U.S. patent application number 13/298492 was filed with the patent office on 2013-05-23 for co-administration of a parvovirus and a cytokine for therapy of pancreatic cancer.
The applicant listed for this patent is Marc Aprahamian, Laurent Daeffler, Nathalia Giese, Svitlana Grekova, Zahari RAYKOV, Jean Rommelaere. Invention is credited to Marc Aprahamian, Laurent Daeffler, Nathalia Giese, Svitlana Grekova, Zahari RAYKOV, Jean Rommelaere.
Application Number | 20130129678 13/298492 |
Document ID | / |
Family ID | 47501037 |
Filed Date | 2013-05-23 |
United States Patent
Application |
20130129678 |
Kind Code |
A1 |
RAYKOV; Zahari ; et
al. |
May 23, 2013 |
CO-ADMINISTRATION OF A PARVOVIRUS AND A CYTOKINE FOR THERAPY OF
PANCREATIC CANCER
Abstract
The application relates to a combination of a parvovirus and a
cytokine, preferably IFN.gamma., for use in treating pancreatic
cancer (PDAC), in particular a terminal stage of this disease.
Inventors: |
RAYKOV; Zahari; (Heidelberg,
DE) ; Grekova; Svitlana; (Heidelberg, DE) ;
Daeffler; Laurent; (Heidelberg, DE) ; Rommelaere;
Jean; (Heidelberg, DE) ; Aprahamian; Marc;
(Limersheim, FR) ; Giese; Nathalia; (Schriesheim,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RAYKOV; Zahari
Grekova; Svitlana
Daeffler; Laurent
Rommelaere; Jean
Aprahamian; Marc
Giese; Nathalia |
Heidelberg
Heidelberg
Heidelberg
Heidelberg
Limersheim
Schriesheim |
|
DE
DE
DE
DE
FR
DE |
|
|
Family ID: |
47501037 |
Appl. No.: |
13/298492 |
Filed: |
November 17, 2011 |
Current U.S.
Class: |
424/85.5 ;
424/278.1; 424/85.4; 424/93.6 |
Current CPC
Class: |
A61K 38/217 20130101;
A61P 37/06 20180101; A61P 35/00 20180101; A61K 38/217 20130101;
A61K 35/768 20130101; A61K 45/06 20130101; A61K 35/768 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/85.5 ;
424/93.6; 424/85.4; 424/278.1 |
International
Class: |
A61K 35/76 20060101
A61K035/76; A61K 31/664 20060101 A61K031/664; A61K 31/436 20060101
A61K031/436; A61P 37/06 20060101 A61P037/06; A61K 38/21 20060101
A61K038/21; A61P 35/00 20060101 A61P035/00 |
Claims
11. A combination of a parvovirus and a cytokine for use in
treating pancreatic cancer.
12. The combination of compounds according to claim 11
characterized in that the use is for treating a terminal stage of
pancreatic cancer.
13. The combination of compounds according to claim 11
characterized in that the use is for treating a terminal stage of
pancreatic cancer characterized by peritoneal carcinosis.
14. The combination of compounds according to claim 11
characterized in that said cytokine is an interferon.
15. The combination of compounds according to claim 14
characterized in that said interferon is IFN-.gamma..
16. The combination of compounds according to claim 11
characterized in that said parvovirus is a rodent parvovirus.
17. The combination of compounds according to claim 16
characterized in that said rodent parvovirus is LuIII, Mouse minute
virus (MMV), Mouse parvovirus (MPV), Rat minute virus (RMV), Rat
parvovirus (RPV), Rat virus (RV) or H1 (H1-PV).
18. The combination of compounds according to claim 11
characterized in that said parvovirus is intratumorally
administered and the cytokine is intraperitoneally
administered.
19. The combination of compounds according to claim 11
characterized in that the combination of compounds further
comprises an immunosuppressive agent.
20. The combination of compounds according to claim 19
characterized in that the immunosuppressive agent is rapamycin or
cyclophosphamide.
21. A method for treating pancreatic cancer comprising
administering an effective amount of a combination of a parvovirus
and a cytokine
22. The method according to claim 21 wherein the pancreatic cancer
is a terminal stage of pancreatic cancer.
23. The method according to claim 22 wherein the terminal stage of
pancreatic cancer is characterized by peritoneal carcinosis.
24. The method according to claim 21 wherein the cytokine is an
interferon.
25. The method according to claim 24 wherein the interferon is
IFN-.gamma..
26. The method according to claim 21 wherein the parvovirus is a
rodent parvovirus.
27. The method according to claim 26 wherein the rodent parvovirus
is LuIII, Mouse minute virus (MMV), Mouse parvovirus (MPV), Rat
minute virus (RMV), Rat parvovirus (RPV), Rat virus (RV) or H1
(H1-PV).
28. The method according to claim 21 wherein the parvovirus is
intratumorally administered and the cytokine is intraperitoneally
administered.
