Note: Descriptions are shown in the official language in which they were submitted.
CA 02422245 2003-03-14
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METHODS FOR THE TREATMENT OF
CELLULAR PROLIFERATIVE DISORDERS
Field of the Invention
[OOOlj The present invention relates to methods of identifying the
susceptibility of cells to
reovirus infection by measuring constitutive mitogen activated protein (MAP)
kinase pathway
signaling. The invention also pertains to methods for using reovirus for the
treatment of cellular
proliferative disorders, and particularly cellular proliferative disorders
wherein the proliferating
cells exhibit constitutive MAP kinase phosphorylation, in mammals. The methods
of the
invention provide for reovirus treatment of mammals to treat proliferative
disorders, which
include tumors in which Ras mutations are not believed to play a significant
role.
References
[0002] The following publications, patent applications, and patents are cited
in this
application:
U.S. Patent No. 5,023,252.
U.S. Patent No. 6,136,307.
U.S. Patent Application Serial No. 09/985,756.
WO 99/08692.
Archer, S.G. et al. ( 1995) Expression of ras p21, p53 and c-erbB-2 in
advanced breast cancer and
response to first line hormonal therapy. Br. J. Cancer 72:1259-66.
Armstrong, G.D. et al. ( 1984) Studies on reovirus receptors of L cells: virus
binding
characteristics and comparison with reovirus receptors of erythrocytes.
Virology 138:37-
48.
Barbacid, M. ( 1987) Ras genes. Annu. Rev. Biochem. 56:779-827.
Baselga, J. et al. ( 1996) Phase II study of weekly intravenous recombinant
humanized anti-
p185HER2 monoclonal antibody in patients with HER2/neu-overexpressing
metastatic
breast cancer. J. Clin. Onc. 14:737-744.
CA 02422245 2003-03-14
PATENT Attorney Docket No. 032775-144
Bos, J.L. ( 1989) Ras oncogenes in human cancer: a review. Cancer Res. 49:4682-
89.
Chandran, K. and Nibert, M.L. ( 1998) Protease cleavage of reovirus capsid
protein mu 1 /mu 1 C is
blocked by alkyl sulfate detergents, yielding a new type of infectious
subvirion particle.
J. Virol. 72:467-75.
Chaubert, P. et al. (1994) K-ras mutations and p53 alterations in neoplastic
and nonneoplastic
lesions associated with longstanding ulcerative colitis. Am. J. Path. 144:767-
75.
Clark, E.A. et al. (1995) Integrins and signal transduction pathways: the road
taken. Science
268:233-239.
Clark, J.W. et al. (1996) Effects of tyrosine kinase inhibitors on the
proliferation of human breast
cancer cell lines and proteins important in the ras signaling pathway. Intl.
J. Cancer
65:186-191.
Cuff, C.F. et al. ( 1998) Enteric reovirus infection as a probe to study
immunotoxicity of the
gastrointestinal tract. Toxicological Sciences 42:99-108.
Di Domenico, M. et al. ( 1996) Estradiol activation of human colon carcinoma-
derived Caco-2
cell growth. Cancer Res. 56:4516-21.
Dokianakis, D.N. et al. (1999) Ras gene activation in malignant cells of human
ovarian
carcinoma peritoneal fluids. Clin. Exp. Metastasis. 17:293-97.
Duncan, R. et al. ( 1991 ) Conformational and functional analysis of the C-
terminal globular head
of the reovirus cell attachment protein. Virology 182:810-19.
Fields, B.N. et al. ( 1996), Fundamental Virology, 3rd Edition, Lippincott-
Raven.
Giman, A.G., Goodman, L.S., Rall, T.W, and Murad, F. (1985) Goodman and
Gilman's The
Pharmacological Basis of Therapeutics, Seventh Edition, MacMillan Press, N.Y.
Harlow, E. and Lane, D. Antibodies: a laboratory manual, Cold Spring Harbor
Laboratory
( 1988).
Harwerth, LM. et al. (1992) Monoclonal antibodies against the extracellular
domain of the erbB-
2 receptor function as partial ligand agonists. J. Biol. Chem. 267:15160-67.
Hudziak et al. (1989) p185HER2 monoclonal antibody has antiproliferative
effects in vitro and
sensitizes human breast tumor cells to tumor necrosis factor. Mol. Cell. Biol.
9:1165-72.
Jackson G.G. and Muldoon R.L. (1973) Viruses causing common respiratory
infection in man.
IV. Reoviruses and Adenoviruses. J. Infect. Dis. 128:81 I-66.
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Jacobs, M.M. et al. (1983) Selenium inhibition of 1,2-dimethylhydrazine-
induced colon
carcinogenesis. Cancer Res. 43:1696-1702.
Janes, P.W. et al. (1994) Activation of the Ras signalling pathway in human
breast cancer cells
overexpressing erbB-2. Oncogene 9:3601-08.
Jardines, L. et al. ( 1993) Neu(c-erbB-2/HER2) and the epidermal growth factor
receptor (EGFR)
in breast cancer. Pathobiology 61:268-82.
Koenders, P.G. et al. ( 1991 ) Epidermal growth factor receptor-negative
tumors are
predominantly confined to the subgroup of estradiol receptor-positive human
primary
breast cancers. Cancer Res. 51:4544-48.
Lee, J.M. et al. (1993) p53 mutations increase resistance to ionizing
radiation. Proc. Nat'1. Acad.
Sci. U.S.A. 90:5742-46.
Lee, P.W. et al. (1981) Characterization of anti-reovirus immunoglobulins
secreted by cloned
hybridoma cell lines. Virology 108:134-46.
Levitzki, A. ( 1994) Signal-transduction therapy. A novel approach to disease
management. Eur.
J. Biochem. 226:1.
Lewin, B., ed. ( 1997) Genes Vl. Oxford University Press and Cell Press, New
York, NY, pp.
1070-81.
Lorentz, O. et al. ( 1999) Downregulation of the colon tumour-suppressor
homeobox gene Cdx-2
by oncogenic Ras. Oncogene. 18:87-92.
Mah, D.C. (1990) The N-terminal quarter of reovirus cell attachment protein
sigma 1 possesses
intrinsic virion-anchoring function. Virology. 179:95-103.
Migliaccio, A. et al. (1996) Tyrosine kinase/p2lras/MAP-kinase pathway
activation by estradiol-
receptor complex in MCF-7 cells. EMBO J. 15:1292-1300.
Migliaccio, A. et al. (1998) Activation of the Src/p2lras/Erk pathway by
progesterone receptor
via cross-talk with estrogen receptor. EMBO J. 17:2008-18.
Mills, N.E. et al. ( 1995) Increased prevalence of K-ras oncogene mutations in
lung
adenocarcinoma. Cancer Res. 55:1444-47.
Nibert, M.L. et al. ( 1995) Reoviruses and their replication in Fields
Virology, 3rd Edition,
Lippencott-Raven Press, pp. 1557-96.
Raybaud-Diogene, H. et al. ( 1997) Markers of radioresistance in squamous cell
carcinomas of
the head and neck: a clinicopathologic and immunohistochemical study. J. Clin.
Oncology I 5:1030-38.
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Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia PA
1Th ed.
( 1985).
Rosen, L. ( 1960) Am. J. Hyg.71:242.
Sabin, A.B. (1959) Science 130:966.
Sebolt-Leopold, J.S. (2000) Development of anticancer drugs targeting the MAP
kinase
pathway. Oncogene 19:6594-99.
