U.S. patent application number 15/972994 was filed with the patent office on 2018-09-13 for use of a combination of myxoma virus and rapamycin for therapeutic treatment.
This patent application is currently assigned to The University of Western Ontario. The applicant listed for this patent is The University of Western Ontario. Invention is credited to John Barrett, Douglas Grant McFadden, Marianne Michelle Stanford.
Application Number | 20180256656 15/972994 |
Document ID | / |
Family ID | 36952904 |
Filed Date | 2018-09-13 |
United States Patent
Application |
20180256656 |
Kind Code |
A1 |
McFadden; Douglas Grant ; et
al. |
September 13, 2018 |
USE OF A COMBINATION OF MYXOMA VIRUS AND RAPAMYCIN FOR THERAPEUTIC
TREATMENT
Abstract
The present invention relates to therapeutic use of a
combination of Myxoma virus, including in combination with
rapamycin. Treatment with rapamycin enhances the ability of Myxoma
virus to selectively infect cells that have a deficient innate
anti-viral response, including cells that are not responsive to
interferon. The combination of rapamycin and Myxoma virus can be
used to treat diseases characterized by the presence of such cells,
including cancer. The invention also relates to therapeutic use of
Myxoma virus that does not express functional M135R.
Inventors: |
McFadden; Douglas Grant;
(Gainesville, FL) ; Barrett; John; (London,
CA) ; Stanford; Marianne Michelle; (Upper Tantallon,
CA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
The University of Western Ontario |
London |
|
CA |
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|
Assignee: |
The University of Western
Ontario
London
CA
|
Family ID: |
36952904 |
Appl. No.: |
15/972994 |
Filed: |
May 7, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14049737 |
Oct 9, 2013 |
9987315 |
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15972994 |
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13679096 |
Nov 16, 2012 |
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14049737 |
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12850599 |
Aug 4, 2010 |
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13679096 |
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11908076 |
Aug 13, 2008 |
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PCT/CA2006/000315 |
Mar 6, 2006 |
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12850599 |
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60658816 |
Mar 7, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 43/00 20180101; A61P 31/00 20180101; C12N 2710/24032 20130101;
A61K 35/768 20130101; A61K 31/436 20130101; A61P 31/12 20180101;
A61K 35/768 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 35/768 20060101
A61K035/768; A61K 31/436 20060101 A61K031/436 |
Claims
1-21. (canceled)
22. A method for inhibiting a human cancer cell, comprising
contacting a human cancer cell that is deficient in an innate
antiviral response and permissive to Myxoma virus replication with
an effective amount of a mutated Myxoma virus comprising a mutated
M135R gene, wherein the mutated M135R gene comprises a mutation in
a coding region or a regulatory region of the M135R gene such that
the mutated M135R gene does not express a functional M135R
protein.
23. The method of claim 22, wherein the innate antiviral response
comprises interferon signaling.
24. The method of claim 22, wherein the mutated M135R gene
comprises a mutation in a coding region of the M135R gene, and
wherein the mutation is a deletion, replacement, or interruption of
the coding region or a segment thereof.
25. The method of claim 24, wherein the mutation is a deletion or
replacement of part or all of the coding region of the M135R
gene.
26. The method of claim 22, wherein the mutated M135R gene
comprises a mutation in the regulatory region of the M135R gene,
and wherein the mutation is a deletion, replacement, or
interruption of the regulatory region or a segment thereof.
27. The method of claim of claim 26, wherein the mutation is a
deletion or replacement of part or all of the regulatory region of
the M135R gene.
28. The method of claim 22, wherein the contacting step comprises
administering the mutated Myxoma virus to a human cancer patient
having cancer cells that are deficient in an innate antiviral
response and permissive to Myxoma virus replication.
29. The method of claim 28, wherein the cancer is selected from the
group consisting of prostate cancer, renal cancer, colon cancer,
lung cancer, ovarian cancer, melanoma, breast cancer, and
osteosarcoma.
30. The method of claim 28, wherein the mutated Myxoma virus is
further modified genetically to express a therapeutic protein.
31. The method of claim 28, wherein the mutated Myxoma virus is
administered to the site of the cancer by injection.
32. The method of claim 28, wherein the human cancer patient is
further administered an anti-cancer agent.
33. The method of claim 28, wherein part or all of the open reading
frame that encodes M135R of the mutated Myxoma virus is
deleted.
34. A pharmaceutical composition, comprising a pharmaceutically
acceptable carrier and a mutated Myxoma virus, which comprises a
mutated M135R gene, wherein (i) part or all of the open reading
frame of the M135R gene is deleted or replaced, or (ii) part or all
of the regulatory region of the M135R gene is deleted or
replaced.
35. The pharmaceutical composition of claim 34, wherein the mutated
Myxoma virus is further modified genetically to express a
therapeutic protein.
36. The pharmaceutical composition of claim 34, wherein the
pharmaceutical composition further comprises a therapeutic
agent.
37. The pharmaceutical composition of claim 36, wherein the
therapeutic agent is rapamycin.
38. A method for producing a mutated Myxoma virus that does not
express functional M135R protein, the method comprising: deleting
or replacing part or whole of (i) the coding region of a M135R
gene, or (ii) the regulatory region of the M135R gene to produce a
mutated Myxoma virus.
39. The method of claim 38, wherein the method further comprises
confirming that the mutated Myxoma virus does not express a
functional M135R protein.
40. The method of claim 39, wherein the method further comprises
contacting the mutated Myxoma virus with a human cancer cell to
determine infectivity and/or cytolytic effects on the human cancer
cell.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/850,599 filed Aug. 4, 2010, which is a
continuation of U.S. patent application Ser. No. 11/908,076, filed
Sep. 7, 2007, which is a .sctn. 371 of PCT/CA06/00315, filed Mar.
6, 2006, which claims the benefit of U.S. Provisional Application
No. 60/658,816, filed Mar. 7, 2005, the disclosures of which are
incorporated herein by reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates generally to therapeutic use
of Myxoma virus and rapamycin.
BACKGROUND OF THE INVENTION
[0003] Current treatments used to treat various types of cancer
tend to work by poisoning or killing the cancerous cell.
Unfortunately, treatments that are toxic to cancer cells typically
tend to be toxic to healthy cells as well. Moreover, the
heterogenous nature of tumours is one of the primary reasons that
effective treatments for cancer remain elusive. Current mainstream
therapies such as chemotherapy and radiotherapy tend to be used
within a narrow therapeutic window of toxicity. These types of
therapies are considered blunt tools that have limited
applicability due to the varying types of tumour cells and the
limited window in which these treatments can be administered.
[0004] Modern anticancer therapies currently being developed
attempt to selectively target tumour cells while being less toxic
to healthy cells, thereby being more likely to leave healthy cells
unaffected.
[0005] Oncolytic viral therapy is one approach that aims to exploit
cellular differences between tumour cells and normal cells. This
therapy uses replication-competent, tumour-selective viral vectors
as anti-cancer agents. The oncolytic virus either specifically
targets cancer cells for infection, or is more suited for efficient
replication in cancer cells versus healthy cells. These
replication-competent, oncolytic viruses are either naturally
occurring or genetically engineered to be a highly selective and
highly potent means of targeting the heterogeneous tumour
population. Since the replication selective oncolytic virus does
not replicate efficiently in normal cells, toxicity to the patient
should be low, particularly in comparison to traditional therapies
such as radiation or chemotherapy.
[0006] Numerous studies have reported oncolytic activity for
various virus strains, with the most promising oncolytic viruses
being a naturally occurring or genetically modified version of
adenovirus, herpes simplex virus 1 ("HSV I"), Reovirus, Vaccinia
Virus, Vesicular Stomatitis Virus ("VSV") or Poliovirus. Modified
oncolytic viruses currently under investigation as anticancer
agents include HSV, adenovirus, Newcastle disease virus ("NDV"),
Reovirus and Vaccinia virus, measles, VSV and poliovirus. Various
oncolytic viruses are in Phase I and Phase II clinical trials with
some showing sustained efficacy. However, it is unknown which
viruses will best fulfill the oncolytic goals of sustained
replication, specificity and potent lytic activity. A completely
efficient candidate for an oncolytic viral vector would be one that
has a short lifecycle, forms mature virions quickly, spreads
efficiently from cell to cell and has a large genome ready for
insertions. As well, evidence suggests that inhibiting the early
innate immune response and slowing the development of Th1 responses
are important for the efficacy of oncolytic therapy. It is clear
that human viruses are highly immunogenic, as measured by the high
level of antibody and T cell responses that are observed in the
normal population for many of the viruses being considered for the
development of oncolytic viruses.
[0007] Clinical work has shown that current oncolytic viruses are
indeed safe, but are not potent enough as monotherapies to be
completely clinically effective. As insufficient or inefficient
infection of tumour cells is usually observed, the current movement
is to arm candidate viruses by genetically engineering them to
express therapeutic transgenes to increase their efficiency. Most
of the above-mentioned oncolytic viruses are also being tested in
combination with other common oncolytic therapies.
[0008] Adenovirus can be easily genetically manipulated and has
well-known associated viral protein function. In addition, it is
associated with a fairly mild disease. The ONYX-015 human
adenovirus (Onyx Pharmaceuticals Inc.) is one of the most
extensively tested oncolytic viruses that has been optimized for
clinical use. It is believed to replicate preferentially in
p53-negative tumours and shows potential in clinical trials with
head and neck cancer patients. However, reports show that ONYX-015
has only produced an objective clinical response in 14% of treated
patients (Nemunaitis J, Khuri F, Ganly I, Arseneau J, Posner M,
Vokes E, Kuhn J, McCarty T, Landers S, Blackburn A, Romel L,
Randlev B, Kaye S, Kirn D. J. Clin. Oncol. 2001 Jan. 15;
19(2):289-98).
[0009] WO96/03997 and WO97/26904 describe a mutant oncolytic HSV
that inhibits tumour cell growth and is specific to neuronal cells.
Further advantages are that the HSV can be genetically modified
with ease, and drugs exist to shut off any unwanted viral
replication. However, the application of such a common human
pathogen is limited, as it is likely that the general population
has been exposed and acquired an immune response to this virus,
which would attenuate the lytic effect of the virus. HSV can also
cause serious side effects or a potentially fatal disease.
[0010] Reovirus type III is associated with relatively mild
diseases and its viral gene function is fairly well understood.
Reovirus type III is currently being developed by Oncolytic Biotech
as a cancer therapeutic which exhibits enhanced replication
properties in cells expressing mutant ras oncogen and
preferentially grows in PKR -/- cells (Strong J. E. and P. W. Lee,
J. Virology, 1996, 70:612-616). However, Reovirus is difficult to
genetically manipulate and its viral replication cannot be easily
shut off.
[0011] VSV is associated with relatively mild diseases and also has
well-known viral gene function. WO99/04026 discloses the use of VSV
as a vector in gene therapy for the expression of wide treatment of
a variety of disorders. However, VSV suffers from the same problems
as the Reovirus in that it is difficult to genetically manipulate
and its viral replication cannot be easily shut off.
[0012] Vaccina virus and Poliovirus are other candidate oncolytic
viruses described in the art but have been associated with a
serious or potentially fatal disease.
[0013] U.S. Pat. No. 4,806,347 discloses the use of gamma
interferon and a fragment of IFN.gamma. against human tumour cells.
WO99/18799 discloses a method of treating disease in a mammal in
which the diseased cells have defects in an interferon-mediated
antiviral response, comprising administering to the mammal a
therapeutically effective amount of an interferon-sensitive,
replication competent clonal virus. It specifically discloses that
VSV particles have toxic activity against tumour cells but that
alleviation of cytotoxicity in normal cells by VSV occurs in the
presence of interferon. WO99/18799 also discloses that NDV-induced
sensitivity was observed with the interferon-treated tumour cells
but that adding interferon to normal cells makes these cells
resistant to NDV. This method aims to make cells sensitive to
interferon by infecting them with interferon sensitive viruses.
SUMMARY OF THE INVENTION
[0014] The present invention is based on the unexpected discovery
that rabbit Myxoma virus, including a novel Myxoma virus that does
not express functional M135R protein, can selectively infect cells,
including human tumour cells, that have a deficient innate
anti-viral response, including those that are non-responsive to
interferon, and that such infection is enhanced by treating such
cells with the drug rapamycin. The term "innate" as used in this
context describes non-antigen specific immune response. Since
Myxoma virus does not replicate efficiently in normal human cells,
the virus can therefore be used as a treatment for various
disorders and conditions characterized by cells that have a
deficient innate anti-viral response, including cells that are
non-responsive to interferon, for example, as an oncolytic
treatment for cancer. The virus can also be used to identify cells
that have a deficient innate anti-viral response and to image these
cells in vivo.
