U.S. patent application number 16/598900 was filed with the patent office on 2020-12-10 for smc combination therapy for the treatment of cancer.
The applicant listed for this patent is Children's Hospital of Eastern Ontario Research Institute Inc.. Invention is credited to Shawn T. BEUG, Robert G. KORNELUK, Eric C. LACASSE, Vera A. TANG.
Application Number | 20200384103 16/598900 |
Document ID | / |
Family ID | 1000005037920 |
Filed Date | 2020-12-10 |
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United States Patent
Application |
20200384103 |
Kind Code |
A1 |
KORNELUK; Robert G. ; et
al. |
December 10, 2020 |
SMC COMBINATION THERAPY FOR THE TREATMENT OF CANCER
Abstract
The present invention includes methods and compositions for
enhancing the efficacy of SMCs in the treatment of cancer. In
particular, the present invention includes methods and compositions
for combination therapies that include an SMC and at least a second
agent that stimulates one or more apoptotic or immune pathways. The
second agent may be, e.g., an immunostimulatory compound or
oncolytic virus.
Inventors: |
KORNELUK; Robert G.;
(Ottawa, CA) ; LACASSE; Eric C.; (Ottawa, CA)
; BEUG; Shawn T.; (Ottawa, CA) ; TANG; Vera
A.; (Ottawa, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Children's Hospital of Eastern Ontario Research Institute
Inc. |
Ottawa |
|
CA |
|
|
Family ID: |
1000005037920 |
Appl. No.: |
16/598900 |
Filed: |
October 10, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15113634 |
Jul 22, 2016 |
10441654 |
|
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PCT/CA2015/000043 |
Jan 26, 2015 |
|
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16598900 |
|
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61931321 |
Jan 24, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/427 20130101;
A61K 2039/55561 20130101; A61K 31/433 20130101; A61K 2039/55588
20130101; A61K 45/06 20130101; A61K 2039/572 20130101; A61K 31/4745
20130101; A61K 31/407 20130101; C12N 7/00 20130101; A61K 31/55
20130101; A61K 35/765 20130101; A61K 39/39 20130101; A61K 39/0011
20130101; A61K 31/404 20130101; A61K 2039/55511 20130101; A61K
2039/55594 20130101; A61K 38/212 20130101; A61K 35/761 20130101;
A61K 39/205 20130101; A61K 2039/5252 20130101; A61K 31/409
20130101; C12N 2760/20134 20130101; A61K 2039/585 20130101; A61K
35/766 20130101; A61K 38/21 20130101 |
International
Class: |
A61K 39/39 20060101
A61K039/39; A61K 31/4745 20060101 A61K031/4745; A61K 31/404
20060101 A61K031/404; A61K 45/06 20060101 A61K045/06; A61K 35/761
20060101 A61K035/761; A61K 35/766 20060101 A61K035/766; A61K 38/21
20060101 A61K038/21; A61K 35/765 20060101 A61K035/765; A61K 31/407
20060101 A61K031/407; A61K 31/409 20060101 A61K031/409; A61K 31/427
20060101 A61K031/427; A61K 31/433 20060101 A61K031/433; A61K 31/55
20060101 A61K031/55; A61K 39/00 20060101 A61K039/00; A61K 39/205
20060101 A61K039/205; C12N 7/00 20060101 C12N007/00 |
Claims
1. A composition comprising an SMC from Table 1 and an
immunostimulatory agent from Table 2 or Table 3, wherein said SMC
and said immunostimulatory agent are provided in amounts that
together are sufficient to treat cancer when administered to a
patient in need thereof.
2. A method for treating a patient diagnosed with cancer, said
method comprising administering to the patient an SMC from Table 1
and an immunostimulatory agent from Table 2 or Table 3, wherein
said SMC and said immunostimulatory agent are administered
simultaneously or within 28 days, 14 days, 10 days, 5 days, 24
hours, or 6 hours of each other in amounts that together are
sufficient to treat said cancer.
3-8. (canceled)
9. The method of claim 2, wherein said SMC is a monovalent SMC.
10. The method of claim 9, wherein said SMC is LCL161,
GDC-0152/RG7419, GDC-0917/CUDC-427, or SM-406/AT-406/Debio1143.
11-12. (canceled)
13. The method of claim 2, wherein said SMC is a bivalent SMC.
14. The method of claim 13, wherein said SMC is AEG40826/HGS1049,
OICR720, TL32711/Birinapant, or SM-1387/APG-1387.
15-17. (canceled)
18. The method of claim 2, wherein said immunostimulatory agent is
a TLR agonist from Table 2, a lipopolysaccharide, a peptidoglycan,
a lipopeptide, a CpG oligodeoxynucleotide, or a virus from Table
3.
19-20. (canceled)
21. The method of claim 18, wherein said CpG oligodeoxynucleotide
is CpG-ODN 2216.
22. The method of claim 18, wherein said immunostimulatory agent is
imiquimod, poly(I:C), or BCG.
23-24. (canceled)
25. The method of any one of claim 2, wherein said
immunostimulatory agent is a virus from Table 3.
26. The method of claim 25, wherein said virus is a vesicular
stomatitis virus (VSV), adenovirus, maraba vesiculovirus, reovirus,
rhabdovirus, vaccinia virus or a variant thereof, or Talimogene
laherparepvec.
27. The method of claim 26, wherein said VSV virus is VSV-M51R,
VSV-M.DELTA.51, VSV-IFN.beta., or VSV-IFN.beta.-NIS.
28-29. (canceled)
30. The method of claim 2, wherein said cancer is refractory to
treatment by an SMC in the absence of an immunostimulatory
agent
31. The method of claim 2, wherein said treatment further comprises
administration of a therapeutic agent comprising an interferon.
32. The method of claim 31, wherein said interferon is a type 1
interferon.
33. The method of claim 2, wherein said cancer is selected from
adrenal cancer, basal cell carcinoma, biliary tract cancer, bladder
cancer, bone cancer, brain cancer, breast cancer, cervical cancer,
choriocarcinoma, colon cancer, colorectal cancer, connective tissue
cancer, cancer of the digestive system, endometrial cancer,
epipharyngeal carcinoma, esophageal cancer, eye cancer, gallbladder
cancer, gastric cancer, cancer of the head and neck, hepatocellular
carcinoma, intra-epithelial neoplasm, kidney cancer, laryngeal
cancer, leukemia, liver cancer, liver metastases, lung cancer,
lymphoma, melanoma, myeloma, multiple myeloma, neuroblastoma,
mesothelioma, neuroglioma, myelodysplastic syndrome, multiple
myeloma, oral cavity cancer, ovarian cancer, paediatric cancer,
pancreatic cancer, pancreatic endocrine tumors, penile cancer,
plasma cell tumors, pituitary adenoma, thymoma, prostate cancer,
renal cell carcinoma, cancer of the respiratory system,
rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer,
small bowel cancer, stomach cancer, testicular cancer, thyroid
cancer, ureteral cancer, and cancer of the urinary system.
34. A composition comprising an SMC from Table 1 and an
immunostimulatory agent, said immunostimulatory agent comprising:
(a) a killed virus, an inactivated virus, or a viral vaccine; or
(b) a first agent that primes an immune response and at least a
second agent that boosts said immune response, wherein said SMC and
said immunostimulatory agent are provided in amounts that together
are sufficient to treat cancer when administered to a patient in
need thereof.
35. The composition of claim 34, wherein said immunostimulatory
agent is an NRRP or a rabies vaccine.
36. (canceled)
37. The composition of claim 36, wherein one or both of said first
agent and said second agent is an oncolytic virus vaccine, or
wherein said first agent is an adenovirus carrying a tumor antigen
and said second agent is a vesiculovirus.
38. (canceled)
39. The composition of claim 37, wherein said vesiculovirus is
selected from Maraba-MG1 carrying the same tumor antigen as said
adenovirus and Maraba-MG1 that does not carry a tumor antigen.
Description
BACKGROUND OF THE INVENTION
[0001] The death of cells by apoptosis (or programmed cell death),
and other cell death pathways, is regulated by various cellular
mechanisms. Inhibitor of apoptosis (IAP) proteins, such as X-linked
IAP (XIAP) or cellular IAP proteins 1 and 2 (cIAP1 and 2), are
regulators of programmed cell death, including (but not limited to)
apoptosis pathways, e.g., in cancer cells. Other forms of cell
death could include, but are not limited to, necroptosis, necrosis,
pyroptosis, and immunogenic cell death. In addition, these IAPs
regulate various cell signaling pathways through their ubiquitin E3
ligase activity, which may or may not be related to cell survival.
Another regulator of apoptosis is the polypeptide Smac. Smac is a
proapoptotic protein released from mitochondria in conjunction with
cell death. Smac can bind to IAPs, antagonizing their function.
Smac mimetic compounds (SMCs) are non-endogenous proapoptotic
compounds capable of carrying out one or more of the functions or
activities of endogenous Smac.
[0002] The prototypical XIAP protein directly inhibits key
initiator and executioner caspase proteins within apoptosis
cascades. XIAP can thereby thwart the completion of apoptotic
programs. Cellular IAP proteins 1 and 2 are E3 ubiquitin ligases
that regulate apoptotic signaling pathways engaged by immune
cytokines. The dual loss of cIAP1 and 2 can cause TNF.alpha.,
TRAIL, and/or IL-1.beta. to become toxic to, e.g., the majority of
cancer cells. SMCs may inhibit XIAP, cIAP1, cIAP2, or other IAPs,
and/or contribute to other proapoptotic mechanisms.
[0003] Treatment of cancer by the administration of SMCs has been
proposed. However, SMCs alone may be insufficient to treat certain
cancers. There exists a need for methods of treating cancer that
improve the efficacy of SMC treatment in one or more types of
cancer.
SUMMARY OF THE INVENTION
[0004] The present invention includes compositions and methods for
the treatment of cancer by the administration of an SMC and an
immunostimulatory, or immunomodulatory, agent. SMCs and
immunostimulatory agents are described herein, including, without
limitation, the SMCs of Table 1 and the immunostimulatory agents of
Tables 2 and 3.
[0005] One aspect of the present invention is a composition
including an SMC from Table 1 and an immunostimulatory agent from
Table 2 or Table 3, such that the SMC and the immunostimulatory
agent are provided in amounts that together are sufficient to treat
cancer when administered to a patient in need thereof.
[0006] Another aspect of the present invention is a method for
treating a patient diagnosed with cancer, the method including
administering to the patient an SMC from Table 1 and an
immunostimulatory agent from Table 2 or Table 3, such that the SMC
and the immunostimulatory agent are administered simultaneously or
within 28 days of each other in amounts that together are
sufficient to treat the cancer.
[0007] In some embodiments, the SMC and the immunostimulatory agent
are administered within 14 days of each other, within 10 days of
each other, within 5 days of each other, within 24 hours of each
other, within 6 hours of each other, or simultaneously.
[0008] In particular embodiments, the SMC is a monovalent SMC, such
as LCL161, SM-122, GDC-0152/RG7419, GDC-0917/CUDC-427, or
SM-406/AT-406/Debio1143. In other embodiments, the SMC is a
bivalent SMC, such as AEG40826/HGS1049, OICR720,
TL32711/Birinapant, SM-1387/APG-1387, or SM-164.
[0009] In particular embodiments, the immunostimulatory agent is a
TLR agonist from Table 2. In certain embodiments, the
immunostimulatory agent is a lipopolysaccharide, peptidoglycan, or
lipopeptide. In other embodiments, the immunostimulatory agent is a
CpG oligodeoxynucleotide, such as CpG-ODN 2216. In still other
embodiments, the immunostimulatory agent is imiquimod or
poly(I:C).
[0010] In particular embodiments, the immunostimulatory agent is a
virus from Table 3. In certain embodiments, the immunostimulatory
agent is a vesicular stomatitis virus (VSV), such as VSV-M51R,
VSV-M.DELTA.51, VSV-IFN.beta., or VSV-IFN.beta.-NIS. In other
embodiments, the immunostimulatory agent is an adenovirus, maraba
vesiculovirus, reovirus, rhabdovirus, or vaccinia virus, or a
variant thereof. In some embodiments, the immunostimulatory agent
is a Talimogene laherparepvec.
[0011] In some embodiments, a composition or method of the present
invention includes a plurality of immunostimulatory or
immunomodulatory agents, including but not limited to interferons,
and/or a plurality of SMCs.
[0012] In some embodiments, a composition or method of the present
invention includes one or more interferon agents, such as an
interferon type 1 agent, an interferon type 2 agent, and/or an
interferon type 3 agent.
[0013] In any method of the present invention, the cancer can be a
cancer that is refractory to treatment by an SMC in the absence of
an immunostimulatory or immunomodulatory agent. In any method of
the present invention, the treatment can further include
administration of a therapeutic agent including an interferon.
[0014] In any method of the present invention, the cancer can be a
cancer that is selected from adrenal cancer, basal cell carcinoma,
biliary tract cancer, bladder cancer, bone cancer, brain cancer,
breast cancer, cervical cancer, choriocarcinoma, colon cancer,
colorectal cancer, connective tissue cancer, cancer of the
digestive system, endometrial cancer, epipharyngeal carcinoma,
esophageal cancer, eye cancer, gallbladder cancer, gastric cancer,
cancer of the head and neck, hepatocellular carcinoma,
intra-epithelial neoplasm, kidney cancer, laryngeal cancer,
leukemia, liver cancer, liver metastases, lung cancer, lymphoma,
melanoma, myeloma, multiple myeloma, neuroblastoma, mesothelioma,
neuroglioma, myelodysplastic syndrome, multiple myeloma, oral
cavity cancer, ovarian cancer, paediatric cancer, pancreatic
cancer, pancreatic endocrine tumors, penile cancer, plasma cell
tumors, pituitary adenoma, thymoma, prostate cancer, renal cell
carcinoma, cancer of the respiratory system, rhabdomyosarcoma,
salivary gland cancer, sarcoma, skin cancer, small bowel cancer,
stomach cancer, testicular cancer, thyroid cancer, ureteral cancer,
and cancer of the urinary system.
[0015] The invention further includes a composition including an
SMC from Table 1 and an immunostimulatory agent. The
immunostimulatory agent may include a killed virus, an inactivated
virus, or a viral vaccine, such that the SMC and the
immunostimulatory agent are provided in amounts that together are
sufficient to treat cancer when administered to a patient in need
thereof. In particular embodiments, the said immunostimulatory
agent is a NRRP or a rabies vaccine. In other embodiments, the
invention includes a composition including an SMC from Table 1 and
an immunostimulatory agent. The immunostimulatory agent may include
a first agent that primes an immune response and at least a second
agent that boosts the immune response, such that the SMC and the
said immunostimulatory agent are provided in amounts that together
are sufficient to treat cancer when administered to a patient in
need thereof. In certain embodiments, one or both of the first
agent and the second agent is an oncolytic virus vaccine. In other
particular embodiments, the first agent is an adenovirus carrying a
tumor antigen and the second agent is a vesiculovirus, such as a
Maraba-MG1 carrying the same tumor antigen as the adenovirus or a
Maraba-MG1 that does not carry a tumor antigen.
[0016] "Neighboring" cell means a cell sufficiently proximal to a
reference cell to directly or indirectly receive an immune,
inflammatory, or proapoptotic signal from the reference cell.
[0017] "Potentiating apoptosis or cell death" means to increase the
likelihood that one or more cells will apoptose or die. A treatment
may potentiate cell death by increasing the likelihood that one or
more treated cells will apoptose, and/or by increasing the
likelihood that one or more cells neighboring a treated cell will
apoptose or die.
[0018] "Endogenous Smac activity" means one or more biological
functions of Smac that result in the potentiation of apoptosis,
including at least the inhibition of cIAP1 and cIAP2. It is not
required that the biological function occur or be possible in all
cells under all conditions, only that Smac is capable of the
biological function in some cells under certain naturally occurring
in vivo conditions.
[0019] "Smac mimetic compound" or "SMC" means a composition of one
or more components, e.g., a small molecule, compound, polypeptide,
protein, or any complex thereof, capable of inhibiting cIAP1 and/or
inhibiting cIAP2. Smac mimetic compounds include the compounds
listed in Table 1.
[0020] To "induce an apoptotic program" means to cause a change in
the proteins or protein profiles of one or more cells such that the
amount, availability, or activity of one or more proteins capable
of participating in an IAP-mediated apoptotic pathway is increased,
or such that one or more proteins capable of participating in an
IAP-mediated apoptotic pathway are primed for participation in the
activity of such a pathway. Inducing an apoptotic program does not
require the initiation of cell death per se: induction of a program
of apoptosis in a manner that does not result in cell death may
synergize with treatment with an SMC that potentiates apoptosis,
leading to cell death.
[0021] "Immunostimulatory agent" means a composition of one or more
components cumulatively capable of inducing an apoptotic or
inflammatory program in one or more cells of a subject, and cell
death downstream of this program being inhibited by at least cIAP1
and cIAP2. An immunostimulatory agent may be, e.g., a TLR agonist
(e.g., a compound listed in Table 2) or a virus (e.g., a virus
listed in Table 3), such as an oncolytic virus.
[0022] "Treating cancer" means to induce the death of one or more
cancer cells in a subject, or to provoke an immune response which
could lead to tumor regression and block tumor spread (metastasis).
Treating cancer may completely or partially abolish some or all of
the signs and symptoms of cancer in a subject, decrease the
severity of one or more symptoms of cancer in a subject, lessen the
progression of one or more symptoms of cancer in a subject, or
mediate the progression or severity of one or more subsequently
developed symptoms.
[0023] "Prodrug" means a therapeutic agent that is prepared in an
inactive form that may be converted to an active form within the
body of a subject, e.g. within the cells of a subject, by the
action of one or more enzymes, chemicals, or conditions present
within the subject.
[0024] By a "low dosage" or "low concentration" is meant at least
5% less (e.g., at least 10%, 20%, 50%, 80%, 90%, or even 95%) than
the lowest standard recommended dosage or lowest standard
recommended concentration of a particular compound formulated for a
given route of administration for treatment of any human disease or
condition.
[0025] By a "high dosage" is meant at least 5% (e.g., at least 10%,
20%, 50%, 100%, 200%, or even 300%) more than the highest standard
recommended dosage of a particular compound for treatment of any
human disease or condition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGS. 1A-1F are a set of graphs and images showing that SMC
synergizes with oncolytic rhabdoviruses to induce cancer cell
death. FIGS. 1A-1F are representative of data from at least three
independent experiments using biological replicates (n=3). FIG. 1A
is a pair of graphs showing the results of Alamar blue viability
assays of cells treated with LCL161 and increasing MOIs of
VSV.DELTA.51. Error bars, mean.+-.s.d. FIG. 1B is a set of
micrographs of cells treated with LCL161 and 0.1 MOI of
VSV.DELTA.51-GFP. FIG. 1C is a pair of graphs showing viability
(Alamar Blue) of cells infected with VSV.DELTA.51 (0.1 MOI) in the
presence of increasing concentrations of LCL161. Error bars,
mean.+-.s.d. FIG. 1D is a pair of graphs showing data from cells
that were infected with VSV.DELTA.51 for 24 hours. Cell culture
supernatant was exposed to virus-inactivating UV light and then
media was applied to new cells for viability assays (Alamar Blue)
in the presence of LCL161. Error bars, mean.+-.s.d. FIG. 1E is a
graph showing the viability of cells co-treated with LCL161 and
non-spreading virus VSV.DELTA.51AG (0.1 MOI). Error bars,
mean.+-.s.d. FIG. 1F is a graph and a pair of images relating to
cells that were overlaid with agarose media containing LCL161,
inoculated with VSV.DELTA.51-GFP in the middle of the well, and
infectivity measured by fluorescence and cytotoxicity was assessed
by crystal violet staining (images were superimposed;
non-superimposed images are in FIG. 11). Error bars,
mean.+-.s.d.
[0027] FIGS. 2A-2E are a set of graphs and images showing that SMC
treatment does not alter the cancer cell response to oncolytic
virus (OV) infection. FIGS. 2A-2E are representative of data from
at least three independent experiments using biological replicates.
FIG. 2A is a pair of graphs showing data from cells that were
pretreated with LCL161 and infected with the indicated MOI of
VSV.DELTA.51. Virus titer was assessed by a standard plaque assay.
FIG. 2B is a pair of graphs and a set of micrographs captured over
time from cells that were treated with LCL161 and VSV.DELTA.51-GFP.
The graphs plot the number of GFP signals over time. Error bars,
mean.+-.s.d. n=12. FIG. 2C, is pair of graphs showing data from an
experiment in which cell culture supernatants from LCL161 and
VSV.DELTA.51 treated cells were processed for the presence of
IFN.beta. by ELISA. Error bars, mean.+-.s.d. n=3. FIG. 2D is a pair
of graphs showing data from an experiment in which cells were
treated with LCL161 and VSV.DELTA.51 for 20 hours and processed for
RT-qPCR to measure interferon stimulated gene (ISG) expression.
Error bars, mean.+-.s.d. n=3. FIG. 2E is a pair of images showing
immunoblots for STAT1 pathway activation performed on cells that
were pretreated with LCL161 and subsequently stimulated with
IFN.beta..
[0028] FIGS. 3A-3H are a set of graphs showing that SMC treatment
of OV-infected cancer cells leads to type 1 interferons (type 1
IFN) and nuclear-factor kappa B (NF-.kappa.b)-dependent production
of proinflammatory cytokines. FIGS. 3A-3H are representative of
data from at least three independent experiments using biological
replicates (n=3). FIG. 3A is a graph showing Alamar blue viability
assay of cells transfected with combinations of nontargeting (NT),
TNF-R1 and DR5 siRNA and subsequently treated with LCL161 and
VSV.DELTA.51 (0.1 MOI) or IFN.beta.. Error bars, mean.+-.s.d. FIG.
3B is a graph showing the viability of cells transfected with NT or
IFNAR1 siRNA and subsequently treated with LCL161 and
VSV.DELTA.51.DELTA.G. Error bars, mean.+-.s.d. FIG. 3C is a graph
showing data from an experiment in which cells were pretreated with
LCL161, infected with 0.5 MOI of VSV.DELTA.51, and cytokine gene
expression was measured by RT-qPCR. Error bars, mean.+-.s.d. FIG.
3D is a chart showing data collected from an experiment in which
cytokine ELISAs were performed on cells transfected with NT or
IFNAR1 siRNA and subsequently treated with LCL161 and 0.1 MOI of
VSV.DELTA.51. Error bars, mean.+-.s.d. FIG. 3E is a graph showing
the viability of cells co-treated with LCL161 and cytokines. Error
bars, mean.+-.s.d. FIG. 3F is a graph showing data from an
experiment in which cells were pretreated with LCL161, stimulated
with 250 U/mL (.about.20 pg/mL) IFN.beta. and cytokine mRNA levels
were determined by RT-qPCR. Error bars, mean.+-.s.d. FIG. 3G is a
pair of graphs showing the results of cytokine ELISAs conducted on
cells treated with LCL161 and 0.1 MOI of VSV.DELTA.51. FIG. 3H is a
graph showing the result of cytokine ELISAs performed on cells
expressing IKK.beta.-DN and treated with LCL161 and VSV.DELTA.51 or
IFN.beta.. Error bars, mean.+-.s.d.
[0029] FIGS. 4A-4G are a set of graphs and images showing that
combinatorial SMC and OV treatment is efficacious in vivo and is
dependent on cytokine signaling. FIG. 4A is a pair of graphs
showing data from an experiment in which EMT6-Fluc tumors were
treated with 50 mg/kg LCL161 (p.o.) and, 5.times.10.sup.8 PFU
VSV.DELTA.51 (i.v.). The left panel depicts tumor growth. The right
panel represents the Kaplan-Meier curve depicting mouse survival.
Error bars, mean.+-.s.e.m. n=5 per group. Log-rank with Holm-Sidak
multiple comparison: **, p<0.01; ***, p<0.001. Representative
data from two independent experiments are shown. FIG. 4B is a
series of representative IVIS images that were acquired from the
experiment of FIG. 4A. FIGS. 4C-4D are sets of immunofluorescence
images of infection and apoptosis in 24 hour treated tumors using
.alpha.-VSV or .alpha.-c-caspase-3 antibodies. FIG. 4E is an image
showing an immunoblot in which protein lysates of tumors from the
corresponding treated mice were immunoblotted with the indicated
antibodies. FIG. 4F is a pair of graphs showing data from an
experiment in which mice bearing EMT6-Fluc tumors were injected
with neutralizing TNF.alpha. or isotype matched antibodies, and
subsequently treated with 50 mg/kg LCL161 (p.o.) and
5.times.10.sup.8 PFU VSV.DELTA.51 (i.v.). The left panel depicts
tumor growth. The right panel represents the Kaplan-Meier curve
depicting mouse survival. Error bars, mean.+-.s.e.m. Vehicle
.alpha.-TNF.alpha., n=5; SMC .alpha.-TNF.alpha., n=5;
vehicle+VSV.DELTA.51, n=5; .alpha.-TNF.alpha., n=5;
SMC+VSV.DELTA.51 .alpha.-TNF.alpha., n=7; SMC+VSV.DELTA.51
.alpha.-IgG, n=7. Log-rank with Holm-Sidak multiple comparison:
***, p<0.001. FIG. 4G is a set of representative IVIS images
that were acquired from the experiment of FIG. 4F.
[0030] FIGS. 5A-5E are a series of graphs and images showing that
small molecule immune stimulators enhance SMC therapy in murine
cancer models. FIG. 5A is a graph showing the results of Alamar
blue viability assays of EMT6 cells which were co-cultured with
splenocytes in a transwell system, and for which the segregated
splenocytes were treated with LCL161 and the indicated TLR
agonists. Error bars, mean.+-.s.d. Representative data from at
least three independent experiments using biological replicates
(n=3) is shown. FIG. 5B is a pair of graphs showing the results of
an experiment in which established EMT6-Fluc tumors were treated
with SMC (50 mg/kg LCL161, p.o.) and poly(I:C) (15 ug i.t. or 2.5
mg/kg i.p.). The left panel depicts tumor growth. The right panel
represents the Kaplan-Meier curve depicting mouse survival.
Vehicle, vehicle+poly(I:C) i.p., n=4; remainder groups, n=5. Error
bars, mean.+-.s.e.m. Log-rank with Holm-Sidak multiple comparison:
**, p<0.01; ***, p<0.001. FIG. 5C is a series of
representative IVIS images that were acquired from the experiment
of FIG. 5B. FIG. 5D is a pair of graphs showing the results of an
experiment in which EMT6-Fluc tumors were treated with LCL161 or
combinations of 200 .mu.g (i.t.) and/or 2.5 mg/kg (i.p.) CpG ODN
2216. The left panel depicts tumor growth. The right panel
represents the Kaplan-Meier curve depicting mouse survival.
Vehicle, n=5; SMC, n=5; vehicle+CpG i.p., n=5; SMC+CpG i.p., n=7;
vehicle+CpG i.t., n=5; SMC+CpG i.t., n=8; vehicle+CpG i.p.+i.t.,
n=5; SMC+CpG i.p.+i.t., n=8. Error bars, mean.+-.s.e.m. Log-rank
with Holm-Sidak multiple comparison: *, p<0.05; **, p<0.01;
***, p<0.001. FIG. 5E is a series of representative IVIS images
that were acquired from the experiment of FIG. 5D.
[0031] FIG. 6 is a graph showing the responsiveness of a panel of
cancer and normal cells to the combinatorial treatment of SMC and
OV. The indicated cancer cell lines (n=28) and non-cancer human
cells (primary human skeletal muscle (HSkM) and human fibroblasts
(GM38)) were treated with LCL161 and increasing VSV.DELTA.51 for 48
hours. The dose required to yield 50% viable cells in the presence
in SMC versus vehicle was determined using nonlinear regression and
plotted as a log EC50 shift toward increasing sensitivity.
Representative data from at least two independent experiments using
biological replicates (n=3) are shown.
[0032] FIG. 7 is pair of graphs showing that SMC and OV
co-treatment is highly synergistic in cancer cells. The graphs show
Alamar blue viability of cells treated with serial dilutions of a
fixed ratio combination mixture of VSV.DELTA.51 and LCL161 (PFU:
.mu.M LCL161). Combination indexes (CI) were calculated using
Calcusyn. Plots represent the algebraic estimate of the CI in
function of the fraction of cells affected (Fa). Error bars,
mean.+-.s.e.m. Representative data from three independent
experiments using biological replicates (n=3) is shown.
[0033] FIG. 8 is a pair of graphs showing that monovalent and
bivalent SMCs synergize with OVs to cause cancer cell death. The
graphs show the result of Alamar blue viability assay of cells
treated with 5 .mu.M monovalent SMCs (LCL161, SM-122) or 0.1 .mu.M
bivalent SMCs (AEG40730, OICR720, SM-164) and VSV.DELTA.51 at
differing MOIs. Error bars, mean.+-.s.d. Representative data from
three independent experiments using biological replicates (n=3) is
shown.