Shackney, S.E. et al. ( 1998) Intracellular coexpression of epidermal growth
factor receptor, Her-
2/neu, and p21 ras in human breast cancers: evidence for the existence of
distinctive
patterns of genetic evolution that are common to tumors from different
patients. Clin.
Cancer Res. 4:913-28.
Sherman, M.E. (2000) Theories of endometrial carcinogenesis: a
multidisciplinary approach.
Mod. Pathol. 13:295-308.
Shirasawa, S. et al. ( 1993) Altered growth of human colon cancer cell lines
disrupted at activated
Ki-ras. Science. 260:85-8.
Slamon, D.J. et al. (1989) Studies of the HER-2/neu proto-oncogene in human
breast and ovarian
cancer. Science 244:707-712.
Spandiodos, D.A. ( 1987) Oncogene activation in malignant transformation: a
study of H-ras in
human breast cancer. Anticancer Res. 7:991-996.
Stanley, N.F. ( 1967) Reoviruses. Br. Med. Bull. 23:150-54.
Strong, J.E. et al. ( 1993) The v-erbB oncogene confers enhanced cellular
susceptibility to
reovirus infection. Virology 197:405-411.
Strong, J.E. et al. ( 1998) The molecular basis of viral oncolysis: usurpation
of the Ras signaling
pathway by reovirus. EMBO J. 17:3351-62.
Turner, D.L. et al. ( 1992) Site-directed mutagenesis of the C-terminal
portion of reovirus protein
sigma 1: evidence for a conformation-dependent receptor binding domain.
Virology.
186:2 I 9-27.
Verbeek, B.S. et al. ( 1996) c-Src protein expression is increased in human
breast cancer. An
immunohistochemical and biochemical analysis. J. Path. 180:383-388.
Ward, Y. et al. (2001) Signal pathways which promote invasion and metastasis:
critical and
distinct contributions of extracellular signal-regulated kinase and Ral-
specific guanine
exchange factor pathways. Mol Cell Biol. 21:5958-69.
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Wiesmuller, L. and Wittinghofer, F. (1989) Signal transduction pathways
involving Ras. Mini
review. Cell Signal. 6:247-67.
Zhou, D.J. et al. ( 1989) Proto-oncogene abnormalities in human breast cancer:
c-ERBB-2
amplification does not correlate with recurrence of disease. Oncogene 4:105-
08.
[0(103] All of the publications, patent applications, and patents, cited above
or elsewhere in
this application, are herein incorporated by reference in their entirety to
the same 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
[0004] 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-
oncogene activity or
inactivation of tumor suppression. It is believed that the interpretation of
these signals ultimately
influences the growth and differentiation of a cell, and that
misinterpretation of these signals can
result in neoplastic growth (neoplasia).
[0005] Genetic alteration of the proto-oncogene Ras is believed to contribute
to
approximately 30% of all human tumors (Wiessmuller and Wittinghofer (1994);
Barbacid
( 1987)). 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, for
example pancreatic cancer (80%), sporadic colorectal carcinomas (40-50%),
human lung
adenocarcinomas ( 15-24%), thyroid tumors (50%) and myeloid leukemia (30%)
(Mills et al.
( 1995); Chaubert et al. ( 1994); Bos ( 1989)), and ovarian carcinomas (20-
47%) (Sherman (2000);
Dokianakis et al. ( 1999)).
[0006] Ras is known to interact with a number of downstream effectors,
including members
of the Raf family (e.g., Rafl, B-raf, and A-raf), members of the RaIGEF family
(e.g., RaIGDS,
RGL1, RGL2, and Rlf), and phosphotidylinositol-3-kinases (PI-3 kinases) (e.g.,
isoforms of
protein kinase C, AKT kinase/protein kinase B, p70-S6 kinase, and RacGEFs).
The mitogen
activated protein (MAP) kinase pathway is perhaps the best studied of the
downstream Ras-
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effector pathways. The MAP kinase pathway (or simply the MAP pathway)
comprises Ras, Raf,
MEK (MAP/ERK kinase, formerly known as MAP kinase kinase), and ERK
(extracelluIar signal
related kinase or MAP kinase), wherein Ras activates Raf, which activates MEK,
which activates
MAP kinase. Phosphorylated MAP kinase (ERK) translocates to the cell nucleus
and modulates
the activity of transcription factors, for example c-Myc and Elk-1, through
further
phosphorylation (Ward et al. (2001 ); Sebolt-Leopold (2000); Lewin ( I997)),
thereby affecting
gene expression.
(000'1] Ras activation also results as a consequence of upstream mitogenic
signaling, notably
by activation of tyrosine kinase receptors, including but not limited to the
epidermal growth
factor (EGF) and platelet-derived growth factor (PDGF) receptors. Tyrosine
kinase receptors
activate Ras through adapter proteins such as Grb2 and Sos (e.g., Lewin (
1997)). These
upstream elements, if amplified or overexpressed, result in elevated Ras
activity and subsequent
activation downstream Ras effectors. An example of activation of elements
upstream of Ras
includes overexpression of c-erbB-2/neu PDGFR (Levitzki ( I 994); Janes et al.
( 1994); Bos
(1989)). HER-2/neu (erbB-2) is overexpressed in approximately 30% of all
breast cancers
(Spandidos (1987); Zhou et al. (I989); Archer et al. (1995)) and is associated
with poor patient
prognosis (Slamon et al. (1989)). When overexpressed in NIH-3T3 cells, HER-2
mediates
transformation; however, there does appear to be a threshold Level of
overexpression that is
required for this transformation to occur (Jardines et al. ( 1993); Clark et
al. ( 1995)). It appears
that this transforming ability of HER-2 is dependent upon Ras activity as cell
lines
overexpressing HER-2 exhibit a dramatic increase in MAP kinase activity, the
latter being
reflective of Ras activity (Japes et al. ( 1994)). The growth factor receptor
EGFR that is closely
related to, but distinct from HER-2, has also been observed to be
overexpressed in breast tumors
and has also been correlated strongly with a poor patient prognosis (Shackney
et al. (1998);
Koenders et al. ( 1991 )).
[0008] Additional elements upstream of Ras have also been implicated in the
etiology of
breast cancer. The non-receptor tyrosine kinase c-Src has been implicated as
being as important
candidate in promoting the progression of breast cancer. A number of studies
indicate a 4- to 30-
fold increase of c-Src activity in primary breast cancer tumors when compared
to normal breast
tissue (Verbeeks et al. (1996); Jacobs et al. (1983)). c-Src has been
suggested to also play an
important role in the transmission of signals from both the estrogen and
progestin receptors via
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the Ras/MAP kinase pathway. A number of groups have observed that the
treatment of breast
tumor derived MCF-7 cells with estradiol results in the activation of c-Src
kinase activity with
resultant activation of MAP kinase (Di Domenico et al. ( 1996); Migliaccio et
al. ( 1996)). More
recently, Migliaccio et al. ( 1998), have demonstrated in T47D breast cancer
cells that progestin
stimulated cell proliferation is dependent upon Src/Ras/MAP kinase signaling.
The fact that
these steroid receptors can utilize Ras for their signaling suggests that Ras
may play a more
pivotal role in the promotion of growth in ER and PR positive breast tumors.
[0009] MEK is also activated in a Ras-independent manner through MEK kinase,
which is
activated through largely unknown or uncharacterized G-proteins (Lewin (
1997)). Accordingly,
MEK is a common factor in Ras-dependent and Ras-independent MAP kinase pathway
actmation.
[0010] It has previously been demonstrated that reovirus is replication
competent in cells that
possess an activated Ras signaling pathway, either through direct mutation of
Ras itself or via
upstream elements that result in its activation (Strong et al. ( 1993); Strong
et al. ( 1998)).