[0015] In one aspect, the present invention provides a method for
inhibiting a cell that has a deficient innate anti-viral response
comprising administering to the cell an effective amount of a
combination of Myxoma virus and rapamycin.
[0016] In one aspect, the invention provides a method for treating
a disease state characterized by the presence of cells that have a
deficient innate anti-viral response, comprising administering to a
patient in need thereof an effective amount of a combination of
Myxoma virus and rapamycin.
[0017] The present invention further provides use of an effective
amount of a combination of Myxoma virus and rapamycin for
inhibiting a cell that has a deficient innate anti-viral response
and for the manufacture of a medicament for inhibiting a cell that
has a deficient innate anti-viral response.
[0018] The present invention further provides use of an effective
amount of a combination of Myxoma virus and rapamycin for treating
a disease state in a patient, wherein the disease state is
characterized by the presence of cells that have a deficient innate
anti-viral response and for the manufacture of a medicament for
treating such a disease state in a patient.
[0019] In another aspect, the present invention provides a
pharmaceutical composition comprising Myxoma virus and rapamycin.
The pharmaceutical composition may be useful for inhibiting a cell
that has a deficient innate anti-viral response or for treating a
disease state characterized by the presence of cells that have a
deficient innate anti-viral response.
[0020] In another aspect, the present invention provides a kit
comprising Myxoma virus, rapamycin and instructions for inhibiting
a cell that has a deficient innate anti-viral response or for
treating a disease state characterized by the presence of cells
that have a deficient innate anti-viral response. The disease
states include cancer and a chronic viral infection.
[0021] The present invention further provides a method of detection
a cell that has a deficient innate anti-viral response, comprising
exposing a population of cells to a combination of Myxoma virus and
rapamycin: allowing the virus to infect a cell that has a deficient
innate anti-viral response; and determining the infection of any
cells of the population of cells by the Myxoma virus.
[0022] The present invention is further based on the unexpected
discovery that rabbit Myxoma virus protein M135R is involved in
eliciting an immune response in rabbits and that a Myxoma virus
strain that does not express functional M135R can kill cells in
vitro, but does not cause myxomatosis disease in animals. Such a
viral strain can be used to treat cells having a deficient innate
anti-viral response, including those that are non-responsive to
interferon, and including treatments given in combination with the
drug rapamycin, without the need for increased containment of the
virus, leading to improved safety.
[0023] In one aspect, the present invention provides a method for
inhibiting a cell that has a deficient innate anti-viral response
comprising administering to the cell an effective amount of Myxoma
virus that does not express functional M135R, optionally in
combination with an effective amount of rapamycin.
[0024] In one aspect, the invention provides a method for treating
a disease state characterized by the presence of cells that have a
deficient innate anti-viral response, comprising administering to a
patient in need thereof an effective amount of Myxoma virus that
does not express functional M135R, optionally in combination with
an effective amount of rapamycin.
[0025] The present invention further provides use of an effective
amount of Myxoma virus that does not express functional M135R,
optionally in combination with an effective amount of rapamycin,
for inhibiting a cell that has a deficient innate anti-viral
response and in the manufacture of a medicament for inhibiting a
cell that has a deficient innate anti-viral response.
[0026] The present invention further provides use of an effective
amount of Myxoma virus that does not express functional M135R,
optionally in combination with an effective amount of rapamycin,
for treating a disease state in a patient, wherein the disease
state is characterized by the presence of cells that have a
deficient innate anti-viral response and in the manufacture of a
medicament for treating such a disease state in a patient.
[0027] In a further aspect, the present invention provides a Myxoma
virus that does not express functional M135R.
[0028] In another aspect, the present invention provides a
pharmaceutical composition comprising Myxoma virus that does not
express functional M135R. The pharmaceutical composition may be
useful for inhibiting a cell that has a deficient innate anti-viral
response or for treating a disease state characterized by the
presence of cells that have a deficient innate anti-viral response.
The pharmaceutical composition may further comprise rapamycin.
[0029] In another aspect, the present invention provides a kit
comprising Myxoma virus that does not express functional M135R and
instructions for inhibiting a cell that has a deficient innate
anti-viral response or for treating a disease state characterized
by the presence of cells that have a deficient innate anti-viral
response. The kit may further comprise rapamycin. The disease state
includes cancer and a chronic viral infection.
[0030] The present invention further provides a method for
detecting a cell that has a deficient innate anti-viral response,
comprising exposing a population of cells to a Myxoma virus that
does not express functional M135R, optionally in combination with
rapamycin; allowing the virus to infect a cell that has a deficient
innate anti-viral response; and determining the infection of any
cells of the population of cells by the Myxoma virus.
[0031] Other aspects and features of the present invention will
become apparent to those of ordinary skill in the art upon review
of the following description of specific embodiments of the
invention in conjunction with the accompanying figures. It should
be understood, however, that the detailed description and the
specific examples while indicating preferred embodiments of the
invention are given by way of illustration only, since various
changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] In the figures, which illustrate embodiments of the present
invention, by way of example only.
[0033] FIG. 1 is a schematic diagram of an interferon mediated
anti-viral signalling scheme induced upon viral infection of a
cell;
[0034] FIG. 2 is a phase contrast micrograph of nonpermissive WT
murine embryonic fibroblasts ("MEFs") after exposure to Myxoma
virus, demonstrating that the MEFs become permissive after
inhibition of interferon .alpha./.beta. with neutralizing
antibody;
[0035] FIG. 3 is a Western blot showing phosphorylation states
(activation) of STAT1 and STAT2 after Myxoma virus infection,
demonstrating that nonpermissive infections of MEF cells is
associated with activation of STAT 1 and STAT 2;
[0036] FIG. 4 is a Western blot showing phosphorylation states
(inactivation) of STAT3, STAT4, STAT5 and STAT6 after Myxoma virus
infection, demonstrating that nonpermissive infections of MEF cells
does not activate any of these species;
[0037] FIG. 5 is a phase contrast micrograph of IFN.alpha./.beta.
R-/- MEFs and STAT1 -/- MEFs, IFN.alpha./.beta. R -/- MEFs and STAT
-/- MEFs after infection with Myxoma virus, showing that
inactivation of IFN/STAT/JAK signalling renders cells permissive
for Myxoma infection;
[0038] FIG. 6 is a Western blot showing phosphorylation states of
PKR in nonpermissive wildtype MEFs after Myxoma virus infection,
demonstrating that PKR is not activated by Myxoma virus
infection;
[0039] FIG. 7 is a Western blot showing phosphorylation states of
PKR in wildtype MEFs either mock infected or pre-infected with
Myxoma virus, showing that Myxoma virus blocks PKR activation in
MEF cells:
[0040] FIG. 8 is a Western blot showing phosphorylation states of
PERK in wildtype MEFs after Myxoma virus infection, demonstrating
that Myxoma virus blocks PERK activation in MEF cells;
[0041] FIG. 9 is a phase contrast micrograph of PKR-/-, RNase L-/-
and Mx1-/- triple knockout after exposure to Myxoma virus, showing
that the antiviral state in MEF cells is mediated by a distinct
pathway;
[0042] FIG. 10 is a phase contrast micrograph of PKR-/-. RNase L-/-
and Mx1-/- triple knockout after exposure to Myxoma virus:
[0043] FIG. 11 is a phase contrast micrograph of PKR-/-, RNase L-/-
and Mx1-/- triple knockout after treatment with neutralizing
antibody to IFN.alpha./.beta. and after exposure to Myxoma
virus;
[0044] FIG. 12 is a Western blot showing phosphorylation levels of
eIF2a and PKR in nonpermissive MEFs after treatment with
neutralizing antibody to IFN.alpha./.beta. and after exposure to
Myxoma virus, showing that eIF2a phosphorylation in nonresponsive
cells is catalysed by a PKR-independent pathway;
[0045] FIG. 13 is a Western blot showing STAT1 phosphorylation
states in PKR-/-. RNase L-/- and Mx1-/- triple knockout after
Myxoma virus infection, indicating normal IFN-induced signalling
responses
[0046] FIG. 14 is a phase contrast micrograph illustrating
subcellular localization of tyrosine-phosphorylated STAT1 in
nonpermissive PKR-/-+RNaseL-/-+Mx1-/- cells at 12 hours
post-infection, indicating that the activated STAT localizes to the
nucleus, as predicted for normal IFN/STAT signalling responses;
[0047] FIG. 15 is a fluorescent image of brains from nude mice
having intracranial gliomas mock-infected or infected with dead or
live Myxoma virus expressing GFP, showing targeting of Myxoma to
the glioma cells;
[0048] FIG. 16 is a fluorescent image and a photograph of a
thin-sectioned mouse glioma infected with Myxoma virus expressing
GFP showing that the Myxoma virus replicated only in tumour
cells;
[0049] FIG. 17 is a phase contrast micrograph of HT29 human tumour
cells, stained with either X-Gal or Crystal violet after infection
with Myxoma virus, showing an example of a non-permissive infection
in human cells;
[0050] FIG. 18 is a phase contrast micrograph of HOP92 human tumour
cells, stained with X-Gal or Crystal Violet after infection with
Myxoma virus, showing an example of a permissive infection of human
cells;
[0051] FIG. 19 is phase contrast micrograph of OVCAR4 human tumour
cells, stained with either X-Gal or Crystal Violet after infection
with Myxoma virus, showing an example of a permissive infection of
human cells;
[0052] FIG. 20 is a phase contrast micrograph of SK-MEL3 human
tumour cells, stained with either X-Gal or Crystal Violet after
infection with Myxoma virus, showing an example of a permissive
infection of human cells;
[0053] FIG. 21 is a phase contrast micrograph of SK-MEL28 human
tumour cells, stained with either X-Gal or Crystal Violet after
infection with Myxoma virus, showing an example of a
semi-permissive infection of human tumour cells;
[0054] FIG. 22 is a phase contrast micrograph of BGMK cells,
stained with either X-Gal or Crystal Violet after infection with
Myxoma virus, showing a typical permissive control infection;
[0055] FIG. 23 is a phase contrast micrograph of positive control
BGMK cells and human tumour lines U87, A172 and U373 infected with
increasing concentrations of Myxoma virus expressing the LacZ
protein, stained with X-Gal, showing that these human glioma cells
were all permissive for Myxoma virus replication;
[0056] FIG. 24 is a graph depicting survival rate of BGMK. U87,
A172 and U373 cells infected with Myxoma virus, 72 hours
post-infection, at increasing concentrations of the virus,
demonstrating the ability of Myxoma to kill all of these cells;
[0057] FIG. 25 is a phase contrast micrograph and fluorescence
micrograph of SF04 1585 astrocytoma cells infected with MV GFP,
showing the infection in primary human glioma cells;
[0058] FIG. 26 is a phase contrast micrograph of U373 glioma cells
infected with Myxoma virus expressing the LacZ protein and stained
with X-Gal, showing infection of these human tumour cells;
[0059] FIG. 27 is a graph depicting the survival rate of SF04 1585
cells infected with MV GFP 48 hours post-infection, showing killing
of these infected human tumour cells;
[0060] FIG. 28 is a fluorescence micrograph of Daoy and D384
medulloblastoma lines infected with Myxoma virus expressing GFP,
showing infection of these human tumour cells.