[0034] FIGS. 9A and 9B are a set of images and graphs showing that
SMC-mediated cancer cell death is potentiated by oncolytic viruses.
FIG. 9A is a series of images showing the results of a virus
spreading assay of cells that were overlaid with 0.7% agarose in
the presence of vehicle or LCL161 and 500 PFU of the indicated
viruses were dispensed in to the middle of the well. Cytotoxicity
was assessed by crystal violet staining. Arrow denotes extension of
the cell death zone from the origin of OV infection. FIG. 9B is a
set of graphs showing the Alamar blue viability of cells treated
with LCL161 and increasing MOIs of VSV.DELTA.51 or Maraba-MG1.
Error bars, mean.+-.s.d. Representative data from two independent
experiments using biological replicates (n=3) is shown.
[0035] FIGS. 10A and 10B are a set of graphs and images showing
that cIAP1, cIAP2 and XIAP cooperatively protect cancer cells from
OV-induced cell death. FIG. 10A shows Alamar blue viability of
cells transfected with nontargeting (NT) siRNA or siRNA targeting
cIAP1, cIAP2 or XIAP, and subsequently treated with LCL161 and 0.1
MOI VSV.DELTA.51 for 48 hours. Error bars, mean.+-.s.d.
Representative data from three independent experiments using
biological replicates (n=3) is shown. FIG. 10B is a representative
siRNA efficacy immunoblots for the experiment of FIG. 10A.
[0036] FIG. 11 is a set of images used for superimposed images
depicted in FIG. 1F. Cells were overlaid with agarose media
containing LCL161, inoculated with VSV.DELTA.51-GFP in the middle
of the well, and infectivity measured by fluorescence and
cytotoxicity was denoted by crystal violet (CV) staining. Note: the
bars represent the same size.
[0037] FIGS. 12A and 12B are a set of images and a graph showing
that SMC treatment does not affect OV distribution or replication
in vivo. FIG. 12A is a set of images showing images from an
experiment in which EMT6-bearing mice were treated with 50 mg/kg
LCL161 (p.o.) and 5.times.10.sup.8 PFU firefly luciferase tagged
VSV.DELTA.51 (VSV.DELTA.51-Fluc) via i.v. injection. Virus
distribution and replication was imaged at 24 and 48 hours using
the IVIS. Red outline denotes region of tumors. Representative data
from two independent experiments are shown. Arrow indicates spleen
infected with VSV.DELTA.51-Fluc. FIG. 12B is a graph showing data
from an experiment in which tumors and tissues at 48 hour
post-infection were homogenized and virus titrations were performed
for each group. Error bars, mean.+-.s.e.m.
[0038] FIGS. 13A and 13B are images showing verification of
siRNA-mediated knockdown of non-targeting (NT), TNFR1, DR5 and
IFNAR1 by immunoblotting. FIG. 13A is an immunoblot showing
knockdown in samples from the experiment of FIG. 3A. FIG. 13B is an
immunoblot showing knockdown in samples from the experiment of FIG.
3B.
[0039] FIGS. 14A-14G are images and graphs showing that SMC
synergizes with OVs to induce caspase-8- and RIP-1-dependent
apoptosis in cancer cells. FIGS. 14A-14G show representative data
from three independent experiments using biological replicates.
FIG. 14A is a pair of images of immunoblots in which immunoblotting
for caspase and PARP activation was conducted on cells pretreated
with LCL161 and subsequently treated with 1 MOI of VSV.DELTA.51.
FIG. 14B is a series of images showing micrographs of caspase
activation that were acquired with cells that were co-treated with
LCL161 and VSV.DELTA.51 in the presence of the caspase-3/7
substrate DEVD-488. FIG. 14C is a graph in which the proportion of
DEVD-488-positive cells from the experiment of FIG. 14B was plotted
(n=12). Error bars, mean.+-.s.d. FIG. 14D is a series of images
from an experiment in which apoptosis was assessed by micrographs
of translocated phosphatidyl serine (Annexin V-CF594, green) and
loss of plasma membrane integrity (YOYO-1, blue) in cells treated
with LCL161 and VSV.DELTA.51. FIG. 14E is a graph in which the
proportion of Annexin V-CF594-positive and YOYO-1-negative
apoptotic cells from the experiment of FIG. 14D was plotted (n=9).
Error bars, mean.+-.s.d. FIG. 14F is a pair of graphs showing
alamar blue viability of cells transfected with nontargeting (NT)
siRNA or siRNA targeting caspase-8 or RIP1, and subsequently
treated with LCL161 and 0.1 MOI of VSV.DELTA.51 (n=3). Error bars,
mean.+-.s.d. FIG. 14G is an image of an immunoblot showing
representative siRNA efficacy for the experiment of FIG. 14F.
[0040] FIGS. 15A and 15B are a set of graphs showing that
expression of TNF.alpha. transgene from OVs potentiates
SMC-mediated cancer cell death further. FIG. 15A is a pair of
graphs showing Alamar blue viability assay of cells co-treated with
5 .mu.M SMC and increasing MOIs of VSV.DELTA.51-GFP or
VSV.DELTA.51-TNF.alpha. for 24 hours. Error bars, mean.+-.s.d. FIG.
15B is a graph showing representative EC50 shifts from the
experiment of FIG. 15A. The dose required to yield 50% viable cells
in the presence in SMC versus vehicle was determined using
nonlinear regression and plotted as EC50 shift. Representative data
from three independent experiments using biological replicates
(n=3).
[0041] FIG. 16 is a set of images showing that oncolytic virus
infection leads to enhanced TNF.alpha. expression upon SMC
treatment. EMT6 cells were co-treated with 5 .mu.M SMC and 0.1 MOI
VSV.DELTA.51-GFP for 24 hours, and cells were processed for the
presence of intracellular TNF.alpha. via flow cytometry. Images
show representative data from four independent experiments.
[0042] FIGS. 17A-17C are a pair of graphs and an image showing that
TNF.alpha. signaling is required for type I IFN induced synergy
with SMC treatment. FIGS. 17A-17C show representative data from at
least three independent experiments using biological replicates
(n=3). FIG. 17A is a graph showing the results of an Alamar blue
viability assay of EMT6 cells transfected with nontargeting (NT) or
TNF-R1 siRNA and subsequently treated with LCL161 and VSV.DELTA.51
(0.1 MOI) or IFN.beta.. Error bars, mean.+-.s.d. FIG. 17B is a
representative siRNA efficacy blot from the experiment of FIG. 17A.
FIG. 17C is a graph showing the viability of EMT6 cells that were
pretreated with TNF.alpha. neutralizing antibodies and subsequently
treated with 5 .mu.M SMC and VSV.DELTA.51 or IFN.beta..
[0043] FIGS. 18A and 18B are a schematic of OV-induced type I IFN
and SMC synergy in bystander cancer cell death. FIG. 18A is a
schematic showing that virus infection in refractory cancer cells
leads to the production of Type 1 IFN, which subsequently induces
expression of IFN stimulated genes, such as TRAIL. Type 1 IFN
stimulation also leads to the NF-.kappa.B-dependent production of
TNF.alpha.. IAP antagonism by SMC treatment leads to upregulation
of TNF.alpha. and TRAIL expression and apoptosis of neighboring
tumor cells. FIG. 18B is a schematic showing that infection of a
single tumor cell results in the activation of innate antiviral
Type 1 IFN pathway, leading to the secretion of Type 1 IFNs onto
neighboring cells. The neighboring cells also produce the
proinflammatory cytokines TNF.alpha. and TRAIL. The singly infected
cell undergoes oncolysis and the remainder of the tumor mass
remains intact. On the other hand, neighboring cells undergo
bystander cell death due upon SMC treatment as a result of the
SMC-mediated upregulation of TNF.alpha./TRAIL and promotion of
apoptosis upon proinflammatory cytokine activation.
[0044] FIGS. 19A and 19B are a graph and a blot showing that SMC
treatment causes minimal transient weight loss and leads to
downregulation of cIAP1/2. FIG. 19A is graph showing weights from
LCL161 treated mice female BALB/c mice (50 mg/kg LCL161, p.o.) that
were recorded after a single treatment (day 0). n=5 per group.
Error bars, mean.+-.s.e.m. FIG. 19B is a blot of samples from an
experiment in which EMT6-tumor bearing mice were treated with 50
mg/kg LCL161 (p.o.). Tumors were harvested at the indicated time
for western blotting using the indicated antibodies.
[0045] FIGS. 20A-20C are a set of graphs showing that SMC treatment
induces transient weight loss in a syngeneic mouse model of cancer.
FIGS. 20A-20C are graphs showing measurements of mouse weights upon
SMC and oncolytic VSV (FIG. 20A), poly(I:C) (FIG. 20B), or CpG
(FIG. 20C) co-treatment in tumor-bearing animals from the
experiments depicted in FIGS. 4A, 5B, and 5D, respectively. Error
bars, mean.+-.s.e.m.
[0046] FIGS. 21A-21D are a series of graphs showing that
VSV.DELTA.51-induced cell death in HT-29 cell is potentiated by SMC
treatment in vitro and in vivo. FIG. 21A is a graph showing data
from an experiment in which cells were infected with VSV.DELTA.51,
the cell culture supernatant was exposed to UV light for 1 hour and
was applied to new cells at the indicated dose in the presence of
LCL161. Viability was ascertained by Alamar blue. Error bars,
mean.+-.s.d. FIG. 21B is a graph showing Alamar blue viability of
cells co-treated with LCL161 and a non-spreading virus
VSV.DELTA.51.DELTA.G (0.1 MOI). Error bars, mean.+-.s.d. Panels a
and b show representative data from three independent experiments
using biological replicates (n=3). FIG. 21C is a pair of graphs
showing data from an experiment in which CD-1 nude mice with
established HT-29 tumors were treated with 50 mg/kg LCL161 (p.o.)
and 1.times.10.sup.8 PFU VSV.DELTA.51 (i.t.). Vehicle, n=5;
VSV.DELTA.51, n=6; SMC, n=6; VSV.DELTA.51+SMC, n=7. The left panel
depicts tumor growth relative to day 0 post-treatment. The right
panel represents the Kaplan-Meier curve depicting mouse survival.
Error bars, mean.+-.s.e.m. Log-rank with Holm-Sidak multiple
comparison: ***, p<0.001. FIG. 21D is a graph showing
measurement of mouse weights upon SMC and OV co-treatment in
tumor-bearing animals. Error bars, mean.+-.s.e.m.
[0047] FIG. 22 is a blot showing that type I IFN signaling is
required for SMC and OV synergy in vivo. EMT6 tumor bearing mice
were treated with vehicle or 50 mg/kg LCL161 for 4 hours, and
subsequently treated with neutralizing IFNAR1 or isotype antibodies
for 20 hours. Subsequently, animals were treated with PBS or
VSV.DELTA.51 for 18 hours. Tumors were processed for Western
blotting with the indicated antibodies.
[0048] FIGS. 23A and 23B are a pair of graphs showing that
oncolytic infection of innate immune cells leads to cancer cell
death in the presence of SMCs. FIG. 23A is a graph showing data
from an experiment in which immune subpopulations were sorted from
splenocytes (CD11b+ F4/80+: macrophage; CD11b+ Gr1+: neutrophil;
CD11b- CD49b+: NK cell; CD11b- CD49b-: T and B cells) and were
infected with 1 MOI of VSV.DELTA.51 for 24 hours. Cell culture
supernatants were applied to SMC-treated ETM6 cells for 24 hours
and EMT6 viability was assessed by Alamar Blue. Error bars,
mean.+-.s.d. FIG. 23B is a chart showing data from an experiment in
which bone marrow derived macrophages were infected with
VSV.DELTA.51 and the supernatant was applied to EMT6 cells in the
presence of 5 .mu.M SMC, and viability was measured by Alamar blue.
Error bars, mean.+-.s.d.
[0049] FIGS. 24A-24H are a series of images of full-length
immunoblots. Immunoblots of FIGS. 24A-24H pertain to (FIG. 24A)
FIG. 2E, (FIG. 24B) FIG. 4E, (FIG. 24C) FIG. 10B, (FIG. 24D) FIGS.
13A and 13B, (FIG. 24E) FIG. 14A, (FIG. 24F) FIG. 14G, (FIG. 24G)
FIG. 19B, and (FIG. 24H) FIG. 17B, respectively.
[0050] FIGS. 25A-25B are a set of graphs showing that
non-replicating rhabdovirus-derived particles (NRRPs) synergize
with SMCs to cause cancer cell death. FIG. 25A is a set of graphs
showing data from an experiment in which EMT6, DBT, and CT-2A
cancer cells were co-treated with the SMC LCL161 (SMC; EMT6: 5
.mu.M, DBT and CT-2A: 15 .mu.M) and different numbers of NRRPs for
48 hr (EMT6) or 72 hr (DBT, CT-2A), and cell viability was assessed
by Alamar Blue. FIG. 25B is a pair of graphs showing data from an
experiment in which ufractionated mouse splenocytes were incubated
with 1 particle per cell of NRRP or 250 .mu.M CpG ODN 2216 for 24
hr. Subsequently, the supernatant was applied to EMT6 cells in a
dose-response fashion, and 5 .mu.M LCL161 was added. EMT6 viability
was assessed 48 hr post-treatment by Alamar blue.
[0051] FIGS. 26A and 26B are a graph and a set of image showing
that vaccines synergize with SMCs to cause cancer cell death. FIG.
26A is a graph showing data from an experiment in which EMT6 cells
were treated with vehicle or 5 .mu.M LCL161 (SMC) and 1000 CFU/mL
BCG or 1 ng/mL TNF.alpha. for 48 hr, and viability was assessed by
Alamar blue. FIG. 26B is a set of representative IVIS images
depicting survival of mice bearing mammary fat pad tumors
(EMT6-Fluc) that were treated twice with vehicle or 50 mg/kg LCL161
(SMC) and PBS intratumorally (i.t.), BCG (1.times.10.sup.5 CFU)
i.t., or BCG (1.times.10.sup.5 CFU) intraperitoneally (i.p.) and
subjected to live tumor bioluminescence imaging by IVIS CCD camera
at various time points. Scale: p/sec/cm2/sr.
[0052] FIGS. 27A and 27B are a pair of graphs and a set of images
showing that SMCs synergize with type I IFN to cause mammary tumor
regression. FIG. 27A is a pair of graphs showing data from an
experiment in which mice were injected with EMT6-Fluc tumors in the
mammary fat pad and were treated at eight days post-implantation
with combinations of vehicle or 50 mg/kg LCL161 (SMC) orally and
bovine serum albumin (BSA), 1 .mu.g IFN.alpha. intraperitoneally
(i.p.), or 2 .mu.g IFN.alpha. intratumorally (i.t.). The left panel
depicts tumor growth. The right panel represents the Kaplan-Meier
curve depicting mouse survival. Error bars, mean.+-.s.e.m. FIG. 27B
is a series of representative IVIS images from the experiment
described in FIG. 27A. Scale: p/sec/cm2/sr.
[0053] FIG. 28 is a graph showing that the expression of type I IFN
from VSV synergizes with SMCs to cause cancer cell death. The graph
shows data from an experiment in which EMT6 cells were co-treated
with vehicle or 5 .mu.M LCL161 (SMC) and differing multiplicity of
infection (MOI) of VSV.DELTA.51-GFP, VSV-IFN.beta., or
VSV-NIS-IFN.beta.. Cell viability was assessed 48 hr post-treatment
by Alamar blue.
[0054] FIG. 29 is a graph showing that non-viral and viral triggers
induce robust expression of TNF.alpha. in vivo. Mice were treated
with 50 mg of poly(I:C) intraperitoneally or with intravenous
injections of 5.times.10.sup.8 PFU VSV.DELTA.51, VSV-mIFN.beta., or
Maraba-MG1. At the indicated times, serum was isolated and
processed for ELISA to quantify the levels of TNF.alpha..
[0055] FIGS. 30A-30C are a set of graphs and images showing that
virally-expressed proinflammatory cytokines synergizes with SMCs to
induce mammary tumor regression. FIG. 30A is a pair of graphs
showing data from an experiment in which mice were injected with
EMT6-Fluc tumors in the mammary fat pad, and were treated at seven
days post-implantation with combinations of vehicle or 50 mg/kg
LCL161 (SMC) orally and PBS, 1.times.10.sup.8 PFU
VSV.DELTA.51-memTNF.alpha. (i.v.), or 1.times.10.sup.8 PFU
VSV.DELTA.51-solTNF.alpha. (i.v.). The left panel depicts tumor
growth. The right panel represents the Kaplan-Meier curve depicting
mouse survival. Error bars, mean.+-.s.e.m. FIG. 30B is a set of
representative bioluminescent IVIS images that were acquired from
the experiment described in FIG. 30A. Scale: p/sec/cm2/sr. FIG. 30C
is a pair of graphs showing data from an experiment in which mice
were injected with CT-26 tumors subcutaneously and were treated 10
days post-implantation with combinations of vehicle or 50 mg/kg
LCL161 orally and either PBS or 1.times.10.sup.8 PFU
VSV.DELTA.51-solTNF.alpha. intratumorally. The left panel depicts
tumor growth. The right panel represents the Kaplan-Meier curve
depicting mouse survival. Error bars, mean.+-.s.e.m.
[0056] FIGS. 31A and 31B are a set of images showing that SMC
treatment leads to down-regulation of cIAP1/2 protein in vivo in an
orthotopic, syngeneic mouse model of glioblastoma. FIG. 31A is an
image showing an immunoblot from an experiment in which CT-2A cells
were implanted intracranially and treated with 50 mg/kg orally of
LCL161 (SMC) and tumors were excised at the indicated time points
and processed for western blotting using antibodies against
cIAP1/2, XIAP, and .beta.-tubulin. FIG. 31B is an image showing an
immunoblot from an experiment in which CT-2A cells were implanted
intracranially and treated with 10 uL of 100 .mu.M LCL161
intratumorally and tumors were excised at the indicated time points
and processed for western blotting using antibodies against
cIAP1/2, XIAP, and .beta.-tubulin.
[0057] FIGS. 32A-32E are a set of graphs and images showing that a
transient proinflammatory response in the brain synergizes with
SMCs to cause glioblastoma cell death. FIG. 32A is a graph showing
data from an experiment in which an ELISA was conducted to
determine the levels of soluble TNF.alpha. from 300 mg of crude
brain protein extract that was derived from mice injected
intraperitoneally (i.p.) with PBS or 50 mg poly(I:C) for 12 or 24
h. Brain protein extracts were obtained by mechanical
homogenization in saline solution. FIG. 32B is a graph showing data
from Alamar blue viability assays of mouse glioblastoma cells
(CT-2A, K1580) that were treated with 70 mg of crude brain
homogenates and 5 .mu.M LCL161 (SMC) in culture for 48 h. Brain
homogenates were obtained from mice that were treated for 12 h with
i.p. injections of poly(I:C), or intravenous injections of
5.times.10.sup.8 PFU VSV.DELTA.51 or VSV-mIFN.beta.. FIG. 32C
represents the Kaplan-Meier curve depicting survival of mice that
received three intracranial treatments of 50 mg poly(I:C).
Treatments were on days 0, 3, and 7. FIG. 32D represents the
Kaplan-Meier curve depicting survival of mice bearing CT-2A
intracranial tumors that received combinations of SMC, VSV.DELTA.51
or poly(I:C). Mice received combinations of three treatments of
vehicle, three treatments of 75 mg/kg LCL161 (oral), three
treatments of 5.times.10.sup.8 PFU VSV.DELTA.51 (i.v.), or two
treatments of 50 mg poly(I:C) (intracranial, i.c.). Mice were
treated on day 7, 10, and 14 post tumor cell implantation with the
different conditions, except for the poly(I:C) treated group that
received i.c. injections on day 7 and 15. Numbers in brackets
denote number of mice per group. FIG. 32E is a series of
representative MRI images of mouse skulls from the experiments
depicted in FIG. 32D, which shows an animal at endpoint and a
representative mouse of the indicated groups at 50 days
post-implantation. Dashed line denotes the brain tumor.
[0058] FIG. 33 is a graph showing that SMCs synergize with type I
IFN to eradicate brain tumors. The graph represents the
Kaplan-Meier curve depicting survival of mice bearing CT-2A that
received intracranial injections of vehicle or 100 .mu.M LCL161
(SMC) with PBS or 1 .mu.g IFN.alpha. at 7 days
post-implantation.
DETAILED DESCRIPTION
[0059] The present invention includes methods and compositions for
enhancing the efficacy of Smac mimetic compounds (SMCs) in the
treatment of cancer. In particular, the present invention includes
methods and compositions for combination therapies that include an
SMC and a second agent that stimulates one or more cell death
pathways that are inhibited by cIAP1 and/or cIAP2. The second agent
may be, e.g., a TLR agonist a virus, such as an oncolytic virus, or
an interferon or related agent.
[0060] The data provided herein demonstrates that treatment with an
immunostimulatory agent and an SMC results in tumor regression and
durable cures in vivo (see, e.g., Example 1). These combination
therapies were well tolerated by mice, with body weight returning
to pre-treatment levels shortly after the cessation of therapy.
Tested combination therapies were able to treat several treatment
refractory, aggressive mouse models of cancer. One of skill in the
art will recognize, based on the disclosure and data provided
herein, that any one or more of a variety of SMCs and any one or
more of a variety of immunostimulatory agents, such as a TLR
agonist, pathogen, or pathogen mimetic, may be combined in one or
more embodiments of the present invention to potentiate apoptosis
and treat cancer.
[0061] While other approaches to improve SMC therapy have been
attempted, very rarely have complete responses been observed,
particularly in aggressive immunocompetent model systems. Some
embodiments of the present invention, including treatment of cancer
with a pathogen mimetic, e.g., a pathogen mimetic having a
mechanism of action partially dependent on TRAIL, can have certain
advantages. First, this approach can evoke TNF.alpha.-mediated
apoptosis and necroptosis: given the plasticity and heterogeneity
of some advanced cancers, treatments that simultaneously induce
multiple distinct cell death mechanisms may have greater efficacy
than those that do not. Second, pathogen mimetics can elicit an
integrated innate immune response that includes layers of negative
feedback. These feedback mechanisms may act to temper the cytokine
response in a manner difficult to replicate using recombinant
proteins, and thus act as a safeguard to this combination therapy
strategy.
SMCs
[0062] An SMC of the present invention may be any small molecule,
compound, polypeptide, protein, or any complex thereof, capable, or
predicted of being capable, of inhibiting cIAP1 and/or cIAP2, and,
optionally, one or more additional endogenous Smac activities. An
SMC of the present invention is capable of potentiating apoptosis
by mimicking one or more activities of endogenous Smac, including
but not limited to, the inhibition of cIAP1 and the inhibition of
cIAP2. An endogenous Smac activity may be, e.g., interaction with a
particular protein, inhibition of a particular protein's function,
or inhibition of a particular IAP. In particular embodiments, the
SMC inhibits both cIAP1 and cIAP2. In some embodiments, the SMC
inhibits one or more other IAPs in addition to cIAP1 and cIAP2,
such as XIAP or Livin/ML-IAP, the single BIR-containing IAP. In
particular embodiments, the SMC inhibits cIAP1, cIAP2, and XIAP. In
any embodiment including an SMC and an immune stimulant, an SMC
having particular activities may be selected for combination with
one or more particular immune stimulants. In any embodiment of the
present invention, the SMC may be capable of activities of which
Smac is not capable. In some instances, these additional activities
may contribute to the efficacy of the methods or compositions of
the present invention.
[0063] Treatment with SMCs can deplete cells of cIAP1 and cIAP2,
through, e.g., the induction of auto- or trans-ubiquitination and
proteasomal-mediated degradation. SMCs can also de-repress XIAP's
inhibition of caspases. SMCs may primarily function by targeting
cIAP1 and 2, and by converting TNF.alpha., and other cytokines or
death ligands, from a survival signal to a death signal, e.g., for
cancer cells.
[0064] Certain SMCs inhibit at least XIAP and the cIAPs. Such
"pan-IAP" SMCs can intervene at multiple distinct yet interrelated
stages of programmed cell death inhibition. This characteristic
minimizes opportunities for cancers to develop resistance to
treatment with a pan-IAP SMC, as multiple death pathways are
affected by such an SMC, and allows synergy with existing and
emerging cancer therapeutics that activate various apoptotic
pathways in which SMCs can intervene.
[0065] One or more inflammatory cytokines or death ligands, such as
TNF.alpha., TRAIL, and IL-1.beta., potently synergize with SMC
therapy in many tumor-derived cell lines. Strategies to increase
death ligand concentrations in SMC-treated tumors, in particular
using approaches that would limit the toxicities commonly
associated with recombinant cytokine therapy, are thus very
attractive. TNF.alpha., TRAIL, and dozens of other cytokines and
chemokines can be upregulated in response to pathogen recognition
by the innate immune system of a subject. Importantly, this ancient
response to microbial pathogens is usually self-limiting and safe
for the subject, due to stringent negative regulation that limits
the strength and duration of its activity.
[0066] SMCs may be rationally designed based on Smac. The ability
of a compound to potentiate apoptosis by mimicking one or more
functions or activities of endogenous Smac can be predicted based
on similarity to endogenous Smac or known SMCs. An SMC may be a
compound, polypeptide, protein, or a complex of two or more
compounds, polypeptides, or proteins.
[0067] In some instances, SMCs are small molecule IAP antagonists
based on an N-terminal tetrapeptide sequence (revealed after
processing) of the polypeptide Smac. In some instances, an SMC is a
monomer (monovalent) or dimer (bivalent). In particular instances,
an SMC includes 1 or 2 moieties that mimic the tetrapeptide
sequence of AVPI from Smac/DIABLO, the second mitochondrial
activator of caspases, or other similar IBMs (e.g., IAP-binding
motifs from other proteins like casp9). A dimeric SMC of the
present invention may be a homodimer or a heterodimer. In certain
embodiments, the dimer subunits are tethered by various linkers.
The linkers may be in the same defined spot of either subunit, but
could also be located at different anchor points (which may be `aa`
position, P1, P2, P3 or P4, with sometimes a P5 group available).
In various arrangements, the dimer subunits may be in different
orientations, e.g., head to tail, head to head, or tail to tail.
The heterodimers can include two different monomers with differing
affinities for different BIR domains or different IAPs.
Alternatively, a heterodimer can include a Smac monomer and a
ligand for another receptor or target which is not an IAP. In some
instances, an SMCs can be cyclic. In some instances, an SMC can be
trimeric or multimeric. A multimerized SMC can exhibit a fold
increase in activity of 7,000-fold or more, such as 10-, 20-, 30-,
40-, 50-, 100-, 200-, 1,000-, 5,000-, 7,000-fold, or more
(measured, e.g., by EC50 in vitro) over one or more corresponding
monomers. This may occur, in some instances, e.g., because the
tethering enhances the ubiquitination between IAPs or because the
dual BIR binding enhances the stability of the interaction.
Although multimers, such as dimers, may exhibit increased activity,
monomers may be preferable in some embodiments. For example, in
some instances, a low molecular weight SMC may be preferable, e.g.,
for reasons related to bioavailability.
[0068] In some instances of the present invention, an agent capable
of inhibiting cIAP1/2 is a bestatin or Me-bestatin analog. Bestatin
or Me-bestatin analogs may induce cIAP1/2 autoubiquitination,
mimicking the biological activity of Smac.
[0069] In certain embodiments of the present invention, an SMC
combination treatment includes one or more SMCs and one or more
interferon agents, such as an interferon type 1 agent, an
interferon type 2 agent, and an interferon type 3 agent.
Combination treatments including an interferon agent may be useful
in the treatment of cancer, such as multiple myeloma.
[0070] In some embodiments, a VSV expressing IFN, and optionally
expressing a gene that enables imaging, such as NIS, the
sodium-iodide symporter, is used in combination with an SMC. For
instance, such a VSV may be used in combination with an SMC, such
as the Ascentage Smac mimetic SM-1387/APG-1387, the Novartis Smac
mimetic LCL161, or Birinapant. Such combinations may be useful in
the treatment of cancer, such as hepatocellular carcinoma or liver
metastases.
[0071] Various SMCs are known in the art. Non-limiting examples of
SMCs are provided in Table 1. While Table 1 includes suggested
mechanisms by which various SMCs may function, methods and
compositions of the present invention are not limited by or to
these mechanisms.
TABLE-US-00001 TABLE 1 Smac mimetic compounds Clinical
Organization; Compound Structure or Reference Status
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Immunostimulatory Agents
[0072] An immunostimulatory or immunomodulatory agent of the
present invention may be any agent capable of inducing a
receptor-mediated apoptotic program that is inhibited by cIAP1 and
cIAP2 in one or more cells of a subject. An immune stimulant of the
present invention may induce an apoptotic program regulated by
cIAP1(BIRC2), cIAP2 (BIRC3 or API2), and optionally, one or more
additional IAPs, e.g., one or more of the human IAP proteins NAIP
(BIRC1), XIAP (BIRC4), survivin (BIRC5), Apollon/Bruce (BIRC6),
ML-IAP (BIRC7 or livin), and ILP-2 (BIRC8). It is additionally
known that various immunomodulatory or immunostimulatory agents,
such as CpGs or IAP antagonists, can change immune cell
contexts.