29. The method according to claim 21 wherein the combination of
compounds further comprises an immunosuppressive agent.
30. The method according to claim 29 wherein the immunosuppressive
agent is rapamycin or cyclophosphamide.
Description
INCORPORATION BY REFERENCE
[0001] All documents cited or referenced herein ("herein cited
documents"), and all documents cited or referenced in herein cited
documents, together with any manufacturer's instructions,
descriptions, product specifications, and product sheets for any
products mentioned herein or in any document incorporated by
reference herein, are hereby incorporated herein by reference, and
may be employed in the practice of the invention. More
specifically, all referenced documents are incorporated by
reference to the same extent as if each individual document was
specifically and individually indicated to be incorporated by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a combination of a
parvovirus and a cytokine, preferably IFN.gamma., for use in
treating pancreatic cancer, in particular a terminal stage of this
disease.
BACKGROUND OF THE INVENTION
[0003] Pancreatic cancer is an aggressive malignancy with one of
the worst outcomes among all cancers. For all stages combined, the
5-year relative survival rate is only 5% (Ahmedin J, Siegel R, Ward
E, Hao Y, Xu J and Thun M. Cancer Statistics 2009. CA Cancer J Clin
2009;59:225-49). The radical surgery (Whipple's operation) is the
only curative option in this aggressive tumor but can be offered to
less than 20% of pancreatic ductal adenocarcinoma cancer (PDAC)
patients. Chemotherapy can be used as adjuvant to surgery or in
advanced stage pancreatic cancer where, in a small group of
patients, it offers real benefit in terms of survival and quality
of life (Katz M H, Fleming J B, Lee J E, Pisters P W. Current
status of adjuvant therapy for pancreatic cancer. Oncologist
2010;15:1205-13). Nevertheless, the therapeutic options for PDAC
patients, especially these with peritoneal carcinosis, are
lacking.
[0004] Novel virus-based anticancer therapies involve the use of
viruses either as replicating oncolytic agents, or as recombinant
vectors for gene transfer (Kirn D H, McCormick F. Replicating
viruses as selective cancer therapeutics Mol Med Today
1996;2:519-527). The autonomous parvoviruses MVMp and H-1 belong to
a group of small (.about.5 kb) non-integrating single-stranded DNA
viruses. Their oncotropic and oncotoxic properties make them
promising candidates for both types of applications (Cornelis J J,
Haag A, Kornfeld C et al. Autonomous parvovirus vectors In:
Cid-Arregui A, Garcia-Garranca A, eds Viral Vectors: Basic Science
and Gene Therapy Natick, M A: Eaton Publishing 2000;97-118).
Recently it could be demonstrated that applying H-1PV as
mono-therapy or as second-line treatment after gemcitabine
chemotherapy, caused the reduction of tumor growth, prolonged the
survival of rats bearing pre-established pancreatic tumors and led
to the suppression of metastases (Angelova A L, Aprahamian M,
Grekova S P, Hajri A, Leuchs B, Giese N A, et al. Improvement of
gemcitabine-based therapy of pancreatic carcinoma by means of
oncolytic parvovirus H-1PV. Clin Cancer Res 2009;15:511-9).
Furthermore, it was found that immunological mechanisms are
involved in the anticancer activity of H-1PV with a strong
correlation between the therapeutic effect of the virus and
IFN-.gamma. expression in the draining lymph nodes of pancreatic
tumors (Grekova S, Aprahamian M, Giese N, Schmitt S, Giese T, Falk
C S, et al. Immune cells participate in the oncosuppressive
activity of parvovirus H-1PV and are activated as a result of their
abortive infection with this agent. Cancer Biol Ther
2011;10:1280-9).
[0005] Despite the impressive results achieved the anticancer
efficacy of the most promising parvovirus candidates for human
clinical applications (including H-1PV) needs to be improved, e.g.,
as regards the extension of life span after diagnosis and as
regards particular tumors like pancreatic tumors.
[0006] Citation or identification of any document in this
application is not an admission that such document is available as
prior art to the present invention.
SUMMARY OF THE INVENTION
[0007] It is an object of the present invention to provide means
for an improved parvovirus-based therapy. According to the
invention this may achieved by the subject matters defined in the
claims.
[0008] Oncolytic viruses with their capacity to specifically
replicate in and kill tumor cells emerged as a novel class of
cancer therapeutics. Rat oncolytic parvovirus (H-1PV) was used to
treat different types of cancer in preclinical settings and was
lately successfully combined with standard gemcitabine chemotherapy
in treating pancreatic ductal adenocarcinoma in rats (PDAC).
[0009] The experiments resulting in the present invention are based
on an idea that the therapeutic properties of H-1PV may be boosted
with IFN.gamma. for the treatment of late incurable stages of PDAC
like peritoneal carcinosis. Rats bearing established orthotopic
pancreatic carcinomas with peritoneal metastases were treated with
a single intratumoral (i.t.) or intraperitoneal (i.p.) injection of
5.times.10.sup.8 plaque forming units of H-1PV with or without
concomitant IFNy application. Intratumoral injection proved to be
more effective than the intraperitoneal route in controlling the
growth of both the primary pancreatic tumors and peritoneal
carcinosis, accompanied by migration of virus from primary to
metastatic deposits.