[0061] FIG. 29 is graphical representations of the rate of virus
production in various cell lines with or without pre-treatment with
rapamycin: BGMK (primate control cell line); RK-13 and RL5 (rabbit
control cell lines); 4T1 and B16F10 (mouse cancer cell lines); HOS,
PC3, 786-0, HCT116, ACHN, MCF-7, M14 and COLO205 (human cancer cell
lines); using wildtype virus vMyxLac and the M-T5 knock out virus
vMyxTSKO as indicated;
[0062] FIG. 30 is photographs of virally infected cell lines,
infected with either vMyxLac or vMyxLacT5-;
[0063] FIG. 31 is graphical representations of the rate of virus
production in various cell lines (BGMK; A9; MCF-7; MDA-MB-435; M14;
and COLO205) with or without pre-treatment with rapamycin;
[0064] FIG. 32 is (A) a schematic alignment of Myxoma virus protein
M135R and Vaccinia virus protein B18R and (B) an amino acid
sequence alignment between M135R and the first 179 amino acids of
B18R;
[0065] FIG. 33 is (A) a Western blot of M135R expressed in BGMK
cells infected with Myxoma virus Lausanne (vMyxLau) and (B) a
Western blot of M135R expressed in BGMK cells infected with vMyxLau
and treated with araC, tunicamycin or monensin;
[0066] FIG. 34 is (A) a fluorescence micrograph of BGMK cells mock
infected or infected with Myxoma virus and stained for M135R and
(B) a Western blot against immunoprecipitations or cell lysates of
cells infected with wildtype Myxoma virus (vMyxgfp) or an M135R
knockout strain (vMyx135KO) using anti-M135R antibody;
[0067] FIG. 35 is (A) is a schematic diagram of the cloning
strategy to produce vMyx135KO. (B) an agarose gel of the PCR insert
product and (C) a Western blot of cells infected with wildtype and
M135R knockout Myxoma virus;
[0068] FIG. 36 is a growth curve of viral foci in BGMK cells
infected with vMyxgfp or vMyx135KO:
[0069] FIG. 37 is light and fluorescent micrographs of rabbit
embryo fibroblasts infected with vMyxgfp or vMyx135KO;
[0070] FIG. 38 is light and fluorescent micrographs of rabbit HIG82
fibroblasts infected with vMyxgfp or vMyx135KO;
[0071] FIG. 39 is light and fluorescent micrographs of human
primary fibroblasts infected with vMyxgfp or vMyx135KO;
[0072] FIG. 40 is a graph of body temperature in rabbits infected
with vMyxLau or vMyx135KO;
[0073] FIG. 41 is a graph of .sup.125I emissions of cells mock
infected or infected with vMyxgfp or vMyx135KO and treated with
.sup.125I-labelled rabbit interferon .alpha./.beta.;
[0074] FIG. 42 is a graph of foci formed by infecting RK13 or BGMK
cells with vMyxgfp or vMyx135KO, in which cells were untreated or
treated with rabbit interferon .alpha./.beta. 24 hours prior to
infection; and
[0075] FIG. 43 is photographs of Western blots using cell lysates
from 786-0 human cancer cells that were pre-treated with either 20
nM rapamycin (R) or with the vehicle control (D), probed using
antibodies directed against the indicated proteins.
DETAILED DESCRIPTION
[0076] Previously, the inventors have discovered that wildtype
Myxoma virus, a virus that normally infects rabbits, can
selectively infect and kill cells, including human cells, that have
a deficient innate anti-viral response, for example, cells that are
non-responsive to interferon, as described in the application
PCT/CA2004/000341, which is herein fully incorporated by reference.
Myxoma virus does not replicate efficiently in normal human cells.
Since many diseases or conditions are characterized by the presence
of cells that have a deficient innate anti-viral response,
including cells that are not responsive to interferon, for example,
cancer. Myxoma virus can be used to treat such diseases and
conditions, including cancer, with low toxicity for normal healthy
cells. Myxoma virus can also be used to treat chronically infected
cells as such cells have a deficient innate anti-viral response.
For example, many viruses encode gene products that function to
inhibit the antiviral, interferon response of cells; Myxoma virus
can selectively infect such cells.
[0077] Myxoma virus ("MV") is the causative agent of myxomatosis in
rabbits. MV belongs to the Leporipoxvirus genus of the Poxviridae
family, the largest of the DNA viruses. MV induces a benign disease
in its natural host, the Sylvilagus rabbit in the Americas.
However, it is a virulent and host-specific poxvirus that causes a
fatal disease in European rabbits, characterized by lesions found
systemically and especially around the mucosal areas. (Cameron C,
Hota-Mitchell S, Chen L, Barrett J, Cao J X. Macaulay C, Willer D,
Evans D, McFadden G. Virology 1999, 264(2): 298-318; Kerr P &
McFadden G. Viral Immunology 2002, 15(2): 229-246).
[0078] MV is a large virus with a double-stranded DNA genome of 163
kb which replicates in the cytoplasm of infected cells (B. N.
Fields, D. M. Knipe, P. M. Howley, Eds., Virology Lippincott Raven
Press, New York, 2nd ed., 1996). MV is known to encode a variety of
cell-associated and secreted proteins that have been implicated in
down-regulation of the host's immune and inflammatory responses and
inhibition of apoptosis of virus-infected cells. MV can be taken up
by all human somatic cells. However, other than in normal somatic
rabbit cells, if the cells have a normal innate anti-viral
response, the virus will not be able to productively infect the
cell, meaning the virus will not be able to replicate and cause
cell death.
[0079] Interferons ("IFNs") are a family of cytokines that are
secreted in response to a variety of stimuli. Interferons bind to
cell surface receptors, activating a signaling cascade that leads
to numerous cellular responses, including an anti-viral response
and induction of growth inhibition and/or apoptotic signals.
Interferons are classified as either type I or type II. Type I IFNs
include IFN-.alpha., -.beta., -.tau., and -.omega., which are all
monomeric; the only type II IFN is IFN-.gamma., a dimer. Twelve
different subtypes of IFN-.alpha. are produced by 14 genes, but all
other IFNs are monogenic (Arduini et al., 1999). IFNs exert direct
anti-tumour activity via the modulation of oncogene expression.
Overexpression of growth-stimulating oncogenes or loss of tumour
suppressor oncogenes can lead to malignant transformation. Some
oncogenes implicated in the genesis of cancer are p53, Rb, PC, NF1,
WT1, DCC.
[0080] Myxoma virus, as well as other oncolytic viruses such as
Reovirus and VSV, needs to bypass the anti-viral defenses that
exist in normal healthy cells in order to be able to replicate
within cells. MV and other oncolytic viruses induce interferon
production, and are generally sensitive to the anti-viral effect of
the IFN pathway. Relevant proteins induced by the IFN anti-viral
response, and which principally affect virus multiplication include
PKR, OAS synthetase and Rnase L nuclease. PKR activates
eIF2.alpha., leading to inhibition of translation and induction of
apoptosis. A schematic representation of the IFN response pathway
is depicted in FIG. 1. In normal cells, MV is directly affected by
PKR and eIF2.alpha..
[0081] Anti-viral response pathways are often disrupted in
cancerous cells. For example, reduced or defective response to IFN
is a genetic defect that often arises during the process of
transformation and tumour evolution. Over 80% of tumour cell lines
do not respond to, or exhibit impaired responses to, interferon.
(Stojdl et al., Cancer Cell (2003) 4: 263-275 and references cited
therein; Wong et al. J Biol Chem. (1997) 272(45):28779-85; Sun et
al. Blood. (1998) 91(2):570-6; Matin et al. Cancer Res. (2001)
61(5):2261-6; Balachandran et al Cancer Cell (2004) 5(1):51-65). As
previously disclosed in PCT/CA2004/000341, MV can infect and kill
cancer cells, including human tumour cells, and without being
limited by any particular theory, it is believed that MV can infect
these cells because they have a deficient innate anti-viral
response.
[0082] Evidence suggests that inhibiting the early innate immune
response and slowing the development of Th1 responses are important
for the efficacy of oncolytic therapy. Although Myxoma virus is a
virulent virus, it is host-specific and has a very narrow host
range; it does not infect humans or mice. Without being limited by
any specific theory, it is believed that since Myxoma virus is a
non-human virus, it should encounter no pre-existing immune
recognition in humans. Therefore, its potential as an oncolytic
virus will be less compromised and Myxoma virus should provide more
potent infection of permissive tumour cells than native human
viruses, and thereby can provide an effective oncolytic treatment
for cancer.
[0083] The Myxoma virus host range gene M-T5 appears to play a
critical role during Myxoma virus infection of many human tumour
cell lines (Sypula et al, (2004) Gene Ther. Mol. Biol. 8:103). The
MT-5 gene encodes an ankyrin repeat protein that is required for
Myxoma replication in rabbit lymphocytes, and Myxoma virus with the
MT-5 gene deleted cannot cause myxomatosis in susceptible rabbits
(Mossman et al, (1996) J. Virol. 70: 4394). Available evidence
suggests that differences in the intracellular signalling within an
infected human tumour cell are critical for distinguishing human
tumour cells that are permissive to Myxoma virus infection and
productive replication (Johnston et al, (2003) J. Virol. 77:
5877).
[0084] Furthermore. Myxoma virus possesses a protein, M135R, which
displays homology to the amino terminus portion of interferon
.alpha./.beta. receptor ("IFN.alpha./.beta.-R"). It has been
suggested that M135R mimics the host IFN.alpha./.beta.-R in order
to prevent IFN.alpha./.beta. from triggering a host anti-viral
response (Barrett et al., Seminars in Immunology (2001) 13:73-84).
The prediction is based on sequence homology to the viral
IFN.alpha./.beta.-R from vaccinia virus, B18R, and it has been
demonstrated that Vaccinia virus ("VV") employs such an immune
evasion strategy. However, M135R is only half the size of VV B18R
and all other IFN.alpha./.beta.-R homologs from sequenced
poxviruses, and in all cases aligns only to the amino terminus half
of the homolog.
[0085] The inventors have discovered that even though
immunofluorescence results suggest that M135R localizes to the cell
surface, attempts to demonstrate the ability of M135R to interact
with IFN.alpha./.beta. have been negative. Despite these results,
the inventors have discovered that deletion of M135R severely
attenuates the ability of Myxoma virus to cause disease in host
animals although Myxoma virus having such a deletion is equally
effective at infecting and killing cells in vitro compared to
wildtype MV. Thus, in one aspect, the present invention relates to
the discovery that Myxoma virus that does not express functional
M135R is useful for treatment of cells having a deficient innate
anti-viral response, including for oncolytic studies, since this
virus provides a safer alternative for oncolytic viral therapy as
no unusual containment strategies should be needed for patients
undergoing treatment.
[0086] In another aspect, the present invention relates to the
discovery that the anti-cancer agent rapamycin acts to enhance the
levels of infectivity of Myxoma virus in human tumour cells which
are permissive for Myxoma virus infection, and that rapamycin
allows replication of certain strains of Myxoma virus in human
tumour cells which, without rapamycin, are restrictive for the
replication of those strains of Myxoma virus. A cell that is
permissive for Myxoma virus infection is a cell that the virus can
enter and in which the virus can productively reproduce. Permissive
cells may have defects or mutations in one or more of the pathways
that involve the proteins PTEN, PDK, AKT, GSK, Raf, mTOR or P70S6K.
A restrictive cell is a cell which is permissive to Myxoma virus
only under certain conditions, but does not allow productive
infection under other conditions. For example, a restrictive cell
may be permissive to wildtype strains of the virus, but does not
allow certain mutant Myxoma strains, for example a strain having
the MT-5 gene knocked out, to productively reproduce. In another
example, a cell restrictive for Myxoma virus may not permit
productive infection of Myxoma virus alone, but when treated with
rapamycin, the same Myxoma virus is able to productively infect the
cell. Abortive cell lines are non-permissive for Myxoma virus
infection, meaning that the virus may be able to enter the cell,
but does not productively infect the cell.
[0087] Thus, rapamycin, when used in combination with Myxoma virus,
enhances the infectivity of Myxoma virus for cells having a
deficient innate anti-viral response. The present invention relates
to the use of rapamycin in combination with Myxoma virus to treat
cells having a deficient innate anti-viral response.
[0088] Rapamycin is a macrocyclic lactone that has been shown to be
the active antifungal compound purified from the soil bacterium
Streptomyces hygroscopicus. Rapamycin as used herein refers to
rapamycin (also referred to as sirolimus) and analogs or
derivatives thereof capable of complexing with FKBP12 and
inhibiting mTOR, including the analogs CCI-779 (also referred to as
cell cycle inhibitor-779 or
rapamycin-42,2,2-bis(hydroxymethyl)-propionic acid) and RAD001
(also referred to as everolimus or
40-O-(2-hydroxyethyl)-rapamycin). Rapamycin, CCI-779 and RAD001 are
commercially available, and rapamycin is available under the name
Rapamune.TM., from Wyeth-Ayerst. The term rapamycin further
includes pharmaceutically acceptable salts and esters of rapamycin,
its hydrates, solvates, polymorphs, analogs or derivatives, as well
as pro-drugs or precursors which are metabolized or converted to
rapamycin or its analogs or derivatives during use, for example
when administered to a patient.
[0089] Rapamycin as an inhibitor of cellular signaling is highly
specific: it enters the cell and binds to a cellular protein known
as FKBP12. The rapamycin/FKBP12 complex then binds to the specific
cellular target mTOR (mammalian Target of Rapamycin). Many cancers
have been shown to develop from an over activity of signaling
molecules such as P13K, or a loss of the tumor suppressor gene
PTEN. Both of these molecules lie upstream of mTOR. mTOR has been
shown to be a central regulator of cell proliferation, growth,
differentiation, migration and survival, and is therefore an ideal
target in stemming the uncontrolled growth of cancer cells. Cancer
cell lines that are sensitive to rapamycin are generally those that
have resulted from an activation of the pathway through mTOR.
[0090] Rapamycins are used primarily in transplant patients as an
alternative or complementary treatment to cyclosporine treatment.