[0073] In some instances, an immune stimulant may be a TLR agonist,
such as a TLR ligand. A TLR agonist of the present invention may be
an agonist of one or more of TLR-1, TLR-2, TLR-3, TLR-4, TLR-5,
TLR-6, TLR-7, TLR-8, TLR-9, and TLR-10 in humans or related
proteins in other species (e.g., murine TLR-1 to TLR-9 and TLR-11
to TLR-13). TLRs can recognize highly conserved structural motifs
known as pathogen-associated microbial patterns (PAMPs), which are
exclusively expressed by microbial pathogens, as well as
danger-associated molecular patterns (DAMPs) that are endogenous
molecules released from necrotic or dying cells. PAMPs include
various bacterial cell wall components such as lipopolysaccharide
(LPS), peptidoglycan (PGN), and lipopeptides, as well as flagellin,
bacterial DNA, and viral double-stranded RNA. DAMPs include
intracellular proteins such as heat shock proteins as well as
protein fragments from the extracellular matrix. Agonists of the
present invention further include, for example, CpG
oligodeoxynucleotides (CpG ODNs), such as Class A, B, and C CpG
ODN's, base analogs, nucleic acids such as dsRNA or pathogen DNA,
or pathogen or pathogen-like cells or virions. In certain
embodiments, the immunostimulatory agent is an agent that mimics a
virus or bacteria or is a synthetic TLR agonist.
[0074] Various TLR agonists are known in the art. Non-limiting
examples of TLR agonists are provided in Table 2. While Table 2
includes suggested mechanisms, uses, or TLR targets by which
various TLR agonists may function, methods and compositions of the
present invention are not limited by or to these mechanisms, uses,
or targets.
TABLE-US-00002 TABLE 2 Immunostimulatory agents: TLR Agonists
Agonist Compound Structure or Reference Compound Type or
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C.sub.19H.sub.23N.sub.7O.sub.4 ##STR00003## or TLR-7/8 CL307 Base
analog TRL-7 or TLR-7/8 Gardi- U.S. Patent Publication No.
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ODN 1585 Ballas ZK. et al., 2001. Divergent therapeutic and
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30,1841-1850 CL401 Formula: C.sub.54H.sub.92N.sub.8O.sub.45
##STR00007## Dual TLR agonist TLR-2 and TLR-7 Adili- poline .TM.
(CL413;) Formula: C.sub.81H.sub.145N.sub.17O.sub.12S ##STR00008##
Dual TLR agonist TLR-2 and TLR-7 CL531 Formula:
C.sub.82H.sub.144N.sub.16O.sub.14S ##STR00009## Dual TLR agonist
TLR-2 and TLR-7 CL572 ( Formula: C.sub.41H.sub.65N.sub.9O.sub.7S
##STR00010## Dual TLR agonist Human TLR-2, mouse TLR-7, an human
TLR-7 Adi- Fectin .TM. (CL347;) Formula:
C.sub.72H.sub.134N.sub.11O.sub.6P ##STR00011## TLR agonist and
nucleic acid carrier TLR-7 CL419 Formula:
C.sub.48H.sub.97N.sub.5O.sub.5S ##STR00012## TLR agonist and
nucleic acid carrier TLR-2 Pamadi- Fectin .TM. (CL553;) Formula:
C.sub.67H.sub.118N.sub.12O.sub.8S ##STR00013## TLR agonist and
nucleic acid carrier TLR-2 and TLR-7 Peptido- TLR ligand; cell
surface location TLR-1/2; glycan (Expert Rev Clin Pharmacol 4(2):
TLR-2/6 275-289, 2011) Diacylated Buwitt-Beckmann u. et al., 2005.
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activation and apoptosis by bacterial TLR ligand; cell surface
location TLR-1/2 lipopeptide lipoproteins through toll-like
receptor-2. Science. 285(5428):736-9. Ozinsky a. et al., 2000. The
repertoire for pattern recognition of pathogens by the innate
immune system is defined by cooperation between toll-like
receptors. PNAS. 97(25):13766-71. 3 Lipopoly- N/A TLR ligand; cell
surface location; TLR-4 saccharide intratumoral administration for
(LPS) treatment of glioma. (see, e.g., Mariani CL, Rajon D, Bova
FJ, Streit WJ. Nonspecific immunotherapy with intratumoral
lipopolysaccharide and zymosan A but not GM-CSF leads to an
effective anti-tumor response in subcutaneous RG-2 gliomas. J.
Neurooncol. 2007; 85(3):231-240. ) CpG 7909 Intravenous
administration for TLR-9 treatment of non-Hodgkin lymphoma. (see,
e.g., Link BK, Ballas ZK, Weisdorf D, et al. Oligodeoxynucleotide
CpG 7909 delivered as intravenous infusion demonstrates immunologic
modulation in patients with previously treated non-Hodgkin
lymphoma. J. Immunother. 2006; 29(5):558-568.) 852A Intravenous
administration for TLR-7 treatment of melanoma and other cancer
[12,55]; (see, e.g., Dudek AZ, Yunis C, Harrison LI, et al. First
in human Phase I trial of 852A, a novel systemic Toll- like
receptor 7 agonist, to activate innate immune responses in patients
with advanced cancer. Olin. Cancer Res. 2007; 13(23):7119-7125';
Dummer R, Hauschild A, Becker JC, et al. An exploratory study of
systemic administration of the Toll-like receptor-7 agonist 852A in
patients with refractory metastatic melanoma. Clin. Cancer Res.
2008; 14(3):856- 864. intravenous administration for treatment of
chronic lymphocytic leukemia (see, e.g., Spaner DE, Shi Y, White D,
et al. A Phase I/II trial of TLR7 agonist immunotherapy in chronic
lymphocytic leukemia. Leukemia. 2010; 24(1):222-226.) Ampligen
Intravenous administration for TLR-3 treatment of chronic fatigue
syndrome [60]; intravenous administration for treatment of HIV
(see, e.g., Thompson KA, Strayer DR, Salvato PD, et al. Results of
a double-blind placebo-controlled study of the double-stranded RNA
drug polyl:polyC12U in the treatment of HIV infection. Eur. J.
Olin. Microbiol. Infect. Dis. 1996; 15(7):580-587. [PubMed:
8874076]) Resiquimod Oral administration for treatment of TLR-7/8
hepatitis C ((see, e.g., Pockros PJ, Guyader D, Patton H, et al.
Oral resiquimod in chronic HCV infection: safety and efficacy in 2
placebo- controlled, double-blind Phase IIa studies. J. Hepatol.
2007; 47(2):174-182.); Topical administration for treatment of
Herpes simplex virus 2 (see, e.g., Mark KE, Corey L, Meng TC, et
al. Topical resiquimod 0.01% gel decreases herpes simplex virus
type 2 genital shedding: a randomized, controlled trial. J. Infect.
Dis. 2007; 195(9):1342-1331.) ANA975 Oral administration for
treatment of TLR-7 hepatitis (see, e.g., Fletcher S,
Steffy K, Averett D. Masked oral prodrugs of Toll-like receptor 7
agonists: a new approach for the treatment of infectious disease.
Curr. Opin. Investig. Drugs. 2006; 7(8):702-708.) Imiquimod
Imidazoquinoline compound; topical TLR-7 (InvivoGen) administration
for treatment of basal cell carcinoma (see, e.g., Schulze HJ,
Cribier B, Requena L, et al. Imiquimod 5% cream for the treatment
of superficial basal cell carcinoma: results from a randomized
vehicle-controlled Phase III study in Europe. Br. J. Dermatol.
2005; 152(5):939-947; Quirk C, Gebauer K, Owens M, Stampone P.
Two-year interim results from a 5-year study evaluating clinical
recurrence of superficial basal cell carcinoma after treatment with
imiquimod 5% cream daily for 6 weeks. Australas. J. Dermatol. 2006;
47(4):258-265.); Topical administration for treatment of squamous
cell carcinoma (see, e.g., Ondo AL, Mings SM, Pestak RM, Shanler
SD. Topical combination therapy for cutaneous squamous cell
carcinoma in situ with 5-fluorouracil cream and imiquimod cream in
patients who have failed topical monotherapy. J. Am. Acad.
Dermatol. 2006; 55(6):1092-1094.) Topical administration for
treatment of melanoma (see, e.g., Turza K, Dengel LT, Harris RC, et
al. Effectiveness of imiquimod limited to dermal melanoma
metastases, with simultaneous resistance of subcutaneous
metastasis. J. Cutan. Pathol. 2009 DOI: 10.1111/j.1600-
0560.2009.01290.x. (Epub ahead of print); (see, e.g., Green DS,
Dalgleish AG, Belonwu N, Fischer MD, Bodman-Smith MD. Topical
imiquimod and intralesional interleukin-2 increase activated
lymphocytes and restore the Th1/Th2 balance in patients with
metastatic melanoma. Br. J. Dermatol. 2008; 159(3):606-614.);
Topical administration for treatment of vulvar intraepithelial
neoplasia (see, e.g., Van Seters M, Van Beurden M, Ten Kate FJ, et
al. Treatment of vulvar intraepithelial neoplasia with topical
imiquimod. N. Engl. J. Med. 2008; 358(14):1465- 1473.); Topical
administration for treatment of cutaneous lymphoma (see, e.g.,
Stavrakoglou A, Brown VL, Coutts I. Successful treatment of primary
cutaneous follicle centre lymphoma with topical 5% imiquimod. Br.
J. Dermatol. 2007; 157(3):620-622.); Topical treatment as Human
papillomavirus (HPV) vaccine (see, e.g., Daayana S, Elkord E,
Winters U, et al. Phase II trial of imiquimod and HPV therapeutic
vaccination in patients with vulval intraepithelial neoplasia. Br.
J. Cancer. 2010; 102(7):1129-1136.); Subcutaneous/intramuscular
administration: New York esophageal squamous cell carcinoma 1
cancer antigen (NY- ESO-1) protein vaccine for melanoma (see, e.g.,
Adams S, O'Neill DW, Nonaka D, et al. Immunization of malignant
melanoma patients with full-length NY-ESO-1 protein using TLR7
agonist imiquimod as vaccine adjuvant. J. Immunol. 2008;
181(1):776-784.) Mono- Subcutaneous/intramuscular TLR-4 phosphoryl
administration for vaccination lipid A against HPV (see, e.g.,
Harper DM, (MPL) Franco EL, Wheeler CM, et al. Sustained efficacy
up to 4.5 years of a bivalent L1 virus-like particle vaccine
against human papillomavirus types 16 and 18: follow-up from
arandomised control trial. Lancet. 2006; 367(9518):1247- 1255.);
Subcutaneous/intramuscular administration for vaccination against
non-small-cell lung cancer (see, e.g., Butts C, Murray N, Maksymiuk
A, et al. Randomized Phase IIB trial of BLP25 liposome vaccine in
stage IIIB and IV non- small-cell lung cancer. J. Clin. Oncol.
2005; 23(27):6674-6681.) CpG 7909 Subcutaneous/intramuscular TLR-9
(i.e., PF- administration for treatment of non- 3512676) small-cell
lung cancer (see, e.g., Manegold C, Gravenor D, Woytowitz D, et al.
Randomized Phase II trial of a Toll-like receptor 9 agonist
oligodeoxynucleotide, PF-3512676, in combination with first-line
taxane plus platinum chemotherapy for advanced-stage non-small-cell
lung cancer. J. Clin. Oncol. 2008; 26(24):3979-3986; Readett, D.;
Denis, L.; Krieg, A.; Benner, R.; Hanson, D. PF-3512676 (CPG 7909)
a Toll-like receptor 9 agonist- status of development for non-small
cell lung cancer (NSCLC). Presented at: 12th World Congress on Lung
Cancer; Seoul, Korea. 2-6 September 2007);
Subcutaneous/intramuscular administration for treatment of
metastatic melanoma (see, e.g., Pashenkov M, Goess G, Wagner C, et
al. Phase II trial of a Toll-like receptor 9-activating
oligonucleotide in patients with metastatic melanoma. J. Olin.
Oncol. 2006; 24(36):5716-5724.; Subcutaneous/intramuscular
administration; Melan-A peptide vaccine for melanoma (see, e.g.,
Speiser DE, Lienard D, Rufer N, et al. Rapid and strong human CD8+
T cell responses to vaccination with peptide, IFA, and CpG
oligodeoxynucleotide 7909. J. Olin. Invest. 2005; 115(3): 739-746;
Appay V, Jandus C, Voelter V, et al. New generation vaccine induces
effective melanoma- specific CD8+ T cells in the circulation but
not in the tumor site. J. Immunol. 2006; 177(3):1670- 1678.);
Subcutaneous/intramuscular administration; NY-ESO-1 protein vaccine
(see, e.g., Valmori D, Souleimanian NE, Tosello V, et al.
Vaccination with NY-ESO-1 protein and CpG in Montanide induces
integrated antibody/Th1 responses and CD8 T cells through cross-
priming. Proc. Natl Acad. Sci. USA. 2007; 104(21):8947-8952.) CpG
1018 Subcutaneous/intramuscular TLR-9 ISS administration for
treatment of lymphoma (see, e.g., Friedberg JW, Kim H, McCauley M,
et al. Combination immunotherapy with a CpG oligonucleotide (1018
ISS) and rituximab in patients with non- Hodgkin lymphoma:
increased interferon-.alpha./.beta.-inducible gene expression,
without significant toxicity. Blood. 2005; 105(2):489- 495;
Friedberg JW, Kelly JL, Neuberg D, et al. Phase II study of a TLR-9
agonist (1018 ISS) with rituximab in patients with relapsed or
refractory follicular lymphoma. Br. J. Haematol. 2009;
146(3):282-291.) Bacillus N/A Intratumoral administration for TLR-2
Calmette- treatment of bladder cancer (see, Guerin e.g., Simons MP,
O'Donnell MA. (BCG) Griffith TS. Role of neutrophils in BCG
immunotherapy for bladder cancer. Urol. Oncol. 2008;
26(4):341-345.) Zymosan A Intratumoral administration for TLR-2
treatment of glioma (see, e.g., Mariani CL, Rajon D, Bova FJ,
Streit WJ. Nonspecific immunotherapy with intratumoral
lipopolysaccharide and zymosan A but not GM-CSF leads to an
effective anti-tumor response in subcutaneous RG-2 gliomas. J.
Neurooncol. 2007; 85(3):231-240.) ##STR00014##
[0075] In other instances, an immune stimulant may be a virus,
e.g., an oncolytic virus. An oncolytic virus is a virus that
selectively infects, replicates, and/or selectively kills cancer
cells. Viruses of the present invention include, without
limitation, adenoviruses, Herpes simplex viruses, measles viruses,
Newcastle disease viruses, parvoviruses, polioviruses, reoviruses,
Seneca Valley viruses, retroviruses, Vaccinia viruses, vesicular
stomatitis viruses, lentiviruses, rhabdoviruses, sindvis viruses,
coxsackieviruses, poxviruses, and others. In particular embodiments
of the present invention, the immunostimulatory agent is a
rhabodvirus, e.g., VSV. Rhabdoviruses can replicate quickly with
high IFN production. In other particular embodiments, the
immunostimulatory agent is a feral member, such as Maraba virus,
with the MG1 double mutation, Farmington virus, Carajas virus.
Viral immunostimulatory agents of the present invention include
mutant viruses (e.g., VSV with a .DELTA.51 mutation in the Matrix,
or M, protein), transgene-modified viruses (e.g., VSV-hIFN.beta.),
viruses carrying -TNF.alpha., -LT.alpha./TNF.beta., -TRAIL, FasL,
-TL1.alpha., chimeric viruses (eg rabies), or pseudotyped viruses
(e.g., viruses pseudotyped with G proteins from LCMV or other
viruses). In some instances, the virus of the present invention
will be selected to reduce neurotoxicity. Viruses in general, and
in particular oncolytic viruses, are known in the art.
[0076] In certain embodiments, the immunostimulatory agent is a
killed VSV NRRP particle or a prime-and-boost tumor vaccine. NRRPs
are wild type VSV that have been modified to produce an infectious
vector that can no longer replicate or spread, but that retains
oncolytic and immunostimulatory properties. NRRPs may be produced
using gamma irradiation, UV, or busulfan. Particular combination
therapies include prime-and-boost with adeno-MAGE3 (melanoma
antigen) and/or Maraba-MG1-MAGE3. Other particular combination
therapies include UV-killed or gamma irradiation-killed wild-type
VSV NRRPs. NRRPs may demonstrate low or absent neurotixicity. NRRPs
may be useful, e.g., in the treatment of glioma, hematological
(liquid) tumors, or multiple myeloma.
[0077] In some instances, the immunostimulatory agent of the
present invention is a vaccine strain, attenuated virus or
microorganism, or killed virus or microorganism. In some instances,
the immunostimulatory agent may be, e.g., BCG, live or dead Rabies
vaccines, or an influenza vaccine.
[0078] Non-limiting examples of viruses of the present invention,
e.g., oncolytic viruses, are provided in Table 3. While Table 3
includes suggested mechanisms or uses for the provided viruses,
methods and compositions of the present invention are not limited
by or to these mechanisms or uses.
TABLE-US-00003 TABLE 3 Immunostimulatory agents Modification(s)/
Strain Description Virus Clinical Trial; Indication; Route; Status;
Reference Oncorine (H101) E1B-55k- Adenovirus Phase 2; SCCHN;
intratumoral (IT); completed; Xu R H, Yuan Z Y, Guan Z Z, Cao Y,
Wang H Q, Hu X H, Feng J F, Zhang Y, Li F, Chen Z T, Wang J J,
Huang J J, Zhou Q H, Song S T. [Phase II clinical study of
intratumoral H101, an E1B deleted adenovirus, in combination with
chemotherapy in patients with cancer]. Ai Zheng. 2003 December;
22(12): 1307-10. Chinese. Oncorine (H101) E3- Adenovirus Phase 3;
SCCHN; IT; Completed; Xia Z J, Chang J H, Zhang L, Jiang W Q, Guan
Z Z, Liu J W, Zhang Y, Hu X H, Wu G H, Wang H Q, Chen Z C, Chen J
C, Zhou Q H, Lu J W, Fan Q X, Huang J J, Zheng X. [Phase III
randomized clinical trial of intratumoral injection of E1B
gene-deleted adenovirus (H101) combined with cisplatin-based
chemotherapy in treating squamous cell cancer of head and neck or
esophagus]. Ai Zheng. 2004 December; 23(12): 1666-70. Chinese.
Onyx-015 E1B-55k- Adenovirus Phase 1; Lung Mets; intravenous (IV);
Completed; Nemunaitis J, Cunningham C, Buchanan A, Blackburn A,
Edelman G, Maples P, Netto G, Tong A, Randlev B, Olson S, Kirn D.
Intravenous infusion of a replication-selective adenovirus
(ONYX-015) in cancer patients: safety, feasibility and biological
activity. Gene Ther. 2001 May; 8(10): 746-59. Onyx-015 E3B-
Adenovirus Phase 1; Glioma; Intracavity; Completed; Chiocca E A,
Abbed K M, Tatter S, Louis D N, Hochberg F H, Barker F, Kracher J,
Grossman S A, Fisher J D, Carson K, Rosenblum M, Mikkelsen T, Olson
J, Markert J, Rosenfeld S, Nabors L B, Brem S, Phuphanich S,
Freeman S, Kaplan R, Zwiebel J. A phase I open-label,
dose-escalation, multi-institutional trial of injection with an
E1B- Attenuated adenovirus, ONYX-015, into the peritumoral region
of recurrent malignant gliomas, in the adjuvant setting. Mol Ther.
2004 November; 10(5): 958-66. Phase 1; Ovarian cancer;
intraperitoneal (IP); Completed; Vasey P A, Shulman L N, Campos S,
Davis J, Gore M, Johnston S, Kirn D H, O'Neill V, Siddiqui N,
Seiden M V, Kaye S B. Phase I trial of intraperitoneal injection of
the E1B-55- kd-gene-deleted adenovirus ONYX-015 (dl1520) given on
days 1 through 5 every 3 weeks in patients with
recurrent/refractory epithelial ovarian cancer. J Clin Oncol. 2002
Mar. 15; 20(6): 1562-9. Phase 1; SCCHN; IT; Completed; Ganly I,
Kirn D, Eckhardt G, Rodriguez G I, Soutar D S, Otto R, Robertson A
G, Park O, Gulley M L, Heise C, Von Hoff D D, Kaye S B. A phase I
study of Onyx-015, an E1B attenuated adenovirus, administered
intratumorally to patients with recurrent head and neck cancer.
Clin Cancer Res. 2000 March; 6(3): 798-806. Erratum in: Clin Cancer
Res 2000 May; 6(5): 2120. Clin Cancer Res 2001 March; 7(3): 754.
Eckhardt S G [corrected to Eckhardt G]. Phase 1; Solid tumors; IV;
Completed; Nemunaitis J, Senzer N, Sarmiento S, Zhang Y A, Arzaga
R, Sands B, Maples P, Tong A W. A phase I trial of intravenous
infusion of ONYX-015 and enbrel in solid tumor patients. Cancer
Gene Ther. 2007 November; 14(11): 885-93. Epub 2007 Aug. 17. Phase
1; Sarcoma; IT; Completed; Galanis E, Okuno S H, Nascimento A G,
Lewis B D, Lee R A, Oliveira A M, Sloan J A, Atherton P, Edmonson J
H, Erlichman C, Randlev B, Wang Q, Freeman S, Rubin J. Phase I-II
trial of ONYX-015 in combination with MAP chemotherapy in patients
with advanced sarcomas. Gene Ther. 2005 March; 12(5): 437-45. Phase
1/2; PanCa; IT; Completed; Hecht J R, Bedford R, Abbruzzese J L,
Lahoti S, Reid T R, Soetikno R M, Kirn D H, Freeman S M. A phase
I/II trial of intratumoral endoscopic ultrasound injection of
ONYX-015 with intravenous gemcitabine in unresectable pancreatic
carcinoma. Clin Cancer Res. 2003 February; 9(2): 555-61. Phase 2;
CRC; IV; Completed; Hamid O, Varterasian M L, Wadler S, Hecht J R,
Benson A 3rd, Galanis E, Uprichard M, Omer C, Bycott P, Hackman R
C, Shields A F. Phase II trial of intravenous CI-1042 in patients
with metastatic colorectal cancer. J Clin Oncol. 2003 Apr. 15; 21
(8): 1498-504. Phase 2; Hepatobiliary; IT; Completed; Makower D,
Rozenblit A, Kaufman H, Edelman M, Lane M E, Zwiebel J, Haynes H,
Wadler S. Phase II clinical trial of intralesional administration
of the oncolytic adenovirus ONYX-015 in patients with hepatobiliary
tumors with correlative p53 studies. Clin Cancer Res. 2003
February; 9(2): 693-702. Phase 2; CRC, PanCa; intra-arteria (IA);
Completed; Reid T, Galanis E, Abbruzzese J, Sze D, Wein L M,
Andrews J, Randlev B, Heise C, Uprichard M, Hatfield M, Rome L,
Rubin J, Kirn D. Hepatic arterial infusion of a replication-
selective oncolytic adenovirus (dl1520): phase II viral,
immunologic, and clinical endpoints. Cancer Res. 2002 Nov. 1;
62(21): 6070-9. Phase 2; SCCHN; IT; Completed; 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. Phase
II trial of intratumoral administration of ONYX-015, a
replication-selective adenovirus, in patients with refractory head
and neck cancer. J Clin Oncol. 2001 Jan. 15; 19(2): 289-98. Phase
2; SCCHN; IT; Completed; Khuri FR, Nemunaitis J, Ganly I, Arseneau
J, Tannock I F, Romel L, Gore M, Ironside J, MacDougall R H, Heise
C, Randlev B, Gillenwater A M, Bruso P, Kaye S B, Hong W K, Kirn D
H. a controlled trial of intratumoral ONYX-015, a
selectively-replicating adenovirus, in combination with cisplatin
and 5-fluorouracil in patients with recurrent head and neck cancer.
Nat Med. 2000 August; 6(8): 879-85. Phase 2; CRC; IV; Completed;
Reid T R, Freeman S, Post L, McCormick F, Sze D Y. Effects of
Onyx-015 among metastatic colorectal cancer patients that have
failed prior treatment with 5-FU/leucovorin. Cancer Gene Ther. 2005
August; 12(8): 673-81. CG7060 PSA control Adenovirus Phase 1;
Prostate cancer; IT; Completed; DeWeese T L, van der Poel H, Li S,
Mikhak B, Drew R, Goemann M, Hamper U, DeJong R, Detorie N,
Rodriguez R, Hauik T, DeMarzo A M, Piantadosi S, Yu D C, Chen Y,
Henderson D R, Carducci M A, Nelson W G, Simons J W. A phase I
trial of CV706, a replication- competent, PSA selective oncolytic
adenovirus, for the treatment of locally recurrent prostate cancer
following radiation therapy. Cancer Res. 2001 Oct. 15; 61 (20):
7464-72. CG7870/CV787 Rat probasin- Adenovirus Phase 1/2; Prostate
cancer; IV; Completed; Small E J, Carducci M A, Burke J M, E1A
Rodriguez R, Fong L, van Ummersen L, Yu D C, Aimi J, Ando D,
Working P, Kirn D, Wilding G. A phase I trial of intravenous
CG7870, a replication- selective, prostate-specific
antigen-targeted oncolytic adenovirus, for the treatment of
hormone-refractory, metastatic prostate cancer. Mol Ther. 2006
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2006 Jan. 1; 12(1): 305-13. Telomelysin hTERT Adenovirus Phase 1;
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I study of telomerase-specific replication competent oncolytic
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24. Ad5-CD/TKrep CD/TK Adenovirus Phase 1; Prostate cancer; IT;
Completed; Freytag S O, Khil M, Stricker H, Peabody J, Menon M,
DePeralta-Venturina M, Nafziger D, Pegg J, Paielli D, Brown S,
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D G, Peabody J, DePeralta-Venturina M, Xia X, Brown S, Lu M, Kim J
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IT; Recruiting Phase 1/2; Glioma; IT; Recruiting Ad5-SSTR/TK- SSTR,
TK, RGD Adenovirus Phase 1; Ovarian cancer; IP; Active; Ramesh N,
Ge Y, Ennist D L, Zhu M, Mina RGD M, Ganesh S, Reddy P S, Yu D C.
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treatment of bladder cancer. Clin Cancer Res. 2006 Jan. 1; 12(1):
305-13. CGTG-102 Ad5/3, GM-CSF Adenovirus Phase 1/2; Solid tumors;
IT; Not open; Koski A, Kangasniemi L, Escutenaire S, Pesonen S,
Cerullo V, Diaconu I, Nokisalmi P, Raki M, Rajecki M, Guse K, Ranki
T, Oksanen M, Holm S L, Haavisto E, Karioja-Kallio A, Laasonen L,
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IT; Recruiting (CVA21) Phase 1; SCCHN; IT; Terminated Phase 1;
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James N D, Love C A, McNeish I, (OncoVEX) virus Medley L C, Michael
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(OncoVEX) virus Talimogene Us11 .uparw. Herpes Phase 1/2; SCCHN;
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laherparepvec simplex Hickey J, Bhide S A, Clarke P M, Renouf L C,
Thway K, Sibtain A, McNeish I A, (OncoVEX) virus Newbold K L,
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IT; Recruiting (Seprehvir) simplex Phase 1; SCCHN; IT; Completed;
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Phase 1; Breast cancer; IT; Completed; Kimata H, Imai T, Kikumori
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Oncol. 2006 August; 13(8): 1078-84. Epub 2006 Jul. 24. Phase 1;
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simplex virus HF10 in recurrent head and neck squamous cell
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Herpes Phase 1; CRC liver mets; IA; Completed; Fong Y, Kim T,
Bhargava A, simplex Schwartz L, Brown K, Brody L, Covey A, Karrasch
M, Getrajdman G, virus Mescheder A, Jarnagin W, Kemeny N. A herpes
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18. MV-CEA CEA Measles Phase 1; Ovarian cancer; IP; Completed;
Galanis E, Hartmann L C, Cliby W A, virus Long H J, Peethambaram P
P, Barrette B A, Kaur J S, Haluska P J Jr, Aderca I, (Edmonston)
Zollman P J, Sloan J A, Keeney G, Atherton P J, Podratz K C, Dowdy
S C, Stanhope C R, Wilson T O, Federspiel M J, Peng K W, Russell S
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Phase 1; Glioma; IT; Recruiting MV-NIS NIS Measles Phase 1;
Myeloma; IV; Recruiting virus Phase 1; Ovarian cancer; IP;
Recruiting (Edmonston) Phase 1; Mesothelioma; IP; Recruiting Phase
1; SCCHN; IT; Not open NDV-HUJ -- Newcastle Phase 1/2; Glioma; IV;
Completed; Freeman A I, Zakay-Rones Z, Gomori J M, disease Linetsky
E, Rasooly L, Greenbaum E, Rozenman-Yair S, Panet A, Libson E,
virus Irving C S, Galun E, Siegal T. Phase I/II trial of
intravenous NDV-HUJ oncolytic virus in recurrent glioblastoma
multiforme. Mol Ther. 2006 January; 13(1): 221-8. Epub 2005 Oct.