[0010] Concomitant i.p. treatment of H-1PV with recIFNy resulted in
improved therapeutic effect yielding an extended animal survival,
compared to i.p. treatment with H-1PV alone. IFNy application
enhanced the H-1PV-induced peritoneal macrophage and splenocyte
responses against tumor cells while causing a significant reduction
in the titers of H1-PV-neutralising antibodies in ascitic fluid.
Thus, IFN.gamma. co-application together with H-1PV might be
considered as a novel therapeutic option to improve the survival of
PDAC patients with peritoneal carcinosis.
[0011] Accordingly, it is an object of the invention to not
encompass within the invention any previously known product,
process of making the product, or method of using the product such
that Applicants reserve the right and hereby disclose a disclaimer
of any previously known product, process, or method. It is further
noted that the invention does not intend to encompass within the
scope of the invention any product, process, or making of the
product or method of using the product, which does not meet the
written description and enablement requirements of the USPTO (35
U.S.C. .sctn.112, first paragraph) or the EPO (Article 83 of the
EPC), such that Applicants reserve the right and hereby disclose a
disclaimer of any previously described product, process of making
the product, or method of using the product.
[0012] It is noted that in this disclosure and particularly in the
claims and/or paragraphs, terms such as "comprises", "comprised",
"comprising" and the like can have the meaning attributed to it in
U.S. Patent law; e.g., they can mean "includes", "included",
"including", and the like; and that terms such as "consisting
essentially of" and "consists essentially of" have the meaning
ascribed to them in U.S. Patent law, e.g., they allow for elements
not explicitly recited, but exclude elements that are found in the
prior art or that affect a basic or novel characteristic of the
invention.
[0013] These and other embodiments are disclosed or are obvious
from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The following detailed description, given by way of example,
but not intended to limit the invention solely to the specific
embodiments described, may best be understood in conjunction with
the accompanying drawings.
[0015] FIG. 1: Impact of IFNy addition or depletion on H-1PV
immunomodulating activity. (A) Macrophages were isolated from the
peritoneal cavity of four groups (n=3) of tumor bearing Lewis rats
treated either with PBS (mock) or with an intratumoral injection of
5.times.10.sup.8 pfu/rat of H-1PV (H-1PV IT) combined either with
an antibody against IFN.gamma. (H-1PV IT+aIFNy) or recombinant
IFN.gamma. (H-1PV IT+recIFN.gamma.). Cells were plated in 48-well
plates at a density of 5.times.10.sup.5 cells per well and
stimulated or not with LPS. TNF.alpha. production in the
supernatants was measured 24 hrs later by ELISA. Average values and
standard deviations are shown. (B) Peritoneal macrophages
(5.times.10.sup.5/well) from the same groups of rats were
cocultured or not with 1.times.10.sup.5 HA-RPC cells at a 5:1 ratio
in 48-well plates and the release of interleukins -10 and -12 was
measured by ELISA. Mean cytokine ratios and standard deviations are
presented. (C) Single cell suspensions of rat splenocytes were
labeled with CFSE, plated in 24 well plates at 1.times.10.sup.6
cells/well and cocultured or not with 2.times.10.sup.5 HA-RPC cells
at a 5:1 ratio. 48 hrs later cells were harvested and processed for
FACS analysis of proliferation. All data were median from three
animals from triplicate wells. Differences were considered
significant at p values below 0.05.
[0016] FIG. 2: Therapeutic effects of H-1PV+IFN.gamma. combination
and virus distribution. (A) Lewis rats (n=28) bearing
simultaneously induced orthotopic tumors and peritoneal metastases
were divided into four groups (n=7) and either left untreated
(Control), inoculated i.t. (H-1PV intratumoral) or i.p (H-1PV
intraperitoneal) with H-1PV in the absence or presence of recIFNy
(H-1PV+IFN.gamma. intraperitoneal). After the sacrifice of two
animals 1 week after treatment, the survival of five rats from each
group was followed up to six months after tumor induction when
animals were sacrificed. Median values were considered significant
at p values below 0.05. (B) Two animals per group were sacrificed 1
week after H-1PV and/or IFN.gamma. treatments. Total RNA was
extracted from visible tumors and metastases, converted to cDNA and
subjected to RT-PCRs to evaluate the presence of H-1PV
DNA/unspliced mRNA and b-actin transcripts, using respective
primers. The abbreviations for the route and type of treatment are
indicated on the figure. The source of the material (Tumour or
Metastasis) is indicated in superscript.