In transplant patients, rapamycin treatment generally has fewer
side effects that cyclosporine A or FK506. In addition,
retrospective studies have indicated that patients on rapamycin
treatment generally develop fewer cancers and have a lower
incidence of CMV (cytomegalovirus; a herpes virus) infection. It is
therefore surprising that rapamycin treatment enhances Myxoma virus
infection of cancer cells, particularly in light of research
postulating that CMV replication should be reduced by rapamycin
(reviewed by Ponticelli: "The pleiotropic effects of mTOR
inhibitors" in J Nephrology 2004; 17: 762). Without being limited
to a particular theory, it is possible that Myxoma virus takes
advantage of aberrant signaling through the mTOR pathway that may
be associated with the neoplastic phenotype of these cells.
Manipulation of this pathway by mTOR inhibitors could then be a
selective advantage to the virus.
[0091] Thus, there is provided a method for inhibiting a cell that
has a deficient innate anti-viral response comprising administering
to the cell an effective amount of Myxoma virus. In a further
embodiment, the virus is administered in combination with an
effective amount of rapamycin.
[0092] The Myxoma virus may be any virus that belongs to the
Leporipoxvirus species of pox viruses that is
replication-competent. The Myxoma virus may be a wild-type strain
of Myxoma virus or it may be a genetically modified strain of
Myxoma virus, including an MT-5 knockout strain of Myxoma. The
Myxoma virus may be a strain that has an attenuated affect in
rabbits, thereby causing lower risk of disease, including a strain
that does not express functional M135 protein, as described
below.
[0093] In a particular embodiment, the Myxoma virus is a Myxoma
virus that does not express functional M135R.
[0094] A Myxoma virus that does not express functional M135R
includes a Myxoma virus that has part, or all, of the open reading
frame that encodes M135R deleted, replaced or interrupted such that
no gene product, no stable gene product, or no functional gene
product is expressed. Such a virus also includes a Myxoma virus
that has part, or all, of the M135R gene regulatory region deleted,
replaced or interrupted such that no protein can be expressed from
the gene encoding M135R. Functional M135R protein is M135R that is
transcribed, translated, folded, post-translationally modified and
localized within the cell, and which allows Myxoma virus to cause
myxomatosis in an infected host. If the M135R protein is not, or
not properly or not sufficiently, transcribed, translated, folded,
post-translationally modified or localized within the cell such
that an infected host does not develop myxomatosis, then no
functional M135R protein is expressed in the cell.
[0095] In a further embodiment, the cell is non-responsive to
interferon.
[0096] In specific embodiments, the cell is a mammalian cancer
cell. In one embodiment the cell is a human cancer cell including a
human solid tumour cell.
[0097] In another embodiment, the cell is chronically infected with
a virus.
[0098] A "combination" of rapamycin and Myxoma virus for
administration may be formulated together in the same dosage form
or may be formulated in separate dosage forms, and the separate
dosage forms may be the same form or different forms, for
administration by the same mode or by different modes of
administration. Furthermore, administration of a combination of
rapamycin and Myxoma virus, when not together in the same dosage
form, means that the rapamycin and Myxoma virus are administered
concurrently to the mammal being treated, and may be administered
at the same time or sequentially in any order or at different
points in time. Thus, rapamycin and Myxoma virus may be
administered separately but sufficiently closely in time so as to
provide the desired therapeutic effect.
[0099] The term "effective amount" as used herein means an amount
effective, at dosages and for periods of time necessary to achieve
the desired result.
[0100] The term "a cell that has a deficient innate anti-viral
response" as used herein refers to a cell that, when exposed to a
virus or when invaded by a virus, does not induce anti-viral
defence mechanisms, which include inhibition of viral replication,
production of interferon, induction of the interferon response
pathway, and apoptosis, which may or may not be mediated by
interferon, and is thereby infectable by MV, alone or in
combination with rapamycin treatment. The term includes a cell that
has a reduced or defective innate anti-viral response upon exposure
to or infection by a virus as compared to a normal cell, for
example, a non-infected, or non-cancer cell. This includes a cell
that is non-responsive to interferon and a cell that has a reduced
or defective apoptotic response or induction of the apoptotic
pathway. The deficiency may be caused by various causes, including
infection, genetic defect, or environmental stress. It will however
be understood that when the deficiency is caused by a pre-existing
infection, superinfection by MV may be excluded and a skilled
person can readily identify such instances. A skilled person can
readily determine without undue experimentation whether any given
cell type has a deficient innate anti-viral response and therefore
infectable by Myxoma virus, either alone or in combination with
rapamycin treatment. For example, VSV is commonly used to measure
an anti-viral response of a cell.
[0101] To assess whether a given cell type, for example a given
cancer cell type, has a deficient innate anti-viral response, a
skilled person can take an explant, grow some of the cells in vitro
and determine infectability by VSV or alternatively, by Myxoma
virus, including Myxoma virus in combination with rapamycin.
[0102] The term "a cell that is non-responsive to interferon" as
used throughout the specification means a cell that does not
respond to the activity of interferon, for example anti-viral or
anti-tumour activity of interferon or that has an abnormal
interferon response, for example, a reduced or ineffective response
to interferon, or abnormal interferon signalling as measured by,
for example, phosphorylation or activation of signalling molecules
such as transcription factors, for example STAT1. For example,
without limitation, the cell may not undergo inhibition of
proliferation or it may not be killed when exposed to interferon
levels sufficient to induce such a response in a cell that is
responsive to interferon. The cell that is non-responsive to
interferon may have a defect in the intracellular signalling
pathway or pathways that are normally activated in the responsive
cells. Typically, susceptibility to infection by VSV is indicative
of non-responsiveness to interferon, and a skilled person can
readily determine whether a particular cell is non-responsive to
interferon by its ability, or lack thereof, to inhibit VSV
infection in the presence of interferon or using other markers of
interferon activity known in the art, for example, the level of
expression of IFN stimulated genes such as PKR, STAT, OAS, MX.
[0103] The term "replication-competent" as used throughout the
specification refers to a virus that is capable of infecting and
replicating within a particular host cell. This includes a virus
which alone is restricted for replication in a particular host
cell, but when the host cell is treated with rapamycin, the virus
can then productively infect that cell.
[0104] The term "a cell" as used herein includes a single cell as
well as a plurality or population of cells.
[0105] Administering an agent to a cell includes both in vitro and
in vive administrations.
[0106] The term "animal" as used herein includes all members of the
animal kingdom, including particularly mammals, especially
humans.
[0107] The term "inhibiting" a cell that has a deficient innate
anti-viral response includes cell death by lysis or apoptosis or
other mechanisms of cell death, in addition to rendering the cell
incapable of growing or dividing or reducing or retarding cell
growth or division.
[0108] The Myxoma virus genome may be readily modified to express
one or more therapeutic transgenes using standard molecular biology
techniques known to a skilled person, and described for example in
Sambrook et al. ((2001) Molecular Cloning: a Laboratory Manual,
3.sup.rd ed., Cold Spring Harbour Laboratory Press). A skilled
person will be able to readily determine which portions of the
Myxoma viral genome can be deleted such that the virus is still
capable of productive infection. For example, non-essential regions
of the viral genome that can be deleted can be deduced from
comparing the published viral genome sequence with the genomes of
other well-characterized viruses (see for example C. Cameron, S.
Hota-Mitchell, L. Chen, J. Barrett, J.-X. Cao, C. Macaulay, D.
Willer, D. Evans, and G. McFadden, Virology (1999) 264:
298-318)).
[0109] The term "therapeutic gene" or "therapeutic transgenes" as
used herein is intended to describe broadly any gene the expression
of which effects a desired result, for example, anti-cancer effect.
For example, the virus may be modified to carry a gene that will
enhance the anti-cancer effect of the viral treatment. Such a gene
may be a gene that is involved in triggering apoptosis, or is
involved in targeting the infected cell for immune destruction,
such as a gene that repairs a lack of response to interferon, or
which results in the expression of a cell surface marker that
stimulates an antibody response, such as a bacterial cell surface
antigen. The virus may also be modified to express genes involved
in shutting off the neoplastic or cancer cell's proliferation and
growth, thereby preventing the cells from dividing. As well, the
virus may be modified to include therapeutic genes, such as genes
involved in the synthesis of chemotherapeutic agents, or it may be
modified to have increased replication levels in cells of the
particular species from which the cells to be inhibited or killed
are derived, for example, human cells. Specific examples of genes
that may be inserted into the Myxoma virus to increase its
anti-cancer effect include the human gene for the TRAIL protein or
the adenoviral gene that encodes the E4 orf4 polypeptide, both of
which proteins are involved in killing human tumour cells.
[0110] It will be understood that therapeutic effect of the Myxoma
virus, including when used in combination with rapamycin, may be
achieved by cell lysis by the virus or by delivery of therapeutic
products by the virus. The inclusion of rapamycin in combination
with the Myxoma virus should allow for enhancement of the effect of
Myxoma virus alone. That is, the Myxoma virus, when administered in
combination with rapamycin should be able to productively infect a
greater number of target cells than Myxoma virus alone, or should
be able to productively infect target cells having a deficient
innate anti-viral response which are restrictive for productive
infection by Myxoma virus in the absence of rapamycin.
[0111] The virus may be prepared using standard techniques known in
the art. For example, the virus may be prepared by infecting
cultured rabbit cells with the Myxoma virus strain that is to be
used, allowing the infection to progress such that the virus
replicates in the cultured cells and can be released by standard
methods known in the art for disrupting the cell surface and
thereby releasing the virus particles for harvesting. Once
harvested, the virus titre may be determined by infecting a
confluent lawn of rabbit cells and performing a plaque assay (see
Mossman et al. (1996) Virology 215:17-30).
[0112] There is also provided a method for treating a disease state
characterized by the presence of cells that have a deficient innate
anti-viral response in a patient in need of such treatment
comprising administering to the patient an effective amount of
Myxoma virus, optionally in combination with rapamycin. The patient
may be any animal, including a mammal, including a human.
[0113] "A disease state characterized by the presence of cells that
have a deficient innate anti-viral response" as used herein refers
to any disease, disorder or condition which is associated with,
related to, or a characteristic of which is, the presence of cells
that have a deficient innate anti-viral response and which disease,
disorder, condition or symptoms thereof may be treated by killing
these cells. For example, the disease state may be cancer. The
disease state may also include chronic infection with a virus.
[0114] "Treating" a disease state refers to an approach for
obtaining beneficial or desired results, including clinical
results. Beneficial or desired clinical results can include, but
are not limited to, alleviation or amelioration of one or more
symptoms or conditions, diminishment of extent of disease,
stabilization of the state of disease, prevention of development of
disease, prevention of spread of disease, delay or slowing of
disease progression, delay or slowing of disease onset,
amelioration or palliation of the disease state, and remission
(whether partial or total). "Treating" can also mean prolonging
survival of a patient beyond that expected in the absence of
treatment. "Treating" can also mean inhibiting the progression of
disease, slowing the progression of disease temporarily, although
more preferably, it involves halting the progression of the disease
permanently.
[0115] In one embodiment, the disease state is cancer. The cancer
may be any type of cancer wherein at least some of the cells,
although not necessarily all of the cells have a deficient innate
anti-viral response. In one embodiment, the cancer may be a cancer
wherein at least some of the cells are non-responsive to
interferon. As used herein, the terms "tumour", "tumour cells",
"cancer" and "cancer cells", (used interchangeably) refer to cells
that exhibit abnormal growth, characterized by a significant loss
of control of cell proliferation or cells that have been
immortalized. The term "cancer" or "tumour" includes metastatic as
well as non-metastatic cancer or tumours. As used herein.
"neoplastic" or "neoplasm" broadly refers to a cell or cells that
proliferate without normal growth inhibition mechanisms, and
therefore includes benign tumours, in addition to cancer as well as
dysplastic or hyperplastic cells.
[0116] A cancer may be diagnosed using criteria generally accepted
in the art, including the presence of a malignant tumor.
[0117] Types of cancer that may be treated according to the present
invention include, but are not limited to, hematopoietic cell
cancers including leukemias and lymphomas, colon cancer, lung
cancer, kidney cancer, pancreas cancer, endometrial cancer, thyroid
cancer, oral cancer, ovarian cancer, laryngeal cancer,
hepatocellular cancer, bile duct cancer, squamous cell carcinoma,
prostate cancer, breast cancer, cervical cancer, colorectal cancer,
melanomas and any other tumours. Solid tumours such as sarcomas and
carcinomas include but are not limited to fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and
other sarcomas, synovioma, mesothelioma, Ewing's tumor,
leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid
malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian
cancer, prostate cancer, hepatocellular carcinoma, squamous cell
carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland
carcinoma, sebaceous gland carcinoma, papillary carcinoma,
papillary adenocarcinomas, medullary carcinoma, bronchogenic
carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,
choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor,
bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma,
medulloblastoma, craniopharyogioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,
melanoma, neuroblastoma and retinoblastoma).