28; Pecora A L, Rizvi N, Cohen G I, Meropol N J, Sterman D,
Marshall J L, Goldberg S, Gross P, O'Neil J D, Groene W S, Roberts
M S, Rabin H, Bamat M K, Lorence R M. Phase I trial of intravenous
administration of PV701, an oncolytic virus, in patients with
advanced solid cancers. J Clin Oncol. 2002 May 1; 20(9): 2251-66.
PV701 -- Newcastle Phase 1; Solid tumors; IV; Completed; Laurie S
A, Bell J C, Atkins H L, Roach J, disease Bamat M K, O'Neil J D,
Roberts M S, Groene W S, Lorence R M. A phase 1 virus clinical
study of intravenous administration of PV701, an oncolytic virus,
using two-step desensitization. Clin Cancer Res. 2006 Apr. 15;
12(8): 2555-62. MTH-68/H -- Newcastle Phase 2; Solid tumors;
Inhalation; Completed; Csatary L K, Eckhardt S, disease Bukosza I,
Czegledi F, Fenyvesi C, Gergely P, Bodey B, Csatary C M. virus
Attenuated veterinary virus vaccine for the treatment of cancer.
Cancer Detect Prev. 1993; 17(6): 619-27. H-1PV -- Parvovirus Phase
1/2; Glioma; IT/IV; Recruiting; Geletneky K, Kiprianova I, Ayache
A, Koch R, Herrero Y Calle M, Deleu L, Sommer C, Thomas N,
Rommelaere J, Schlehofer J R. Regression of advanced rat and human
gliomas by local or systemic treatment with oncolytic parvovirus
H-1 in rat models. Neuro Oncol. 2010 August; 12(8): 804-14. doi:
10.1093/neuonc/noq023. Epub 2010 Mar. 18. PVS-RIPO IRES Poliovirus
Phase 1; Glioma; IT; Recruiting; Goetz C, Gromeier M. Preparing an
oncolytic (Sabin) poliovirus recombinant for clinical application
against glioblastoma multiforme. Cytokine Growth Factor Rev. 2010
April-June; 21(2-3): 197-203. doi: 10.1016/j.cytogfr.2010.02.005.
Epub 2010 Mar. 17. Review. Reolysin -- Reovirus Phase 1/2; Glioma;
IT; Completed; Forsyth P, Roldan G, George D, Wallace C, (Dearing)
Palmer C A, Morris D, Cairncross G, Matthews M V, Markert J,
Gillespie Y, Coffey M, Thompson B, Hamilton M. A phase I trial of
intratumoral administration of reovirus in patients with
histologically confirmed recurrent malignant gliomas. Mol Ther.
2008 March; 16(3): 627-32. doi: 10.1038/sj.mt.6300403. Epub 2008
Feb. 5. Phase 1; Peritoneal cancer; IP; Recruiting Phase 1; Solid
tumors; IV; Completed; Vidal L, Pandha H S, Yap T A, White C L,
Twigger K, Vile R G, Melcher A, Coffey M, Harrington K J, DeBono J
S. A phase I study of intravenous oncolytic reovirus type 3 Dearing
in patients with advanced cancer. Clin Cancer Res. 2008 Nov. 1;
14(21): 127-37. doi: 10.1158/1078-0432.CCR-08-0524. Phase 1; Solid
tumors; IV; Recruiting Phase 1; CRC; IV; Recruiting Phase 2;
Sarcoma; IV; Completed Phase 2; Melanoma; IV; Suspended Phase 2;
Ovarian, peritoneal cancer; IV; Recruiting Phase 2; Pancreatic
cancer; IV; Recruiting Phase 2; SCCHN; IV; Not recruiting Phase 2;
Melanoma; IV; Recruiting Phase 2; Pancreatic cancer; IV; Recruiting
Phase 2; Lung cancer; IV; Recruiting Phase 3; SCCHN; IV; Recruiting
NTX-010 Seneca Phase 2; Small cell lung cancer; IV; Recruiting;
PMID: 17971529 Valley virus Toca 511 CD Retrovirus Phase 1/2;
Glioma; IT; Recruiting; Tai C K, Wang W
J, Chen T C, Kasahara N. Single-shot, multicycle suicide gene
therapy by replication-competent retrovirus vectors achieves
long-term survival benefit in experimental glioma. Mol Ther. 2005
November; 12(5): 842-51. JX-594 GM-CSF Vaccinia Phase 1; CRC; IV;
Recruiting (Wyeth strain) JX-594 TK(-) Vaccinia Phase 1; Solid
tumors; IV; Completed (Wyeth Phase 1; HCC; IT; Completed; Park B H,
Hwang T, Liu T C, Sze D Y, Kim J S, strain) Kwon H C, Oh S Y, Han S
Y, Yoon J H, Hong S H, Moon A, Speth K, Park C, Ahn Y J, Daneshmand
M, Rhee B G, Pinedo H M, Bell J C, Kirn D H. Use of a targeted
oncolytic poxvirus, JX-594, in patients with refractory primary or
metastatic liver cancer: a phase I trial. Lancet Oncol. 2008 June;
9(6): 533-42. doi: 10.1016/S1470-2045(08)70107-4. Epub 2008 May 19.
Erratum in: Lancet Oncol. 2008 July; 9(7): 613. Phase 1; Pediatric
solid tumors; IT; Recruiting Phase 1; Melanoma; IT; Completed;
Hwang T H, Moon A, Burke J, Ribas A, Stephenson J, Breitbach C J,
Daneshmand M, De Silva N, Parato K, Diallo J S, Lee Y S, Liu T C,
Bell J C, Kirn D H. A mechanistic proof-of-concept clinical trial
with JX-594, a targeted multi-mechanistic oncolytic poxvirus, in
patients with metastatic melanoma. Mol Ther. 2011 October; 19(10):
1913-22. doi: 10.1038/mt.2011.132. Epub 2011 Jul. 19. Phase 1/2;
Melanoma; IT; Completed; Mastrangelo M J, Maguire H C Jr, Eisenlohr
L C, Laughlin C E, Monken C E, McCue P A, Kovatich A J, Lattime E
C. Intratumoral recombinant GM-CSF-encoding virus as gene therapy
in patients with cutaneous melanoma. Cancer Gene Ther. 1999
September-October; 6(5): 409-22. Phase 2; HCC; IT; Not recruiting,
analyzing data Phase 2B; HCC; IV; Recruiting Phase 1/2; CRC; IV/IT;
Recruiting Phase 2; CRC; IT; Not yet recruiting vvDD-CDSR TK-,
VGF-, Vaccinia Phase 1; Solid tumors; IT/IV; Recruiting; McCart J
A, Mehta N, Scollard D, LacZ, CD, (Western Reilly R M, Carrasquillo
J A, Tang N, Deng H, Miller M, Xu H, Libutti S K, Somatostatin R
Reserve) Alexander H R, Bartlett D L. Oncolytic vaccinia virus
expressing the human somatostatin receptor SSTR2: molecular imaging
after systemic delivery using 111In-pentetreotide. Mol Ther. 2004
September; 10(3): 553-61. GL-ONC1 Renilla Vaccinia Phase 1; Solid
tumors; IV; Recruiting, Gentschev I, Muller M, Adelfinger M,
luciferase Weibel S, Grummt F, Zimmermann M, Bitzer M, Heisig M,
Zhang Q, Yu Y A, Chen N G, Stritzker J, Lauer U M, Szalay A A.
Efficient colonization and therapy of human hepatocellular
carcinoma (HCC) using the oncolytic vaccinia virus strain GLV-1h68.
PLoS One. 2011; 6(7): e22069. doi: 10.1371/journal.pone.0022069.
Epub 2011 Jul. 11. (GLV-h68) GFP, .beta.-gal Vaccinia Phase 1/2;
Peritoneal carcinomatosis; IP; Recruiting Lister
.beta.-glucoronidase Vaccinia Phase 1/2; SCCHN; IV; Recruiting
VSV-hIFN.beta. IFN-.beta. Vesicular Phase 1; HCC; IT; Recruiting
stomatitis virus (Indiana) DNX-2401 DNAtrix Adenovirus See, e.g.,
Molecular Therapy 21(10): 1814-1818, 2013 and Journal of Vascular
and Interventional Radiology 24(8): 1115-1122, 2013 Toca511 Tocagen
Lentivirus See, e.g., Molecular Therapy 21 (10): 1814-1818, 2013
and Journal of Vascular and Interventional Radiology 24(8):
1115-1122, 2013 HSV T-VEC HSV See, e.g., Molecular Therapy 21(10):
1814-1818, 2013 and Journal of Vascular and Interventional
Radiology 24(8): 1115-1122, 2013 H-1 Parvovirus See, e.g.,
Molecular Therapy 21 (10): 1814-1818, 2013 and ParvOryx Journal of
Vascular and Interventional Radiology 24(8): 1115-1122, 2013
VACV-TRAIL (see work of Vaccinia See, e.g., Molecular Therapy 21
(10): 1814-1818, 2013 and Karolina Autio virus Journal of Vascular
and Interventional Radiology 24(8): and Suvi 1115-1122, 2013
Parvainen, Helsinki) VACV-CD40L (see work of Vaccinia See, e.g.,
Molecular Therapy 21 (10): 1814-1818, 2013 and Karolina Autio virus
Journal of Vascular and Interventional Radiology 24(8): and Suvi
1115-1122, 2013 Parvainen, Helsinki) Maraba (see work of Dave
Rhabdovirus Preclinical/Clinical Candidate Stojdl, and John Bell)
Maraba- (see work of Dave Rhabdovirus MG1 Stojdl, and John Bell)
Maraba (see work of Dave Rhabdovirus Preclinical/Clinical Candidate
MG1- Stojdl, Brian hMAGE-A3 Litchy and John Bell) Sindbis
Preclinical/Clinical Candidate virus Coxsackievirus
Preclinical/Clinical Candidate A21 MYXV Poxvirus
Preclinical/Clinical Candidate Chan W M, Rahman M M, McFadden G.
Oncolytic myxoma virus: the path to clinic. Vaccine. 2013 Sep. 6;
31(39): 4252-8. doi: 10.1016/j.vaccine.2013.05.056. Epub 2013 May
29. WT VSV The parental rWT Recombinant VSV used as oncolytic agent
against cancer(see, e.g., see, e.g., (`Rose lab`) VSV for most J
Gen Virol/93(12): 2529-2545, 2012; Lawson N D, Stillman E A, Whitt
M A, VSV-based OVs. Rose J K. Recombinant vesicular stomatitis
viruses from DNA. Proc Natl Acad The L gene and Sci USA. 1995 May
9; 92(10): 4477-81. Erratum in: Proc Natl Acad Sci USA the
N-terminal 49 1995 Sep. 12; 92(19): 9009.) residues of the N gene
are derived from the Mudd- Summers strain, the rest is from the San
Juan strain (both Indiana serotype) VSV-WT-XN2 Derivative of
Recombinant VSV used as oncolytic agent against cancer (see, e.g.,
Hastie E, (or XN1) rWT VSV (`Rose Grdzelishvili V Z. Vesicular
stomatitis virus as a flexible platform for oncolytic lab`).
Generated virotherapy against cancer. J Gen Virol. 2012 December;
93(Pt 12): 2529-45. doi: using pVSV-XN2 (or 10.1099/vir.0.046672-0.
Epub 2012 Oct. 10.; Schnell M J, Buonocore L, pVSV-XN1), a
Kretzschmar E, Johnson E, Rose J K. Foreign glycoproteins expressed
from full-length VSV recombinant vesicular stomatitis viruses are
incorporated efficiently into virus plasmid containing particles.
Proc Natl Acad Sci USA. 1996 Oct. 15; 93(21): 11359-65.) uniqueXhol
and Nhel sites flanked by VSV transcription start and stop signals
between G and L genes. pVSV-XN2 (or pVSV-XN1) is commonly used to
generate recombinant VSVs encoding an extra gene WT VSV Alternative
rWT Recombinant VSV used as oncolytic agent against cancer (see,
e.g., Hastie E, (`Wertz lab`) VSV. The N, P, Grdzelishvili V Z.
Vesicular stomatitis virus as a flexible platform for oncolytic M
and L genes virotherapy against cancer. J Gen Virol. 2012 December;
93(Pt 12): 2529-45. doi: originate from 10.1099/vir.0.046672-0.
Epub 2012 Oct. 10; Whelan S P, Ball L A, Barr J N, the San Juan
Wertz G T. Efficient recovery of infectious vesicular stomatitis
virus entirely from strain; G gene cDNA clones. Proc Natl Acad Sci
USA. 1995 Aug. 29; 92(18): 8388-92.) from the Orsay strain (both
Indiana serotype). Rarely used in OV studies VSV-WT-GFP, WT VSV
encoding Recombinant VSV used as oncolytic agent against cancer
(see, e.g., Hastie E, -RFP, -Luc, reporter genes Grdzelishvili V Z.
Vesicular stomatitis virus as a flexible platform for oncolytic
-LacZ (between G and virotherapy against cancer. J Gen Virol. 2012
December; 93(Pt 12): 2529-45. doi: L) to track
10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Fernadez et al.,
"Genetically virus infection. Engineered Vesicular Stomatitis Virus
in Gene Therapy: Application for Based on pVSV- Treatment of
Malignant Disease", J Virol 76: 895-904 (2002); Lan Wu, Tian-gui
XN2. Toxicity Huang, Marcia Meseck, Jennifer Altomonte, Oliver
Ebert, Katsunori Shinozaki, similar to Adolfo Garcia-Sastre, John
Fallon, John Mandeli, and Savio L. C. Woo. Human VSV-WT Gene
Therapy. June 2008, 19(6): 635-647) VSV-G/GFP GFP sequence fused
Recombinant VSV used as oncolytic agent against cancer (see, e.g.,
Hastie E, to VSV G gene is Grdzelishvili V Z. Vesicular stomatitis
virus as a flexible platform for oncolytic inserted between
virotherapy against cancer. J Gen Virol. 2012 December; 93(Pt 12):
2529-45. doi: the WT G and L 10.1099/vir.0.046672-0. Epub 2012 Oct.
10; Dalton, K. P. & Rose, J. K. (2001). genes (in addition
Vesicular stomatitis virus glycoprotein containing the entire green
fluorescent to WT G). Toxicity protein on its cytoplasmic domain is
incorporated efficiently into virus particles. similar to that
Virology 279, 414-421.) of VSV-WT VSV-rp30 Derivative of
Recombinant VSV used as oncolytic agent against cancer (see, e.g.,
Hastie E, VSV-G/GFP. Grdzelishvili V Z. Vesicular stomatitis virus
as a flexible platform for oncolytic Generated by virotherapy
against cancer. J Gen Virol. 2012 December; 93(Pt 12): 2529-45.
doi: positive selection 10.1099/vir.0.046672-0. Epub 2012 Oct. 10;
Wollmann, G., Tattersail, P. & van on glioblastoma den Pol, A.
N. (2005). Targeting human glioblastoma cells: comparison of nine
cells and viruses with oncolytic potential. J Virol 79, 6005-6022.)
contains two silent mutations and two missense mutations, one in P
and one in L. `rp30` indicates 30 repeated passages VSV-p1-GFP, VSV
expressing Recombinant VSV used as oncolytic agent against cancer
(see, e.g., Hastie E, VSV-p1-RFP GFP or red Grdzelishvili V Z.
Vesicular stomatitis virus as a flexible platform for oncolytic
fluorescent virotherapy against cancer. J Gen Virol. 2012 December;
93(Pt 12): 2529-45. doi: protein (RFP or 10.1099/vir.0.046672-0.
Epub 2012 Oct. 10; Wollmann, G., Rogulin, V., Simon, dsRed)
reporter I., Rose, J. K. & van den Pol, A. N. (2010). Some
attenuated variants of gene at position vesicular stomatitis virus
show enhanced oncolytic activity against human 1. Attenuated
glioblastoma cells relative to normal brain cells. J Virol 84,
1563-1573.) because all VSV genes are moved downward, to positions
2-6. Safe and still effective as an OV VSV-dG-GFP Similar to
Recombinant VSV used as oncolytic agent against cancer (see, e.g.,
Hastie E, (or RFP) VSV-p1-GFP or Grdzelishvili V Z. Vesicular
stomatitis virus as a flexible platform for oncolytic
(replication- VSV-p1-RFP virotherapy against cancer. J Gen Virol.
2012 December; 93(Pt 12): 2529-45. doi: defective) described above,
10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Wollmann, G., Rogulin,
V., Simon, but with the G I., Rose, J. K. & van den Pol, A. N.
(2010). Some attenuated variants of gene deleted. vesicular
stomatitis virus show enhanced oncolytic activity against human
Cannot generate glioblastoma cells relative to normal brain cells.
J Virol 84, 1563-1573.) a second round of infection. Poor ability
to kill tumor cells VSV-.DELTA.P, Each virus cannot Recombinant VSV
used as oncolytic agent against cancer (see, e.g., Hastie E,
-.DELTA.L, -.DELTA.G replicate alone Grdzelishvili V Z. Vesicular
stomatitis virus as a flexible platform for oncolytic (semi-
because of one virotherapy against cancer. J Gen Virol. 2012
December; 93(Pt 12): 2529-45. doi: replication- VSV gene deleted,
10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Muik, A., Dold, C.,
Gei.beta., Y., Volk, competent) but when viruses A., Werbizki, M.,
Dietrich, U. & von Laer, D. (2012). Semireplication-competent
co-infect, they vesicular stomatitis virus as a novel platform for
oncolytic virotherapy. J Mol show good Med (Berl) 90, 959-970.)
replication, safety and oncolysis (especially the combination of
VSV.DELTA.G/VSV.DELTA.L). VSV.DELTA.P and VSV.DELTA.L contain dsRed
in place of the corresponding viral gene. VSV.DELTA.G contains GFP
gene in place of G VSV-M51R M mutant; the Recombinant VSV used as
oncolytic agent against cancer (see, e.g., Hastie E, M51R mutation
was Grdzelishvili V Z. Vesicular stomatitis virus as a flexible
platform for oncolytic introduced into M virotherapy against
cancer. J Gen Virol. 2012 December; 93(Pt 12): 2529-45. doi:
10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Kopecky, S. A.,
Willingham, M. C. & Lyles, D. S. (2001). Matrix protein and
another viral component contribute to induction of apoptosis in
cells infected with vesicular stomatitis virus. J Virol 75,
12169-12181.) VSV-.DELTA.M51, M mutant; the Recombinant VSV used as
oncolytic agent against cancer (see, e.g., Hastie E,
VSV-.DELTA.M51- .DELTA.M51 mutation Grdzelishvili V Z. Vesicular
stomatitis virus as a flexible platform for oncolytic GFP, -RFP,
was introduced virotherapy against cancer. J Gen Virol. 2012
December; 93(Pt 12): 2529-45. doi: -FLuc, -Luc, into M. In
10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Stojdl, D. F., Lichty,
B. D., -LacZ addition, some tenOever, B. R., Paterson, J. M.,
Power, A. T., Knowles, S., Marius, R., recombinants Reynard, J.,
Poliquin, L. & other authors (2003). VSV strains with defects
in encode a their ability to shutdown innate immunity are potent
systemic anti-cancer reporter gene agents. Cancer Cell 4, 263-275.;
Power, A. T. & Bell, J. C. (2007). Cell-based between the G
delivery of oncolytic viruses: a new strategic alliance for a
biological strike and L against cancer. Mol Ther 15, 660-665.; Wu,
L., Huang, T. G., Meseck, M., Altomonte, J., Ebert, O., Shinozaki,
K., Garci{acute over ( )}a-Sastre, A., Fallon, J., Mandeli, J.
& Woo, S. L. (2008). rVSV(MD51)-M3 is an effective and safe
oncolytic virus for cancer therapy. Hum Gene Ther 19, 635-647.)
VSV-*Mmut M mutant; VSV Recombinant VSV used as oncolytic agent
against cancer (see, e.g., Hastie E, with a single Grdzelishvili V
Z. Vesicular stomatitis virus as a flexible platform for oncolytic
mutation or virotherapy against cancer. J Gen Virol. 2012 December;
93(Pt 12): 2529-45. doi: combination 10.1099/vir.0.046672-0. Epub
2012 Oct. 10; Hoffmann, M., Wu, Y. J., Gerber, of mutations at M.,
Berger-Rentsch, M., Heimrich, B., Schwemmle, M. & Zimmer, G.
(2010). the following M Fusion-active glycoprotein G mediates the
cytotoxicity of vesicular stomatitis positions: M33A, virus M
mutants lacking host shut-off activity. J Gen Virol 91, 2782-2793.)
M51R, V221F and S226R VSV-M6PY > M mutant; the Recombinant VSV
used as oncolytic agent against cancer (see, e.g., Hastie E,
A4-R34E M51R mutation Grdzelishvili V Z. Vesicular stomatitis virus
as a flexible platform for oncolytic and other was introduced
virotherapy against cancer. J Gen Virol. 2012 December; 93(Pt 12):
2529-45. doi: M mutants into the M gene, 10.1099/vir.0.046672-0.
Epub 2012 Oct. 10; Irie, T., Carnero, E., Okumura, A., and, in
addition, Garci{acute over ( )}a-Sastre, A. & Harty, R. N.
(2007). Modifications of the PSAP region of the mutations the
matrix protein lead to attenuation of vesicular stomatitis virus in
vitro and in in the PSAP motif vivo. J Gen Virol 88, 2559-2567.)
(residues 37- 40) of M VSV-M(mut) M mutant; VSV Recombinant VSV
used as oncolytic agent against cancer (see, e.g., Hastie E, M
residues 52- Grdzelishvili V Z. Vesicular stomatitis virus as a
flexible platform for oncolytic 54 are mutated virotherapy against
cancer. J Gen Virol. 2012 December; 93(Pt 12): 2529-45. doi: from
DTY to AAA. 10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Heiber, J.
F. & Barber, G. N. M(mut) cannot (2011). Vesicular stomatitis
virus expressing tumor suppressor p53 is a highly block nuclear
attenuated, potent oncolytic agent. J Virol 85, 10440-10450.) mRNA
export VSV-G5, -G5R, G mutant; Recombinant VSV used as oncolytic
agent against cancer (see, e.g., Hastie E, -G6, -G6R VSV-expressing
Grdzelishvili V Z. Vesicular stomatitis virus as a flexible
platform for oncolytic mutant G with virotherapy against cancer. J
Gen Virol. 2012 December; 93(Pt 12): 2529-45. doi: amino acid
10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Janelle, V., Brassard,
F., Lapierre, substitutions at P., Lamarre, A. & Poliquin, L.
(2011). Mutations in the glycoprotein of vesicular various
positions stomatitis virus affect cytopathogenicity: potential for
oncolytic virotherapy. J (between residues Virol 85, 6513-6520.)
100 and 471). Triggers type I IFN secretion as the M51R, but
inhibits cellular transcription and host protein translation like
WT VSV-CT1 G mutant; the Recombinant VSV used as oncolytic agent
against cancer (see, e.g., Hastie E, cytoplasmic tail of
Grdzelishvili V Z. Vesicular stomatitis virus as a flexible
platform for oncolytic the G protein was virotherapy against
cancer. J Gen Virol. 2012 December; 93(Pt 12): 2529-45. doi:
truncated from 29 10.1099/vir.0.046672-0. Epub 2012 Oct. 10;
Ozduman, K., Wollmann, G., to 1 aa. Decreased Ahmadi, S. A. &
van den Pol, A. N. (2009). Peripheral immunization blocks
neuropathology, but lethal actions of vesicular stomatitis virus
within the brain. J Virol 83, 11540- marginal oncolytic 11549.;
Wollmann, G., Rogulin, V., Simon, I., Rose, J. K. & van den
Pol, A. N. efficacy (2010). Some attenuated variants of vesicular
stomatitis virus show enhanced oncolytic activity against human
glioblastoma cells relative to normal brain cells. J Virol 84,
1563-1573.) VSV-CT9- G mutant; the Recombinant VSV used as
oncolytic agent against cancer (see, e.g., Hastie E, M51
cytoplasmic tail Grdzelishvili V Z. Vesicular stomatitis virus as a
flexible platform for oncolytic of VSV-G was virotherapy against
cancer. J Gen Virol. 2012 December; 93(Pt 12): 2529-45. doi:
reduced from 29 10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Ozduman,
K., Wollmann, G., to 9 aa, also has Ahmadi, S. A. & van den
Pol, A. N. (2009). Peripheral immunization blocks .DELTA.M51
mutation. lethal actions of vesicular stomatitis virus within the
brain. J Virol 83, 11540- Attenuated 11549.; Wollmann, G., Rogulin,
V., Simon, I., Rose, J. K. & van den Pol, A. N. neurotoxicity
and (2010). Some attenuated variants of vesicular stomatitis virus
show enhanced good OV abilities oncolytic activity against human
glioblastoma cells relative to normal brain cells. J Virol 84,
1563-1573.) VSV- Foreign Recombinant VSV used as oncolytic agent
against cancer (see, e.g., Hastie E, DV/F(L289A) glycoprotein; VSV
Grdzelishvili V Z. Vesicular stomatitis virus as a flexible
platform for oncolytic (same as expressing the virotherapy against
cancer. J Gen Virol. 2012 December; 93(Pt 12): 2529-45. doi:
rVSV-F) NDV fusion 10.1099/vir.0.046672-0. Epub 2012 Oct. 10;
Ebert, O., Shinozaki, K., Kournioti, protein gene C., Park, M. S.,
Garci{acute over ( )}a-Sastre, A. & Woo, S. L. (2004). Syncytia
induction between G and L. enhances the oncolytic potential of
vesicular stomatitis virus in virotherapy for The L289A mutation
cancer. Cancer Res 64, 3265-3270.) in this protein allows it to
induce syncytia alone (without NDV HN protein) VSV-S-GP Foreign
Recombinant VSV used as oncolytic agent against cancer (see, e.g.,
Hastie E, glycoprotein; Grdzelishvili V Z. Vesicular stomatitis
virus as a flexible platform for oncolytic VSV with the virotherapy
against cancer. J Gen Virol. 2012 December; 93(Pt 12): 2529-45.
doi: native G gene 10.1099/vir.0.046672-0. Epub 2012 Oct. 10;
Bergman, I., Griffin, J. A., Gao, Y. deleted and &
Whitaker-Dowling, P. (2007). Treatment of implanted mammary tumors
with replaced with a recombinant vesicular stomatitis virus
targeted to Her2/neu. Int J Cancer 121, modified 425-430.)
glycoprotein protein (GP) from Sindbis virus (SV). Also expressing
mouse GM-CSF and GFP (between SV GP and VSV L). The modified GP
protein recognizes the Her2 receptor, which is overexpressed on
many breast cancer cells VSV-.DELTA.G- Foreign Recombinant VSV used
as oncolytic agent against cancer (see, e.g., Hastie E, SV5-F
glycoprotein; VSV Grdzelishvili V Z. Vesicular stomatitis virus as
a flexible platform for oncolytic G gene is replaced virotherapy
against cancer. J Gen Virol. 2012 December; 93(Pt 12): 2529-45.
doi: with the fusogenic 10.1099/vir.0.046672-0. Epub 2012 Oct. 10;
Chang, G., Xu, S., Watanabe, M., simian parainfluenza Jayakar, H.
R., Whitt, M. A. & Gingrich, J. R. (2010). Enhanced oncolytic
virus 5 fusion activity of vesicular stomatitis virus encoding
SV5-F protein against prostate protein (SV5-F) cancer. J Urol 183,
1611-1618.) gene VSV-FAST, Foreign Recombinant VSV used as
oncolytic agent against cancer (see, e.g., Hastie E,
VSV-(.DELTA.M51)- glycoprotein; VSV Grdzelishvili V Z. Vesicular
stomatitis virus as a flexible platform for oncolytic FAST or
VSV-M.DELTA.51 virotherapy against cancer. J Gen Virol. 2012
December; 93(Pt 12): 2529-45. doi: expressing the p14
10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Brown, C. W.,
Stephenson, K. B.,
FAST protein of Hanson, S., Kucharczyk, M., Duncan, R., Bell, J. C.
& Lichty, B. D. (2009). The reptilian reovirus p14 FAST protein
of reptilian reovirus increases vesicular stomatitis virus (between
VSV G and neuropathogenesis. J Virol 83, 552-561.) L) VSV-LCMV-GP
Foreign Recombinant VSV used as oncolytic agent against cancer
(see, e.g., Hastie E, (replication- glycoprotein; VSV Grdzelishvili
V Z. Vesicular stomatitis virus as a flexible platform for
oncolytic defective) lacking the G gene virotherapy against cancer.