However, it would be useful to be able to treat cells with other types of
proliferative disorders,
for example disorders not associated with activated Ras, using oncolytic
reovirus.
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SUMMARY OF THE INVENTION
[0011] The present invention is directed to methods of determining the
susceptibility of cells
to reovirus infection by measuring constitutive MAP kinase pathway signaling
in the cell,
wherein the presence of constitutive signaling indicates susceptibility to
infection by reovirus.
According to one aspect of the invention, MAP kinase pathway signaling is
measured by
determining the state of phosphorylation of MEK, for example using an antibody
specific for
phosphorylated MEK (phospho-MEK). The transformed, reovirus-susceptible cells
may harbor
normal Ras and cell activation may not be a result of Ras activation. In one
aspect of the
invention, cell transformation results from activation or mutation of one or
more elements in the
MAP kinase pathway, including but not limited to Raf, MEK, and MAP kinase. In
another
aspect of the invention, cell transformation results from activation or
mutation of MEK kinase.
[0012] Cells used in the practice of the invention may be comprised in a
biological specimen,
particularly one obtained from a mammal suspected of having a proliferative
disorder. Examples
of biological specimens useful for practicing the invention include but are
not limited to cell
pellets from cultured immortalized cells lines, cultured primary tumor cells,
tumor biopsy
specimens, and post-mortem tumor specimens. Examples of proliferative
disorders include but
are not limited to neurofibromatosis, lung cancer, prostate cancer, colorectal
cancer, thyroid
cancer, renal cancer, adrenal cancer, liver cancer, pancreatic cancer, breast
cancer and central,
peripheral nervous system cancer, or a hematopoietic neoplasm. The
proliferative disorder may
be a neoplasm, including a solid neoplasm. The neoplasm may be metastatic.
[0013] The invention may be practiced using a mammal. In one aspect of the
invention, the
mammal is selected from the group consisting of dogs, cats, sheep, goats,
cattle, horses, pigs,
mice, non-human primates, and humans. The mammal may be immunocompetent or
immunocompromised.
[0014] Another aspect of the invention is the treatment of a proliferative
disorder in a
mammal, which disorder is characterized by constitutive MEK phosphorylation.
The method
comprises administering to the proliferating cells in the mammal an effective
amount of one or
more reoviruses under conditions that result in substantial lysis of the
proliferating cells. The
method may further comprising a step selected from the group consisting of:
administering to
the proliferating cells in said mammal an effective amount of an immune
suppressive agent;
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removing B-cells or T-cells from said mammal; removing anti-reovirus
antibodies from said
mammal; removing antibodies from said mammal; administering anti-antireovirus
antibodies to
said mammal; andlor suppressing the immune system of the mammal.
[0015] The reovirus used to practice the invention may be a mammalian reovirus
or an avian
reovirus. The reovirus may be a human reovirus, including but not limited to
serotype 1
reovirus, serotype 2 reovirus and serotype 3 reovirus. The reovirus may be one
or more
recombinant reovirus. The reovirus may be a field isolate. One or more
different strain and/or
types of reovirus may be administered. The reovirus may be immunoprotected or
encapsulated
in a micelle. The reovirus may be treated with a protease prior to
administration. In one aspect
of the invention, approximately 1 to 10'5 plaque forming units of reovirus/kg
body weight are
administered. The reovirus may be administered in a single dose or in more
than one dose. An
effective amount of a chemotherapeutic agent may be administered along with
the reovirus.
[0016] The reovirus may be administered by one or more of a number of
different routes,
including but not limited to intravascular, intrathecal, intravenous,
intramuscular, subcutaneous,
intraperitoneal, topical, oral, rectal, vaginal, nasal, and intratumoral. The
reovirus may also be
administered by injection into or near the solid neoplasm. Administration of
reovirus may be
preceded by surgical removal of the substantially all of the neoplasm,
followed by administration
of the reovirus to the surgical site in an amount sufficient to result in
substantial oncolysis of any
remaining neoplastic cells.
[0017] Also provided is a pharmaceutical composition comprising an effective
amount of a
reovirus and a pharmaceutically acceptable excipient. The pharmaceutical
composition may
comprise an immunosuppressant or immunoinhibitant, along with a reovirus and a
pharmaceutically acceptable excipient. Kits comprising a reovirus and,
optionally, an
immunosuppressant or immunoinhibitant are also provided.
[0018] In another aspect of the invention, suspected tumorigenic cells are
identified in a
biological specimen or population of cells by lysing the cells, incubating the
cell material with
phospho-MEK-specific antibody, and detecting the presence of the phospho-MEK-
specific
antibody. To facilitate antibody detection, the phospho-MEK-specific antibody
may be
conjugated to a tag, including but not limited to a fluorescent tag, a
radioactive tag, and an
enzyme (e.g., horseradish peroxidase or alkaline phosphatase).
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[0019] In another aspect of the invention, constitutive MEK phosphorylation is
determined
using a MEK-specific antibody that does not recognize the phosphorylation
status of MEK.
Total MEK is determined by immunoblot analysis, immunoprecipitation,
radioimmunoassay
(RIA), or 2D-electrophoreses. Total MEK is determined, for example, in cell
lysates from
normal cells and in cell lysates from cells with a suspected proliferative
disorder. The relative
amount of phospho-MEK is then determined by pulse labeling cells, for example,
with a [32P]- or
[3sP]-containing molecule, prior to determining total MEK using a MEK-specific
antibody. The
relative amount of label incorporated into MEK is them compared in normal
cells and suspected
tumorigenic cells, normalized for the total amount of MEK in the respective
specimen. These
assays are performed in the absence of mitogens, thus an increased proportion
of phospho-MEK
in biological specimens comprising cells with suspected proliferative
disorders, compared to the
relative amount of phospho-MEK in specimens comprising normal cells, indicates
constitutive
MEK phosphorylation, the presence of a cell proliferative disorder, and
susceptibility to reovirus
infection.
[0020] The methods and pharmaceutical compositions of the invention provide an
effective
means to treat neoplasia, without the side effects associated with other forms
of cancer therapy.
When used, inhibition or suppression of the immune system increases the
availability of reovirus
to infect and lyse proliferating cells which exhibit constitutive MEK
phosphorylation because
anti-reovirus antibodies are not formed. Because reovirus is not known to be
associated with
disease, any safety concerns associated with deliberate administration of a
virus are minimized.
[0021] 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
[0022] Figure 1 shows the results of immunoblot analyses used to determine
relative
amounts of GTP-bound Ras, total Ras, phosphorylated MEK, and total MEK, in
various normal
and tumorigenic colon (A) and ovarian (B) cells.
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[0023] Figure 2 shows the change in tumor size (mm2) over time (days) in
animals receiving
colon (A-C) or ovarian (D) tumor xenografts followed by live (open circles) or
UV-inactivated
(closed circles) reovirus.
DETAILED DESCRIPTION OF THE INVENTION
A. Definitions
[0024] Administration to a proliferating cell or neoplasm: Administration of
reovirus 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. Routes of
inoculation include but are not limited to direct tumor injection;
intravenous, intravascular,
intrathecal, intramuscular, subcutaneous, intraperitoneal, topical, orally,
rectal, vaginal, or nasal
administration; or by inhalation spray.
[0025] Anti-antireovirus antibodies: Antibodies directed against anti-reovirus
antibodies.
[0026] Antibody specific for MEK or MEK-specific antibody: An antibody that
specifically
recognizes phosphorylated and non-phosphorylated forms of MEK.