[0017] FIG. 3: Macrophage activation after H-1PV+IFN.gamma.
combined treatment. Macrophages were isolated from the peritoneal
cavity of the H-1PV and H-1PV+IFN.gamma. intraperitoneally injected
rats, plated at 5.times.10.sup.5/well in 48 well plates and
cocultured or not with 1.times.10.sup.5 HA-RPC cells. The ratios of
TNF.alpha. and IL-10 in supernatants were determined 24 hrs later
by ELISA.
[0018] FIG. 4: Influence of IFN.gamma. application on the
generation of virus neutralizing antibodies. (A) Serum and ascitic
fluid were collected from all groups of virus-treated rats (see
FIG. 2) and the titers of virus neutralizing antibodies
(.alpha.H-1PV) were determined using cytotoxicity protection assay
on NB324K cells. The titers are expressed as the percentage of
antivirus protection offered by serum or ascitis dilutions compared
to mock infected cells. (B) Two groups of metastasis bearing rats
were treated with two intraperitoneal injections of H-1PV
(3.times.10.sup.8 pfu/injection per animal) spanning four weeks
between them, with or without intermediate recIFN.gamma. i.p.
inoculation. Titers of .alpha.H-1PV in ascitic fluids were
determined 10-30 days after the second H-1PV i.p. injection and
expressed as indicated above.
[0019] FIG. 5: Release of TNF.alpha. from PDAC and PBMC cocultures
after H-1PV+IFN.gamma. treatment. The indicated pancreatic cancer
cell lines were seeded into 10 cm.sup.2 dishes at
1.5.times.10.sup.6 cells/dish and infected or not with H-1PV at an
MOI of 10 pfu/cell. 24 hpi cells were harvested and plated onto
pre-isolated PBMCs in 48 well plates at a ratio of PDAC:PBMC 1:5.
The cocultures were treated or not with 50 UI/ml of human
recombinant IFN.gamma. and the release of TNF.alpha. was measured
in supernatants 24 hrs later by ELISA. Mock or H-1PV infected (MOI
10) monocultures of PBMCs served as controls. The indicated values
are average of at least three independent experiments. SD values
are shown.
DETAILED DESCRIPTION OF THE INVENTION
[0020] Thus, the present invention provides a combination of a
parvovirus and a cytokine, preferably a parvovirus and a cytokine
as separate entities, e.g. in separate containers, for use in
treating pancreatic cancer.
[0021] This combination of compounds is particularly useful for
treating a terminal stage of pancreatic cancer. "Terminal stage"
means a disease that cannot be cured or adequately treated and that
is reasonably expected to result in the death of the patient within
a relatively short period of time, e.g. within some weeks or
months. The combination of compounds is suitable for treating in
particular an incurable stage, like peritoneal carcinosis.
Peritoneal carcinosis represents the advanced evolutive stage of
several tumors that develop into abdominal organs, such as colon,
ovary, appendix, stomach, pancreas and liver. When the disease
increases, the tumoral cells reach and affect the membrane covering
the same organs (visceral peritoneum). Once this "barrier" has been
passed, the affected cells are able to move into the abdominal
cavity, carried by the peritoneal fluid. Even in mesothelioma cases
that affect directly the peritoneum, tumoral cells can break off
the membrane and fall into the peritoneal fluid. The tumoral cells
present into the liquid can die or survive feeding on substances
contained in the same liquid. These cells tend to accumulate in
those points of greater liquid readsorption, creating agglomerates
that grow more and more, spreading into the whole abdomen and
originating the carcinosis.
[0022] The term "parvovirus" as used herein comprises wild-type or
modified replication-competent derivatives thereof, as well as
related viruses or vectors based on such viruses or derivatives.
Suitable parvoviruses, derivatives, etc. as well as cells which can
be used for actively producing said parvoviruses and which are
useful for therapy, are readily determinable within the skill of
the art based on the disclosure herein, without undue empirical
effort. Rodent parvoviruses are preferred. Particularly preferred
are the following rodent parvoviruses: H1 (H1-PV), LuIII, Mouse
minute virus (MMV), Mouse parvovirus (MPV), Rat minute virus (RMV),
Rat parvovirus (RPV) and Rat virus (RV).
[0023] Patients treatable by the combination of agents according to
the invention include humans as well as non-human animals. Examples
of the latter include, without limitation, animals such as cows,
sheep, pigs, horses, dogs, and cats.
[0024] As used herein, the term "cytokine" relates to a category of
signalling molecules that are used extensively in cellular
communication. They comprise proteins, peptides, or glycoproteins.
The term cytokine encompasses a large family of polypeptide
regulators that are produced widely throughout the body by cells of
diverse embryological origin. The action of cytokines may be
autocrine, paracrine, and endocrine. All cytokines are critical to
the development and functioning of both the innate and adaptive
immune response. They are often secreted by immune cells that have
encountered a pathogen, thereby activating and recruiting further
immune cells to increase the system's response to the pathogen.