J Gen Virol. 2012 December; 93(Pt 12): 2529-45. doi: was
pseudotyped with 10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Muik,
A., Kneiske, I., Werbizki, M., the non-neurotropic Wilflingseder,
D., Giroglou, T., Ebert, O., Kraft, A., Dietrich, U., Zimmer, G.
& glycoprotein of other authors (2011). Pseudotyping vesicular
stomatitis virus with lymphocytic LMCV choriomeningitis virus
glycoproteins enhances infectivity for glioma cells and minimizes
neurotropism. J Virol 85, 5679-5684.) VSV-H/F, Foreign Recombinant
VSV used as oncolytic agent against cancer (see, e.g., Hastie E,
-.alpha.EGFR, -.alpha.FR, glycoprotein; VSV Grdzelishvili VZ.
Vesicular stomatitis virus as a flexible platform for oncolytic
-.alpha.PSMA lacking the G gene virotherapy against cancer. J Gen
Virol. 2012 December; 93(Pt 12): 2529-45. doi: (replication- was
pseudotyped 10.1099/vir.0.046672-0. Epub 2012 Oct. 10;
Ayala-Breton, C., Barber, G. N., defective) with the MV F and
Russell, S. J. & Peng, K. W. (2012). Retargeting vesicular
stomatitis virus using H displaying measles virus envelope
glycoproteins. Hum Gene Ther 23, 484-491.) single-chain antibodies
(scFv) specific for epidermal growth factor receptor, folate
receptor, or prostate membrane-specific antigen. Retargeted VSV to
cells that expressed the targeted receptor VSV- let- microRNA
target; Recombinant VSV used as oncolytic agent against cancer
(see, e.g., Hastie E, 7wt the let-7 Grdzelishvili V Z. Vesicular
stomatitis virus as a flexible platform for oncolytic microRNA
targets virotherapy against cancer. J Gen Virol. 2012 December;
93(Pt 12): 2529-45. doi: are inserted into 10.1099/vir.0.046672-0.
Epub 2012 Oct. 10; Edge, R. E., Falls, T. J., Brown, C. the 3'-UTR
of W., Lichty, B. D., Atkins, H. & Bell, J. C. (2008). A let-7
microRNA-sensitive VSV M vesicular stomatitis virus demonstrates
tumor-specific replication. Mol Ther 16, 1437-1443.) VSV-124,
microRNA target; Recombinant VSV used as oncolytic agent against
cancer (see, e.g., Hastie E, -125, -128, VSV recombinants
Grdzelishvili V Z. Vesicular stomatitis virus as a flexible
platform for oncolytic -134 (M or with neuron-specific virotherapy
against cancer. J Gen Virol. 2012 December; 93(Pt 12): 2529-45.
doi: L mRNA) microRNA (miR-124, 10.1099/vir.0.046672-0. Epub 2012
Oct. 10; Kelly, E. J., Nace, R., Barber, G. N. 125, 128 or 134)
& Russell, S. J. (2010). Attenuation of vesicular stomatitis
virus encephalitis targets inserted through microRNA targeting. J
Virol 84, 1550-1562.) in the 3'-UTR of VSV M or L mRNA VSV-mp53,
Cancer suppressor; Recombinant VSV used as oncolytic agent against
cancer (see, e.g., Hastie E, VSV- M(mut)- VSV [WT or Grdzelishvili
V Z. Vesicular stomatitis virus as a flexible platform for
oncolytic mp53 M(mut)] virotherapy against cancer. J Gen Virol.
2012 December; 93(Pt 12): 2529-45. doi: expressing the
10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Heiber, J. F. &
Barber, G. N. murine p53 gene. (2011). Vesicular stomatitis virus
expressing tumor suppressor p53 is a highly M(mut) has attenuated,
potent oncolytic agent. J Virol 85, 10440-10450.) residues 52-54 of
the M protein changed from DTY to AAA VSV- Suicide gene;
Recombinant VSV used as oncolytic agent against cancer (see, e.g.,
Hastie E, C:U VSV expressing Grdzelishvili V Z. Vesicular
stomatitis virus as a flexible platform for oncolytic E. coli
CD/UPRT, virotherapy against cancer. J Gen Virol. 2012 December;
93(Pt 12): 2529-45. doi: catalysing the 10.1099/vir.0.046672-0.
Epub 2012 Oct. 10; Porosnicu, M., Mian, A. & Barber,
modification of G. N. (2003). The oncolytic effect of recombinant
vesicular stomatitis virus is 5-fluorocytosine enhanced by
expression of the fusion cytosine deaminase/uracil into
phosphoribosyltransferase suicide gene. Cancer Res 63, 8366-8376.)
chemotherapeutic 5-FU VSV-C Suicide gene; Recombinant VSV used as
oncolytic agent against cancer (see, e.g., Hastie E, VSV-M.DELTA.51
Grdzelishvili V Z. Vesicular stomatitis virus as a flexible
platform for oncolytic expressing virotherapy against cancer. J Gen
Virol. 2012 December; 93(Pt 12): 2529-45. doi: CD/UPRT
10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Leveille, S., Samuel,
S., Goulet, M. L. & Hiscott, J. (2011). Enhancing VSV oncolytic
activity with an improved cytosine deaminase suicide gene strategy.
Cancer Gene Ther 18, 435-443.) VSV- Suicide gene; Recombinant VSV
used as oncolytic agent against cancer (see, e.g., Hastie E,
(M.DELTA.51)- VSV-M.DELTA.51 Grdzelishvili V Z. Vesicular
stomatitis virus as a flexible platform for oncolytic NIS
expressing the virotherapy against cancer. J Gen Virol. 2012
December; 93(Pt 12): 2529-45. doi: human NIS
10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Goel, A., Carlson, S.
K., Classic, K. gene (for L., Greiner, S., Naik, S., Power, A. T.,
Bell, J. C. & Russell, S. J. (2007). `radiovirotherapy`
Radioiodide imaging and radiovirotherapy of multiple myeloma using
with 131I) VSV(D51)-NIS, an attenuated vesicular stomatitis virus
encoding the sodium iodide symporter gene. Blood 110, 2342-2350.)
VSV- TK Suicide gene; Recombinant VSV used as oncolytic agent
against cancer (see, e.g., Hastie E, VSV expressing Grdzelishvili V
Z. Vesicular stomatitis virus as a flexible platform for oncolytic
TK; can improve virotherapy against cancer. J Gen Virol. 2012
December; 93(Pt 12): 2529-45. doi: oncolysis if
10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Fernandez, M.,
Porosnicu, M., used with non- Markovic, D. & Barber, G. N.
(2002). Genetically engineered vesicular toxic prodrug stomatitis
virus in gene therapy: application for treatment of malignant
disease. ganciclovir J Virol 76, 895-904.) VSV Immunomodulation;
Recombinant VSV used as oncolytic agent against cancer (see, e.g.,
Hastie E, -mIFN.beta., VSV expressing the Grdzelishvili V Z.
Vesicular stomatitis virus as a flexible platform for oncolytic
-hIFN.beta., murine (m), human virotherapy against cancer. J Gen
Virol. 2012 December; 93(Pt 12): 2529-45. doi: VSV-rIFN.beta. (h)
or rat (r) IFN- 10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Jenks,
N., Myers, R., Greiner, S. .beta. gene M., Thompson, J., Mader, E.
K., Greenslade, A., Griesmann, G. E., Federspiel, M. J., Rakela, J.
& other authors (2010). Safety studies on intrahepatic or
intratumoral injection of oncolytic vesicular stomatitis virus
expressing interferonb in rodents and nonhuman primates. Hum Gene
Ther 21, 451-462.; Obuchi, M., Fernandez, M. & Barber, G. N.
(2003). Development of recombinant vesicular stomatitis viruses
that exploit defects in host defense to augment specific oncolytic
activity. J Virol 77, 8843-8856.) VSV- Immunomodulation;
Recombinant VSV used as oncolytic agent against cancer (see, e.g.,
Hastie E, IL4 VSV expressing Grdzelishvili V Z. Vesicular
stomatitis virus as a flexible platform for oncolytic IL-4
virotherapy against cancer. J Gen Virol. 2012 December; 93(Pt 12):
2529-45. doi: 10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Fernandez,
M., Porosnicu, M., Markovic, D. & Barber, G. N. (2002).
Genetically engineered vesicular stomatitis virus in gene therapy:
application for treatment of malignant disease. J Virol 76,
895-904.) VSV- VSV expressing Naik S, Nace R, Federspiel M J,
Barber G N, Peng K W, Russell S J. Curative IFN- IFNb and thyroidal
one-shot systemic virotherapy in murine myeloma. Leukemia. 2012 NIS
sodium iodide August; 26(8): 1870-8. doi: 10.1038/leu.2012.70. Epub
2012 Mar. 19. symporter VSV- Immunomodulation; Recombinant VSV used
as oncolytic agent against cancer (see, e.g., Hastie E, IL12 VSV
expressing Grdzelishvili V Z. Vesicular stomatitis virus as a
flexible platform for oncolytic IL-12 virotherapy against cancer. J
Gen Virol. 2012 December; 93(Pt 12): 2529-45. doi:
10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Shin, E. J., Wanna, G.
B., Choi, B., Aguila, D., III, Ebert, O., Genden, E. M. & Woo,
S. L. (2007a). Interleukin-12 expression enhances vesicular
stomatitis virus oncolytic therapy in murine squamous cell
carcinoma. Laryngoscope 117, 210-214.) VSV- Immunomodulation;
Recombinant VSV used as oncolytic agent against cancer (see, e.g.,
Hastie E, IL23 VSV expressing Grdzelishvili V Z. Vesicular
stomatitis virus as a flexible platform for oncolytic IL-23.
virotherapy against cancer. J Gen Virol. 2012 December; 93(Pt 12):
2529-45. doi: Significantly 10.1099/vir.0.046672-0. Epub 2012 Oct.
10; Miller, J. M., Bidula, S. M., Jensen, attenuated in the T. M.
& Reiss, C. S. (2010). Vesicular stomatitis virus modified with
single CNS, but effective chain IL-23 exhibits oncolytic activity
against tumor cells in vitro and in vivo. Int OV J Infereron
Cytokine Mediator Res 2010, 63-72.) VSV- Immunomodulation;
Recombinant VSV used as oncolytic agent against cancer (see, e.g.,
Hastie E, IL28 VSV expressing Grdzelishvili V Z. Vesicular
stomatitis virus as a flexible platform for oncolytic IL-28, a
member virotherapy against cancer. J Gen Virol. 2012 December;
93(Pt 12): 2529-45. doi: of the type III 10.1099/vir.0.046672-0.
Epub 2012 Oct. 10; Wongthida, P., Diaz, R. M., Galivo, IFN
(IFN-.lamda.) F., Kottke, T., Thompson, J., Pulido, J., Pavelko,
K., Pease, L., Melcher, A. & family Vile, R. (2010). Type III
IFN interleukin-28 mediates the antitumor efficacy of oncolytic
virus VSV in immune-competent mouse models of cancer. Cancer Res
70, 4539-4549.) VSV- Immunomodulation; Recombinant VSV used as
oncolytic agent against cancer (see, e.g., Hastie E, opt.hIL-15
VSV-M.DELTA.51 Grdzelishvili V Z. Vesicular stomatitis virus as a
flexible platform for oncolytic expressing a virotherapy against
cancer. J Gen Virol. 2012 December; 93(Pt 12): 2529-45. doi: highly
secreted 10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Stephenson, K.
B., Barra, N. G., version of human Davies, E., Ashkar, A. A. &
Lichty, B. D. (2012). Expressing human interleukin- IL-15 15 from
oncolytic vesicular stomatitis virus improves survival in a murine
metastatic colon adenocarcinoma model through the enhancement of
antitumor immunity. Cancer Gene Ther 19, 238-246.) VSV-
Immunomodulation; Recombinant VSV used as oncolytic agent against
cancer (see, e.g., Hastie E, CD40L VSV expressing Grdzelishvili V
Z. Vesicular stomatitis virus as a flexible platform for oncolytic
CD40L, a member virotherapy against cancer. J Gen Virol. 2012
December; 93(Pt 12): 2529-45. doi: of the tumor
10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Galivo, F., Diaz, R. M.,
necrosis factor Thanarajasingam, U., Jevremovic, D., Wongthida, P.,
Thompson, J., Kottke, T., (TNF) family of Barber, G. N., Melcher,
A. & Vile, R. G. (2010). Interference of CD40L- cell-surface
mediated tumor immunotherapy by oncolytic vesicular stomatitis
virus. Hum
molecules Gene Ther 21, 439-450.) VSV- Immunomodulation;
Recombinant VSV used as oncolytic agent against cancer (see, e.g.,
Hastie E, Flt3L VSV-M.DELTA.51 Grdzelishvili V Z. Vesicular
stomatitis virus as a flexible platform for oncolytic expressing
the virotherapy against cancer. J Gen Virol. 2012 December; 93(Pt
12): 2529-45. doi: soluble form of 10.1099/vir.0.046672-0. Epub
2012 Oct. 10; Leveille, S., Goulet, M. L., Lichty, the human Flt3L,
B. D. & Hiscott, J. (2011). Vesicular stomatitis virus
oncolytic treatment a growth factor interferes with
tumor-associated dendritic cell functions and abrogates tumor
activating DCs antigen presentation. J Virol 85, 12160-12169.)
VSV/hDCT Immunomodulation; Recombinant VSV used as oncolytic agent
against cancer (see, e.g., Hastie E, VSV-M.DELTA.51 Grdzelishvili V
Z. Vesicular stomatitis virus as a flexible platform for oncolytic
expressing hDCT virotherapy against cancer. J Gen Virol. 2012
December; 93(Pt 12): 2529-45. doi: 10.1099/vir.0.046672-0. Epub
2012 Oct. 10; Boudreau, J. E., Bridle, B. W., Stephenson, K. B.,
Jenkins, K. M., Brunellie{grave over ( )} re, J., Bramson, J. L.,
Lichty, B. D. & Wan, Y. (2009). Recombinant vesicular
stomatitis virus transduction of dendritic cells enhances their
ability to prime innate and adaptive antitumor immunity. Mol Ther
17, 1465-1472.) VSV- Immunomodulation; Recombinant VSV used as
oncolytic agent against cancer (see, e.g., Hastie E, hgp100 VSV
expressing Grdzelishvili V Z. Vesicular stomatitis virus as a
flexible platform for oncolytic hgp100, an altered virotherapy
against cancer. J Gen Virol. 2012 December; 93(Pt 12): 2529-45.
doi: self-TAA against 10.1099/vir.0.046672-0. Epub 2012 Oct. 10;
Wongthida, P., Diaz, R. M., Galivo, which tolerance is F., Kottke,
T., Thompson, J., Melcher, A. & Vile, R. (2011). VSV oncolytic
well-established virotherapy in the B16 model depends upon intact
MyD88 signaling. Mol Ther in C57BL/6 mice 19, 150-158.) VSV-
Immunomodulation; Recombinant VSV used as oncolytic agent against
cancer (see, e.g., Hastie E, ova VSV expressing Grdzelishvili V Z.
Vesicular stomatitis virus as a flexible platform for oncolytic
chicken ovalbumin virotherapy against cancer. J Gen Virol. 2012
December; 93(Pt 12): 2529-45. doi: (for B16ova cancer
10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Diaz, R. M., Galivo, F.,
Kottke, T., model) Wongthida, P., Qiao, J., Thompson, J., Valdes,
M., Barber, G. & Vile, R. G. (2007). Oncolytic
immunovirotherapy for melanoma using vesicular stomatitis virus.
Cancer Res 67, 2840-2848.) VSV-gG Immunomodulation; Recombinant VSV
used as oncolytic agent against cancer (see, e.g., Hastie E, VSV
expressing Grdzelishvili V Z. Vesicular stomatitis virus as a
flexible platform for oncolytic EHV-1 glycoprotein virotherapy
against cancer. J Gen Virol. 2012 December; 93(Pt 12): 2529-45.
doi: G, a broad- 10.1099/vir.0.046672-0. Epub 2012 Oct. 10;
Altomonte, J., Wu, L., Chen, L., spectrum viral Meseck, M., Ebert,
O., Garci{acute over ( )}a-Sastre, A., Fallon, J. & Woo, S. L.
(2008). chemokine-binding Exponential enhancement of oncolytic
vesicular stomatitis virus potency by protein vector-mediated
suppression of inflammatory responses in vivo. Mol Ther 16,
146-153.) VSV- Immunomodulation; Recombinant VSV used as oncolytic
agent against cancer (see, e.g., Hastie E, UL141 VSV expressing
Grdzelishvili V Z. Vesicular stomatitis virus as a flexible
platform for oncolytic a secreted form virotherapy against cancer.
J Gen Virol. 2012 December; 93(Pt 12): 2529-45. doi: of the human
10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Altomonte, J., Wu, L.,
Meseck, M., cytomegalovirus Chen, L., Ebert, O., Garcia-Sastre, A.,
Fallon, J., Mandeli, J. & Woo, S. L. UL141 protein, (2009).
Enhanced oncolytic potency of vesicular stomatitis virus through
known to inhibit vector-mediated inhibition of NK and NKT cells.
Cancer Gene Ther 16, 266- the function of 278.) NK cells by
blocking the ligand of NK cell- activating receptors VSV-
Immunomodulation; Recombinant VSV used as oncolytic agent against
cancer (see, e.g., Hastie E, (.DELTA.51)-M3 VSV-M.DELTA.51
Grdzelishvili V Z. Vesicular stomatitis virus as a flexible
platform for oncolytic expressing the virotherapy against cancer. J
Gen Virol. 2012 December; 93(Pt 12): 2529-45. doi: murine
10.1099/vir.0.046672-0. Epub 2012 Oct. 10; Wu, L., Huang, T.
G.,Meseck, M., gammaherpesvirus- Altomonte, J., Ebert, O.,
Shinozaki, K., Garci{acute over ( )}a-Sastre, A., Fallon, J.,
Mandeli, 68 chemokine- J. & Woo, S. L. (2008). rVSV(MD51)-M3 is
an effective and safe oncolytic virus binding protein for cancer
therapy. Hum Gene Ther 19, 635-647.) M3 HSV-1 Genome and
Herpesviridae Clinical phase I/II; Glioma; Wollmann et al.
Oncolytic virus therapy for Structure: ds glioblastoma multiforme:
concepts and candidates. Cancer J. 2012 DNA; Enveloped
January-February; 18(1): 69-81 Representative Host: Human NDV
Genome and Paramyxoviridae Clinical phase I/II; Glioma; Wollmann et
al. Oncolytic virus therapy for Structure: ss glioblastoma
multiforme: concepts and candidates. Cancer J. 2012 (-) RNA;
January-February; 18(1): 69-81 Enveloped Representative Host: Avian
Adeno Genome and Adenoviridae Clinical phase I; Glioma; Wollmann et
al. Oncolytic virus therapy for Structure: ds glioblastoma
multiforme: concepts and candidates. Cancer J. 2012 DNA; Naked
January-February; 18(1): 69-81 Representative Host: Human Reo
Genome and Reoviridae Clinical phase I; Glioma; Wollmann et al.
Oncolytic virus therapy for Structure: ds glioblastoma multiforme:
concepts and candidates. Cancer J. 2012 RNA; Naked
January-February; 18(1): 69-81 Representative Host: Mammalian
Vaccinia Genome and Poxviridae Preclinical in vivo; Glioma;
Wollmann et al. Oncolytic virus therapy for Structure: ds
glioblastoma multiforme: concepts and candidates. Cancer J. 2012
DNA; Enveloped January-February; 18(1): 69-81 Representative Host:
Cow/horse, others Polio Genome and Picornaviridae Clinical phase I;
Glioma; Wollmann et al. Oncolytic virus therapy for Structure: ss
glioblastoma multiforme: concepts and candidates. Cancer J. 2012
(+) RNA; January-February; 18(1): 69-81 Naked Representative Host:
Human VSV Genome and Rhabdoviridae Preclinical in vivo; Glioma;
Wollmann et al. Oncolytic virus therapy for Structure: ss (-)
glioblastoma multiforme: concepts and candidates. Cancer J. 2012
RNA; Enveloped January-February; 18(1): 69-81 Representative Host:
Livestock/ mosquito MVM Genome and Parvoviridae Preclinical in
vitro; Glioma; Wollmann et al. Oncolytic virus therapy for
Structure: ss glioblastoma multiforme: concepts and candidates.
Cancer J. 2012 DNA; Naked January-February; 18(1): 69-81
Representative Host: Mouse Sindbis Genome and Togaviridae
Preclinical in vitro; Glioma; Wollmann et al. Oncolytic virus
therapy for Structure: ss (+) glioblastoma multiforme: concepts and
candidates. Cancer J. 2012 RNA; Enveloped January-February; 18(1):
69-81 Representative Host: Mammalian/ mosquito PRV Genome and
Herpesviridae Preclinical in vitro; Glioma; Wollmann et al.
Oncolytic virus therapy for Structure: ds glioblastoma multiforme:
concepts and candidates. Cancer J. 2012 DNA; Enveloped
January-February; 18(1): 69-81 Representative Host: Pig Measles
Genome and Paramyxoviridae Clinical phase I; Glioma; Wollmann et
al. Oncolytic virus therapy for Structure: ss (-) glioblastoma
multiforme: concepts and candidates. Cancer J. 2012 RNA; Enveloped
January-February; 18(1): 69-81 Representative Host: Human Myxoma
Genome and Poxviridae Preclinical in vivo; Glioma; Wollmann et al.
Oncolytic virus therapy for Structure: ds glioblastoma multiforme:
concepts and candidates. Cancer J. 2012 DNA; Enveloped
January-February; 18(1): 69-81 Representative Host: Rabbit H1PV
Genome and Parvoviridae Clinical phase I; Glioma; Wollmann et al.
Oncolytic virus therapy for Structure: ss glioblastoma multiforme:
concepts and candidates. Cancer J. 2012 DNA; Naked
January-February; 18(1): 69-81 Representative Host: Rat SVV Genome
and Picornaviridae Preclinical in vitro; Glioma; Wollmann et al.
Oncolytic virus therapy for Structure: ss glioblastoma multiforme:
concepts and candidates. Cancer J. 2012 (+) RNA; January-February;
18(1): 69-81 Naked Representative Host: Pig HSV (G207)I Phase I;
Malignant glioma; IT injection; Wollmann et al. Oncolytic virus
therapy for glioblastoma multiforme: concepts and candidates.
Cancer J. 2012 January-February; 18(1): 69-81; Markert J M, Medlock
M D, Rabkin S D, et al. Conditionally replicating herpes simplex
virus mutant, G207 for the treatment of malignant glioma: results
of a phase I trial. Gene Ther. 2000; 7: 867Y874. Phase I; Malignant
glioma; IT injection; Wollmann et al. Oncolytic virus therapy for
glioblastoma multiforme: concepts and candidates. Cancer J. 2012
January-February; 18(1): 69-81; Markert J M, Liechty P G, Wang W,
et al. Phase Ib trial of mutant herpes simplex virus G207
inoculated pre-and post-tumor resection for recurrent GBM. Mol
Ther. 2009; 17: 199Y207. Phase I; Malignant glioma; IT injection;
Wollmann et al. Oncolytic virus therapy for glioblastoma
multiforme: concepts and candidates. Cancer J. 2012
January-February; 18(1): 69-81 HSV (1716) Phase II; Malignant
glioma; IT injection; Wollmann et al. Oncolytic virus therapy for
glioblastoma multiforme: concepts and candidates. Cancer J. 2012
January-February; 18(1): 69-81; Rampling R, Cruickshank G,
Papanastassiou V, et al. Toxicity evaluation of
replication-competent herpes simplex virus (ICP 34.5 null mutant
1716) in patients with recurrent malignant glioma. Gene Ther. 2000;
7: 859Y866. Phase I; Malignant glioma; IT injection; Wollmann et
al. Oncolytic virus therapy for glioblastoma multiforme: concepts
and candidates. Cancer J. 2012 January-February; 18(1): 69-81;
Papanastassiou V, Rampling R, Fraser M, et al. The potential for
efficacy of the modified (ICP 34.5(j)) herpes simplex virus HSV1716
following intratumoral injection into human malignant glioma: a
proof of principle study. Gene Ther. 2002; 9: 398Y406. Phase I;
Malignant glioma; IT injection; Wollmann et al. Oncolytic virus
therapy
for glioblastoma multiforme: concepts and candidates. Cancer J.
2012 January-February; 18(1): 69-81; Harrow S, Papanastassiou V,
Harland J, et al. HSV1716 injection into the brain adjacent to
tumor following surgical resection of high-grade glioma: safety
data and long-term survival. Gene Ther. 2004; 11: 1648Y1658. Phase
II; Malignant glioma; Wollmann et al. Oncolytic virus therapy for
glioblastoma multiforme: concepts and candidates. Cancer J. 2012
January-February; 18(1): 69-81 HSV Phase I; Malignant glioma;
Wollmann et al. Oncolytic virus therapy for (G4721) glioblastoma
multiforme: concepts and candidates. Cancer J. 2012
January-February; 18(1): 69-81 HSV Phase I; Malignant glioma;
Wollmann et al. Oncolytic virus therapy for (M032) glioblastoma
multiforme: concepts and candidates. Cancer J. 2012
January-February; 18(1): 69-81 AdV (ONYX- Phase I; Malignant
glioma; injection to tumor resection cavity; Wollmann et al. 015)
Oncolytic virus therapy for glioblastoma multiforme: concepts and
candidates. Cancer J. 2012 January-February; 18(1): 69-81; Chiocca
E A, Abbed K M, Tatter S, et al. A phase I open-label,
dose-escalation, multi-institutional trial of injection with an
E1BAttenuated adenovirus, ONYX-015, into the peritumoral region of
recurrent malignant gliomas, in the adjuvant setting. Mol Ther.
2004; 10: 958Y966. AdV Phase I; Malignant glioma; Wollmann et al.
Oncolytic virus therapy for (Delta24- glioblastoma multiforme:
concepts and candidates. Cancer J. 2012 RGD) January-February;
18(1): 69-81 ReoV Phase I; Malignant glioma; IT injection; Wollmann
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concepts and candidates. Cancer J. 2012 January-February; 18(1):
69-81; Forsyth P, Roldan G, George D, et al. A phase I trial of
intratumoral administration of reovirus in patients with
histologically confirmed recurrent malignant gliomas. Mol Ther.
2008; 16: 627Y632. Phase I; Malignant glioma; Convection enhanced;
Wollmann et al. Oncolytic virus therapy for glioblastoma
multiforme: concepts and candidates. Cancer J. 2012
January-February; 18(1): 69-81 NDV Phase I/II; Malignant glioma;
IV; Wollmann et al. Oncolytic virus therapy for (HUJ) glioblastoma
multiforme: concepts and candidates. Cancer J. 2012
January-February; 18(1): 69-81; Freeman A I, Zakay-Rones Z, Gomori
J M, et al. Phase I/II trial of intravenous NDV-HUJ oncolytic virus
in recurrent glioblastoma multiforme. Mol Ther. 2006; 13: 221Y228.
Phase I/II; Malignant glioma; IV; Wollmann et al. Oncolytic virus
therapy for glioblastoma multiforme: concepts and candidates.
Cancer J. 2012 January-February; 18(1): 69-81 NDV Case
Studies/Series; Malignant glioma; IV; Wollmann et al. Oncolytic
virus (MTH-68) therapy for glioblastoma multiforme: concepts and
candidates. Cancer J. 2012 January-February; 18(1): 69-81; Csatary
L K, Bakacs T. Use of Newcastle disease virus vaccine (MTH- 68/H)
in a patient with high-grade glioblastoma. JAMA. 1999; 281:
1588Y1589. Case Studies/Series; Malignant glioma; IV; Wollmann et
al. Oncolytic virus therapy for glioblastoma multiforme: concepts
and candidates. Cancer J. 2012 January-February; 18(1): 69-81;
Csatary L K, Gosztonyi G, Szeberenyi J, et al. MTH-68/H oncolytic
viral treatment in human high-grade gliomas. J Neurooncol. 2004;
67: 83Y93. Case Studies/Series; Malignant glioma; IV; Wollmann et
al. Oncolytic virus therapy for glioblastoma multiforme: concepts
and candidates. Cancer J. 2012 January-February; 18(1): 69-81;
Wagner S, Csatary C M, Gosztonyi G, et al. Combined treatment of
pediatric high-grade glioma with the oncolytic viral strain
MTH-68/H and oral valproic acid. APMIS. 2006; 114: 731Y743. Measles
Phase I; Malignant glioma; IT injection; Wollmann et al. Oncolytic
virus therapy (MV- CEA) for glioblastoma multiforme: concepts and
candidates. Cancer J. 2012 January-February; 18(1): 69-81 H1 Phase
I; Malignant glioma; IT injection; Wollmann et al. Oncolytic virus
therapy H1PV for glioblastoma multiforme: concepts and candidates.