[0027] Antibody specific for phosphorylated MEK or phospho-MEK-specific
antibody: An
antibody that specifically recognizes a phosphorylated form of MEK, for
example, the antibody
MEK 1 /2.
[0028] Anti-reovirus antibody: An antibody which binds to reovirus.
[0029] Biological specimen: Any cell- or cell material-comprising specimen
obtained
directly or indirectly from animals, including but not limited to cultured
immortalized cells,
cultured primary cells, tumor biopsy specimens, and post-mortem tumor
specimens.
[0030] Constitutive MEK phosphorylation: MEK phosphorylation in the absence of
mitogens. Herein expressed as "the level of constitutive MEK phosphorylation
or "the "relative
amount of MEK phosphorylation" with respect to total MEK (i.e., phosphorylated
and non-
phosphorylated MEK) or using equivalent terminology.
[0031] Elements in the MAP kinase pathway: Factors in the MAP kinase pathway,
including
but not limited to Ras, Raf, MEK, and MAP kinase.
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[0032] Elements in the Ras pathway: Factors in the Ras pathway, including but
not limited
to the EGF and PDGF receptors, Grb2, Sos, and Ras.
[0033] Elements upstream of MEK: Factors in the Ras, MAP kinase, or any other
pathways
that directly or indirectly activate or suppress MEK, for example, through
phosphorylation.
Examples include but are not limited to ligands that activate MEK-containing
signal transduction
pathways, cell surface receptors such as tyrosine kinase receptors (e.g., the
EGF and PDGF
receptors), Grb2, Sos, Ras, Raf, and MEK kinase.
[0034) Humanized antibodies: Antibody molecules in which the amino acid
sequence in the
non-antigen binding regions has been altered so that the antibody more closely
resembles a
human antibody, and still retains its original binding ability.
[0035] Immunosuppressants or immune suppressive agents: Include conventional
immunosuppressants, immunoinhibitors, antibodies, and conditions such as
radiation therapy or
HIV infection which result in compromise of the immune system.
[0036] Mammal suspected of having a proliferative disorder: A mammal having a
proliferative disorder or tumor, that has been diagnosed with a proliferative
disorder or tumor,
that has been previously diagnosed with a proliferative disorder or tumor, or
that has had
substantially all of a tumor surgically removed and is suspected of harboring
some residual
tumor cells.
[0037] MAP kinase pathway (or MAP pathway): The mitogen-activated protein
(MAP)
kinase pathway is a downstream Ras-effector pathway comprising (in order of
activation) Ras,
Raf, MEK (MAP/ERK kinase, formerly known as MAP kinase kinase), ERK
(extracellular
signal related kinase or MAP kinase), and transcription factors, such as c-Myc
and Elk-1.
[0038] MAPK, MAP kinase: MAP (mitogen-activated protein) kinase or ERK
(extracellular
signal related kinase).
(0039] MEK kinase. Mitogen-activated extracellular signal-regulated kinase. A
kinase
activated by G-proteins, likely in response to activation of serpetine
receptors. MEK kinase is
part of a MAP kinase pathway-independent method of MEK activation.
[0040] MEK: MAP/ERK kinase, formerly called MAP kinase kinase.
[0041] Naturally-occurring reovirus: Reovirus that can be isolated from a
source in nature
and has not been intentionally modified by humans in the laboratory. For
example, reovirus
from a field source or a patient.
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[0042] Neoplasm: An abnormal benign or malignant 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. 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 hematopoietic 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.
[0043] Normal cells: Cells not manifesting proliferative disorders or a
transformed
phenotype.
[0044] Normal mammal: An animal not manifesting a cell proliferative disorder.
[0045] Normal Ras: Ras having no mutations associated with cell
transformation.
[0046] Phospho-MAPK: Phosphorylated-MAP kinase.
[0047] Phospho-MEK: Phosphorylated MEK..
[0048] Proliferating cell: A cell that is proliferating more rapidly than
normal cells.
[0049] Proliferative disorder: Any cellular disorder in which the cells
proliferate more
rapidly than normal tissue growth. Proliferative disorders include but are not
limited to
neoplasms. Other proliferative disorders include, but are not limited to
neurofibromatosis.
[0050] Pulse labeling cells: The addition of a labeling agent, typically a
radioactive isotope-
containing precursor molecule for cell macromolecule (e.g., protein or nucleic
acid), to growing
cells, causing the cells to incorporate label into macromolecules that are
synthesized during the
labeling period. Common labeling agents include but are not limited to [35S]-
methionine, [35S]-
cysteine and b' or ( [32P]-nucleotides and ribonucleotides.
[0051] Biological specimens that have been pulse-labeled may be referred to as
"labeled"
while corresponding or equivalent biological specimens that have not been
pulse-labeled may be
referred to as "unlabeled."
[0052) Ras activation: Activation of Ras, including but not limited to
activation by upstream
elements in the Ras pathway.
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[0053] Ras pathway: In mammals, the Ras signal transduction pathway involves
(in order of
activation) tyrosine receptor kinases such as the EGF and PDGF receptors, an
adaptor protein
such as Grb2, Sos, and Ras (a monomeric guanine nucleotide-binding protein).
GTP-bound Ras
may then activate downstream Ras effector pathways, including but not limited
to the MAP
kinase pathway.
[0054] Reovirus or reoviruses: 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.
[0055] The life cycle and biology of reoviruses make them ideally suited for
practicing the
embodiments of the invention. 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
( 1959)). The term "reovirus" refers to all viruses classified in the reovirus
genus.
[0056] The human reovirus consists of three serotypes: type 1 (strain Lang or
T1L), type 2
(strain Jones, T2J) and type 3 (strain bearing or strain Abney, T3D): The
three serotypes are
easily identifiable on the basis of neutralization and hemagglutinin-
inhibition assays (Sabin
( 1959); Fields et al. ( I 996); Rosen ( 1960); Stanley ( 1967)).
[0057] 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
and Muldoon
( 1973); Stanley ( 1974)).
[0058] Resistance to reovirus infection: Those cells that do not demonstrate
induction of
cytopathic effects, viral protein synthesis, and/or virus production following
exposure to
reovirus.
[0059] Substantial lysis: Lysis of at least 10%, more preferably at least 30%,
and most
preferably at least 60%, of proliferating cells. The percentage of lysis can
be determined for
tumor cells by measuring the reduction in the size of the tumor in the mammal
or the lysis of the
tumor cells in vitro.
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[0060] Susceptible to reovirus infection: Those cells that demonstrate
induction of
cytopathic effects, viral protein synthesis, and/or virus production following
exposure to
reovirus.
[0061] Total MEK or Total level of MEK: The amount or level of phosphorylated
MEK in
addition to non-phosphorylated MEK in biological specimen.
[0062] Unit dosage forms: Physically discrete units suitable as unitary
dosages for human
subjects and other mammals, each unit containing a predetermined quantity of
reovirus
calculated to produce the desired therapeutic effect, in association with a
suitable pharmaceutical
excipient.
B. Detailed Description of T'he Invention
[0063] As described herein, Applicants have discovered that cells which
exhibit constitutive
MAP kinase pathway phosphorylation, independent of the presence of Ras
mutation of the
presence of elevated levels of GTP-bound Ras, are susceptible to reovirus
infection. Resistance
of cells to reovirus infection indicates that infection of the cells with the
virus does not result in
significant viral production or yield. 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.
[0064] In the treatment methods of the invention, reovirus is administered to
proliferating
cells in the individual mammal exhibiting constitutive MEK phosphorylation. In
one
embodiment of this invention a course of reovirus therapy is administered one
or more times.
