, demonstrated that treatment of U-87
tumors with a single intratumoral injection of 1.0.times.10.sup.7
PFUs of 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), in comparison with treatment
of tumors with a single intratumoral injection of the same amount
of UV-inactivated reovirus (closed circles, n=5).
[0207] 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 rumor cells was due to direct lysis by the
virus, the same mechanism of cell killing as bv reovirus in
vitro.
[0208] 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.
[0209] 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.
Infectious virus was found to be present in the blood at a
concentration of 1.times.10.sup.5 PFUs/ml (data not shown).
[0210] 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.times.0.5 cm. The left-side tumors were then injected with a
single dose (1.times.10.sup.7 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.
[0211] Results, shown in FIG. 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 reovirus-treated animals (closed circles and
squares). Subsequent imnmunofluorescent 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
[0212] 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-1687), MIAPACA-2 (ATCC deposit CRL-1420), PANC-1 (ATCC deposit
CRL-1469), AsPC-1 (ATCC deposit CRL-1682) and Hs766T (ATCC deposit
HTB-134).
[0213] Five of these six cell lines demonstrated susceptibility to
reovirus infection including Capan-1, MIAPACA-2, PANC-1, AsPC-1 and
Hs766T, whereas BxPC3 demonstrated little infectability as assayed
by virus-induced cytopathological effects, immunofluorescence and
[.sup.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-1, MIAPACA-2, PANC-1 and AsPC-1) whereas the one lacking
such susceptibility (BxPC3) has been shown to lack such a mutation
(Berrozpe, G., et al. (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
[0214] 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.times.10.sup.6 PFUs ras-transformned 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.times.10.sup.8
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.times.10.sup.7 PFUs) every second
day. Mock-treated animals received an equivalent amount of
UV-inactivated virus.
[0215] FIG. 5 demonstrates that rebvirus 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.
[0216] To assess the effects of previous reovirus exposure on tumor
repression and regression, one-half of a test group was challenged
with reovirus (intramuscular injection of 1.0.times.10.sup.8 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.
[0217] FIG. 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.
[0218] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims.
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