Cancer J. 2012 January-February; 18(1): 69-81 Polio Phase I;
Malignant glioma; convection-enahnced IT injection; Wollmann et al.
(PVS- RIPO) Oncolytic virus therapy for glioblastoma multiforme:
concepts and candidates. Cancer J. 2012 January-February; 18(1):
69-81
Cancers
[0079] The methods and compositions of the present invention may be
used to treat a wide variety of cancer types. One of skill in the
art will appreciate that, since cells of many if not all cancers
are capable of receptor-mediated apoptosis, the methods and
compositions of the present invention are broadly applicable to
many if not all cancers. The combinatorial approach of the present
invention is efficacious in various aggressive, treatment
refractory tumor models. In particular embodiments, for example,
the cancer treated by a method of the present invention may be
adrenal cancer, basal cell carcinoma, biliary tract cancer, bladder
cancer, bone cancer, brain and other central nervous system (CNS)
cancer, breast cancer, cervical cancer, choriocarcinoma, colon
cancer, colorectal cancer, connective tissue cancer, cancer of the
digestive system, endometrial cancer, epipharyngeal carcinoma,
esophageal cancer, eye cancer, gallbladder cancer, gastric cancer,
cancer of the head and neck, hepatocellular carcinoma,
intra-epithelial neoplasm, kidney cancer, laryngeal cancer,
leukemia, liver cancer, liver metastases, lung cancer, lymphomas
including Hodgkin's and non-Hodgkin's lymphomas, melanoma, myeloma,
multiple myeloma, neuroblastoma, mesothelioma, neuroglioma,
myelodysplastic syndrome, multiple myeloma, oral cavity cancer
(e.g. lip, tongue, mouth, and pharynx), ovarian cancer, paediatric
cancer, pancreatic cancer, pancreatic endocrine tumors, penile
cancer, plasma cell tumors, pituitary adenomathymoma, prostate
cancer, renal cell carcinoma, cancer of the respiratory system,
rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer,
small bowel cancer, stomach cancer, testicular cancer, thyroid
cancer, ureteral cancer, cancer of the urinary system, and other
carcinomas and sarcomas. Other cancers are known in the art.
[0080] The cancer may be a cancer that is refractory to treatment
by SMCs alone. The methods and compositions of the present
invention may be particularly useful in cancers that are refractory
to treatment by SMCs alone. Typically, a cancer refractory to
treatment with SMCs alone may be a cancer in which IAP-mediated
apoptotic pathways are not significantly induced. In particular
embodiments, a cancer of the present invention is a cancer in which
one or more apoptotic pathways are not significantly induced, i.e.,
is not activated in a manner such that treatment with SMCs alone is
sufficient to effectively treat the cancer. For instance, a cancer
of the present invention can be a cancer in which a
cIAP1/2-mediated apoptotic pathway is not significantly
induced.
[0081] A cancer of the present invention may be a cancer refractory
to treatment by one or more immunostimulatory agents. In particular
embodiments, a cancer of the present invention may be a cancer
refractory to treatment by one or more immunostimulatory agents
(absent an SMC) and also refractory to treatment by one or more
SMCs (absent an immunostimulatory agent).
Formulations and Administration
[0082] In some instances, delivery of a naked, i.e. native form, of
an SMC and/or immunostimulatory agent may be sufficient to
potentiate apoptosis and/or treat cancer. SMCs and/or
immunostimulatory agents may be administered in the form of salts,
esters, amides, prodrugs, derivatives, and the like, provided the
salt, ester, amide, prodrug or derivative is suitably
pharmacologically effective, e.g., capable of potentiating
apoptosis and/or treating cancer.
[0083] Salts, esters, amides, prodrugs and other derivatives of an
SMC or immunostimulatory agent can be prepared using standard
procedures known in the art of synthetic organic chemistry. For
example, an acid salt of SMCs and/or immunostimulatory agents may
be prepared from a free base form of the SMC or immunostimulatory
agent using conventional methodology that typically involves
reaction with a suitable acid. Generally, the base form of the SMC
or immunostimulatory agent is dissolved in a polar organic solvent,
such as methanol or ethanol, and the acid is added thereto. The
resulting salt either precipitates or can be brought out of
solution by addition of a less polar solvent. Suitable acids for
preparing acid addition salts include, but are not limited to, both
organic acids, e.g., acetic acid, propionic acid, glycolic acid,
pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid,
maleic acid, fumaric acid, tartaric acid, citric acid, benzoic
acid, cinnamic acid, mandelic acid, methanesulfonic acid,
ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and
the like, as well as inorganic acids, e.g., hydrochloric acid,
hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and
the like.
[0084] An acid addition salt can be reconverted to the free base by
treatment with a suitable base. Certain typical acid addition salts
of SMCs and/or immunostimulatory agents, for example, halide salts,
such as may be prepared using hydrochloric or hydrobromic acids.
Conversely, preparation of basic salts of SMCs and/or
immunostimulatory agents of the present invention may be prepared
in a similar manner using a pharmaceutically acceptable base, such
as sodium hydroxide, potassium hydroxide, ammonium hydroxide,
calcium hydroxide, trimethylamine, or the like. Certain typical
basic salts include, but are not limited to, alkali metal salts,
e.g., sodium salt, and copper salts.
[0085] Preparation of esters may involve functionalization of,
e.g., hydroxyl and/or carboxyl groups that are present within the
molecular structure of SMCs and/or immunostimulatory agents. In
certain embodiments, the esters are acyl-substituted derivatives of
free alcohol groups, i.e., moieties derived from carboxylic acids
of the formula RCOOH where R is alky, and preferably is lower
alkyl. Esters may be reconverted to the free acids, if desired, by
using conventional hydrogenolysis or hydrolysis procedures.
[0086] Amides may also be prepared using techniques known in the
art. For example, an amide may be prepared from an ester using
suitable amine reactants or prepared from an anhydride or an acid
chloride by reaction with ammonia or a lower alkyl amine.
[0087] An SMC or immunostimulatory agent of the present invention
may be combined with a pharmaceutically acceptable carrier
(excipient) to form a pharmacological composition. Pharmaceutically
acceptable carriers can contain one or more physiologically
acceptable compound(s) that act, e.g., to stabilize the
composition, increase or decrease the absorption of the SMC or
immunostimulatory agent, or improve penetration of the blood brain
barrier (where appropriate). Physiologically acceptable compounds
may include, e.g., carbohydrates (e.g., glucose, sucrose, or
dextrans), antioxidants (e.g. ascorbic acid or glutathione),
chelating agents, low molecular weight proteins, protection and
uptake enhancers (e.g., lipids), compositions that reduce the
clearance or hydrolysis of the active agents, or excipients or
other stabilizers and/or buffers. Other physiologically acceptable
compounds, particularly of use in the preparation of tablets,
capsules, gel caps, and the like include, but are not limited to,
binders, diluents/fillers, disintegrants, lubricants, suspending
agents, and the like. In certain embodiments, a pharmaceutical
formulation may enhance delivery or efficacy of an SMC or
immunostimulatory agent.
[0088] In various embodiments, an SMC or immunostimulatory agent of
the present invention may be prepared for parenteral, topical,
oral, nasal (or otherwise inhaled), rectal, or local
administration. Administration may occur, for example,
transdermally, prophylactically, or by aerosol.
[0089] A pharmaceutical composition of the present invention may be
administered in a variety of unit dosage forms depending upon the
method of administration. Suitable unit dosage forms, include, but
are not limited to, powders, tablets, pills, capsules, lozenges,
suppositories, patches, nasal sprays, injectibles, implantable
sustained-release formulations, and lipid complexes.
[0090] In certain embodiments, an excipient (e.g., lactose,
sucrose, starch, mannitol, etc.), an optional disintegrator (e.g.
calcium carbonate, carboxymethylcellulose calcium, sodium starch
glycollate, crospovidone, etc.), a binder (e.g. alpha-starch, gum
arabic, microcrystalline cellulose, carboxymethylcellulose,
polyvinylpyrrolidone, hydroxypropylcellulose, cyclodextrin, etc.),
or an optional lubricant (e.g., talc, magnesium stearate,
polyethylene glycol 6000, etc.) may be added to an SMC or
immunostimulatory agent and the resulting composition may be
compressed to manufacture an oral dosage form (e.g., a tablet). In
particular embodiments, a compressed product may be coated, e.g.,
to mask the taste of the compressed product, to promote enteric
dissolution of the compressed product, or to promote sustained
release of the SMC or immunostimulatory agent. Suitable coating
materials include, but are not limited to, ethyl-cellulose,
hydroxymethylcellulose, polyoxyethylene glycol, cellulose acetate
phthalate, hydroxypropylmethylcellulose phthalate, and Eudragit
(Rohm & Haas, Germany; methacrylic-acrylic copolymer).
[0091] Other physiologically acceptable compounds that may be
included in a pharmaceutical composition including an SMC or
immunostimulatory agent may include wetting agents, emulsifying
agents, dispersing agents or preservatives that are particularly
useful for preventing the growth or action of microorganisms.
Various preservatives are well known and include, for example,
phenol and ascorbic acid. The choice of pharmaceutically acceptable
carrier(s), including a physiologically acceptable compound,
depends, e.g., on the route of administration of the SMC or
immunostimulatory agent and on the particular physio-chemical
characteristics of the SMC or immunostimulatory agent.
[0092] In certain embodiments, one or more excipients for use in a
pharmaceutical composition including an SMC or immunostimulatory
agent may be sterile and/or substantially free of undesirable
matter. Such compositions may be sterilized by conventional
techniques known in the art. For various oral dosage form
excipients, such as tablets and capsules, sterility is not
required. Standards are known in the art, e.g., the USP/NF
standard.
[0093] An SMC or immunostimulatory agent pharmaceutical composition
of the present invention may be administered in a single or in
multiple administrations depending on the dosage, the required
frequency of administration, and the known or anticipated tolerance
of the subject for the pharmaceutical composition with respect to
dosages and frequency of administration. In various embodiments,
the composition may provide a sufficient quantity of an SMC or
immunostimulatory agent of the present invention to effectively
treat cancer.
[0094] The amount and/or concentration of an SMC or
immunostimulatory agent to be administered to a subject may vary
widely, and will typically be selected primarily based on activity
of the SMC or immunostimulatory agent and the characteristics of
the subject, e.g., species and body weight, as well as the
particular mode of administration and the needs of the subject,
e.g., with respect to a type of cancer. Dosages may be varied to
optimize a therapeutic and/or prophylactic regimen in a particular
subject or group of subjects.
[0095] In certain embodiments, an SMC or immunostimulatory agent of
the present invention is administered to the oral cavity, e.g., by
the use of a lozenge, aerosol spray, mouthwash, coated swab, or
other mechanism known in the art.
[0096] In certain embodiments, an SMC or immunostimulatory agent of
the present invention may be administered systemically (e.g.,
orally or as an injectable) in accordance with standard methods
known in the art. In certain embodiments, the SMC or
immunostimulatory agent may be delivered through the skin using a
transdermal drug delivery systems, i.e., transdermal "patches,"
wherein the SMCs or immunostimulatory agents are typically
contained within a laminated structure that serves as a drug
delivery device to be affixed to the skin. In such a structure, the
drug composition is typically contained in a layer or reservoir
underlying an upper backing layer. The reservoir of a transdermal
patch includes a quantity of an SMC or immunostimulatory agent that
is ultimately available for delivery to the surface of the skin.
Thus, the reservoir may include, e.g., an SMC or immunostimulatory
agent of the present invention in an adhesive on a backing layer of
the patch or in any of a variety of different matrix formulations
known in the art. The patch may contain a single reservoir or
multiple reservoirs.
[0097] In particular transdermal patch embodiments, a reservoir may
comprise a polymeric matrix of a pharmaceutically acceptable
contact adhesive material that serves to affix the system to the
skin during drug delivery. Examples of suitable skin contact
adhesive materials include, but are not limited to, polyethylenes,
polysiloxanes, polyisobutylenes, polyacrylates, and polyurethanes.
Alternatively, the SMC and/or immunostimulatory agent-containing
reservoir and skin contact adhesive are present as separate and
distinct layers, with the adhesive underlying the reservoir which,
in this case, may be either a polymeric matrix as described above,
a liquid or hydrogel reservoir, or another form of reservoir known
in the art. The backing layer in these laminates, which serves as
the upper surface of the device, preferably functions as a primary
structural element of the patch and provides the device with a
substantial portion of flexibility. The material selected for the
backing layer is preferably substantially impermeable to the SMC
and/or immunostimulatory agent and to any other materials that are
present.
[0098] Additional formulations for topical delivery include, but
are not limited to, ointments, gels, sprays, fluids, and creams.
Ointments are semisolid preparations that are typically based on
petrolatum or other petroleum derivatives. Creams including an SMC
or immunostimulatory agent are typically viscous liquids or
semisolid emulsions, e.g. oil-in-water or water-in-oil emulsions.
Cream bases are typically water-washable and include an oil phase,
an emulsifier, and an aqueous phase. The oil phase, also sometimes
called the "internal" phase, of a cream base is generally comprised
of petrolatum and a fatty alcohol, e.g., cetyl alcohol or stearyl
alcohol; the aqueous phase usually, although not necessarily,
exceeds the oil phase in volume, and generally contains a
humectant. The emulsifier in a cream formulation is generally a
nonionic, anionic, cationic, or amphoteric surfactant. The specific
ointment or cream base to be used may be selected to provide for
optimum drug delivery according to the art. As with other carriers
or vehicles, an ointment base may be inert, stable, non-irritating,
and non-sensitizing.
[0099] Various buccal and sublingual formulations are also
contemplated.
[0100] In certain embodiments, administration of an SMC or
immunostimulatory agent of the present invention may be parenteral.
Parenteral administration may include intraspinal, epidural,
intrathecal, subcutaneous, or intravenous administration. Means of
parenteral administration are known in the art. In particular
embodiments, parenteral administration may include a subcutaneously
implanted device.
[0101] In certain embodiments, it may be desirable to deliver an
SMC or immunostimulatory agent to the brain. In embodiments
including system administration, this could require that the SMC or
immunostimulatory agent cross the blood brain barrier. In various
embodiments this may be facilitated by co-administering an SMC or
immunostimulatory agent with carrier molecules, such as cationic
dendrimers or arginine-rich peptides, which may carry an SMC or
immunostimulatory agent over the blood brain barrier.
[0102] In certain embodiments, an SMC or immunostimulatory agent
may be delivered directly to the brain by administration through
the implantation of a biocompatible release system (e.g., a
reservoir), by direct administration through an implanted cannula,
by administration through an implanted or partially implanted drug
pump, or mechanisms of similar function known the art. In certain
embodiments, an SMC or immunostimulatory agent may be systemically
administered (e.g., injected into a vein). In certain embodiments,
it is expected that the SMC or immunostimulatory agent will be
transported across the blood brain barrier without the use of
additional compounds included in a pharmaceutical composition to
enhance transport across the blood brain barrier.
[0103] In certain embodiments, one or more an SMCs or
immunostimulatory agents of the present invention may be provided
as a concentrate, e.g., in a storage container or soluble capsule
ready for dilution or addition to a volume of water, alcohol,
hydrogen peroxide, or other diluent. A concentrate of the present
invention may be provided in a particular amount of an SMC or
immunostimulatory agent and/or a particular total volume. The
concentrate may be formulated for dilution in a particular volume
of diluents prior to administration.
[0104] An SMC or immunostimulatory agent may be administered orally
in the form of tablets, capsules, elixirs or syrups, or rectally in
the form of suppositories. The compound may also be administered
topically in the form of foams, lotions, drops, creams, ointments,
emollients, or gels. Parenteral administration of a compound is
suitably performed, for example, in the form of saline solutions or
with the compound incorporated into liposomes. In cases where the
compound in itself is not sufficiently soluble to be dissolved, a
solubilizer, such as ethanol, can be applied. Other suitable
formulations and modes of administration are known or may be
derived from the art.
[0105] An SMC or immunostimulatory agent of the present invention
may be administered to a mammal in need thereof, such as a mammal
diagnosed as having cancer. An SMC or immunostimulatory agent of
the present invention may be administered to potentiate apoptosis
and/or treat cancer.
[0106] A therapeutically effective dose of a pharmaceutical
composition of the present invention may depend upon the age of the
subject, the gender of the subject, the species of the subject, the
particular pathology, the severity of the symptoms, and the general
state of the subject's health.
[0107] The present invention includes compositions and methods for
the treatment of a human subject, such as a human subject having
been diagnosed with cancer. In addition, a pharmaceutical
composition of the present invention may be suitable for
administration to an animal, e.g., for veterinary use. Certain
embodiments of the present invention may include administration of
a pharmaceutical composition of the present invention to non-human
organisms, e.g., a non-human primates, canine, equine, feline,
porcine, ungulate, or lagomorphs organism or other vertebrate
species.
[0108] Therapy according to the invention may be performed alone or
in conjunction with another therapy, e.g., another cancer therapy,
and may be provided at home, the doctor's office, a clinic, a
hospital's outpatient department, or a hospital. Treatment
optionally begins at a hospital so that the doctor can observe the
therapy's effects closely and make any adjustments that are needed
or it may begin on an outpatient basis. The duration of the therapy
depends on the type of disease or disorder being treated, the age
and condition of the subject, the stage and type of the subject's
disease, and how the patient responds to the treatment.
[0109] In certain embodiments, the combination of therapy of the
present invention further includes treatment with a recombinant
interferon, such as IFN-.alpha., IFN-.beta., IFN-.gamma., pegylated
IFN, or liposomal interferon. In some embodiments, the combination
of therapy of the present invention further includes treatment with
recombinant TNF-.alpha., e.g., for isolated-limb perfusion. In
particular embodiments, the combination therapy of the present
invention further includes treatment with one or more of a
TNF-.alpha. or IFN-inducing compound, such as DMXAA, Ribavirin, or
the like. Additional cancer immunotherapies that may be used in
combination with present invention include antibodies, e.g.,
monoclonal antibodies, targeting CTLA-4, PD-1, PD-L1, PD-L2, or
other checkpoint inhibitors.
[0110] Routes of administration for the various embodiments
include, but are not limited to, topical, transdermal, nasal, and
systemic administration (such as, intravenous, intramuscular,
subcutaneous, inhalation, rectal, buccal, vaginal, intraperitoneal,
intraarticular, ophthalmic, otic, or oral administration). As used
herein, "systemic administration" refers to all nondermal routes of
administration, and specifically excludes topical and transdermal
routes of administration.
[0111] In any of the above embodiments, the route of administration
may be optimized based on the characteristics of the SMC or
immunostimulatory agent. In some instances, the SMC or
immunostimulatory agent is a small molecule or compound. In other
instances, the SMC or immunostimulatory agent is a nucleic acid. In
still other instances, the immunostimulatory agent may be a cell or
virus. In any of these or other embodiments, appropriate
formulations and routes of administration will be selected in
accordance with the art.
[0112] In the embodiments of the present invention, an SMC and an
immunostimulatory agent are administered to a subject in need
thereof, e.g., a subject having cancer. In some instances, the SMC
and immunostimulatory agent will be administered simultaneously. In
some embodiments, the SMC and immunostimulatory agent may be
present in a single therapeutic dosage form. In other embodiments,
the SMC and immunostimulatory agent may be administered separately
to the subject in need thereof. When administered separately, the
SMC and immunostimulatory agent may be administered simultaneously
or at different times. In some instances, a subject will receive a
single dosage of an SMC and a single dosage of an immunostimulatory
agent. In certain embodiments, one or more of the SMC and
immunostimulatory agent will be administered to a subject in two or
more doses. In certain embodiments, the frequency of administration
of an SMC and the frequency of administration of an
immunostimulatory agent are non-identical, i.e., the SMC is
administered at a first frequence and the immunostimulatory agent
is administered at a second frequency.
[0113] In some embodiments, an SMC is administered within one week
of the administration of an immunostimulatory agent. In particular
embodiments, an SMC is administered within 3 days (72 hours) of the
administration of an immunostimulatory agent. In still more
particular embodiments, an SMC is administered within 1 day (24
hours) of the administration of an immunostimulatory agent.
[0114] In particular embodiments of any of the methods of the
present invention, the SMC and immunostimulatory agent are
administered within 28 days of each other or less, e.g., within 14
days of each other. In certain embodiments of any of the methods of
the present invention, the SMC and immunostimulatory agent are
administered, e.g., simultaneously or within 1 minute, 5 minutes,
10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6
hours, 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 4 days, 8
days, 10 days, 12 days, 16 days, 20 days, 24 days, or 28 days of
each other. In any of these embodiments, the first administration
of an SMC of the present invention may precede the first
administration of an immunostimulatory agent of the present
invention. Alternatively, in any of these embodiments, the first
administration of an SMC of the present invention may follow the
first administration of an immunostimulatory agent of the present
invention. Because an SMC and/or immunostimulatory agent of the
present invention may be administered to a subject in two more
doses, and because, in such instances, doses of the SMC and
immunostimulatory agent of the present invention may be
administered at different frequencies, it is not required that the
period of time between the administration of an SMC and the
administration of an immunostimulatory agent remain constant within
a given course of treatment or for a given subject.
[0115] One or both of the SMC and the immunostimulatory agent may
be administered in a low dosage or in a high dosage. In embodiments
in which the SMC and immunostimulatory agent are formulated
separately, the pharmacokinetic profiles for each agent can be
suitably matched to the formulation, dosage, and route of
administration, etc. In some instances, the SMC is administered at
a standard or high dosage and the immunostimulatory agent is
administered at a low dosage. In some instances, the SMC is
administered at a low dosage and the immunostimulatory agent is
administered at a standard or high dosage. In some instances, both
of the SMC and the immunostimulatory agent are administered at a
standard or high dosage. In some instances, both of the SMC and the
immunostimulatory agent are administered at a low dosage.
[0116] The dosage and frequency of administration of each component
of the combination can be controlled independently. For example,
one component may be administered three times per day, while the
second component may be administered once per day or one component
may be administered once per week, while the second component may
be administered once per two weeks. Combination therapy may be
given in on-and-off cycles that include rest periods so that the
subject's body has a chance to recover from effects of
treatment.
Kits
[0117] In general, kits of the invention contain one or more SMCs
and one or more immunostimulatory agents. These can be provided in
the kit as separate compositions, or combined into a single
composition as described above. The kits of the invention can also
contain instructions for the administration of one or more SMCs and
one or more immunostimulatory agents.
[0118] Kits of the invention can also contain instructions for
administering an additional pharmacologically acceptable substance,
such as an agent known to treat cancer that is not an SMC or
immunostimulatory agent of the present invention.
[0119] The individually or separately formulated agents can be
packaged together as a kit. Non-limiting examples include kits that
contain, e.g., two pills, a pill and a powder, a suppository and a
liquid in a vial, two topical creams, ointments, foams etc. The kit
can include optional components that aid in the administration of
the unit dose to subjects, such as vials for reconstituting powder
forms, syringes for injection, customized IV delivery systems,
inhalers, etc. Additionally, the unit dose kit can contain
instructions for preparation and administration of the
compositions. The kit may be manufactured as a single use unit dose
for one subject, multiple uses for a particular subject (at a
constant dosage regimen or in which the individual compounds may
vary in potency as therapy progresses); or the kit may contain
multiple doses suitable for administration to multiple subjects
("bulk packaging"). The kit components may be assembled in cartons,
blister packs, bottles, tubes, and the like.
[0120] The dosage of each compound of the claimed combinations
depends on several factors, including: the administration method,
the disease (e.g., a type of cancer) to be treated, the severity of
the disease, and the age, weight, and health of the person to be
treated. Additionally, pharmacogenomic (the effect of genotype on
the pharmacokinetic, pharmacodynamic or efficacy profile of a
therapeutic) information about a particular subject may affect the
dosage regimen or other aspects of administration.
EXAMPLES
Example 1: Smac Mimetics Prime Tumors for Destruction by the Innate
Immune System
[0121] Smac mimetic compounds are a class of apoptosis sensitizing
drugs that have proven safe in cancer patient Phase I trials.
Stimulating an innate anti-pathogen response may generate a potent
yet safe inflammatory "cytokine storm" that would trigger death of
tumors treated with Smac mimetics. The present example demonstrates
that activation of innate immune responses via oncolytic viruses
and adjuvants, such as poly(I:C) and CpG, induces bystander death
of cancer cells treated with Smac mimetics in a manner mediated by
IFN.beta., TNF.alpha. or TRAIL. This therapeutic strategy may lead
to durable cures, e.g., in several aggressive mouse models of
cancer. With these and other innate immune stimulants having
demonstrated safety in human clinical trials, the data provided
herein points strongly towards their combined use with Smac
mimetics for treating cancer.
[0122] The present example examines whether stimulating the innate
immune system using pathogen mimetics would be a safe and effective
strategy to generate a cytokine milieu necessary to initiate
apoptosis in tumors treated with an SMC. We report here that
non-pathogenic oncolytic viruses, as well as mimetics of microbial
RNA or DNA, such as poly (I:C) and CpG, induce bystander killing of
cancer cells treated with an SMC that is dependent either upon
IFN.beta., TNF.alpha., or TRAIL production. Importantly, this
therapeutic strategy was tolerable in vivo and led to durable cures
in several aggressive mouse models of cancer.
SMC Therapy Sensitizes Cancer Cells to Bystander Cell Death During
Oncolytic Virus Infection
[0123] Oncolytic viruses (OVs) are emerging biotherapies for cancer
currently in phase I-III clinical evaluation. One barrier to OV
therapy may be the induction of type I IFN- and
NF.kappa.B-responsive cytokines by the host, which orchestrate an
antiviral state in tumors. It was examined whether we could harness
those innate immune cytokines to induce apoptosis in cancer cells
pretreated with an SMC. To begin, a small panel of tumor-derived
and normal cell lines (n=30) was screened for responsiveness to the
SMC LCL161 and the oncolytic rhabdovirus VSV.DELTA.51. We chose
LCL161 because this compound is the most clinically advanced drug
in the SMC class, and VSV.DELTA.51 because it is known to induce a
robust antiviral cytokine response. In 15 of the 28 cancer cell
lines tested (54%), SMC treatment enhanced sensitivity the EC50 of
VSV.DELTA.51 by 10-10,000 fold (FIG. 6, and representative examples
in FIGS. 1A and 1B). Similarly, low dose of VSV.DELTA.51 reduced
the EC50 of SMC therapy from undetermined levels (>2500 nM) to
4.5 and 21.9 nM in two representative cell lines: the mouse mammary
carcinoma EMT6 and the human glioblastoma SNB75 cells, respectively
(FIG. 1C). Combination index analyses determined that the
interaction between SMC therapy and VSV.DELTA.51 was synergistic
(FIG. 7). Experiments using four other SMCs and five other
oncolytic viruses showed that a spectrum of monovalent and bivalent
SMCs synergize with VSV.DELTA.51 (FIG. 8). We find that the
oncolytic rhabdoviruses, VSV.DELTA.51 and Maraba-MG1, are superior
in eliciting bystander killing in synergizing with SMCs, compared
to HSV, reovirus, vaccinia and wild-type VSV platforms, all of
which have elaborate mechanisms to disarm aspects of innate immune
signalling (FIGS. 9A AND 9B). Genetic experiments using
RNAi-mediated silencing demonstrated that both XIAP and the cIAPs
must be inhibited to obtain synergy with VSV.DELTA.51 (FIGS. 10A,
10B, and 24C). In stark contrast to the results in tumor-derived
cell lines, non-cancer GM38 primary human skin fibroblasts and HSkM
human skeletal myoblasts were unaffected by VSV.DELTA.51 and SMC
combination therapy (FIG. 6). Taken together, these data indicate
that oncolytic VSV synergizes with SMC therapy in a tumor-selective
fashion.
[0124] To determine if VSV.DELTA.51 elicits bystander cell death in
IAP-depleted neighbouring cells not infected by the virus, cells
were treated with SMCs prior to infection with a low dose of
VSV.DELTA.51 (MOI=0.01 infectious particles per cell). We assessed
whether conditioned media derived from cells infected with
VSV.DELTA.51 (which was subsequently inactivated by UV light) could
induce death when transferred to a plate of virus naive cancer
cells treated with an SMC. The conditioned media induced cell death
only when the cells were co-treated with an SMC (FIG. 1D). We also
found that a low-dose of a pseudo-typed G-less strain of
VSV.DELTA.51 (MOI=0.1), containing a deletion of the gene encoding
for its glycoprotein (VSV.DELTA.51.DELTA.G) that limits the virus
to a single round of infection, was toxic to an entire plate of
cancer cells treated with an SMC (FIG. 1E). Finally, we performed a
cytotoxicity assay in cells overlaid with agarose, used to retard
the spread of VSV.DELTA.51 expressing a fluorescent tag, and
observed dramatic cell death in SMC treated cells outside of the
zone of virus infection (FIGS. 1F and 11). Overall, these results
indicate that VSV.DELTA.51 infection leads to the release of at
least one soluble factor that can potently induce bystander cell
death in neighboring, uninfected, cancer cells treated with
SMCs.