[0065] In the methods of the invention, reovirus is administered to
proliferating cells in the
individual mammal exhibiting constitutive MAP kinase phosphorylation.
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 bearing 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 bearing.
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).
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A combination of different serotypes and/or different strains of reovirus,
such as reovirus from
different species of animal, can be used. The reovirus may be naturally
occurring or modified.
[0066] The reovirus may be modified but still capable of lytically infecting a
mammalian cell
exhibiting constitutive MAP kinase pathway phosphorylation. 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 (Chandran and Nibert ( 1998)) to reduce or
prevent an immune
response from a mammal which has developed immunity to the reovirus. For
example, the
virion may be treated with chymotrypsin in the presence of micelle forming
concentrations of
alkyl sulfate detergents to generate a new infectious subviral particle
(ISVP). An ISVP may be
used either alone or in combination with whole virus to provide an agent that
is either poorly
recognized or has not been previously prevented by the patient's immune
system.
[0067) The reovirus may be a recombinant reovirus resulting from the
recombination/
reassortment of genomic segments from two or more genetically distinct
reoviruses.
Recombination/reassortment of reovirus genomic segments may occur in nature
following
infection of a host organism with at least two genetically distinct
reoviruses. Recombinant
virions can also be generated in cell culture, for example, by co-infection of
permissive host cells
with genetically distinct reoviruses (Nibert et al. ( 1995)).
[0068] Accordingly, the invention contemplates the use of recombinant reovirus
resulting
from reassortment of genome segments from two or more genetically distinct
reoviruses,
including but not limited to, human reovirus, such as type 1 (e.g., strain
Lang), type 2 (e.g., strain
Jones), and type 3 (e.g., strain bearing or strain Abney), non-human mammalian
reoviruses, or
avian reovirus. The invention further contemplates the use of recombinant
reoviruses resulting
from reassortment of genome segments from two or more genetically distinct
reoviruses wherein
at least one parental virus is genetically engineered, comprises one or more
chemically
synthesized genomic segment, has been treated with chemical or physical
mutagens, or is itself
the result of a recombination event. The invention further contemplates the
use of recombinant
reovirus that have undergone recombination in the presence of chemical
mutagens, including but
not limited to dimethyl sulfate and ethidium bromide, or physical mutagens,
including but not
limited to ultraviolet light and other forms of radiation.
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[0069] The invention further contemplates the use of recombinant viruses that
comprise
deletions or duplications in one or more genome segments, that comprise
additional genetic
information as a result of recombination with a host cell genome, or that
comprise synthetic
genes.
[0070] The reovirus may be modified by incorporation of mutated coat proteins,
such as for
example, 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 COS 1 cells will result in
the incorporation of
the mutated protein into the reovirus virion particle (Turner et al. ( 1992);
Duncan et al. ( 1991 );
Mah et al. ( 1990)).
(0071] The reovirus is preferably a reovirus modified to reduce or eliminate
an immune
reaction to the reovirus. Such a modified reovirus is termed an
"immunoprotected reovirus".
The modifications could include packaging of the reovirus in a liposome, a
micelle or other
vehicle to mask the reovirus from the host 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. In addition to
reducing or
eliminating immune responses, the modifications may also reduce non-specific
uptake of the
virus in normal tissues. As discussed above, reovirus is capable of binding to
a multitude of cell
types, presumably due to the ubiquitous nature of its receptor. Therefore, by
masking the
reovirus, non-specific binding and uptake can be reduced.
[0072] It has previously been shown that reovirus could be used as an
effective oncolytic
agent in a mouse model (WO 99/08692). It has additionally been shown that
cellular
susceptibility to reovirus infection can be determined by measuring
constitutive MAP signaling
of the cell, wherein the presence of elevated levels of phosphorylated MAP
kinase indicates
activation of Ras, for example by one or more mutations in the Ras gene or by
activation of
upstream elements in the Ras pathway (U.S. Patent Application Serial No.
09/985,756).
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[0073] Evidence is presented herein to show that reovirus is useful as an
oncolytic agent
against various colon and ovarian tumors and that the susceptibility of these
cells to reovirus
infection is not a function of activation or overexpression of regulatory
elements in the Ras
pathway, or the existence of activating mutations in Ras itself.
[0074) The invention is based, inter alia, on the observation that reovirus
infected different
human colon and ovarian cancer cells independent of whether the cells harbored
mutated Ras or
whether Ras was activated in the cells. For example, Caco-2, DLD-1, HCT-116,
HT-29, and
SW-48 are all human colon cancer cell lines. While DLD-1 and HCT-116 are known
to harbor
activating mutations of K-ras (Shirasawa et al. ( 1993)), Caco-2 and HT-29
harbor normal Ras
(Lorentz et al. ( 1999)). Reovirus was able to infect all cell lines,
independent of the presence of
activating Ras mutations. Moreover, the ability of reovirus to infect the
different cells did not
correspond with the levels of GTP-bound Ras in the cells, indicating that
reovirus susceptibility
can result from activation or overexpression of downstream elements in the Ras
pathway, in
addition to Ras or upstream elements of the Ras pathway.
[0075] In all cases, the cells that were susceptible to reovirus infection
demonstrated elevated
constitutive levels of phosphorylated MEK (i.e., MAP/ERK kinase), based on
immunoblot
analysis using a phospho-MEK-specific antibody designated MEK1/2. A
constitutive level of
phospho-MEK is that which is present in the absence of a mitogen. These
results suggest that an
assay for detecting the relative levels of phospho-MEK, compared with non-
phophorylated
MEK, can be useful in identifying cells that are susceptible to reovirus
infection.
[0076] One embodiment of the invention is the identification of reovirus
susceptible cells
using a phospho-MEK-specific antibody, for example, in the phospho-MEK
immunoblot assay
described above and in the following Examples. This assay is useful for
screening cells
comprised in a biological specimen, for example, cultured immortalized cells
lines, cultured
primary tumor cells, tumor biopsy specimens, and post-mortem tumor specimens
for
susceptibility to reovirus. In one particular embodiment of the invention,
tumor biopsy
specimens or cultured primary tumor cells can be screened for reovirus
susceptibility to
determine whether reovirus administration could be used to treat the
particular tumors of an
animal.
[0077] In another embodiment of the invention, the phospho-MEK immunoblot
assay could
be used to determine whether non-tumor cells of the same animal were resistant
to reovirus
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infection, thereby determining whether reovirus could be administered to the
animal without
concern about infecting non-tumor cells. In a preferred embodiment of the
invention, tumor cells
and cells adjacent to the tumor could be screened for reovirus susceptibility,
to confirm that the
tumor cells would be specifically infected upon the administration of
reovirus.
[0078] In one embodiment of the invention, phospho-MEK-specific antibodies are
used to
determine the level of phospho-MEK in a specimen and MEK-specific antibodies
are used to
determine the total amount of MEK in a specimen. This embodiment is useful,
for example, for
determining the relative amount of phospho-MEK, compared to the total amount
of MEK, in a
specimen. Multiple specimens may be analyzed in this manner wherein relative
levels of
phospho-MEK in different specimens are normalized for total MEK, allowing for
meaningful
comparison of phospho-MEK levels among different specimens.