[0118] In another embodiment, the disease state is a chronic viral
infection.
[0119] The chronically infecting virus may be any virus that
infects and replicates in cells of an animal in a persistent manner
over a prolonged period so as to cause a pathological condition.
The chronically infecting virus may be a virus that is associated
or correlated with the development of cancer.
[0120] A chronic infection with a virus may be diagnosed using
standard methods known in the art. For example, a chronic viral
infection may be detected by the presence of anti-viral antibodies
in the patient or a positive test for the presence of viral RNA or
DNA in cells of the patient.
[0121] When administered to a patient, an effective amount of the
Myxoma virus, and optionally the combination of Myxoma virus with
rapamycin, is the amount required, at the dosages and for
sufficient time period, for the virus to alleviate, improve,
mitigate, ameliorate, stabilize, prevent the spread of, slow or
delay the progression of or cure the disease. For example, it may
be an amount sufficient to achieve the effect of reducing the
number of or destroying cancerous cells or neoplastic cells, or
reducing the number of or destroying cells chronically infected
with a virus, or inhibiting the growth and/or proliferation of such
cells.
[0122] The effective amount to be administered to a patient can
vary depending on many factors such as the pharmacodynamic
properties of the Myxoma virus and the optionally rapamycin, the
modes of administration, the age, health and weight of the patient,
the nature and extent of the disease state, the frequency of the
treatment and the type of concurrent treatment, if any, and the
virulence and titre of the virus.
[0123] One of skill in the art can determine the appropriate amount
of Myxoma virus for administration based on the above factors. The
virus may be administered initially in a suitable amount that may
be adjusted as required, depending on the clinical response of the
patient. The effective amount of virus can be determined
empirically and depends on the maximal amount of the virus that can
be administered safely, and the minimal amount of the virus that
produces the desired result.
[0124] Myxoma virus may be administered to the patient using
standard methods of administration. In one embodiment, the virus is
administered systemically. In another embodiment, the virus is
administered by injection at the disease site. In a particular
embodiment, the disease state is a solid tumour and the virus is
administered by injection at the tumour site. In various
embodiments, the virus may be administered orally or parenterally,
or by any standard method known in the art.
[0125] To produce the same clinical effect when administering the
virus systemically as that achieved through injection of the virus
at the disease site, administration of significantly higher amounts
of virus may be required. However, the appropriate dose level
should be the minimum amount that would achieve the desired
result.
[0126] The concentration of virus to be administered will vary
depending on the virulence of the particular strain of Myxoma that
is to be administered and on the nature of the cells that are being
targeted. In one embodiment, a dose of less than about 10.sup.9
plaque forming units ("pfu") is administered to a human patient. In
various embodiments, between about 10' to about 10.sup.9 pfu,
between about 10.sup.2 to about 10.sup.7 pfu, between about
10.sup.3 to about 10.sup.6 pfu, or between about 10.sup.4 to about
10.sup.5 pfu may be administered in a single dose.
[0127] One of skill in the art can also determine, using the above
factors, the appropriate amount of rapamycin to administer to a
patient. The effective amount of rapamycin can be determined
empirically and will depend on the amount and strain of virus being
administered, the maximum amount of rapamycin that can be safely
administered and the minimal amount of rapamycin that can be
administered in order to achieve an enhancement of the infectivity
of Myxoma virus.
[0128] Rapamycin may be administered to the patient using standard
methods of administration. In one embodiment, the rapamycin is
administered systemically. In another embodiment, the rapamycin is
administered by injection at the disease site. In a particular
embodiment, the disease state is a solid tumour and the rapamycin
is administered by injection at the tumour site.
[0129] In various embodiments, the rapamycin may be administered
orally or parenterally, or by any standard method known in the
art.
[0130] The total amount of rapamycin may be administered in a
single dose or in multiple doses spread out over 1 day or several
days. The frequency and duration of administration of doses can be
readily determined. The schedule of dosing will depend on the
length of time that the Myxoma virus is to be administered. For
example, rapamycin may be administered once to a patient, or may be
administered 2 to 4 times per day.
[0131] In various embodiments, the dose of rapamycin may be from
about 0.01 to about 250 mg per kg of body weight per day, from
about 0.01 to 50 mg per kg of body weight per day, from about 0.05
to 10 mg per kg of body weight per day, or from about 0.1 to 7.5 mg
per kg of body weight per day.
[0132] Effective amounts of a combination of Myxoma virus and
rapamycin can be given repeatedly, depending upon the effect of the
initial treatment regimen. Administrations are typically given
periodically, while monitoring any response. It will be recognized
by a skilled person that lower or higher dosages than those
indicated above may be given, according to the administration
schedules and routes selected.
[0133] The Myxoma virus, optionally in combination with rapamycin,
may be administered as a sole therapy or may be administered in
combination with other therapies, including chemotherapy, radiation
therapy or other anti-viral therapies. For example, the Myxoma
virus, optionally in combination with rapamycin, may be
administered either prior to or following surgical removal of a
primary tumour or prior to, concurrently with or following
treatment such as administration of radiotherapy or conventional
chemotherapeutic drugs. In one embodiment, the Myxoma virus,
optionally in combination with rapamycin can be administered in
combination with, or in a sequential fashion with, other oncolytic
viruses, which may demonstrate specificity for varying tumour cell
types.
[0134] To aid in administration, the Myxoma virus, optionally in
combination together with rapamycin, may be formulated as an
ingredient in a pharmaceutical composition. Therefore, in a further
embodiment, there is provided a pharmaceutical composition
comprising Myxoma virus, and optionally rapamycin, and a
pharmaceutically acceptable diluent. The invention in one aspect
therefore also includes such pharmaceutical compositions for use in
inhibiting a cell that has a deficient innate anti-viral response
or treating a disease state characterized by the presence of cells
that have a deficient innate anti-viral response. The compositions
may routinely contain pharmaceutically acceptable concentrations of
salt, buffering agents, preservatives and various compatible
carriers. For all forms of delivery, the recombinant Myxoma virus
may be formulated in a physiological salt solution.
[0135] The pharmaceutical compositions may additionally contain
additional therapeutic agents, such as additional anti-cancer
agents. In one embodiment, the compositions include a
chemotherapeutic agent. The chemotherapeutic agent, for example,
may be substantially any agent which exhibits an oncolytic effect
against cancer cells or neoplastic cells of the patient and that
does not inhibit or diminish the tumour killing effect of the
Myxoma virus. For example, the chemotherapeutic agent may be,
without limitation, an anthracycline, an alkylating agent, an alkyl
sulfonate, an aziridine, an ethylenimine, a methylmelamine, a
nitrogen mustard, a nitrosourea, an antibiotic, an antimetabolite,
a folic acid analogue, a purine analogue, a pyrimidine analogue, an
enzyme, a podophyllotoxin, a platinum-containing agent or a
cytokine. Preferably, the chemotherapeutic agent is one that is
known to be effective against the particular cell type that is
cancerous or neoplastic.
[0136] The proportion and identity of the pharmaceutically
acceptable diluent is determined by chosen route of administration,
compatibility with a live virus, and where applicable compatibility
with the chemical stability of rapamycin, and standard
pharmaceutical practice. Generally, the pharmaceutical composition
will be formulated with components that will not significantly
impair the biological properties of the live Myxoma virus, or cause
degradation of or reduce the stability or efficacy of the rapamycin
where included.
[0137] The pharmaceutical composition can be prepared by known
methods for the preparation of pharmaceutically acceptable
compositions suitable for administration to patients, such that an
effective quantity of the active substance or substances is
combined in a mixture with a pharmaceutically acceptable vehicle.
Suitable vehicles are described, for example, in Remington's
Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack
Publishing Company, Easton, Pa., USA 1985). On this basis, the
compositions include, albeit not exclusively, solutions of the
Myxoma virus, optionally with rapamycin, in association with one or
more pharmaceutically acceptable vehicles or diluents, and
contained in buffer solutions with a suitable pH and iso-osmotic
with physiological fluids.
[0138] The pharmaceutical composition may be administered to a
patient in a variety of forms depending on the selected route of
administration, as will be understood by those skilled in the art.
The composition of the invention may be administered orally or
parenterally. Parenteral administration includes intravenous,
intraperitoneal, subcutaneous, intramuscular, transepithelial,
nasal, intrapulmonary, intrathecal, rectal and topical modes of
administration. Parenteral administration may be by continuous
infusion over a selected period of time.
[0139] The pharmaceutical composition may be administered orally,
for example, with an inert diluent or with an assimilable carrier,
or it may be enclosed in hard or soft shell gelatin capsules, or it
may be compressed into tablets. For oral therapeutic
administration, the Myxoma virus may be incorporated, optionally
together with rapamycin, with an excipient and be used in the form
of ingestible tablets, buccal tablets, troches, capsules, elixirs,
suspensions, syrups, wafers and the like.
[0140] Solutions of Myxoma virus, optionally together with
rapamycin, may be prepared in a physiologically suitable buffer.
Under ordinary conditions of storage and use, these preparations
contain a preservative to prevent the growth of microorganisms, but
that will not inactivate the live virus. A person skilled in the
art would know how to prepare suitable formulations. Conventional
procedures and ingredients for the selection and preparation of
suitable formulations are described, for example, in Remington's
Pharmaceutical Sciences and in The United States Pharmacopeia: The
National Formulary (USP 24 NF19) published in 1999.
[0141] In different embodiments, the composition is administered by
injection (subcuteanously, intravenously, intramuscularly, etc.)
directly at the disease site, such as a tumour site, or by oral
administration, alternatively by transdermal administration.
[0142] The forms of the pharmaceutical composition suitable for
injectable use include sterile aqueous solutions or dispersion and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions, wherein the term sterile does
not extend to the live Myxoma virus itself that is to be
administered. In all cases the form must be sterile and must be
fluid to the extent that easy syringability exists.
[0143] The dose of the pharmaceutical composition that is to be
used depends on the particular condition being treated, the
severity of the condition, the individual patient parameters
including age, physical condition, size and weight, the duration of
the treatment, the nature of concurrent therapy (if any), the
specific route of administration and other similar factors that are
within the knowledge and expertise of the health practioner. These
factors are known to those of skill in the art and can be addressed
with minimal routine experimentation.
[0144] The Myxoma virus, optionally in combination with rapamycin,
or pharmaceutical compositions comprising the Myxoma virus and
rapamycin, either together in the same formulation or different
formulations, may also be packaged as a kit, containing
instructions for use of Myxoma virus and rapamycin, including the
use of Myxoma virus, or use of Myxoma virus in combination with
rapamycin, to inhibit a cell that has a deficient innate anti-viral
response, or use of Myxoma virus, or use of Myxoma virus in
combination with rapamycin, to treat a disease state characterized
by the presence of cells that have a deficient innate anti-viral
response, in a patient in need thereof. The disease state may be
cancer, or it may be a chronic viral infection.
[0145] The present invention also contemplates the use of Myxoma
virus, optionally in combination with rapamycin, for inhibiting a
cell that has a deficient innate anti-viral response. In one
embodiment, the cell is non-responsive to interferon. There is
further provided use of Myxoma virus, optionally in combination
with rapamycin, for treating a disease state characterized by the
presence of cells that have a deficient innate anti-viral response,
in a patient in need thereof. In one embodiment the disease state
is cancer.
[0146] There is also provided use of Myxoma virus, optionally in
combination with rapamycin, in the manufacture of a medicament, for
inhibiting a cell that has a deficient innate anti-viral response,
or for treating a disease state characterized by the presence of
cells that have a deficient innate anti-viral response in a patient
in need thereof.
[0147] MV can selectively infect cells in or derived from animals
other than the natural host of MV, from a population of cells,
which have a deficient innate anti-viral response. This ability of
MV provides for the use of MV in detecting cells from a population
of cells, either in culture or in an animal, that have a deficient
innate anti-viral response, including cells that are non-responsive
to interferon. Such cells may otherwise not be easily detectable,
for example certain cancer cells that have not yet advanced to
palpable tumour, or have not yet induced noticeable symptoms in the
animal.
[0148] Thus, in one embodiment, there is provided a method for
detecting cells that have a deficient innate anti-viral response in
a patient, comprising administering to the patient Myxoma virus
modified to express a detectable marker, optionally in combination
with rapamycin; allowing the virus to infect a cell that has a
deficient innate anti-viral response in the patient; and detecting
the cell expressing the detectable marker in the patient.