Relying on the assays shown in Examples 2 to 5 the person skilled
in the art is in a position to select cytokines that show
beneficial effects when administrated according to the present
invention.
[0025] Preferably, the cytokine of the present invention is an
interferon. All interferons (IFNs) are natural cell-signalling
proteins produced by the cells of the immune system of most
vertebrates in response to challenges such as viruses, parasites
and tumor cells. Interferons are produced by a wide variety of
cells in response to the presence of double-stranded RNA, a key
indicator of viral infection. Interferons assist the immune
response by inhibiting viral replication within host cells,
activating natural killer cells and macrophages, increasing antigen
presentation to lymphocytes, and inducing the resistance of host
cells to viral infection. All interferons in general have several
effects in common and, accordingly, the results obtained by use of
IFN-.gamma. in combination with a parvovirus, preferably H1-PV,
might apply to further interferons. Interferons are antiviral and
possess antioncogenic properties, macrophage and natural killer
cell activation, and enhancement of major histocompatibility
complex glycoprotein classes I and II, and thus presentation of
foreign (microbial) peptides to T cells. The production of
interferons is induced in response to microbes such as viruses and
bacteria and their products (viral glycoproteins, viral RNA,
bacterial endotoxin, bacterial flagella, CpG sites), as well as
mitogens and other cytokines, for example interleukin 1,
interleukin 2, interleukin-12, tumor necrosis factor and
colony-stimulating factor, that are synthesised in the response to
the appearance of various antigens in the body. Their metabolism
and excretion take place mainly in the liver and kidneys. They
rarely pass the placenta but they can cross the blood-brain
barrier.
[0026] There are three major classes of interferons that have been
described for humans:
[0027] (a) Interferon type I: The type I interferons present in
humans are IFN-.alpha., IFN-.beta. and IFN-.omega..
[0028] (b) Interferon type II: In humans this is IFN-.gamma..
[0029] (c) Interferon type III: Signal through a receptor complex
consisting of IL10R2 (also called CRF2-4) and IFNLR1 (also called
CRF2-12).
[0030] In a preferred embodiment of the present invention, the
interferon is interferon-.gamma. (IFN.gamma.).
[0031] Preferably, for the therapeutic use of the present invention
the parvovirus and the cytokine are present in an effective dose
and combined with a pharmaceutically acceptable carrier.
"Pharmaceutically acceptable" is meant to encompass any carrier,
which does not interfere with the effectiveness of the biological
activity of the active ingredients and that is not toxic to the
patient to whom it is administered. Examples of suitable
pharmaceutical carriers are well known in the art and include
phosphate buffered saline solutions, water, emulsions, such as
oil/water emulsions, various types of wetting agents, sterile
solutions etc. Such carriers can be formulated by conventional
methods and can be administered to the subject at an effective
dose.
[0032] An "effective dose" refers to amounts of the active
ingredients that are sufficient to affect the course and the
severity of the tumor, leading to the reduction or remission of
such pathology. An "effective dose" useful for treating and/or
preventing these diseases may be determined using methods known to
one skilled in the art (see for example, Fingl et al., The
Pharmocological Basis of Therapeutics, Goodman and Gilman, eds.
Macmillan Publishing Co., New York, pp. 1-46 ((1975)).
[0033] Preferred doses of the parvovirus are in the range of about
10.sup.8 to 10.sup.9 pfu (single injection) in rats and of the
cytokine, in particular IFNy, in the range of about 10.sup.5 to
10.sup.6 IU (single injection). For humans the preferred effective
dose of the parvovirus is approximately 10.sup.11 pfu and of the
cytokine (e.g. IFN.gamma.) about 2.times.10.sup.6 to
10.sup.8IU.
[0034] Additional pharmaceutically compatible carriers can include
gels, bioasorbable matrix materials, implantation elements
containing the therapeutic agent, or any other suitable vehicle,
delivery or dispensing means or material(s).
[0035] Administration of the compounds may be effected by different
ways, e.g. by intravenous, intraperetoneal, subcutaneous,
intramuscular, topical or intradermal administration. The route of
administration, of course, depends on the kind of therapy and the
kind of compounds contained in the pharmaceutical composition. The
dosage regimen of the parvovirus and the cytokine is readily
determinable within the skill of the art, by the attending
physician based on patient data, observations and other clinical
factors, including for example the patient's size, body surface
area, age, sex, the particular parvovirus to be administered, the
time and route of administration, the tumor type and
characteristics, general health of the patient, and other drug
therapies to which the patient is being subjected.