[0079] For example, using a MEK-specific antibody that recognizes
phosphorylated and
unphosphorylated MEK, the total amount of MEK in a biological specimen
suspected of
comprising cells with a proliferative disorder and in a biological specimen
comprising normal
cells is determined by standard antibody-based assays. The amounts of phospho-
MEK in the
same or equivalent biological specimens are determined using a phospho-MEK-
specific
antibody. The relative amounts of phosph-MEK, normalized for the total amount
of MEK, are
then compared in the biological specimen suspected of comprising cells with a
proliferative
disorder and in the biological specimen comprising normal cells; wherein an
increased relative
amount of phospho-MEK in the biological specimen suspected of comprising cells
with a
proliferative disorder is indicative of a cell proliferative disorder and
susceptibility to reovirus
infection.
[0080] In another embodiment of the invention, the relative amount of phospho-
MEK in a
specimen, in the absence of mitogen, is determined using only a MEK-specific
antibody. This
embodiment of the invention is particularly useful when the biological
specimens comprise
cultured cells or biological specimen from animals into which radionuclides
may be
administered. As above, the total amount of MEK in each biological specimen is
determined
using a MEK-specific antibody. However, in this embodiment, the amounts of
phospho-MEK in
each biological are determined by pulse labeling the specimens, for example,
with 32P Or 33P,
then quantitating the amount of labeled MEK. As above, the relative amounts of
phospho-MEK
in each biological specimen, normalized for the total amount of MEK in the
respective biological
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specimens, indicate the levels of constitutive MEK phosphorylation in each
specimen. An
increased proportion of phospho-MEK in the biological specimen suspected of
comprising cells
with a proliferative disorder indicates susceptibility to reovirus infection.
[0081] For example, using a MEK-specific antibody that recognizes
phosphorylated and
unphosphorylated forms of MEK, the total amount of MEK in a biological
specimen suspected
of comprising cells with a proliferative disorder and in a biological specimen
comprising normal
cells is determined by standard antibody-based assays. The amounts of phospho-
MEK in the
same biological specimens are determined using a MEK-specific antibody in
equivalent
biological specimens that have been pulse labeled with a suitable radionuclide
or other labeling
agent. The relative amounts of phosph-MEK, normalized for the total amount of
MEK, are then
compared in the biological specimen suspected of comprising cells with a
proliferative disorder
and in the biological specimen comprising normal cells; wherein an increased
relative amount of
phospho-MEK in the biological specimen suspected of comprising cells with a
proliferative
disorder is indicative of a cell proliferative disorder and susceptibility to
reovirus infection.
[0082] The MEK-specific and phospho-MEK-specific antibodies described herein
can be
used in combination with virtually any antibody-based assay known in the art.
These assays
include but are not limited to, immunoblot analysis, immunoprecipitation,
radioimmunoassay,
and 2D-electrophoresis.
[0083] The results described above and in the Examples further suggest that
elevated levels
of phospho-MEK are more consistently associated with tumor cells than elevated
levels of GTP-
bound. Accordingly, a phospho-MEK-specific biological assay is likely to be
more reliable in
identifying tumor cells (i.e., cells with proliferative disorders) than assays
based on the level of
GTP-bound Ras in a cell or biological specimen. Accordingly, in another
embodiment of the
invention, the phospho-MEK-specific and MEK-specific antibodies and assays
described above
and in the Examples, below, are useful for identifying tumor cells, present,
for example, in
immortalized cells lines, cultured primary tumor cells, tumor biopsy
specimens, and post-mortem
tumor specimens. In a preferred embodiment of the invention, the assays
described herein can
be used to determine both (i) whether biological specimens comprise cells
having elevated levels
of phospho-MEK, indicating the presence of tumorigenic cells in the biological
specimens, and
(ii) whether the cells are susceptible to reovirus infection.
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[0084] The phospho-MEK-specific or MEK-specific antibodies of the invention,
may be
conjugated with a tag to facilitate the detection of the antibody. Antibody
tags are well known in
the art and include but are not limited to fluorescent tags (e.g., green
fluorescent protein or
fluorescein isothiocyanate), radioactive tags (e.g., [35S] or (~ZSI]), and
enzyme tags (e.g.,
horseradish peroxidase or alkaline phosphatase). The presence of a tag
facilitates visualization
and/or quantitation of the antibody and/or decreases the number of steps
required to visualize the
antibody, thereby simplifying the practice of the instant invention.
[0085] The instant invention is useful, for example, for detecting a variety
of proliferative
disorders, in which a subpopulation of cells proliferate more rapidly than
normal cells or normal
tissues. Cell proliferative disorders include neoplasms. Other proliferative
disorders include, but
are not limited to neurofibromatosis.
[0086] Upon determining that a population of cells has a proliferative
disorder associated
with constitutive phosphorylation of MEK, reovirus is then 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, Garner 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 solid neoplasm,
where it then is carned 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).
[0087] 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
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cells resulting in lysis of the cells. Where the mammals to be treated have
higher titers of anti-
reovirus antibodies, more reovirus must be administered to be effective.
[0088] Immunocompetent mammals previously exposed to a reovirus subtype may
have
developed humoral and/or cellular immunity to that reovirus subtype.
Nevertheless, it has been
found that direct injection of the reovirus into a solid tumor in
immunocompetent mammals will
result in the lysis of the neoplastic cells. On the other hand, when the
reovirus is administered
systemically to immunocompetent mammals, the mammals may produce an immune
response to
the reovirus. Such an immune response may be avoided if the reovirus is of a
subtype to which
the mammal has not developed immunity, or the reovirus has been modified as
previously
described herein such that it is immunoprotected, for example, by protease
digestion of the outer
capsid or packaging in a micelle.
[0089] Alternatively, it is contemplated that the immunocompetency of the
mammal against
the reovirus may be suppressed either by the prior or co-administration of
pharmaceuticals
known in the art to suppress the immune system in general (Cuff et al. (1998))
or alternatively
the administration of such immunoinhibitors as anti-antireovirus antibodies.
The humoral
immunity of the mammal against reovirus may also be temporarily reduced or
suppressed by
plasmaphoresis of the mammal's 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.
[0090] It is contemplated that the reovirus may be administered to
immunocompetent
mammals immunized against the reovirus in conjunction with the administration
of
immunosuppressants and/or immunoinhibitors. Such immunosuppressants and
immunoinhibitors are known to those of skill in the art and include such
agents as cyclosporin,
rapamycin, tacrolimus, mycophenolic acid, azathioprine and their analogs, and
the like. Other
agents are known to have immunosuppressant properties as well (see, e.g.,
Gilman et al. at 1242,
the disclosure of which is incorporated herein by reference). Such
immunoinhibitors also
include anti-antireovirus antibodies. Such antibodies can be made by methods
known in the art
(see, e.g., Harlow and Lane ( 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.
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[0091] This invention also includes pharmaceutical compositions containing, as
the active
ingredient, one or more immunosuppressants or immunoinhibitors and one or more
of the
reoviruses associated with pharmaceutically acceptable carriers or excipients.
In making the
compositions of this invention, the active ingredients/ immunosuppressant or
immunoinhibitor
and reovirus are 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.
[0092] 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.
[0093] For preparing solid compositions such as tablets, the principal active
ingredients/
immunosuppressant or immunoinhibitor and reovirus are 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.
[0094] 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
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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.
[0095] 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.
[0096] 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.
[0097] Another preferred formulation employed in the methods of the present
invention
employs transdermal delivery devices ("patches"). Such transdermal patches may
be used to
provide continuous or discontinuous infusion of the reovirus of the present
invention in
controlled amounts. The construction and use of transdermal patches for the
delivery of
pharmaceutical agents is well known in the art. See, for example, U.S. Patent
5,023,252, herein
incorporated by reference. Such patches may be constructed for continuous,
pulsatile, or on
demand delivery of pharmaceutical agents.