SMC Therapy does not Impair the Cellular Innate Immune Response to
Oncolytic VSV
[0125] The cellular innate immune response to an RNA virus
infection in mammalian tumor cells can be initiated by members of a
family of cytosolic (RIG-I-like receptors, RLRs) and endosomal
(toll-like receptors, TLRs) viral RNA sensors. Once triggered,
these receptors can seed parallel IFN-response factor (IRF) 3/7 and
nuclear-factor kappa B (NF-.kappa.B) cell signalling cascades.
These signals can culminate in the production of IFNs and their
responsive genes as well as an array of inflammatory chemokines and
cytokines. This prompts neighboring cells to preemptively express
an armament of antiviral genes and also aids in the recruitment and
activation of cells within the innate and adaptive immune systems
to ultimately clear the virus infection. The cIAP proteins have
recently been implicated in numerous signalling pathways downstream
of pathogen recognition, including those emanating from RLRs and
TLRs. Accordingly, it was examined whether SMC therapy alters the
antiviral response to oncolytic VSV infection in tumor cells and in
mice. To begin, the effect of SMC therapy on VSV.DELTA.51
productivity and spread was evaluated. Single-step and multi-step
growth curves of VSV.DELTA.51 productivity revealed that SMC
treatment does not affect the growth kinetics of VSV.DELTA.51 in
EMT6 or SNB75 cells in vitro (FIG. 2A). Moreover, analysis through
time-lapse microscopy demonstrates that SMC treatment does not
alter VSV.DELTA.51 infectivity in or spread through tumor cells
(FIG. 2B). Furthermore, viral replication and spread in vivo were
analyzed by determining tumor load using IVIS imaging and tissue
virus titration. No differences in viral kinetics were found upon
SMC treatment in EMT6 tumor-bearing mice (FIGS. 12A and 12B). As
EMT6 and SNB75 cells both have functional type I IFN responses that
regulate the VSV life cycle, these data provide strong, albeit
indirect, evidence that SMC therapy does not affect the antiviral
signalling cascades in cancer cells.
[0126] To probe deeper, IFN.beta. production was measured in EMT6
and SNB75 cells treated with VSV.DELTA.51 and SMCs. This experiment
revealed that the SMC treated cancer cells respond to VSV.DELTA.51
by secreting IFN.beta. (FIG. 2C), although at slightly lower levels
as compared to VSV.DELTA.51 alone. It was asked whether the
dampened IFN.beta. secretion from SMC treated cells had any bearing
on the induction of downstream IFN stimulated genes (ISGs).
Quantitative RT-PCR analyses of a small panel of ISGs in cells
treated with VSV.DELTA.51 and SMC revealed that IAP inhibition had
no bearing on ISG gene expression in response to an oncolytic VSV
infection (FIG. 2D). Consistent with this finding, western blot
analyses indicated that SMCs do not alter the activation of
Jak/Stat signalling downstream of IFN.beta. (FIGS. 2E and 24A).
Collectively, these data suggest that SMCs do not impede the
ability of tumor cells to sense and respond to an infection from
VSV.DELTA.51.
IFN.beta. Orchestrates Bystander Cell Death During SMC and
Oncolytic VSV Co-Therapy
[0127] SMCs sensitize a number of cancer cell lines towards caspase
8-dependant apoptosis induced by TNF.alpha., TRAIL, and IL-1.beta..
As RNA viruses can trigger the production of these cytokines as
part of the cellular antiviral response, the involvement of
cytokine signaling in SMC and OV induced cell death was
investigated. To start, the TNF receptor (TNF-R1) and/or the TRAIL
receptor (DR5) were silenced and synergy between SMC and
VSV.DELTA.51 was assayed. This experiment revealed that TNF.alpha.
and TRAIL are not only involved, but collectively are indispensable
for bystander cell death (FIGS. 3A-3H, 13A, and 24D). Consistent
with this finding, western blot and immunofluorescence experiments
revealed strong activation of the extrinsic apoptosis pathway, and
RNAi knockdown experiments demonstrated a requirement for both
caspase-8 and Rip1 in the synergy response (FIGS. 14A-14G, 24E, and
24F). Moreover, engineering TNF.alpha. into VSV.DELTA.51 improved
synergy with SMC therapy by an order of magnitude (FIGS. 15A and
15B).
[0128] Next, the type I IFN receptor (IFNAR1) was silenced and it
was found, unexpectedly, that IFNAR1 knockdown prevented the
synergy between SMC therapy and oncolytic VSV (FIGS. 3B, 13B, and
24D). It was predicted that IFNAR1 knockdown would dampen but not
completely suppress bystander killing, as TRAIL is a
well-established ISG that is responsive to type I IFN28. TNF.alpha.
and IL-1.beta. are considered to be independent of IFN signaling,
but they are nevertheless responsive to NF-.kappa.B signaling
downstream of virus detection. This result suggests the possibility
of a non-canonical type I IFN-dependant pathway for the production
of TNF.alpha. and/or IL-1.beta.. Indeed, when the mRNA expression
of IFN.beta., TRAIL, TNF.alpha., and IL-1.beta. were probed during
an oncolytic VSV infection, a significant temporal lag was found
between the induction of IFN.beta. and that of both TRAIL and
TNF.alpha. (FIG. 3C). This data also suggests that TNF.alpha.--like
TRAIL--may be induced secondary to IFN.beta.. To prove this
concept, IFNAR1 was silenced before treating cells with
VSV.DELTA.51. IFNAR1 knockdown completely abrogated the induction
of both TRAIL and TNF.alpha. by oncolytic VSV (FIG. 3D). Moreover,
synergy with SMC was recapitulated using recombinant type I IFNs
(IFN.alpha./.beta.) and type II IFN (IFN.gamma.), but not type III
IFNs (IL28/29) (FIG. 3E). Taken together, these data indicate that
type I IFN is required for the induction of TNF.alpha. and TRAIL
during a VSV.DELTA.51 infection of tumor cells. Moreover, the
production of these cytokines is responsible for bystander killing
of neighboring, uninfected SMC-treated cells.
[0129] To explore the non-canonical induction of TNF.alpha.
further, the mRNA expression levels of TRAIL and TNF.alpha. in
SNB75 cells treated with recombinant IFN.beta. were measured. Both
cytokines were induced by IFN.beta. treatment (FIG. 3F), and ELISA
experiments confirmed the production of their respective protein
products in the cell culture media (FIG. 3G). Interestingly, there
was a significant time lag between the induction of TRAIL and that
of TNF.alpha.. As TRAIL is a bona fide ISG and TNF.alpha. is not,
this result raised the possibility that TNF.alpha. is not induced
by IFN.beta. directly, but responds to a downstream ISG
up-regulated by IFN.beta.. Thus, quantitative RT-PCR was performed
on 176 cytokines in SNB75 cells and 70 that were significantly
up-regulated by IFN.beta. were identified (Table 4). The role of
these ISGs in the induction of TNF.alpha. by IFN.beta. is currently
being investigated. It is also intriguing that SMC treatment
potentiated the induction of both TRAIL and TNF.alpha. by IFN.beta.
in SNB75 cells (FIGS. 3F and 3G). Furthermore, using a
dominant-negative construct of IKK, it was found that the
production of these inflammatory cytokines downstream of IFN.beta.
was dependent, at least in part, on classical NF-.kappa.B
signalling (FIG. 3H). In EMT6 cells, SMC treatment was found to
enhance cellular production of TNF.alpha. (5- to 7-fold percentage
increase) upon VSV infection (FIG. 16). Finally, it was also
demonstrated that blocking TNF-R1 signalling (with antibodies or
siRNA) prevents EMT6 cell death in the presence of SMC and
VSV.DELTA.51 or IFN.beta. (FIGS. 17A-17C and 24H). The relationship
between type I IFN and TNF.alpha. is complex, having either
complimentary or inhibitory effects depending on the biological
context. However, without limiting the present invention to any
particular mechanism of action, a simple working model can be
proposed as follows: Tumor cells infected by an oncolytic RNA virus
up-regulate type I IFN, and this process is not affected by SMC
antagonism of the IAP proteins. Those IFNs in turn signal to
neighboring, uninfected cancer cells to express and secrete
TNF.alpha. and TRAIL, a process that is enhanced by SMC treatment,
which consequently induces autocrine and paracrine programmed cell
death in uninfected tumor cells exposed to SMC (FIGS. 18A and
18B).
TABLE-US-00004 TABLE 4 VSV IFN.beta. Gene Name Gene Identification
25465.4 1017.8 CCL8 Chemokine (C-C motif) ligand 8 13388.9 44.9
IL29 Interleukin 29 (interferon, lambda 1) 5629.3 24.3 IFNB1
Interferon, beta 1, fibroblast 1526.8 16.2 TNFSF15 Tumor necrosis
factor (ligand) superfamily, member 15 847 24.6 CCL5 Chemokine (C-C
motif) ligand 5 747.7 17.2 CCL3 Chemokine (C-C motif) ligand 3
650.9 60.6 TNFSF10 Tumor necrosis factor (ligand) superfamily,
member 10 421.3 296.1 IL12A Interleukin 12A 289.3 10.7 TNFSF18
Tumor necrosis factor (ligand) superfamily, member 18 255.3 18.8
CCL7 Chemokine (C-C motif) ligand 7 154.2 19.2 IL6 Interleukin 6
(interferon, beta 2) 150.8 12.9 IL1RN Interleukin 1 receptor
antagonist 108.1 25.5 CCL20 Chemokine (C-C motif) ligand 20 78.6
6.2 CXCL1 Chemokine (C-X-C motif) ligand 1 64.7 14.8 CCL2 Chemokine
(C-C motif) ligand 2 62.5 14.5 CCL4 Chemokine (C-C motif) ligand 4
55.6 1.2 CXCL3 Chemokine (C-X-C motif) ligand 3 55.2 4.3 TNF Tumor
necrosis factor (TNF superfamily, member 2) 48.8 4.3 IGF1
Insulin-like growth factor 1 (somatomedin C) 48.4 2.8 CXCL2
Chemokine (C-X-C motif) ligand 2 38.5 3.8 CCL11 Chemokine (C-C
motif) ligand 11 37.5 3.8 HGF Hepatocyte growth factor 36.5 75.1
NGFB Nerve growth factor, beta polypeptide 32.9 4 FGF14 Fibroblast
growth factor 14 24.7 25.6 FGF20 Fibroblast growth factor 20 21.5
16.4 IL1B Interleukin 1, beta 20 36.3 CSF2 Colony stimulating
factor 2 (granulocyte-macrophage) 18.3 2.6 GDF3 Growth
differentiation factor 3 17.2 2 CCL28 Chemokine (C-C motif) ligand
28 12 2.1 CCL22 Chemokine (C-C motif) ligand 22 11.3 2.5 CCL17
Chemokine (C-C motif) ligand 17 10.5 2 CCL13 Chemokine (C-C motif)
ligand 13 10.5 15.3 IL20 Interleukin 20 9.7 22.8 FGF16 Fibroblast
growth factor 16 8.8 3.6 TNFSF14 Tumor necrosis factor (ligand)
superfamily, member 14 8.2 2.7 FGF2 Fibroblast growth factor 2
(basic) 7.1 8.1 BDNF Brain-derived neurotrophic factor 7.1 9.7 IL1A
Interleukin 1, alpha 7.1 10.9 ANGPT4 Angiopoietin 4 7 1.5 TGFB3
Transforming growth factor, beta 3 7 5.8 IL22 Interleukin 22 6.9
9.7 IL1F5 Interleukin 1 family, member 5 (delta) 6.7 2.4 IFNW1
Interferon, omega 1 6.6 12.6 IL11 Interleukin 11 6.6 25.1 IL1F8
Interleukin 1 family, member 8 (eta) 6.3 -1.3 EDA Ectodysplasin A
5.9 8 FGF5 Fibroblast growth factor 5 5.8 5 VEGFC Vascular
endothelial growth factor C 5.2 4.9 LIF Leukemia inhibitory factor
5 1.3 CCL25 Chemokine (C-C motif) ligand 25 4.9 8.3 BMP3 Bone
morphogenetic protein 3 4.9 1.6 IL17C Interleukin 17C 4.8 -2.3
TNFSF7 CD70 molecule 4.3 2.5 TNFSF8 Tumor necrosis factor (ligand)
superfamily, member 8 4.3 2.5 FASLG Fas ligand (TNF superfamily,
member 6) 4.2 2.7 BMP8B Bone morphogenetic protein 8b 4.2 6 IL7
Interleukin 7 4.1 5.2 CCL24 Chemokine (C-C motif) ligand 24 4 -2.2
INHBE Inhibin, beta E 4 5.8 IL23A Interleukin 23, alpha subunit p19
3.8 -1.1 IL17F Interleukin 17F 3.7 2.9 CCL21 Chemokine (C-C motif)
ligand 21 3.5 8.5 CSF1 Colony stimulating factor 1 (macrophage) 3.5
3 IL15 Interleukin 15 3.4 5.7 NRG2 Neuregulin 2 3.3 N/A INHBB
Inhibin, beta B 3.3 N/A LTB Lymphotoxin beta (TNF superfamily,
member 3) 3.3 N/A BMP7 Bone morphogenetic protein 7 3 -3.8 IL1F9
Interleukin 1 family, member 9 2.9 6.1 IL12B Interleukin 12B 2.8
6.2 FLT3LG Fms-related tyrosine kinase 3 ligand 2.7 3 FGF1
Fibroblast growth factor 1 (acidic) 2.5 -2 CXCL13 Chemokine (C-X-C
motif) ligand 13 2.4 2.2 IL17B Interleukin 17B 2.3 7.8 GDNF Glial
cell derived neurotrophic factor 2.3 -1.7 GDF7 Growth
differentiation factor 7 2.3 -2.4 LTA Lymphotoxin alpha (TNF
superfamily, member 1) 2.2 1.7 LEFTY2 Left-right determination
factor 2 2.1 5 FGF19 Fibroblast growth factor 19 2.1 9.8 FGF23
Fibroblast growth factor 23 2.1 4.8 CLC Cardiotrophin-like cytokine
factor 1 2.1 3 ANGPT1 Angiopoietin 1 2 10.6 TPO Thyroid peroxidase
2 2.1 EFNA5 Ephrin-A5 1.9 6.4 IL1F10 Interleukin 1 family, member
10 (theta) 1.9 7.6 LEP Leptin (obesity homolog, mouse) 1.8 3 IL5
Interleukin 5 (colony-stimulating factor, eosinophil) 1.8 5.7 IFNE1
Interferon epsilon 1 1.8 2.7 EGF Epidermal growth factor
(beta-urogastrone) 1.7 3.4 CTF1 Cardiotrophin 1 1.7 -1.9 BMP2 Bone
morphogenetic protein 2 1.7 3 EFNB2 Ephrin-B2 1.6 1 FGF8 Fibroblast
growth factor 8 (androgen-induced) 1.6 -2 TGFB2 Transforming growth
factor, beta 2 1.5 -1.6 BMP8A Bone morphogenetic protein 8a 1.5 3.3
NTF5 Neurotrophin 5 (neurotrophin 4/5) 1.5 1 GDF10 Growth
differentiation factor 10 1.5 1.5 TNFSF13B Tumor necrosis factor
(ligand) superfamily, member 13b 1.5 2.5 IFNA1 Interferon, alpha 1
1.4 -1.3 INHBC Inhibin, beta C 1.4 2.8 FGF7 Galactokinase 2 1.4 3.3
IL24 Interleukin 24 1.4 -1.1 CCL27 Chemokine (C-C motif) ligand 27
1.3 1.9 FGF13 Fibroblast growth factor 13 1.3 1.4 IFNK Interferon,
kappa 1.3 2 ANGPT2 Angiopoietin 2 1.3 7.6 IL18 Interleukin 18
(interferon-gamma-inducing factor) 1.3 7 NRG1 Neuregulin 1 1.3 4.9
NTF3 Neurotrophin 3 1.2 15 FGF10 Fibroblast growth factor 10 1.2
1.9 KITLG KIT ligand 1.2 -1.3 IL17D Interleukin 17D 1.2 1.1 TNFSF4
Tumor necrosis factor (ligand) superfamily, member 4 1.2 1.3 VEGFA
Vascular endothelial growth factor 1.1 2.4 FGF11 Fibroblast growth
factor 11 1.1 -1.4 IL17E Interleukin 17E 1.1 -2.1 TGFB1
Transforming growth factor, beta 1 1 3.1 GH1 Growth hormone 1 -1
6.1 IL9 Interleukin 9 -1 -2.5 EFNB3 Ephrin-B3 -1 1.8 VEGFB Vascular
endothelial growth factor B -1 -1.2 IL1F7 Interleukin 1 family,
member 7 (zeta) -1 -2.1 GDF11 Growth differentiation factor 11 -1.1
1.3 ZFP91 Zinc finger protein 91 homolog (mouse) -1.2 -1.1 BMP6
Bone morphogenetic protein 6 -1.2 -1.2 AMH Anti-Mullerian hormone
-1.3 -1 LEFTY1 Left-right determination factor 1 -1.3 2.4 EFNA3
Ephrin-A3 -1.3 -1.3 LASS1 LAG1 longevity assurance homolog 1 -1.5 1
EFNA4 Ephrin-A4 -1.8 1.3 PDGFD DNA-damage inducible protein 1 -1.8
1.8 IL10 Interleukin 10 -1.9 1.6 GDF5 Growth differentiation factor
5 -1.9 1.3 EFNA2 Ephrin-A2 -1.9 -1.5 EFNB1 Ephrin-B1 -1.9 -1.4 GDF8
Growth differentiation factor 8 -1.9 1.6 PDGFC Platelet derived
growth factor C -2.2 2.4 TSLP Thymic stromal lymphopoietin -2.3
-1.5 BMP10 Bone morphogenetic protein 10 -2.4 -4.6 CXCL12 Chemokine
(C-X-C motif) ligand 12 -2.5 4 IFNG Interferon, gamma -2.6 1.2 EPO
Erythropoietin -2.7 -2.1 GAS6 Growth arrest-specific 6 -2.9 2.9 PRL
Prolactin -2.9 -2.1 BMP4 Bone morphogenetic protein 4 -2.9 -5.7
INHA Inhibin, alpha -3 -1.3 GDF9 Growth differentiation factor 9
-3.1 -1.5 FGF18 Fibroblast growth factor 18 -3.2 N/A IL17
Interleukin 17 -3.2 -1.1 IL26 Interleukin 26 -3.4 1.2 EFNA1
Ephrin-A1 -3.8 -1.1 FGF12 Fibroblast growth factor 12 -4 -2.3 FGF9
Fibroblast growth factor 9 (glia-activating factor) -4.5 1.4 CCL26
Chemokine (C-C motif) ligand 26 -8 9.7 CCL19 Chemokine (C-C motif)
ligand 19 N/A N/A BMP15 Bone morphogenetic protein 15 N/A N/A CCL15
Chemokine (C-C motif) ligand 14 N/A N/A CCL16 Chemokine (C-C motif)
ligand 16 N/A N/A CCL18 Chemokine (C-C motif) ligand 18 N/A N/A
CCL23 Chemokine (C-C motif) ligand 23 N/A N/A CD40LG CD40 ligand
(TNF superfamily) N/A N/A CSF3 Colony stimulating factor 3
(granulocyte) N/A N/A CXCL5 Chemokine (C-X-C motif) ligand 5 N/A
N/A FGF4 Fibroblast growth factor 4 N/A N/A FGF6 Fibroblast growth
factor 6 N/A N/A GH2 Growth hormone 2 N/A N/A IL2 Interleukin 2 N/A
N/A IL21 Interleukin 21 N/A N/A IL28A Interleukin 28A (interferon,
lambda 2) N/A N/A INHBA Inhibin, beta A N/A N/A NRG3 Neuregulin 3
N/A N/A TNFSF11 Tumor necrosis factor (ligand) superfamily, member
11 N/A N/A TNFSF13 Tumor necrosis factor (ligand) superfamily,
member 13 N/A 6.5 NRG4 Neuregulin 4 N/A 6.1 IL3 Interleukin 3
(colony-stimulating factor, multiple) N/A 1.8 TNFSF9 Tumor necrosis
factor (ligand) superfamily, member 9
Oncolytic VSV Potentiates SMC Therapy in Preclinical Animal Models
of Cancer
[0130] To evaluate SMC and oncolytic VSV co-therapy in vivo, the
EMT6 mammary carcinoma was used as a syngeneic, orthotopic model.
Preliminary safety and pharmacodynamic experiments revealed that a
dose of 50 mg/kg LCL161 delivered by oral gavage was well tolerated
and induced cIAP1/2 knockdown in tumors for at least 24 hrs, and up
to 48-72 hours in some cases (FIGS. 19A, 19B, and 24G). When tumors
reached .about.100 mm.sup.3, we began treating mice twice weekly
with SMC and VSV.DELTA.51, delivered systemically. As single
agents, SMC therapy led to a decrease in the rate of tumor growth
and a modest extension in survival, while VSV.DELTA.51 treatments
had no bearing on tumor size or survival (FIGS. 4A and 4B). In
stark contrast, combined SMC and VSV.DELTA.51 treatment induced
dramatic tumor regressions and led to durable cures in 40% of the
treated mice. Consistent with the bystander killing mechanism
elucidated in vitro, immunofluorescence analyses revealed that the
infectivity of VSV.DELTA.51 was transient and limited to small foci
within the tumor (FIG. 4C), whereas caspase-3 activation was
widespread in the SMC and VSV.DELTA.51 co-treated tumors (FIG. 4D).
Furthermore, immunoblots with tumor lysates demonstrated activation
of caspase-8 and -3 in doubly-treated tumors (FIGS. 4E, 24B, and
24G). While the animals in the combination treatment cohort
experienced weight loss, the mice fully recovered following the
last treatment (FIG. 20A).
[0131] To confirm these in vivo data in another model system, the
human HT-29 colorectal adenocarcinoma xenograft model was tested in
nude (athymic) mice. HT-29 is a cell line that is highly responsive
to bystander killing by SMC and VSV.DELTA.51 co-treatment in vitro
(FIGS. 21A and 21B). Similar to our findings in the EMT6 model
system, combination therapy with SMC and VSV.DELTA.51 induced tumor
regression and a significant extension of mouse survival (FIG.
21C). In contrast, neither monotherapy had any effect on HT-29
tumors. Furthermore, there was no additional weight loss in the
double treated mice compared to SMC treated mice (FIG. 21D). These
results indicate that the synergy is highly efficacious in a
refractory xenograft model and that the adaptive immune response
does not have a major role initially in the efficacy of SMC and OV
co-therapy.
Role of the Innate Antiviral Responses and Immune Effectors in
Co-Treatment Synergy
[0132] It was next determined whether oncolytic VSV infection
coupled with SMC treatment leads to TNF.alpha.- or
IFN.beta.-mediated cell death in vivo. It was investigated whether
blocking TNF.alpha. signalling via neutralizing antibodies would
affect SMC and VSV.DELTA.51 synergy in the EMT6 tumor model.
Compared to isotype matched antibody controls, the application of
TNF.alpha. neutralizing antibodies reverted the tumor regression
and decreased the survival rate to values close to the control and
single treatment groups (FIGS. 4F and 4G). This demonstrates that
TNF.alpha. is required in vivo for the anti-tumor combination
efficacy of SMC and oncolytic VSV.
[0133] To investigate the role of IFN.beta. signaling in the SMC
and OV combination paradigm, Balb/c mice bearing EMT6 tumors were
treated with IFNAR1 blocking antibodies. Mice treated with the
IFNAR1 blocking antibody succumbed to viremia within 24-48 hours
post infection. Prior to death, tumors were collected at 18-20
hours after virus infection, and the tumors were analyzed for
caspase activity. Even though these animals with defective type I
IFN signaling were ill due to a large viral burden, the excised
tumors did not demonstrate signs of caspase-8 activity and only
showed minimal signs of caspase-3 activity (FIG. 22) in contrast to
the control group, which showed the expected activation of caspases
within the tumor (FIG. 22). These results support the hypothesis
that intact type I IFN signaling is required to mediate the
anti-tumor effects of the combination approach.
[0134] To assess the contribution of innate immune cells or other
immune mediators to the efficacy of OV/SMC combination therapy,
treating EMT6 tumors was first attempted in immunodeficient
NOD-scid or NSG (NOD-scid-IL2Rgamma.sup.null) mice. However,
similar to the IFNAR1 depletion signaling studies, these mice also
died rapidly due to viremia. Therefore, the contribution of innate
immune cells was addressed by employing an ex vivo splenocyte
culture system as a surrogate model. Innate immune populations that
have the capacity to produce TNF.alpha. were positively selected
and further sorted from naive splenocytes. Macrophages (CD11b+
F4/80+), neutrophils (CD11b+ Gr1+), NK cells (CD11b- CD49b+) and
myeloid-negative (lymphoid) population (CD11b- CD49-) were
stimulated with VSV.DELTA.51, and the conditioned medium was
transferred to EMT6 cells to measure cytotoxicity in the presence
of SMC. These results show that VSV.DELTA.51-stimulated macrophages
and neutrophils, but not NK cells, are capable of producing factors
that lead to cancer cell death in the presence of SMCs (FIG. 23A).
Primary macrophages from bone marrow were also isolated and these
macrophages also responded to oncolytic VSV infection in a
dose-dependent manner to produce factors which kill EMT6 cells
(FIG. 23B). Altogether, these findings demonstrate that multiple
innate immune cell populations can respond to mediate the observed
anti-tumor effects, and that macrophages are the most likely
effectors of this response.
Immune Adjuvants Poly(I:C) and CpG Potentiate SMC Therapy In
Vivo
[0135] It was next investigated whether synthetic TLR agonists,
which are known to induce an innate proinflammatory response, would
synergize with SMC therapy. EMT6 cells were co-cultured with mouse
splenocytes in a transwell insert system, and the splenocytes were
treated with SMC and agonists of TLR 3, 4, 7 or 9. All of the
tested TLR agonists were found to induce the bystander death of SMC
treated EMT6 cells (FIG. 5A). The TLR4, 7, and 9 agonists LPS,
imiquimod, and CpG, respectively, required splenocytes to induce
bystander killing of EMT6 cells, presumably because their target
TLR receptors are not expressed in EMT6 cells. However, the TLR3
agonist poly(I:C) led to EMT6 cell death directly in the presence
of SMCs. Poly(I:C) and CpG were next tested in combination with SMC
therapy in vivo. These agonists were chosen as they have proven to
be safe in humans and are currently in numerous mid to late stage
clinical trials for cancer. EMT6 tumors were established and
treated as described above. While poly(I:C) treatment had no
bearing on tumor growth as a single agent, combination with SMCs
induced substantial tumor regression and, when delivered
intraperitoneally, led to durable cures in 60% of the treated mice
(FIGS. 5B and 5C). Similarly, CpG monotherapy had no bearing on
tumor size or survival, but when combined with SMC therapy led to
tumor regressions and durable cures in 88% of the treated mice
(FIGS. 5D and 5E). Importantly, these combination therapies were
well tolerated by the mice, and their body weight returned to
pre-treatment levels shortly after the cessation of therapy (FIGS.
20B and 20C). Taken together with the oncolytic VSV results, the
data demonstrate that a series of clinically advanced innate immune
adjuvants strongly and safely synergize with SMC therapy in vivo,
inducing tumor regression and durable cures in several treatment
refractory, aggressive mouse models of cancer.
Example 2: Inactivated Viral Particles, Cancer Vaccines, and
Stimulatory Cytokines Synergize with SMCs to Kill Tumors
[0136] The use of current cancer immunotherapies, such as BCG
(Bacillus Calmette-Guerin), recombinant interferon (e.g.
IFN.alpha.), and recombinant Tumor Necrosis Factor (e.g. TNF.alpha.
used in isolated limb perfusion for example), and the recent
clinical use of biologics (e.g. blocking antibodies) to immune
checkpoint inhibitors that overcome tumor-mediated suppression of
the immune system (such as anti-CTLA-4 and anti-PD-1 or PDL-1
monoclonal antibodies) highlight the potential of `cancer
immunotherapy` as an effective treatment modality. As shown in
Example 1, we have demonstrated the robust potential of non-viral
immune stimulants to synergize with SMCs (FIGS. 5A-5E). To expand
on these studies, we also examined for the potential of SMCs to
synergize with non-replicating rhabdovirus-derived particles
(called NRRPs), which are UV-irradiated VSV particles that retain
their infectious and immunostimulatory properties without the
ability to replicate and spread. To assess if NRRPs directly
synergize with SMCs, we co-treated various cancer cell lines, EMT6,
DBT, and CT-2A, with SMCs and differing levels of NRRPs, and
assessed cell viability by Alamar blue. We observed that NRRPs
synergize with SMCs in these cancer cell lines (FIG. 25A). To
assess if NRRPs can induce a potent proinflammatory response, we
treated fractionated mouse splenocytes with NRRPs (or synthetic CpG
ODN 2216 as a positive control), transferred the cell culture
supernatants to EMT6 cells in culture in a dose-response fashion,
and treated the cells with vehicle or SMC. We observed that the
immunogenicity of NRRPs is at a similar level of CpG, as there was
a considerable proinflammatory response, which led to a high degree
of EMT6 cell death in the presence of SMCs (FIG. 25B). As the
treatment of CpG and SMC in the EMT6 tumor model resulted in a 88%
cure rate (FIG. 5D), these findings suggest that the combination of
SMCs and NRRPs can be highly synergistic in vivo.