[0149] The infected cells may be detected using any conventional
method for visualizing diagnostic images. The method of detection
will depend on the particular detectable marker that is used. For
example, cells infected with Myxoma virus genetically modified to
express a fluorescent protein may be detected using fluorescence
digital imaging microscopy. Other methods include computed
tomography (CT), whole body scan such as position emission
tomography (PET), magnetic resonance imaging (MRI), and sonography.
Skilled artisans will be able to determine the appropriate method
for detecting a particular detectable marker.
[0150] The detectable marker includes, but is not limited to, any
marker for which genes for its expression or synthesis can be
inserted into the Myxoma genome so as to result in expression or
synthesis of the marker within cells that are infected by the
modified virus. For example, in one embodiment, the detectable
marker may be a fluorescent protein. The infected cells may be
detected at a suitable time interval after administration of the
modified virus to the patient, so as to allow for the virus to
infect any cells that have a deficient innate anti-viral response,
and to express the detectable marker in such cells at levels that
would allow for detection. For example, detection may occur
anywhere between 2 and 20 days following administration to the
patient of the virus genetically modified to express a fluorescent
protein.
[0151] The detecting method may be carried out repeatedly at
intervals in a patient in order to monitor the presence of cells
that have a deficient innate anti-viral response in that patient.
For example, the method for detecting such cells using Myxoma virus
may be carried out on a patient at 6 month, 1 year or 2 year
intervals, as is necessary, depending on the nature of the cells
that has a deficient innate anti-viral response and the nature of
any disease state caused as a result of the presence of such cells
in a patient. Repeating the method over a time period allows for
monitoring of the progression or remission of disease state, or the
spread of disease within the body of the patient.
[0152] Myxoma virus is capable of selectively infecting cells that
have a deficient innate anti-viral response, and can be used as an
indicator of such a deficiency in cells. Thus, cells removed from a
patient may be assayed for deficiency in innate anti-viral response
using the methods of the present invention. Such determination may
indicate, when combined with other indicators, that the patient may
be suffering from a particular disease state, for example,
cancer.
[0153] In one embodiment therefore, there is provided a method for
detecting in a sample a cell that has a deficient innate anti-viral
response comprising culturing the cell, exposing cultured cells to
Myxoma virus, optionally in combination with rapamycin; and
determining infectivity of cells by Myxoma virus.
[0154] The cells may be removed from a subject, including a human
subject, using known biopsy methods. The biopsy method will depend
on the location and type of cell that is to be tested.
[0155] Cells are cultured according to known culturing techniques,
and are exposed to MV, and optionally rapamycin, by adding live
Myxoma virus, and optionally rapamycin, to the culture medium.
Where Myxoma virus is added in combination with rapamycin, the
virus and rapamycin may be added either simultaneously or
sequentially. The multiplicity of infection ("MOI"), including in
the presence of rapamycin, may be varied to determine an optimum
MOI for a given cell type, density and culture technique, and a
particular rapamycin concentration, using a positive control cell
culture that is known to be infected upon exposure to MV.
[0156] The amount of rapamycin, and the timing of addition of
rapamycin and Myxoma virus to the cultured cells may be varied
depending on cell type, method of culturing and strain of virus.
Such parameters can be readily tested and adjusted with minimal
testing using routine methods.
[0157] Infectivity of the cultured cells by MV, including in the
presence of rapamycin, may be determined by various methods known
to a skilled person, including the ability of the MV to cause cell
death. It may also involve the addition of reagents to the cell
culture to complete an enzymatic or chemical reaction with a viral
expression product. The viral expression product may be expressed
from a reporter gene that has been inserted into the MV genome.
[0158] In one embodiment the MV may be modified to enhance the ease
of detection of infection state. For example, the MV may be
genetically modified to express a marker that can be readily
detected by phase contrast microscopy, fluorescence microscopy or
by radioimaging. The marker may be an expressed fluorescent protein
or an expressed enzyme that may be involved in a colorimetric or
radiolabelling reaction. In another embodiment the marker may be a
gene product that interrupts or inhibits a particular function of
the cells being tested.
[0159] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1: Infection of Mouse and Human Cell Lines with Myxoma
Virus
[0160] Virus Strains
[0161] Viral strains used include wildtype MV, MV modified to
express either green fluorescence protein ("GFP") or
.beta.-galactosidase ("LacZ"), and killed ("dead") MV. Viruses were
prepped and titred using standard techniques.
[0162] Cell Strains
[0163] Mouse experiments were performed using mouse embryo
fibroblasts ("MEFs") derived from a wild-type mouse, and from the
following mouse knockouts: IFN.alpha./.beta. receptor homozygous
knockout; STAT1 homozygous knockout; PKR heterozygous; RNaseL
heterozygous knockout; Mx1 heterozygous knockout; triple
PKR/RNaseL/Mx1 homozygous knockout.
[0164] Human experiments were performed on BGMK control cells and
human tumour cell lines HT29, HOP92, OVCAR4, OVCAR5, SK-MEL3,
SK-MEL28, M14, SKOV3, PC3, DU145, CAKI-1, 786-0, T47D, MDAMB 435,
SF04, U87. A172, U373, Daoy and D384 as described in Stojdl et al.,
Cancer Cell (2003) 4: 263-275.
[0165] Methods
[0166] Generally, assays and experiments were performed as
described in Lalani et al. Virology (1999) 256: 233-245; Johnston
et al. J Virology (2003) 77(13): 7682-7688; and Sypula et al. Gen
Ther Mol Biol (2004) 8: 103.
[0167] For the in vivo mouse studies, nude mice were implanted with
intracranial human gliomas U87. 15 days after implantation, mice
were intratumourally injected with live or dead MV GFP, at a titre
of 5.times.10.sup.6, or mock-infected. 72 hours post-infection,
animals were sacrificed, the brains removed, embedded in OCT
(Optimal Cutting Temperature compound), and frozen sections were
cut. Myxoma-GFP was visualized in whole brain sections by
fluorescence microscopy. Sections were then fixed and stained with
H&E (hemotoxylin and eosin) to visualize the tumor.
[0168] For human tumour cell assays, the tumours were trypsonized
and plated immediately after surgery and infected with virus the
next day at an MOI of 0.1, 1.0 or 10. Data was gathered regarding
cytotoxicity and viral expression using phase microscopy and
fluorescent microscopy, respectively, at 24 and 48 hours
post-infection. Assays using the yellow tetrazolium salt MTT were
performed to quantify the % cell survival (as a percentage of cells
surviving mock infection) at 48, 72 or 96 hours post-infection.
[0169] Human pediatric medulloblastoma cell lines, Daoy and D384,
were infected with 10 M.O.I. of Myxoma-GFP. 72 hours after
infection, cell viability was measured using MTT.
[0170] Results: Infections of Mouse Cell Lines Previous research
showed that some clones of mouse 3T3 cells transfected with
chemokine receptors were infectable by Myxoma virus while other
clones were not. To investigate whether Myxoma virus tropism in
other mouse cells was dependent on any particular receptors, we
exploited primary mouse embryo fibroblasts (MEFs) from wild-type
(WT) mice and various gene knock-outs.
[0171] Since IFNs play a key role in mounting anti-viral responses,
we hypothesized that the restrictive phenotype was related to the
"antiviral state" mediated by IFN. Disruption of the chain of
events of the IFN system, neutralizing circulating IFN with
antibodies or generating IFN receptor negative mice, or mice with
deleted genes in the intracellular pathway of signal transmission,
would severely compromise the host's resistance to the Myxoma virus
which typically does not infect normal mouse cells.
[0172] In order to test this hypothesis we needed to demonstrate if
the non-infectivity of Myxoma virus in the nonpermissive cells was
due to the antiviral action of IFNs. Various MEF cell types having
knock-outs of one or more proteins involved in intracellular IFN
signaling response were tested for the effect of MV infection on
the IFN pathway.
[0173] Experiments performed on primary MEFs demonstrated that
wildtype ("WT") MEFs are not infectable by Myxoma virus. The MEFs
are fully infectable by Myxoma virus when the IFN pathway is
blocked by neutralizing antibody to IFN.alpha./.beta. (FIG. 2).
However, MEFs exposed to neutralizing antibodies to IFN.gamma.
remained nonpermissive. This outlined the importance of
IFN.alpha./3 but not IFN.gamma. in creating a permissive
environment for Myxoma virus to infect MEFs in vitro. Different
intracellular signaling pathways for IFN.alpha./.beta. and
IFN.gamma. have been identified in the literature. However, both
IFN.alpha./.beta. and IFN.gamma. likely play an important role in
infected hosts, unlike cultured fibroblasts. We predict that human
tumors deficient in either IFN.alpha./.beta. and/or IFN.gamma.
pathway will be susceptible to Myxoma virus infection in vivo.
[0174] We examined the activity of STAT1 and STAT2 in nonpermissive
WT MEFs that were infected with MV. The results shown in FIG. 3
indicated that STAT1 and STAT2 were activated. Further study showed
that STAT3, STAT4 and STAT5 are not activated (FIG. 4).
[0175] In order to confirm the importance of the IFN.alpha./.beta.
intracellular pathway in maintaining a nonpermissive state in MEFs,
genetic deletion studies were performed to provide disruptions in
the IFN.alpha./.beta. receptors and in the intracellular cascade.
Genetic deletion of IFN receptors or JAK1 or STAT1 was performed.
MV was used to infect WT MEFs, IFN.alpha./.beta. R-/- MEFs and
STAT1 -/- MEFs. IFN.alpha./.beta. R-/- MEFs and STAT1 -/- MEFs were
permissive to MV demonstrating the IFN.alpha./.beta. and STAT1
signalling cascades are critical for MV infection (FIG. 5).
[0176] Protein Kinase R (PKR) is an enzyme induced in a wide
variety of cells by IFN.alpha./.beta.. This kinase, in the presence
of dsRNA, undergoes autophosphorylation and then phosphorylates
several cellular proteins including eukaryotic protein synthesis
initiation factor (eIF-2.alpha.) whose phosphorylation can induces
an inhibition of protein translation and apoptosis. PKR is also
indicated in the activation of RNaseL. We examined the activation
of PKR in nonpermissive MEFs following MV infection. PKR is not
phosphorylated in nonpermissive MEFs in which the antiviral state
is well established (FIG. 6). Furthermore MV infection inhibits PKR
phosphorylation (FIG. 7). In addition, PERK (PKR-like, ER kinase)
is not phosphorylated in the primary WT MEFs following Myxoma virus
infection (FIG. 8).
[0177] MV was use to infect MEFs with single gene knockouts of PKR,
RNaseL or Mx1 (FIG. 9). It was discovered that PKR, RNaseL and Mx1
are nonessential for maintaining nonpermissiveness for Myxoma virus
infection. To further confirm the nonessential role of PKR, RNaseL
and Mx1 a Triple knockout of PKR-/-, RNase L-/- and Mx1-/- in MEFs
was performed. A PKR-/-, RNase L-/- and Mx1-/- triple knockout does
not support Myxoma virus infection (FIG. 10), however MEFs with a
triple KO of PKR, RNaseL and Mx1 treated with a neutralizing
antibody to Interferon .alpha./.beta. becomes permissive to Myxoma
virus infection (compare FIGS. 10 and 11). These experiments
demonstrate that PKR, RNaseL and Mx1 are not essential in mediating
the nonpermissiveness of MEFs to MV.
[0178] Further studies were performed to examine the activation of
eIF-2.alpha. and PKR in nonpermissive wildtype MEFs and permissive
IFN.alpha./.beta. R-/- MEFs and STAT1 -/-MEFs after MV infection.
After MV infection, eIF-2.alpha. is phosphorylated in nonpermissive
and permissive MEFs although PKR is not phosphorylated in either
case (FIG. 12). This demonstrates that without the involvement of
PKR and PERK, the antiviral state is mediated by another pathway
that causes eIF2.alpha. phosphorylation.
[0179] STAT1 is both serine- and tyrosine-phosphorylated following
Myxoma infections in nonpermissive PKR, RNaseL and Mx1 Triple KO
MEFs (FIG. 13). Subcellular localization of tyrosine-phosphorylated
STAT1 in nonpermissive PKR-/-+RNaseL-/-+Mx1-/-MEFs following Myxoma
virus infection is also shown (FIG. 14).
[0180] In summary, these results indicate that a parallel
PKR/PERK-independent antiviral pathway involving IFN/STAT1 is
critical for poxvirus tropism. Furthermore, eIF2.alpha.
phosphorylation is the best marker for the antiviral action by
INF.