[0098] Other suitable formulations for use in the present invention can be
found in
Remington's Pharmaceutical Sciences, the disclosure of which is incorporated
herein by
reference.
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[0099] The immunosuppressant or immunoinhibitor and reovirus or the
pharmaceutical
composition comprising the immunosuppressant or immunoinhibitor and reovirus
may be
packaged into convenient kits providing the necessary materials packaged into
suitable
containers. Such kits may comprise, for example, one or more types or strains
of reovirus
suspended or lyophilized in a suitable pharmaceutical excipient, any necessary
sterile or
sterilizable solutions and/or reagents for resuspending or otherwise preparing
the reovirus for
administration to an animal, syringes or alternative delivery devices for
administering the
reovirus, and instructions for the use of the kit. It is contemplated that the
kits may also include,
for example, chemotherapeutic agents, antibodies, immunosuppressant agents,
and any
additional pharmacological agents disclosed herein.
[00100] The immunosuppressant or immunoinhibitor is administered in an
appropriate
amount and using an appropriate schedule of administration sufficient to
result in
immunosuppression or immunoinhibition of the mammal's immune system. Such
amounts and
schedules are well known to those of skill in the art.
[00101] 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.U pfu/kg
body weight to
about 10'5 pfu/kg body weight, more preferably from about 102 pfu/kg body
weight to about 10'3
pfu/kg body weight. For example, for treatment of a human, approximately 102
to 10" 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.
[00102] The immunosuppressant or immunoinhibitor and reovirus can be
administered in a
single dose, or multiple doses (i.e., more than one dose). The multiple doses
can be administered
CA 02422245 2003-03-14
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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.
[00103] The compositions are preferably formulated in a unit dosage form, each
dosage
containing an appropriate amount of immunosuppressant or immunoinhibitor and
from about 102
pfus to about 10'3 pfus of the reovirus.
[00104] As mentioned above, 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 in immunocompetent animals. When animals are rendered
immunosuppressed or
immunodeficient in some way, systemic administration of reovirus will be more
effective in
producing oncolysis.
[00105] 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.
[00106] It is contemplated that the reovirus may be administered in
conjunction with or in
addition to radiation therapy which renders the mammal immunosuppressed.
[00107) 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, mitomycin C,
methotrexate, hydroxyurea, cyclophosphamide, 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.
[00108] The reovirus and immunosuppressants 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 metastatic tumors in a mammal comprising
administering an
effective amount of a reovirus to the immunosuppressed mammal.
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PATENT Attorney Docket No. 032775-144
[00109) The practitioner will recognize that other viruses that selectively
lyse Ras-activated
cells may also be used to practice the invention. For example, the viruses
described in U.S.
Patent Application Publication No. 20010048919 may be used to practice the
invention.
[00110] The practitioner will understand that for the purposes of the instant
disclosure, "ras"
or "Ras" refer to the Ras gene or the Ras polypeptide as appropriate in
context (as is the case
with other gene/protein names, including but not limited to, Ral, Raf, MEK,
and ERK). These
terms are used for convenience and no limitations of the scope of the
invention should be
construed based on specific instances of capitalization or italics. Moreover,
the practitioner will
understand that "MAP kinase," MAPK," and "MEK" refer to MAP kinase and "Ras-
MAP
pathway," Ras-MAP kinase pathway," "Ras-MAPK pathway," "MAP pathway," "MAP
kinase
pathway," and "MAPK pathway" all refer to the same biochemical pathway unless
other
otherwise dictated by context.
Utilit
[00111] The diagnostic methods of the present invention may be used to
identify the
susceptibility of cells to reovirus infection by measuring constitutive MEK
phosphorylation.
This will be useful to determine in what cases reovirus treatment of cellular
proliferative
disorders is likely to be effective.
[00112) The reoviruses and immunosuppressants of the present invention may be
used for a
variety of purposes. They may be used in methods for treating proliferative
disorders which
exhibit constitutive MEK phosphorylation in a mammal. They 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.
[00113) 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.
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PATENT Attorney Docket No. 032775-144
EXAMPLES
[00114] In the examples below, all temperatures are in degrees Celsius (unless
otherwise
indicated) and all percentages are weight percentages (also unless otherwise
indicated).
[00115] In the examples below, the following abbreviations have the following
meanings. If
an abbreviation is not defined, it has its generally accepted meaning:
~,M - micromolar
mM - millimolar
M - molar
ml - milliliter
~,l - microliter
mg - milligram
~ g - microgram
PAGE - polyacrylamide gel electrophoresis
FBS - fetal bovine serum
DTT dithiothrietol
SDS - sodium dodecyl sulfate
PBS - phosphate buffered saline
DMEM - Dulbecco's modified Eagle's medium
MEM - modified Eagle's medium
2-ME - 2-mercaptoethanol
MOI or moi multiplicity of infection
-
PFL1 or pfu plaque forming units)
-
MAPK - MAP (mitogen-activated protein) kinase
or ERK
phospho-MAPK phosphorylated-MAP kinase
-
MEK - MAP/ERK kinase, formerly called MAP kinase
kinase
phospho-MEK phosphorylated MEK
-
ERK - extracellular signal related kinase or
MAP kinase
HRP - horseradish-peroxidase
EGFR - epidermal growth factor receptor
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PATENT Attorney Docket No. 032775-144
MEK kinase - mitogen-activated extracellular signal-regulated
kinase
SCID - severe combined immune deficiency
i.v. - intravenous
i.p. intraperitoneal
i.m. - intramuscular
s.c. - subcutaneous
EXAMPLE 1. Use of reovirus as an oncolytic went in cells harborin;~K-ras or a
normal
Ras proto oncog_ene
[00116] Normal human colon fibroblasts (CCD-lBCo), human colon cancer cells
(Caco-2,
DLD-1, HCT-116, HT-29, and SW-48), normal human ovarian fibroblasts (NOV-31),
and
human ovarian cancer cells (MDAH 2774, PA-1, SKOV3, and SW626) were obtained
from the
ATCC (Rockville, MD) and maintained according to ATCC protocols. Cells were
infected at an
MOI of 20 pfu/cell in six-well plates. Reovirus infections and assays for
reovirus replication
were performed essentially as described in U.S. Patent No. 6,136,307,
incorporated by reference
in its entirety.
[00117] Morphological changes following infection were nominal in normal colon
and
ovarian cells (i.e., CCD-lBCo and NOV-31) while >95% of the cells of each
tumor cell line were
destroyed by reovirus infection. Pulse labeling of infected cells with [35S]-
methionine, followed
by analysis by SDS-PAGE of the labeled proteins, demonstrated minimal reovirus
protein
synthesis in normal cells and significant levels of reovirus protein synthesis
in cancers cells,
providing additional evidence that cell death was the result of reovirus
replication.
[00118] HCT-116 and DLD-1 are known to possess activating mutations of K-ras
while Caco-
2 and HT-29 possess a normal Ras proto oncogene. The status of Ras in the
ovarian cancer cell
lines was unknown although Ras mutations occur in only about 20-47% of ovarian
cancers
Sherman (2000); Dokianakis et al. ( 1999)). Thus the ability of reovirus to
infect all five colon
and all four ovarian cancer cell lines tested in this study suggests that an
activating Ras mutation
is not essential for reovirus infectivity.