[0036] If the parvovirus comprises infectious virus particles with
the ability to penetrate through the blood-brain barrier, treatment
can be performed or at least initiated by intravenous injection of
the parvovirus, e.g., H1 virus. As another specific administration
technique, the parvovirus (virus, vector and/or cell agent)
containing composition can be administered to the patient from a
source implanted in the patient. For example, a catheter, e.g., of
silicone or other biocompatible material, can be connected to a
small subcutaneous reservoir (Rickham reservoir) installed in the
patient during tumor removal or by a separate procedure, to permit
the parvovirus containing composition to be injected locally at
various times without further surgical intervention. The parvovirus
or derived vectors containing composition can also be injected into
the tumor by stereotactic surgical techniques or by neuronavigation
targeting techniques. Administration of the parvovirus containing
compositions can also be performed by continuous infusion of viral
particles or fluids containing viral particles through implanted
catheters at low flow rates using suitable pump systems, e.g.,
peristaltic infusion pumps or convection enhanced delivery (CED)
pumps.
[0037] As yet another method of administration of the parvovirus
containing composition is from an implanted article constructed and
arranged to dispense the parvovirus containing composition to the
desired cancer tissue. For example, wafers can be employed that
have been impregnated with the parvovirus containing composition,
e.g., parvovirus H1, wherein the wafer is attached to the edges of
the resection cavity at the conclusion of surgical tumor removal.
Multiple wafers can be employed in such therapeutic intervention.
Cells that actively produce the parvovirus, e.g., parvovirus H1, or
H1 vectors, can be injected into the tumor, or into the tumoral
cavity after tumor removal.
[0038] Preferably, the parvovirus and the cytokine are administered
as separate compounds. The administration of the cytokine, when
administered separately, can be accomplished in a variety of ways.
A preferred route of administration of the parvovirus is
intratumoral administration. A preferred route of administration of
the cytokine is intraperitoneal administration. The combination of
both routes of administration shows synergistic effects.
[0039] The therapeutic efficacy of the combination of compounds
according to the present invention can be further improved by
co-administration of an immunosuppressive agent like rapamycin or
cyclophosphamide.
[0040] Although the present invention and its advantages have been
described in detail, it should be understood that various changes,
substitutions and alterations can be made herein without departing
from the spirit and scope of the invention as defined in the
appended claims.
[0041] The present invention will be further illustrated in the
following Examples which are given for illustration purposes only
and are not intended to limit the invention in any way.
EXAMPLES
Example 1
Materials and Methods
[0042] (A) Cells and reagents. Human pancreatic carcinoma cell
lines from primary (Panc-1, MiaPaCa-2, B.times.PC-3) or metastatic
(Capan 1, T3M4, AsPC-1, Colo357) tumors, were obtained from ATCC
(Manassas, Va.) and grown in RPMI 1640 medium supplemented with 10%
fetal calf serum (FCS). The HA-RPC cell line (ATCC, LGC Standards,
Wesel Germany) derived from a chemically induced pancreatic ductal
adenocarcinoma in Lewis rats was grown in DMEM supplemented with
10% FCS. Human NB324K cells (ATCC, LGC Standards, Wesel Germany)
used for cytotoxicity protection assays were cultured in MEM medium
with 5% FCS. All media were supplemented with penicillin (100 U/ml)
and streptomycin (100 .mu.g/ml). Lyophilized recombinant rat and
human IFNy were obtained from Biomol GMBH (Hamburg, Germany) and
reconstituted in sterile deionized water. The mouse monoclonal
antibody clone DB-1 with specificity against murine IFN.gamma.
(.alpha.IFN.gamma.) was produced in bulk amount by NatuTec GmbH
(Frankfurt, Germany). Where indicated, in some experiments cells
were stimulated using LPS at final concentration of 5 .mu.g/ml.
[0043] For the isolation of peritoneal macrophages rats received an
i.p. injection of 4 ml of 4% Thioglycolate solution in PBS three
days before sacrifice. After sacrificing the animals, 40 ml of
sterile PBS were instilated in the peritoneal cavity and recovered
using a syringe. The cells were collected by centrifugation and
plated in DMEM containing 10% FCS and antibiotics.
[0044] Peripheral blood mononuclear cells (PBMC) were isolated from
the heparinized blood of randomly selected healthy donors by
differential centrifugation over Histopaque (Sigma) and cultured in
RPMI with 10% FCS and antibiotics. Peripheral blood macrophages
were enriched by adherence to plastic surface. Buffy coats were
obtained from the blood bank of IKTZ Heidelberg.
[0045] (B) Virus-neutralizing antibody detection. Serial dilutions
of the sera of experimental animals were made in MEM and mixed with
an equal volume of H-1PV virus suspension (corresponding to
2.times.10.sup.4 pfu/well). After incubation for 30 min. at
37.degree. C., the mixture was inoculated onto NB324K cells plated
in 96-well plates (2.times.10.sup.3 cells/well). The cell survival
rates were assessed after 72 h using a MTT
(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide)
assay.
[0046] (C) Semi-quantitative RT-PCR. For RT-PCR total RNA was
extracted from pancreatic tumors or metastatic nodules of treated
animals, reverse transcribed into cDNA, and PCRs for H-1PV and
B-actin were performed using previously described primer sequences
and conditions (Grekova S, Aprahamian M, Giese N, Schmitt S, Giese
T, Falk C S, et al. Immune cells participate in the oncosuppressive
activity of parvovirus H-1PV and are activated as a result of their
abortive infection with this agent. Cancer Biol Ther
2011;10:1280-9).