[0181] Results: Human Tumour Studies
[0182] We studied the ability of MV to infect human tumour cells in
an in vivo system. Nude mice were injected with human glioma cells,
and subsequently developed intracranial gliomas. Live virus was
able to infect these human tumours cells but did not infect
surrounding cells (FIG. 15). The localization of fluorescent signal
from GFP to the tumour is depicted in FIG. 16.
[0183] Given that many human tumours are non-responsive to
interferon, and that the tumour cells do not have normal IFN
signaling cascades compared to those found in normal human cells,
studies were performed to investigate the effect of Myxoma virus on
human tumours. The results are summarized below.
[0184] Initially, Myxoma virus was used to study the infectivity
and cytolytic effects on various control and human tumour cell
lines: BGMK. HT29, HOP92, OVCAR4, SK-MEL3, and SK-MEL28. MV
demonstrated various infectivity and cytolytic results: HT29 (FIG.
17) HOP92 (FIG. 18), OVCAR4 (FIG. 19) SK-MEL3 (FIG. 20), SK-MEL28
(FIG. 21) and BGMK (FIG. 22).
[0185] Additional tumour cells were tested and Table 1 below
classifies the various tumour types tested as permissive or
non-permissive.
TABLE-US-00001 TABLE 1 Myxoma Virus Trophism for Human Tumour Cells
Non- Cell Line Cell Origin Species Permissive Permissive BGMK
Kidney Monkey X RK-13 Kidney Rabbit X RL5 T-Lymphocyte Rabbit X HOS
Osteosarcoma Human X PC3 Prostate cancer Human X Caki-1 Renal
cancer Human X HCT116 Colon cancer Human X 786-0 Renal cancer Human
X SK-OV-3 Ovarian cancer Human X ACHN Renal cancer Human X HOP92
Lung cancer Human X SK-MEL3 Melanoma Human X SK- Melanoma Human X
MEL28 OVCAR4 Ovarian cancer Human X OVCAR5 Ovarian cancer Human X
DU145 Prostate cancer Human X A498 Renal cancer Human X T47D Breast
cancer Human X Colo205 Colon cancer Human X HT29 Colon cancer Human
X MDAMB Breast cancer Human X 435 M14 Melanoma Human X MCF7 Breast
cancer Human X SK-MEL5 Melanoma Human X
[0186] Various human tumour lines demonstrated varying
responsiveness to infection with increasing concentrations of
MV-LacZ. For example, U373 cells required higher virus titres to
achieve the levels of cell killing achieved with lower virus titres
in U87 (FIG. 23 and FIG. 24). Myxoma efficiently infected
astrocytoma cells (FIG. 25), and glioma cells (FIG. 26). Myxoma was
effective at 48 hours post-infection at killing human astrocytoma
and pediatric medulloblastoma cells (FIGS. 27 and 28).
Example 2: Effect of Rapamycin on the Kinetics of Myxoma Virus
Replication in Restrictive Cell Lines
[0187] Virus Strains
[0188] Viral strains used include wildtype MV ("vMyxLac"), and MV
modified to have the MT-5 gene knocked out ("vMyxLacT5-"). Viruses
were prepped and titred using standard techniques.
[0189] Cell Strains
[0190] Human experiments were performed on BGMK primate control
cells, RK-13 rabbit control cells and normal human fibroblasts A9,
restrictive human tumour cell lines 786-0 (renal), ACHN (renal),
HCT116 (colon), MCF-7 (breast), MDA-MB-435 (breast), M14 (melanoma)
and COLO205 (colon).
[0191] Methods
[0192] Generally, assays and experiments were performed as
described in Lalani et al. Virology (1999) 256: 233-245: Johnston
et al. J Virology (2003) 77(13): 7682-7688; and Sypula et al. Gen
Ther Mol Biol (2004) 8: 103.
[0193] For viral growth curves, cells were grown in vitro in a
monolayer, and pretreated with 20 nM rapamycin or a control (1:5000
dilution of DMSO) prior to infection with virus.
[0194] Samples of indicated cell lines infected with the indicated
viral strain were collected at 72 hours post infection and lysed.
The virus contained within the cell lysates was titrated and used
to infect BGMK monolayers. At 48 hours post infection, cells were
fixed and stained using X-gal.
[0195] Results
[0196] Myxoma virus has been previously demonstrated by the
inventors to be able to infect and replicate in many types of human
tumor cells (Sypula et al. (2004) Gene Ther. Mol. Biol. 8:103).
This rabbit specific virus can preferentially infect a majority
(approximately 70%) of human cancer cell lines from the NCI
reference collection. In addition, the host range gene M-T5 was
found to play a critical role during Myxoma virus infection of many
of these cell lines.
[0197] In the present investigation of potential intracellular
molecules that may be affecting the ability of Myxoma to
selectively replicate within human tumour cells, the effect of
rapamycin was tested.
[0198] As seen in FIG. 29, the ability of Myxoma virus to replicate
and spread following a low multiplicity of infection (MOI) was
performed using a multistep growth curve, using BGMK (control
primate cell line); RK-13 and RL5 (control rabbit cell lines); 4T1
and B16F10 (mouse cancer cell lines); HOS and PC3 (permissive human
cancer cell lines); 786-0, HCT116 and ACHN (restrictive human
cancer cell lines); MCF-7, M14 and COLO205 (abortive human cancer
cell lines). Both wild type vMyxLac and the M-T5 knock out virus
vMyxT5KO were tested to investigate the ability of both viruses to
infect and spread throughout the monolayer in the presence and
absence of pre-treatment with rapamycin. Virus titre was assessed
by foci formation on BGMK cells. Cells were pretreated with 20 nM
rapamycin or appropriate vehicle control (1:5000 dilution of DMSO)
for 6 hours before infection.
[0199] As demonstrated, rapamycin has no effect on control BGMK
cells, nor on either of the rabbit cell lines tested, including the
RL-5 cells, which are non permissive for the MT-5 knock out virus.
However, rapamycin does enhance the replication of myxoma virus in
mouse tumour cell lines, and marginally in permissive (Type 1) cell
lines, such as PC-3. Rapamycin has less of an effect on highly
permissive cells such as HOS cells, likely due to the fact that
such cell lines are already maximally permissive for the Myxoma
virus. The greatest effect with rapamycin was observed in the
restrictive (Type II) cell lines (786-0, HCT116 and ACHN), which
are permissive for wildtype virus but non-permissive for the
vMyxT5KO strain. Some effect was seen even in abortive (Type III)
cell lines MCF-7 and COLO205, although not in abortive cell line
M14.
[0200] Samples of the BGMK and 786-0 infected cells were then
collected and lysed, and the isolated virus was used to infect
monolayers of BGMK cells (FIG. 30). Virally infected cells were
visualized using X-Gal staining.
[0201] Pretreatment of tumour cells that are "restrictive" for
Myxoma infection, i.e. those cells that permit the replication of
the wild type Myxoma virus but not the MT-5 knock-out virus, with
rapamycin resulted in a restoration of the ability of Myxoma virus
to replicate in these cancer cell lines, which include renal, colon
and ovarian cancer cell lines (FIGS. 29 and 30).
[0202] In addition, the treatment with rapamycin enhanced the
ability of the wild type virus to replicate in these same cells,
but not control rabbit or primate cells. These results indicate
that rapamycin acts to enhance Myxoma virus infection. In addition,
rapamycin appears to influence the ability of cancer cells that are
poorly infectable by this virus to permit virus replication.
[0203] Subsequent experiments examined the effect of rapamycin
treatment on human tumour cells that could not support wild type
Myxoma virus infection (FIG. 31). The pretreatment had little
effect on control primate cells or normal human fibroblasts, yet
could enhance virus infectivity in several cell lines, including
the breast cancer cell line MCF-7. As several of the human tumour
cell lines remained resistant to rapamycin treatment, as well as
the control cell lines, it is unlikely that rapamycin treatment
could permit Myxoma virus to productively infect non-transformed
tissue.
Example 3: Myxoma Virus M135KO Variant as an Improved Oncolytic
Virus Candidate
[0204] M135R is Expressed from Myxoma Virus as an Early Gene
[0205] Myxoma virus encodes a protein (M135R) identified from the
sequencing of the MV genome (Cameron et al. Virology (1999) 264:
298-318) predicted to mimic the host IFN.alpha./O receptor and
prevent IFN.alpha./.beta. from triggering a host anti-viral
response (Barrett et al. Seminars in Immunology (2001) 13:7384).
This prediction is based on sequence homology to the viral
IFN.alpha./.beta. receptor homolog from vaccinia virus (B18R),
which virus has been demonstrated to employ such an immune evasion
strategy (Symons et al. Cell (1995) 81:551-560). However M135R is
only half the size of VV B18R and all other IFN.alpha./.beta.-R
homologs sequenced from poxviruses, and in all cases aligns only to
the amino terminus half of poxviral IFN.alpha./.beta.-R homologs.
FIG. 32 indicates the predicted structure and sequence similarity
between M135R from MV and B18R from VV. Only the first 179 amino
acid residues of B18R are shown in the sequence alignment. Table 2
indicates the % identity between M135R and the indicated poxviral
IFN.alpha./.beta.-R homologs. Numbers above the diagonal represent
% identity and numbers below the diagonal represent % similarity
between any two species. The numbers in brackets across the top
represent the number of amino acids in the putative proteins.
Comparison was done between the predicted full length copy of M135R
(178 amino acids) and the first 178 residues of each homolog
only.
TABLE-US-00002 TABLE 2 Comparison of M135R to Other Poxviral
Homologs % Identity Myxoma Vaccinia Variola Monkeypox Cowpox
Ectromelia Camelpox YLDV Swinepox LSDV species (178) (351) (354)
(352) (351) (358) (355) (351) (344) (360) Myxoma -- 24 21 24 23 21
22 20 18 17 Vaccinia 39 -- 80 93 90 84 79 20 23 25 Variola 36 88 --
79 87 88 79 19 24 24 Monkeypox 38 95 87 -- 86 83 78 20 20 26 Cowpox
38 93 93 91 -- 92 88 21 23 25 Ectromelia 35 89 93 87 94 -- 87 17 21
25 Camelpox 34 86 94 85 92 93 -- 17 23 24 YLDV 38 37 37 37 38 34 34
-- 23 28 Swinepox 32 39 39 36 39 35 37 38 -- 25 LSDV 32 39 39 41 38
39 36 43 38 --
[0206] Peptides against predicted immunogenic regions of M135R were
synthesized and used to generate polyclonal antibodies in rabbits
that were used in western blot analysis, immuno-precipitations and
immuno-fluorescence. Immunoblotting confirmed that M135R is
synthesized as an early gene whose expression can be detected as
early as three hours post infection (FIG. 33A; lane 1: mock
infected BGMK cells; lanes 2-6: BGMK cells infected with vMyxLau 0,
3, 6, 18 and 36 hours post infection, respectively). Treatment of
infected cells with AraC indicates that synthesis of M135R was not
altered by inhibition of late protein expression and is therefore
an early gene (FIG. 33B). However treatment with tunicamycin
indicates that M135R is N-linked glycosylated, likely at the single
site predicted from the sequence (FIG. 33B). Monensin treatment
suggests that there is no O-linked glycosylation. For the results
shown in FIG. 33, BGMKs were infected at an moi of 10 with Myxoma
virus. Cells were treated with AraC at a concentration of 40
.mu.g/ml, tunicamycin at 1 .mu.g/ml and monensin at .mu.g/ml, or
were untreated, at the times indicated. M135R was detected with a
peptide antibody.
[0207] M135R Encodes a Signal Sequence but is not Secreted
[0208] Sequence analysis of M135R indicates the presence of a
predicted signal sequence (FIG. 32B). However there is also a
predicted transmembrane domain at the carboxy terminus (FIG. 32B).
Immunoblots of supernatants from infected BGMK cells indicate that
M135R is not secreted. However, M135R is easily detected in whole
cell lysates (FIG. 33). To test whether the signal sequence
functioned to drive M135R to the cell surface, we deleted the
transmembrane domain and cloned the mutant into a baculovirus
expression system. Comparison of AcM135R and Ac135.DELTA.TM
infected supernatants indicated that full length M135R is found in
the cell lysate there is no evidence of secretion. In contrast
Ac135.DELTA.TM is secreted and confirms that the signal sequence
functions to drive M135R into the extracellular environment (data
not shown).
[0209] M13SR Protein Localizes to the Surface of Infected Cells
[0210] The observation that M135R has a functional signal sequence
as well as a transmembrane domain prompted us to test the
localization of M135R. Two pieces of evidence indicate that M135R
localizes to the cell surface. First, when BGMKs were seeded onto
glass coverslips and infected with vMyxLau (moi of 10) for 24 hours
then M135R was detected by immunostaining with affinity purified
anti-M135R followed by FITC-conjugated secondary antibody (FIG.