[0137] Our success in finding synergy between SMCs and live or
inactivated single-stranded RNA oncolytic rhabdoviruses (e.g.,
VSV.DELTA.51, Maraba-MG1, and NRRPs) suggested that a clinic
approved attenuated vaccine may be able to synergize with SMCs. To
test this possibility, we assessed the ability to synergize with
SMCs of the cancer biologic, the vaccine for tuberculosis
mycobacterium, BCG, which is typically used to treat bladder cancer
in situ due to the high local production of TNF.alpha.. Indeed, the
combination of SMC and BCG potently synergises to kill EMT6 cells
in vitro (FIG. 26A). These findings were similarly extended in
vivo; we observed significant tumor regression with combined
treatment of an oral SMC and BCG administered locally or
systemically (i.e., either given intratumorally or
intraperitoneally, respectively) (FIG. 26B). These findings attest
to the applicability of approved vaccines for combination cancer
immunotherapies with SMCs.
Type I IFN Synergizes with SMCs In Vivo
[0138] The effects of viruses, and likely other TLR agonists and
vaccines, appear to be mediated, in part, by type I IFN production,
which is controlled by various signaling mechanism, including mRNA
translation. Our findings raised the distinct possibility of
combining SMC treatment with existing immunotherapies, such as
recombinant IFN, as an effective approach to treat cancer. To
explore the potential of this combination, we conducted two
treatment regimens of SMC and either intraperitoneal or
intratumoral injections of recombinant IFN.alpha. in the syngeneic
orthotopic EMT6 mammary carcinoma model. While treatment of
IFN.alpha. had no effect on EMT6 tumor growth or overall survival,
SMC treatment slightly extended mouse survival and had a cure rate
of 17% (FIG. 27A). However, the combined treatment of SMC and
intraperitoneal or intratumoral injections of IFN.alpha.
significantly delayed tumor growth and extended survival of
tumor-bearing mice, resulting in cure rates of 57% and 86%,
respectively (FIG. 27A) These results support the hypothesis that
direct stimulation with type I IFN can synergize with SMCs to
eradicate tumors in vivo.
Assessment of Additional Oncolytic Rhabdoviruses for the Potential
of Synergy with SMCs
[0139] While VSV.DELTA.51 is a preclinical candidate, the oncolytic
rhabdoviruses VSV-IFN.beta. and Maraba-MG1 are currently undergoing
clinical testing in cancer patients. As shown in Example 1, we have
demonstrated that Maraba-MG1 synergizes with SMCs in vitro (FIG.
9A). We also confirmed that SMCs synergized with the clinical
candidates, VSV-IFN.beta. and VSV-NIS-IFN.beta. (i.e. carrying the
imaging gene, NIS, sodium iodide symporter), in EMT6 cells (FIG.
28). To assess whether these viruses can induce a profinflammatory
state in vivo, we treated infected mice i.v. with 5.times.10.sup.8
PFU of VSV.DELTA.51, VSV-IFN.beta., and Maraba-MG1 and measured the
level of TNF.alpha. from the serum of infected mice. In all cases,
there was a transient, but robust increase of TNF.alpha. from
oncolytic virus infection at 12 hrs post-infection, which was
barely detectable by 24 hr (FIG. 29). This makes sense as these
infections are self-limiting in immunocompetent hosts. These
results suggest that the clinical candidate oncolytic rhabdoviruses
have the potential to synergize with SMCs in a fashion similar to
VSV.DELTA.51.
[0140] As shown in Example 1, we documented that a form of
VSV.DELTA.51 that was engineered to express full-length TNF.alpha.
can enhance oncolytic virus induced death in the presence of SMC
(FIGS. 15A and 15B). To expand on these findings, we also
engineered VSV.DELTA.51 to express a form of TNF.alpha. that had
its intracellular and transmembrane components replaced with the
secretory signal from human serum albumin
(VSV.DELTA.51-solTNF.alpha.). Compared to full-length TNF.alpha.
(memTNF.alpha.), solTNF.alpha. is constitutively secreted from host
cells, while the memTNF.alpha. form may be anchored on plasma
membrane (and still capable of inducing cell death in a juxtacrine
manner) or is released due to endogenous processing by
metalloproteases (such as ADAM17) to kill cells in a paracrine
fashion. We assessed whether either forms of TNF.alpha. from
oncolytic VSV infected cells will synergize with SMC in the
orthotopic syngeneic mammary cancer model, EMT6. As expected,
treatment with SMC slightly delayed EMT6 tumor growth rates and
slightly extended the survival of tumor bearing mice, and the
combination of vehicle with either VSV.DELTA.51-memTNF.alpha. or
VSV.DELTA.51-solTNF.alpha. had no impact on overall survival or
tumor growth rates (FIGS. 30A and 30B). On the other hand, virally
expressed TNF.alpha. significantly slowed tumor growth rates and
led to increases in the survival rates of 30% and 70%,
respectively. Notably, the 40% tumor cure rate from combined SMC
and VSV.DELTA.51 (FIG. 4A) required four treatments and a dose of
5.times.10.sup.8 PFU of VSV.DELTA.51. However, the combination of
TNF.alpha.-expressing oncolytic VSV and SMC resulted in a higher
cure rate and was accomplished with two treatment regimens at a
virus dose of 1.times.10.sup.8 PFU. To assess whether this
treatment strategy can be applied to other refractory syngeneic
models, we assessed whether VSV.DELTA.51-solTNF.alpha. synergizes
with SMCs in a subcutaneous model of the mouse colon carcinoma cell
line, CT-26. As expected, we did not observe an impact of tumor
growth rates or survival with VSV.DELTA.51-solTNF.alpha. and
observed a modest decrease of the tumor growth rate and a slight
extension of survival (FIG. 30C). However, we were able to further
delay tumor growth and extend survival of these tumor bearing mice
with the combined treatment of SMC and VSV.DELTA.51-solTNF.alpha..
Hence, the inclusion of a TNF.alpha. transgene within oncolytic
viruses is a significant advantage for the combination of SMC. One
could easily envisage the inclusion of other death ligand
transgenes, such as TRAIL, FasL, or lymphotoxin, into viruses to
synergize with SMCs.
Exploring the Potential of SMCs to Eradicate Brain Tumors
[0141] The combination of SMCs with immune stimulatory agents is
applicable to many different types of cancer, including brain
malignancies for which effective therapies are lacking and for
which immunotherapies hold promise. As a first step, we determined
whether SMCs can cross the blood-brain-barrier (BBB) in a mouse
model of brain tumors, as the BBB is a significant barrier to drug
entry into the brain. We observed the SMC-induced degradation of
cIAP1/2 proteins in intracranial CT-2A tumors several hours after
drug administration, indicative that SMCs are capable of crossing
the BBB to antagonize cIAP1/2 and potentially XIAP within brain
tumors (FIG. 31A). We also demonstrated that the direct injection
of SMC (10 .mu.L of a 100 .mu.M solution) intracranially can result
in the potent down-regulation of both cIAP1/2 and XIAP proteins
(FIG. 31B), which is a direct consequence of SMC-induced
autoubiquitination of the IAPs or the result of tumor cell death
induction in the case of XIAP loss. As a second step, we wished to
determine whether systemic stimulation of immune stimulants can led
to a proinflammatory response in the brain of naive mice. Indeed,
we observed marked up-regulation of TNF.alpha. levels from the
brain from mice that were intraperitoneally injected with the viral
mimic, poly(I:C), a TLR3 agonist (FIG. 32A). We followed up this
finding by extracting crude protein lysates from the brains of mice
that were treated with poly(I:C) or with the clinical candidate
oncolytic rhabdoviruses VSV.DELTA.51, VSV-IFN.beta., or Maraba-MG1,
and then applied these lysates onto CT-2A or K1580 glioblastoma
cells in the presence of SMCs. We observed that the stimulation of
an innate immune response with these non-viral synthetic or
biologic viral agents resulted in enhanced cell death in the
presence of SMCs with these two glioblastoma cell lines (FIG. 32B).
As a third step, we also confirmed that poly(I:C) could be directly
administered intracranially without overt toxicities, which may
provide an even increased cytokine induction at the site of tumors
(FIG. 32C). Finally, we assessed whether the direct immune
stimulation within the brain or systemic stimulation would lead to
durable cures in SMC-treated mouse models of brain cancer. The
combination of SMCs orally and poly(I:C) intracranially or
VSV.DELTA.51 i.v. results in the near complete survival of CT-2A
bearing mouse gliomas (FIGS. 32D and 32E), with an expected
survival rate of 86 and 100%, respectively. As a follow-up to the
observed synergy between SMC and intracranial treatment of
poly(I:C), we also assessed the potential for treatment of CT-2A
gliomas with direct, simultaneous intracranial injections of SMC
and recombinant human IFN.alpha. (B/D). Indeed, we observed a
marked positive impact of mouse survival with the combined
treatment, with a cure rate of 50% (FIG. 33). Importantly, the
single or combined SMC or IFN.alpha. treatment did not result in
any overt neurotoxicity in these tumor bearing mice. Overall, these
results reveal that multiple modes of SMC treatment can synergize
with a multitude of locally or systemically administered innate
immunostimulants to kill cancer cell in vitro and to eradicate
tumors in animal models of cancer.
Methods
Reagents
[0142] Novartis provided LCL161 (Houghton, P. J. et al. Initial
testing (stage 1) of LCL161, a SMAC mimetic, by the Pediatric
Preclinical Testing Program. Pediatr Blood Cancer 58: 636-639
(2012); Chen, K. F. et al. Inhibition of Bcl-2 improves effect of
LCL161, a SMAC mimetic, in hepatocellular carcinoma cells.
Biochemical Pharmacology 84: 268-277 (2012)). SM-122 and SM-164
were provided by Dr. Shaomeng Wang (University of Michigan, USA)
(Sun, H. et al. Design, synthesis, and characterization of a
potent, nonpeptide, cellpermeable, bivalent Smac mimetic that
concurrently targets both the BIR2 and BIR3 domains in XIAP. J Am
Chem Soc 129: 15279-15294 (2007)). AEG40730 (Bertrand, M. J. et al.
cIAP1 and cIAP2 facilitate cancer cell survival by functioning as
E3 ligases that promote RIP1 ubiquitination. Mol Cell 30: 689-700
(2008)) was synthesized by Vibrant Pharma Inc (Brantford, Canada).
OICR720 was synthesized by the Ontario Institute for Cancer
Research (Toronto, Canada) (Enwere, E. K. et al. TWEAK and cIAP1
regulate myoblast fusion through the noncanonical NF-kappaB
signalling pathway. Sci Signal 5: ra75 (2013)). IFN.alpha.,
IFN.beta., IL28 and IL29 were obtained from PBL Interferonsource
(Piscataway, USA). All siRNAs were obtained from Dharmacon (Ottawa,
Canada; ON TARGETplus SMARTpool). CpG-ODN 2216 was synthesized by
IDT (5'-gggGGACGATCGTCgggggg-3' (SEQ ID NO: 1), lowercase indicates
phosphorothioate linkages between these nucleotides, while italics
identify three CpG motifs with phosphodiester linkages). Imiquimod
was purchased from BioVision Inc. (Milpitas, USA). poly(I:C) was
obtained from InvivoGen (San Diego, USA). LPS was from Sigma
(Oakville, Canada).
Cell Culture
[0143] Cells were maintained at 37.degree. C. and 5% CO2 in DMEM
media supplemented with 10% heat inactivated fetal calf serum,
penicillin, streptomycin, and 1% non-essential amino acids
(Invitrogen, Burlington, USA). All of the cell lines were obtained
from ATCC, with the following exceptions: SNB75 (Dr. D. Stojdl,
Children's Hospital of Eastern Ontario Research Institute) and
SF539 (UCSF Brain Tumor Bank). Cell lines were regularly tested for
mycoplasma contamination. For siRNA transfections, cells were
reverse transfected with Lipofectamine RNAiMAX (Invitrogen) or
DharmaFECT I (Dharmacon) for 48 hours as per the manufacturer's
protocol.
Viruses
[0144] The Indiana serotype of VSV.DELTA.51 (Stojdl, D. F. et al.
VSV strains with defects in their ability to shutdown innate
immunity are potent systemic anti-cancer agents. Cancer Cell 4(4),
263-275 (2003)) was used in this study and was propagated in Vero
cells. VSV.DELTA.51-GFP is a recombinant derivative of VSV.DELTA.51
expressing jellyfish green fluorescent protein. VSV.DELTA.51-Fluc
expresses firefly luciferase. VSV.DELTA.51 with the deletion of the
gene encoding for glycoprotein (VSV.DELTA.51.DELTA.G) was
propagated in HEK293T cells that were transfected with pMD2-G using
Lipofectamine2000 (Invitrogen). To generate the
VSV.DELTA.51-TNF.alpha. construct, full-length human TNF.alpha.
gene was inserted between the G and L viral genes. All VSV.DELTA.51
viruses were purified on a sucrose cushion. Maraba-MG1, VVDD-B18R-,
Reovirus and HSV1 ICP34.5 were generated as previously described
(Brun, J. et al. Identification of genetically modified Maraba
virus as an oncolytic rhabdovirus. Mol Ther 18, 1440-1449 (2010);
Le Boeuf, F. et al. Synergistic interaction between oncolytic
viruses augments tumor killing. Mol Ther 18, 888-895 (2011); Lun,
X. et al. Efficacy and safety/toxicity study of recombinant
vaccinia virus JX-594 in two immunocompetent animal models of
glioma. Mol Ther 18, 1927-1936 (2010)). Generation of adenoviral
vectors expressing GFP or co-expressing GFP and dominant negative
IKK.beta. was as previously described 16.
In Vitro Viability Assay
[0145] Cell lines were seeded in 96-well plates and incubated
overnight. Cells were treated with vehicle (0.05% DMSO) or 5 .mu.M
LCL161 and infected with the indicated MOI of OV or treated with
250 U/mL IFN.beta., 500 U/mL IFN.alpha., 500 U/mL IFN.gamma., 10
ng/mL IL28, or 10 ng/mL IL29 for 48 hours. Cell viability was
determined by Alamar blue (Resazurin sodium salt (Sigma)) and data
was normalized to vehicle treatment. The chosen sample size is
consistent with previous reports that used similar analyses for
viability assays. For combination indices, cells were seeded
overnight, treated with serial dilutions of a fixed combination
mixture of VSV.DELTA.51 and LCL161 (5000:1, 1000:1 and 400:1 ratios
of PFU VSV.DELTA.51: .mu.M LCL161) for 48 hours and cell viability
was assessed by Alamar blue. Combination indices (CI) were
calculated according to the method of Chou and Talalay using
Calcusyn (Chou, T. C. & Talaly, P. A simple generalized
equation for the analysis of multiple inhibitions of
Michaelis-Menten kinetic systems. J Biol Chem 252, 6438-6442
(1977)). An n=3 of biological replicates was used to determine
statistical measures (mean with standard deviation or standard
error).
Spreading Assay
[0146] A confluent monolayer of 786-0 cells was overlaid with 0.7%
agarose in complete media. A small hole was made with a pipette in
the agarose overlay in the middle of the well where 5.times.103 PFU
of VSV.DELTA.51-GFP was administered. Media containing vehicle or 5
.mu.M LCL161 was added on top of the overlay, cells were incubated
for 4 days, fluorescent images were acquired, and cells were
stained with crystal violet.
Splenocyte Co-Culture
[0147] EMT6 cells were cultured in multiwell plates and overlaid
with cell culture inserts containing unfractionated splenocytes.
Briefly, single-cell suspensions were obtained by passing mouse
spleens through 70 .mu.m nylon mesh and red blood cells were lysed
with ACK lysis buffer. Splenocytes were treated for 24 hr with
either 0.1 MOI of VSV.DELTA.51.DELTA.G, 1 .mu.g/mL poly(I:C), 1
.mu.g/mL LPS, 2 .mu.M imiquimod, or 0.25 .mu.M CpG prior in the
presence of 1 .mu.M LCL161. EMT6 cell viability was determined by
crystal violet staining. An n=3 of biological replicates was used
to determine statistical measures (mean, standard deviation).
Cytokine Responsiveness Bioassay
[0148] Cells were infected with the indicated MOI of VSV.DELTA.51
for 24 hours and the cell culture supernatant was exposed to UV
light for 1 hour to inactive VSV.DELTA.51 particles. Subsequently,
the UV-inactivated supernatant was applied to naive cells in the
presence of 5 .mu.M LCL161 for 48 hours. Cell viability was
assessed by Alamar blue. An n=3 of biological replicates was used
to determine statistical measures (mean, standard deviation).
Microscopy
[0149] To measure caspase-3/7 activation, 5 .mu.M LCL161, the
indicated MOI of VSV.DELTA.51, and 5 .mu.M CellPlayer Apoptosis
Caspase-3/7 reagent (Essen Bioscience, Ann Arbor, USA) were added
to the cells. Cells were placed in an incubator outfitted with an
IncuCyte Zoom microscope with a 10.times. objective and
phase-contrast and fluorescence images were acquired over a span of
48 hours. Alternatively, cells were treated with 5 .mu.M LCL161 and
0.1 MOI of VSV.DELTA.51-GFP and SMC for 36 hours and labeled with
the Magic Red Caspase-3/7 Assay Kit (ImmunoChemsitry Technologies,
Bloomington, USA). To measure the proportion of apoptotic cells, 1
.mu.g/mL Annexin V-CF594 (Biotium, Hayward, USA) and 0.2 .mu.M
YOYO-1 (Invitrogen) was added to SMC and VSV.DELTA.51 treated
cells. Images were acquired 24 hours post-treatment using the
IncuCyte Zoom. Enumeration of fluorescence signals was processed
using the integrated object counting algorithm within the IncuCyte
Zoom software. An n=12 (caspase-3/7) or n=9 (Annexin V, YOYO-1) of
biological replicates was used to determine statistical measures
(mean, standard deviation).
Multiple Step Growth Curves
[0150] Cells were treated with vehicle or 5 .mu.M LCL161 for 2
hours and subsequently infected at the indicated MOI of
VSV.DELTA.51 for 1 hour. Cells were washed with PBS, and cells were
replenished with vehicle or 5 .mu.M LCL161 and incubated at
37.degree. C. Aliquots were obtained at the indicated times and
viral titers assessed by a standard plaque assay using African
green monkey VERO cells.
Western Immunoblotting
[0151] Cells were scraped, collected by centrifugation and lysed in
RIPA lysis buffer containing a protease inhibitor cocktail (Roche,
Laval, Canada). Equal amounts of soluble protein were separated on
polyacrylamide gels followed by transfer to nitrocellulose
membranes. Individual proteins were detected by western
immunoblotting using the following antibodies: pSTAT1 (9171),
caspase-3 (9661), caspase-8 (9746), caspase-9 (9508), DR5 (3696),
TNF-R1 (3736), cFLIP (3210), and PARP (9541) from Cell Signalling
Technology (Danvers, USA); caspase-8 (1612) from Enzo Life Sciences
(Farmingdale, USA); IFNAR1 (EP899) and TNF-R1 (19139) from Abcam
(Cambridge, USA); caspase-8 (AHZ0502) from Invitrogen; cFLIP (clone
NF6) from Alexis Biochemicals (Lausen, Switzerland); RIP1 (clone
38) from BD Biosciences (Franklin Lakes, USA); and E7 from
Developmental Studies Hybridoma Bank (Iowa City, USA). Our rabbit
anti-rat IAP1 and IAP3 polyclonal antibodies were used to detect
human and mouse cIAP1/2 and XIAP, respectively. AlexaFluor680
(Invitrogen) or IRDye800 (Li-Cor, Lincoln, USA) were used to detect
the primary antibodies, and infrared fluorescent signals were
detected using the Odyssey Infrared Imaging System (Li-Cor).
RT-qPCR
[0152] Total RNA was isolated from cells using the RNAEasy Mini
Plus kit (Qiagen, Toronto, Canada). Two-step RT-qPCR was performed
using Superscript III (Invitrogen) and SsoAdvanced SYBR Green
supermix (BioRad, Mississauga, Canada) on a Mastercycler ep
realplex (Eppendorf, Mississauga, Canada). All primers were
obtained from realtimeprimers.com. An n=3 of biological replicates
was used to determine statistical measures (mean, standard
deviation).
ELISA
[0153] Cells were infected with virus at the indicated MOI or
treated with IFN.beta. for 24 hours and clarified cell culture
supernatants were concentrated using Amicon Ultra filtration units.
Cytokines were measured with the TNF.alpha. Quantikine high
sensitivity, TNF.alpha. DuoSet, TRAIL DuoSet (R&D Systems,
Minneapolis, USA) and VeriKine IFN.beta. (PBL Interferonsource)
assay kits. An n=3 of biological replicates was used to determine
statistical analysis.
EMT6 Mammary Tumor Model
[0154] Mammary tumors were established by injecting 1.times.105
wild-type EMT6 or firefly luciferase-tagged EMT6 (EMT6-Fluc) cells
in the mammary fat pad of 6-week old female BALB/c mice. Mice with
palpable tumors (.about.100 mm.sup.3) were co-treated with either
vehicle (30% 0.1 M HCl, 70% 0.1 M NaOAc pH 4.63) or 50 mg/kg LCL161
per os and either i.v. injections of either PBS or 5.times.108 PFU
of VSV.DELTA.51 twice weekly for two weeks. For poly(I:C) 25 and
SMC treatments, animals were treated with LCL161 twice a week and
either BSA (i.t.), 20 ug poly(I:C) i.t. or 2.5 mg/kg poly(I:C) i.p.
four times a week. The SMC and CpG group was injected with 2 mg/kg
CpG (i.p.) and the next day was followed with CpG and SMC
treatments. The CpG and SMC treatments were repeated 4 days later.
Treatment groups were assigned by cages and each group had min
n=4-8 for statistical measures (mean, standard error; Kaplan-Meier
with log rank analysis). The sample size is consistent with
previous reports that examined tumor growth and mouse survival
following cancer treatment. Blinding was not possible. Animals were
euthanized when tumors metastasized intraperitoneally or when the
tumor burden exceeded 2000 mm.sup.3. Tumor volume was calculated
using (.pi.)(W).sup.2(L)/4 where W=tumor width and L=tumor length.
Tumor bioluminescence imaging was captured with a Xenogen2000 IVIS
CCD-camera system (Caliper Life Sciences Massachusetts, USA)
following i.p. injection of 4 mg luciferin (Gold Biotechnology, St.
Louis, USA).
HT-29 Subcutaneous Tumor Model
[0155] Subcutaneous tumors were established by injecting
3.times.106 HT-29 cells in the right flank of 6-week old female
CD-1 nude mice. Palpable tumors (.about.200 mm3) were treated with
five intratumoral injections (i.t.) of PBS or 1.times.108 PFU of
VSV.DELTA.51. Four hours later, mice were administered vehicle or
50 mg/kg LCL161 per os. Treatment groups were assigned by cages and
each group had min n=5-7 for statistical measures (mean, standard
error; Kaplan-Meier with log rank analysis). The sample size is
consistent with previous reports that examined tumor growth and
mouse survival following cancer treatment. Blinding was not
possible. Animals were euthanized when tumor burden exceeded 2000
mm.sup.3. Tumor volume was calculated using (.pi.)(W).sup.2(L)/4
where W=tumor width and L=tumor length.
[0156] All animal experiments were conducted with the approval of
the University of Ottawa Animal Care and Veterinary Service in
concordance with guidelines established by the Canadian Council on
Animal Care.
Antibody-Mediated Cytokine Neutralization
[0157] For neutralizing TNF.alpha. signaling in vitro, 25 .mu.g/mL
of .alpha.-TNF.alpha. (XT3.11) or isotype control (HRPN) was added
to EMT6 cells for 1 hour prior to LCL161 and VSV.DELTA.51 or
IFN.beta. co-treatment for 48 hours. Viability was assessed by
Alamar blue. For neutralizing TNF.alpha. in the EMT6-Fluc tumor
model, 0.5 mg of .alpha.-TNF.alpha. or .alpha.-HRPN was
administered 8, 10 and 12 days post-implantation. Mice were treated
with 50 mg/kg LCL161 (p.o.) on 8, 10 and 12 days post-implantation
and were infected with 5.times.108 PFU VSV.DELTA.51 i.v. on days 9,
11 and 13. For neutralization of type I IFN signalling, 2.5 mg of
.alpha.-IFNAR1 (MAR1-5A3) or isotype control (MOPC-21) were
injected into EMT6-tumor bearing mice and treated with 50 mg/kg
LCL161 (p.o.) for 20 hours. Mice were infected with 5.times.108 PFU
VSV.DELTA.51 (i.v.) for 18-20 hours and tumors were processed for
Western blotting. All antibodies were from BioXCell (West Lebanon,
USA).
Flow Cytometry and Sorting
[0158] EMT6 cells were co-treated with 0.1 MOI of VSV.DELTA.51-GFP
and 5 .mu.M LCL161 for 20 hours. Cells were trypsinized,
permeabilized with the CytoFix/CytoPerm kit (BD Biosciences) and
stained with APC-TNF.alpha. (MP6-XT22) (BD Biosciences). Cells were
analyzed on a Cyan ADP 9 flow cytometer (Beckman Coulter,
Mississauga, Canada) and data was analyzed with FlowJo (Tree Star,
Ashland, USA).
[0159] Splenocytes were enriched for CD11b using the EasySep CD11b
positive selection kit (StemCell Technologies, Vancouver, Canada).
CD49+ cells were enriched using the EasySep CD49b positive
selection kit (StemCell Technologies) from the CD11b- fraction.
CD11b+ cells were stained with F4/80-PE-Cy5 (BM8, eBioscience) and
Gr1-FITC (RB6-8C5, BD Biosciences) and further sorted with MoFlo
Astrios (Beckman Coulter). Flow cytometry data was analyzed using
Kaluza (Beckman Coulter). Isolated cells were infected with
VSV.DELTA.51 for 24 hours and clarified cell culture supernatants
were applied to EMT6 cells for 24 hours in the presence of 5 .mu.M
LCL161.
Bone Marrow Derived Macrophages
[0160] Mouse femurs and radius were removed and flushed to remove
bone marrow. Cells were cultured in RPMI with 8% FBS and 5 ng/ml of
M-CSF for 7 days. Flow cytometry was used to confirm the purity of
macrophages (F4/80+ CD11b+).
Immunohistochemistry
[0161] Excised tumors were fixed in 4% PFA, embedded in a 1:1
mixture of OCT compound and 30% sucrose, and sectioned on a
cryostat at 12 .mu.m. Sections were permeablized with 0.1% Triton
X-100 in blocking solution (50 mM Tris-HCl pH 7.4, 100 mM L-lysine,
145 mM NaCl and 1% BSA, 10% goat serum). .alpha.-cleaved caspase 3
(C92-605, BD Pharmingen, Mississauga, Canada) and polyclonal
antiserum VSV (Dr. Earl Brown, University of Ottawa, Canada) were
incubated overnight followed by secondary incubation with
AlexaFluor-coupled secondary antibodies (Invitrogen).
Statistical Analysis
[0162] Comparison of Kaplan-Meier survival plots was conducted by
log-rank analysis and subsequent pairwise multiple comparisons were
performed using the Holm-Sidak method (SigmaPlot, San Jose, USA).
Calculation of EC.sub.50 values was performed in GraphPad Prism
using normalized nonlinear regression analysis. The EC.sub.50 shift
was calculated by subtracting the log.sub.10 EC.sub.50 of
SMC-treated and VSV.DELTA.51-infected cells from log.sub.10
EC.sub.50 of vehicle treated cells infected by VSV.DELTA.51. To
normalize the degree of SMC synergy, the EC.sub.50 value was
normalized to 100% to compensate for cell death induced by SMC
treatment alone.
OTHER EMBODIMENTS
[0163] All publications, patent applications, and patents mentioned
in this specification are herein incorporated by reference.
[0164] While the invention has been described in connection with
the specific embodiments, it will be understood that it is capable
of further modifications. Therefore, this application is intended
to cover any variations, uses, or adaptations of the invention that
follow, in general, the principles of the invention, including
departures from the present disclosure that come within known or
customary practice within the art.
* * * * *
References