[0047] (D) ELISA. Measurement of rat TNF.gamma.; IL-10, IL-12 and
human TNF.alpha. release was done using commercially available
ELISA kits from eBioscience (Frankfurt, Germany) as described by
manufacturer.
[0048] (E) FACS determination of splenocytes' proliferation index.
Rat spleens were pressed against a mesh to obtain single cell
suspensions and splenocytes were adjusted to a concentration of
5.times.10.sup.6/ml in PBS. The stock 5 mM CFSE solution was
diluted at 1/1000 in PBS (a final concentration of 5 .mu.M), added
to lymphocytes pellet and mixed rapidly. After incubation for 5
minutes at room temperature 10 volumes of PBS containing 5% FSC
were added and the cells were centrifuged. Washes in PBS/FCS were
repeated 3 times. Labeled splenocytes were co-cultured with HA-RPC
cells or alone as a control. After 72 h of incubation, cells were
collected, washed and measured for CFSE fluorescence using
FACSCalibur (BD, California, USA). The proliferation index was
calculated based on the level of reduction in fluorescence
intensity of the cultures.
[0049] (F) Animal studies. The orthotopic rat model using HA-RPC
cells has been previously described (5). For the induction of
metastasis a cell suspension was prepared in phosphate-buffered
saline (PBS) out of subcutaneous tumors preformed by implantation
of HA-RPC cells and injected intraperitoneally to Lewis rats at
3.times.10.sup.6 cells in 500 .mu.l per animal.
[0050] Rat recIFN.gamma. was injected in 3 consecutive weeks at 30
000 UI/week i.p. in a 100 .mu.l volume for a total dose of 90 000
UI/animal. The aIFNy antibody was applied at the same times at 0.8
mg/animal for a total dose of 2.4 mg. Ascitic fluid was obtained
using a peritoneal puncture under aerosol anesthesia at the time
before animal sacrifice.
[0051] (G) Statistical methods. Means and standard deviations were
calculated from at least three animals in triplicate wells in vitro
experiments. Statistical differences were assessed using Student's
t test and Wilcoxon test. For in vivo mortality data assessment,
experimental groups were compared with log-rank test.
Example 2
RecIFN.gamma. Contributes to the Immunomodulating Anticancer Effect
of H-1PV
[0052] As a first step the potential impact of interferon-.gamma.
on the immunomodulating features of parvovirus H-1PV in pancreatic
cancer was established. Therefore, H-1PV was applied in tumors
raised orthotopically through injection of HA-RPC cells in the
pancreatic tail of three groups of rats using a PBS-treated group
as control. Virus treatment was also combined either with
intraperitoneal recombinant rat interferon (recIFN.gamma.) or with
a neutralizing antibody against it (.alpha.IFN.gamma.). Three days
later animals were sacrificed to perform immunological profiling of
splenocytes and peritoneal macrophages. The cells were cultured for
48 hours either alone (no treat), together with HA-RPC rat
pancreatic cancer cells (the cell line used for initiating the
tumors) or LPS. Different parameters related to the anticancer
immune response, like the production of TNF.alpha. (FIG. 1A), the
IL-12/IL-10 ratio (FIG. 1B) of cytokines released by macrophages,
as well as the proliferation capacity of splenocytes (FIG. 1C) were
analyzed. The supernatants of macrophages isolated from rats, in
which H-1PV was combined with recIFNy contained up to 1 ng more
TNF.alpha., compared to those obtained from animals treated with
virus only. The lowest levels of TNF.alpha. release were measured
either in the non-treated mock (0.2 and 0.5 ng) or when rats were
treated with a neutralizing antibody against interferon gamma (0.3
and 1 ng). A similar pattern of effects of recIFN.gamma. and
.alpha.IFN.gamma. was detected when comparing the IL-12/IL-10
ratios of the different macrophage cultures. In addition, the
combination of H-1PV with recombinant interferon gamma caused a
significant twofold increase in the proliferative potential of
splenocytes both spontaneously and in the presence of tumor
cells.
[0053] These data pointed that H-1PV application alone can activate
peritoneal macrophages or combined with recIFNy i.p. introduction
could change the activation status of immune cells both in spleen
and in the peritoneal cavity leading to predominance of
immuno-stimulatory cytokines (TNF.alpha., IL-12) over the
immunosuppressive factors (IL-10). The decrease of the
above-mentioned immunological parameters upon depletion of IFNy,
especially in the case of peritoneal macrophages, confirmed our
assumption that this cytokine plays a role in stimulating the
innate immune system as part of the immunomodulating effect of
oncolytic H-1PV.