34A). M135R staining pattern indicates localization to the cell
surface of infected cells. vMyxLau is a true wildtype strain of
Myxoma virus which has not been altered by insertion of the
.beta.-gal or EGFP gene.
[0211] The second piece of evidence for cell surface localization
M135R follows biotinylation of cell surface proteins of GHOST cells
infected with either vMyxgfp or vMyx135KO. Twenty-four hours post
infection cell lysates were prepared. Streptavidin agarose beads
were mixed with 500 .mu.g of total cellular protein from cell
lysates for 45 minutes. The beads were washed and separated on a
15% PAGE-SDS gel and then probed with anti-M135R. 50 .mu.g of total
protein from the infected cell lysates were run as controls.
Immunoprecipitation of biotinylated surface proteins indicates that
m135R is at the surface of infected cells (FIG. 34B).
[0212] M135R is Non-Essential for Myxoma Virus Replication In
Vitro
[0213] To test the ability of M135R to act as a virulence factor we
constructed a recombinant virus in which M135R was deleted and
replaced by a cassette encoding EGFP and gpt under VV early/late
promoters (460 nucleotides, or 86% of the orf was deleted). The
cloning strategy and cassette is shown in FIG. 35A. The recombinant
was plaque-purified by selecting virus clones expressing EGFP. The
purity of the recombinant was confirmed by PCR (FIG. 35B; Lane 1 is
the 1 Kb plus DNA ladder, Lane 2 and 3 are PCR products from two
purified vMyx135KO clones. The PCR product represents the region
into which the M135R coding region has been deleted and the
EGFP/gpt marker has been inserted. Lane 2 is plaque 1 and Lane 3 is
plaque 2. Lane 4 represents the same region and covers the native,
uninterrupted M135R locus.). Immunoblotting of BGMK cells infected
with either vMyxLau or vMyx135KO confirmed that vMyx135KO had lost
M135R expression (FIG. 35C; time course of expression of M135R:
Lane 1 is uninfected BGMK cells. Lanes 2-6 represent BGMK cells
infected with vMyxLau at times 0 (lane 2), 3 (lane 3), 6 (lane 4),
18 (lane 5), and 36 hours post infection (lane 6). Lanes 7 and 8
represent BGMK cells infected with vMyx135KO at 6 (lane 7) and 18
(lane 8) hours post infection. Lane 9 is a positive control with
M135R expressed in AcNPV.).
[0214] Single step growth curves were used to test the ability of
vMyx135KO to replicate in BGMK cells. BGMK cells were infected with
vMyxgfp or vMyx135KO at an moi of 5 and cells were collected at the
times indicated. Virus titres were determined on BGMK cells. There
was no difference in the replication pattern between vMyxgfp and
vMyx135KO (FIG. 36). These results indicate that M135R is not
required for replication in vitro.
[0215] During our studies of the ability of another gene of Myxoma
to influence Myxoma replication in rabbit primary embryo
fibroblasts (REFs), we used vMyx135KO as a knockout control and
observed a curious phenomenon. Infection of the REFs with vMyxgfp
resulted in a normal focus of infection however vMyx135KO produced
a plaque-like zone of infection (FIG. 37). When we tested other
cells to confirm this phenotype we were able to replicate the
plaque formation in other rabbit fibroblasts (HIG-82, FIG. 38) and
human primary fibroblasts (ccd922-sk, FIG. 39).
[0216] M135R is a Critical Virulence Factor for Pathogenesis in
Rabbits
[0217] We next tested the ability of vMyx135KO to produce
myxomatosis in lab rabbits. In contrast to the animals injected
with vMyxLau or vMyxgfp which developed normal myxomatosis and had
to be euthanized between days 9 and 10 post injection, the rabbits
injected with vMyx135KO recovered completely (Table 3). To confirm
that loss of M135R caused the attenuation of vMyx135KO we generated
a revertant virus in which M135R was restored and we tested the
ability of this revertant (vMyx135REV) to restore the ability to
produce myxomatosis. All four treated groups of rabbits responded
in a similar manner for the first six days following injection of
the respective viruses (Table 3). We observed a large, red, raised
lesion at the site of injection in all treatment groups by 4 days
post infection. However beginning at day 6 and continuing over the
next 3-4 days the differences between the different viruses became
evident. Those animals injected with the wildtype or revertant
virus had numerous secondary lesions in the ears, eyes and nose
which were not observed in the animals injected with vMyx135KO
(Table 3). We conclude that loss of M135R drastically attenuated MV
in animal models and indicates that M135R is a critical virulence
factor.
TABLE-US-00003 TABLE 3 Pathogenesis of vMyx135KO Compared to
Wildtype Controls Observations and Time of onset (number + days
indicates first appearance in days post injection) Lausanne
vMyx135KO vMyx135REV Clinical Signs (4 animals) (6 animals) (3
animals) inoculation 2 days: red, 4 days: 11-16 mm 3 days: small
red lump, site visible slightly red, raised, dark slightly raised
raised centre 4 days: red, dark centre satellites 4 days 6 days:
just 6 days: 5-10 visible beginning, over increasing to 30-40
satellites course of infection visible by day 8 very few observed
conjunctival none observed 9 days: single rabbit none observed
inflammation discharge from eye anogenital 7 days: swelling 7 days:
redness, edema swelling secondary 6-7 days: first 7 days: few small
red 6 days: first observed as red lesions around eyes then spots
not yet lesions, areas on eyelids, clearly ears ears eyes lesion by
day 7 respiratory little or none little or none little or none
difficulty lesion 11 days: 25 mm, regression black, scabby
satellites losing colour and becoming scabby 13 days: scab
beginning to separate from healthy tissue two animals all animals
recovered three animals euthanized euthanized day 9 day 10 two
animals euthanized day 10
[0218] The temperature of rabbits was taken daily for the three
days preceeding the study. This was considered the baseline body
temperatures of the animals. We continued to take the temperatures
daily of each animal for the duration of the study. However there
was no difference in body temperature between the treatment groups
(FIG. 40). This suggests that M135R does not play a role in the
febrile response of infected animals.
[0219] M135R does not Bind or Inhibit Rabbit IFN.alpha./.beta.
[0220] The sequence of M135R is similar to the vaccinia B18R, an
IFN.alpha./.beta. receptor mimic. We tested the ability of M135R to
bind rabbit type 1 IFN. We first iodinated rabbit IFN (5 .mu.g,
using Iodobeads) and tested the ability of vMyx135KO infected cells
to bind .sup.125I-rabbit IFN in comparison to cells infected with
vMyxgfp (moi of 10). Cells were collected, washed and counted in a
gamma counter. Deletion of M135R did not affect IFN.alpha./.beta.
binding to infected cells and we did not observe any difference in
the amount of IFN bound to the cell surface of either RK13 or BGMK
cells (FIG. 41). As well, treatment of RK13 or BGMK cells with
exogenous rabbit type1 IFN did not affect infection of cells by
vMyx135KO (FIG. 42; cells were seeded in 12 well dishes and
infected with the indicated virus at an moi of 0.01; fluorescent
foci were counted 72-96 hours post infection; 200 units of rabbit
IFN.alpha./.beta. was either added 24 hours prior to infection or
cells were untreated). This same result was observed when cells
were pretreated 24 h before infection to induce an anti-viral state
in the cell. We did not notice any significant difference in the
foci formed following infection in either RK13 or BGMK cells (data
not shown). This phenomenon was also true if cells were treated
with human IFNA/D (data not shown). As well, we were unable to
observe any binding when Ac135.DELTA.TM supernatants were applied
to rabbit IFN .alpha./.beta. adhered to a BIAcore chip (data not
shown).
Example 3: Molecular Consequences of Inhibiting mTOR in the Context
of Myxoma Virus Infection
[0221] Western Blot analysis (FIG. 43) was performed using cell
lysates from 786-0 cells, a Type II cancer cell line where
rapamycin enhances myxoma virus infection. Lysates were collected
16 hours post infection with either vMyxLac or vMyxT5KO at an MOI
of 3, or without virus infection. Indicated lanes contain protein
from cells that were pretreated with 20 nM rapamycin (designated R)
or appropriate vehicle control (1:5000 dilution of DMSO, designated
D) for 6 hours before infection. The blots were probed using
primary antibodies directed against the indicated proteins.
[0222] As demonstrated, myxoma virus infection affects many of the
signaling pathways that converge on mTOR, the physiologic target of
rapamycin. In the context of infection with either wild type
(vMyxLac) or MT-S deficient (vMyxT5KO) virus, where rapamycin has a
beneficial effect on virus replication, global effects are observed
in many of these signaling molecules that would not be predictable
based on treatment with rapamycin alone (see mock infected lanes).
These effects include an increase in the kinase activity of AKT-1,
Raf-1, GSK-3 and mTOR itself, as well as a decrease in the kinase
activity of PTEN and p70S6K. This data indicate that these pathways
are likely to play a role in myxoma virus permissiveness in human
cancer cells lines.
[0223] As can be understood by one skilled in the art, many
modifications to the exemplary embodiments described herein are
possible. The invention, rather, is intended to encompass all such
modification within its scope, as defined by the claims.
[0224] Although various embodiments of the invention are disclosed
herein, many adaptations and modifications may be made within the
scope of the invention in accordance with the common general
knowledge of those skilled in this art. Such modifications include
the substitution of known equivalents for any aspect of the
invention in order to achieve the same result in substantially the
same way. All technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the
art of this invention, unless defined otherwise.
[0225] All reference cited herein are fully incorporated by
reference
Sequence CWU 1
1
21178PRTMyxoma virus 1Met Val Phe Ile Phe Ile Ile Thr Cys Val Cys
Leu Val Thr Arg Ser 1 5 10 15 Cys Gly Gly Gly Leu Glu Asp Asp Ile
Asp Arg Ile Phe Gln Lys Arg 20 25 30 Tyr Asn Glu Leu Ser Gln Pro
Ile Lys Arg Asn Met Arg Thr Leu Cys 35 40 45 Lys Phe Arg Gly Ile
Thr Ala Thr Met Phe Thr Glu Gly Glu Ser Tyr 50 55 60 Leu Ile Gln
Cys Pro Ile Ile His Asp Tyr Val Leu Arg Ala Leu Tyr 65 70 75 80 Asp
Leu Val Glu Gly Ser Tyr Thr Val Arg Trp Glu Arg Glu Thr Glu 85 90
95 Asp Asp Val Glu Ser Val Asp Pro Lys Leu Val Lys Gly Thr Leu Leu
100 105 110 Tyr Leu Gln Pro Asn Ala Ser Ser Ile Gly Thr Tyr Leu Cys
Thr Leu 115 120 125 His Asp Asn Arg Gly Met Cys Tyr Gln Ser Val Ala
His Val Ile Arg 130 135 140 Arg Pro Lys Met Gln Cys Val Lys His Ala
His Thr Thr Ser Asp Ser 145 150 155 160 Asn Leu Trp Ile Tyr Leu Ala
Ile Leu Ala Val Leu Ile Ser Leu Gly 165 170 175 Val Leu
2179PRTVaccinia virus 2Met Thr Met Lys Met Met Val His Ile Tyr Phe
Val Ser Leu Leu Leu 1 5 10 15 Leu Leu Phe His Ser Tyr Ala Ile Asp
Ile Glu Asn Glu Ile Thr Glu 20 25 30 Phe Phe Asn Lys Met Arg Asp
Thr Leu Pro Ala Lys Asp Ser Lys Trp 35 40 45 Leu Asn Pro Ala Cys
Met Phe Gly Gly Thr Met Asn Asp Ile Ala Ala 50 55 60 Leu Gly Glu
Pro Phe Ser Ala Lys Cys Pro Pro Ile Glu Asp Ser Leu 65 70 75 80 Leu
Ser His Arg Tyr Lys Asp Tyr Val Val Lys Trp Glu Arg Leu Glu 85 90
95 Lys Asn Arg Arg Arg Gln Val Ser Asn Lys Arg Val Lys His Gly Asp
100 105 110 Leu Trp Ile Ala Asn Tyr Thr Ser Lys Phe Ser Asn Arg Arg
Tyr Leu 115 120 125 Cys Thr Val Thr Thr Lys Asn Gly Asp Cys Val Gln
Gly Ile Val Arg 130 135 140 Ser His Ile Arg Lys Pro Pro Ser Cys Ile
Pro Lys Thr Tyr Glu Leu 145 150 155 160 Gly Thr His Asp Lys Tyr Gly
Ile Asp Leu Tyr Cys Gly Ile Leu Tyr 165 170 175 Ala Lys His
* * * * *