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EXAMPLE 2. Use of a pho~ho-MEK-specific antibody to determine the
phosphorylation status of MEK in cells harboring K-ras or a normal Ras
proto-oncog_ene
[00119] The cells of Example 1 were assayed for relative levels of GTP-bound
Ras compared
to normal Ras, and phosphorylated MEK compared to nonphosphorylated MEK. The
amount of
GTP-bound Ras was determined by incubating cell lysates from each of the cell
lines with the
Raf 1 Ras-binding domain conjugated to agarose beads. The beads and associated
polypeptides
were collected by centrifugation, washed, resuspended in Laemmli sample
buffer, and heated.
The resulting samples were resolved by SDS-PAGE and subjected to immunoblot
analysis using
Ras-specific antibodies. The total amount of Ras was determined by subjecting
cell lysates to
immunoblot analysis without first using Raf 1 Ras-binding domain-conjugated
beads to select
for GTP-bound Ras.
[00120] Relative amounts of phosphorylated MEK were determined by performing
immunoblot analysis on cell lysates using either a MEK-specific antibody or a
phospho-MEK-
specific antibody and determining the relative amounts of phosphorylated MEK
in each
specimen.
[00121] The results of immunoblot analysis are shown in Figure 1. The relative
levels of
GTP-bound-Ras and phospho-MEK are significantly higher in all tumor cell lines
compared to
normal cell lines. However, the relative levels of phospho-MEK do not in all
cases correspond
to the relative levels of GTP-bound Ras in the same cell lysates. For example,
HCT-116 and
SW626 cells show high levels of GTP-bound-Ras but intermediate levels of
phospho-MEK.
Conversely, DLD-l and MDAH 2774 cells show intermediate levels of GTP-bound-
Ras but high
level of phospho-MEK. These results suggest that phosphorylation of MEK is not
solely a
function of Ras activation and that overexpression and/or activation of other
elements may also
affect MEK phosphorylation and susceptibility to reovirus infection.
EXAMPLE 3. Use of a MEK-specific antibody to determine the relative amount of
phospho-MEK in cells
CA 02422245 2003-03-14
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[00122] MEK phosphorylation is also determined using a MEK-specific antibody
that does
not recognize the phosphorylation status of MEK. Total MEK is determined by
immunoblot
analysis, immunoprecipitation, radioimmunoassay (RIA), 2D-electrophoreses, or
any other
antibody-based detection method known in the art. Total MEK is determined, for
example, in
cell lysates from normal cells and in cell lysates from cells with a suspected
proliferative
disorder.
[00123) The relative amount of phospho-MEK is then determined by pulse
labeling cells, for
example, with a [32P]- or [33PJ-containing molecule, prior to determining
total MEK using a
MEK-specific antibody. The relative amount of label incorporated into MEK is
them compared
in normal cells and suspected tumorigenic cells and normalized for the total
amount of MEK in
the respective specimens. These assays are performed in the absence of
mitogens, thus an
increased proportion of phospho-MEK in specimens comprising cells with
suspected
proliferative disorders, compared to the relative amount of phospho-MEK in
specimens
comprising normal cells, indicates constitutive MEK phosphorylation.
[00124] Constitutive MEK phosphorylation is indicative of a cell proliferative
disorder as well
as susceptibility to reovirus infection. In this manner, a practitioner can
determine whether a
certain population of cells has a proliferative disorder and whether such
cells can be destroyed by
reovirus infection.
EXAMPLE 4. Use of reovirus as an oncolytic agent in tumors resulting from
colon or
ovarian cancer xenografts.
[00125] Colon tumor xenografts were established by implanting DLD-1, HCT-116,
or HT-29
cells subcutaneously in 6-8 week-old female SCID/NOD mice (n = 6-7) obtained
from the Cross
Cancer Institute (Edmonton, Alberta, Canada). Established tumors of 20-25 mm2
were injected
with about 10' pfu live reoviruses or UV-inactivated reovirus and tumor size
was monitored for
about 4 weeks.
[00126] Ovarian tumor xenografts were established in a manner similar to the
above by
implanting SKOV3 cells in the hind flanks of 6-8 week-old female CD-1 nude
mice (n = 6-7)
obtained from the Charles River Laboratory (Montreal, Quebec, Canada).
Established tumors of
20-25 mm2 were injected with about Sx 108 pfu live reoviruses or UV-
inactivated reovirus. The
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injection was repeated two weeks later. Tumor size was monitored for up to
about 7 weeks
following the first intratumoral injection.
[00127] Figure 2 shows the change in tumor size over the course of the
experiment. In all
cases, tumor size in animals receiving intratumoral live reovirus injections
(open circles) was
significantly reduced compared to tumor size in animals receiving intratumoral
UV-inactivated
reovirus injections (closed circles). The maximum reduction in tumor sizes at
the termination of
the experiment were about 10-fold, 4-fold, 6-fold, and 15-fold in the cases of
HCT-116 (Fig.
2A), HT-29 (Fig. 2B), DLD-1 (Fig. 2C), and SKOV3 (Fig. 2D) cells,
respectively.
[00128] These data established that intratumoral injection of reovirus reduces
the size of
tumors derived from cells harboring K-ras (DLD-1 and HCT-116 cells) or normal
Ras (HT-29
cells), further suggesting that reovirus susceptibility, and the efficacy of
reovirus administration
in reducing tumor size, is not dependent upon the presence of Ras mutations.
EXAMPLE 5. Use of reovirus as an oncolytic went in ascites ovarian cancer
model.
Description of the model, animals, and inoculations:
[00129] Data obtained from the experiment described in Example 2 indicate that
MDAH 2774
cells possess high levels of phospho-MEK but only intermediate levels of GTP-
bound Ras
(Figure 1 ), suggesting that the high levels of phospho-MEK in these cells
results from
upregulation or overexpression of a Ras pathway regulatory element other than,
or in addition to,
Ras itself. The introduction of these cells into the peritoneal cavity of
mice, and the subsequent
spread of the cells to distant loci, approximate the clinical manifestations
of stage III and IV
(based on the International Federation of Gynecology and Obstetrics
classification system)
ovarian cancer.
[00130) 6-8 week-old female CD-I mice (n = 20) obtained from the Charles River
Laboratory
were injected i.p. with about 2x 106 MDAH 2774 ovarian carcinoma cells.
Animals then
received either live or UV-inactivated reovirus (Sx 10$ pfu) at days 5 and 19
days following
MDAH 2774 injection (n = 10 for each group). Body weight was monitored weekly
and mice
were sacrificed if distressed or if tumor mass exceeded 20% of body weight.
Histology and immunohistochemis
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[00131] For histological analysis, tumors or remaining masses were f xed in
10% neutral
buffered formalin and imbedded in paraffin. Sections were immersed in xylene
followed by
decreasing concentrations of ethanol. Sections were exposed to 3% hydrogen
peroxide in
methanol for about 15 minutes to inactivate endogenous peroxidase activity.
Following washes,
sections were incubated with a primary rabbit anti-reovirus polyclonal
antibody, washed, then
developed using HRP-conjugated secondary antibody by methods well known to
those skilled in
the art. Sections were counterstained with hematoxylin prior to analysis.
Results:
[00132] Mice receiving UV-inactivated reovirus rapidly gained weight (e.g.,
were 15-20%
heavier than mice receiving live reovirus at 4 weeks following MDAH 2774
injection) and died
by day 42 following MDAH 2774 injection. In contrast, 90% of mice receiving
live reovirus
appeared healthy even after 100 days when they were eventually sacrificed.
These results
suggested that reovirus was effective in treating a stage II1/IV ovarian
cancer-like disease caused
by tumor cells having elevated levels of phospho-MEK that was not a result of
activating
mutation in the Ras polypeptide or in activation or overexpression of upstream
elements in the
Ras/MAP kinase pathway.
[00133] 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.
33