Example 3
RecIFN.gamma. Improves the Therapeutic Potential of H-1PV for the
Treatment of PDAC Peritoneal Carcinosis
[0054] Since the results obtained were encouraging a combination of
recombinant IFN.gamma. and H-1PV parvovirus was used for the
treatment of one of the most lethal complications of pancreatic
cancer in humans, namely the spread of the tumor to the peritoneal
cavity. To mimic this situation tumors both in the pancreas and in
the peritoneal cavity of Lewis rats were induced. Two weeks later,
the rats were randomly divided into four groups, in which H-1PV was
applied through two different routes (intratumoral or
intraperitoneal). In one group the virus i.p. inoculation was
combined with recIFN.gamma. using the same route (FIG. 2A
protocol). Animal survival was followed (FIG. 2A) confirming that
H-1PV intratumoral injection was still most effective to protect
rats against PDAC with two animals remaining tumor free more than
six months after treatment (5). H-1PV could significantly improve
the survival of rats upon peritoneal application compared to the
control group but was still less effective in comparison to the
i.t. route. Notably, the combination with recIFN.gamma. could
significantly improve the effect of the virus extending the median
survival from 83 to 96 days (FIG. 2A).
[0055] Two animals per group were sacrificed one week after
treatment to analyze virus presence by RT-PCR (FIG. 2B). The
distribution of viral DNA signals showed that (i) the virus could
migrate from the primary tumor after intratumoral application
(HIT.sup.Tu) to metastasis (HIT.sup.M) in the peritoneal cavity,
(ii) it can infect metastasis upon intraperitoneal inoculation
(HIP.sup.M), and (iii) that upon i.p. combination with H-1PV
IFN.gamma. does not change significantly the virus levels in
metastases (compare HIP.sup.M and HIFN.sup.M).
[0056] Isolation of peritoneal macrophages from mock, H-1PV or
H-1PV with IFN.gamma. intraperitoneally treated rats showed that
the ratio between TNF.alpha. and IL-10 produced was significantly
increased in the presence of recombinant IFN.gamma. when
macrophages were cocultured with HA-RPC cells, speaking in favor of
phagocytes' activation (FIG. 3).
[0057] In conclusion, intratumoral application of H-1PV seems to
have a superior effect compared to intraperitoneal inoculation for
the treatment of PDAC. In case intraperitoneal inoculations of the
virus are performed at the stage of advanced metastatic disease, a
combination with IFNy can be very favorable.
Example 4
RecIFN.gamma. Cotreatment Reduces the Titers of H-1PV Neutralizing
Antibodies in Ascitic Fluid
[0058] One of the major functions of IFNy is its ability to prime
the cellular (through Th1 cells/cytokines) and to down-modulate the
humoral (through Th2 cells/cytokines) immune response. Therefore,
it was assumed that the combination of IFN.gamma. and oncolytic
H-1PV may also reduce the titers of neutralizing antibodies
produced against the virus. In order to address this hypothesis,
serum from peripheral blood and ascitic fluid from the peritoneal
cavity of rats participating in the above-mentioned experiment were
collected and the titers of .alpha.H-1PV antibodies were
determined, using a cytotoxicity protection assay on
virus-sensitive cells. It was found that in the first experiment
performed, no evident difference could be detected in the titer of
.alpha.H-1PV in animal sera irrespective of the virus inoculation
route and IFN.gamma. treatment (FIG. 4A upper pannel). Similarly,
the inoculation route had no impact on the antiviral titers in
ascitic fluid collected in the time-frame (20 to 40 days) after
virus treatment. On the other hand, co-application of recIFN.gamma.
together with H-1PV caused a significant reduction (from 1:5000 to
1:1280) in the titers of .alpha.H-1PV in the ascitic fluid of the
animals most probably due to the stronger effect of i.p. applied
IFN.gamma..
[0059] Then, in a modified experimental setting it was tested
whether the IFN.gamma.-provoked drop in antiviral antibodies within
ascites would increase the levels of H-1PV DNA in metastases when
this cytokine is applied before a second virus inoculation. First,
it was noticed that the titers of .alpha.H-1PV in ascitic fluid
collected within 10 to 30 days after this second H-1PV i.p.
injection (FIG. 4B) were much higher than the ones induced by a
single H-1PV i.p. application (FIG. 4A). This effect was most
probably due to boosting of the immune system related to the
repeated virus application. Interestingly, when IFN.gamma. was
applied before the second H-1PV inoculation (H-1+IFN.gamma.) it was
noticed that .alpha.H-1PV titers remained similar to the ones
observed in fluids from animals subjected to a single virus
inoculation (compare FIG. 4B with FIG. 4A, lower panel), suggesting
that the cytokine has inhibited the overproduction of .alpha.H-1PV
triggered by the second virus injection.
[0060] The H-1PV transduction level of metastasis in the two groups
of rats after the second virus application (SFIG. 1) was also
evaluated. Unfortunately, IFN.gamma. treatment had no positive
impact on the amounts of viral DNA in metastasis despite the
reduction of antiviral antibodies (FIG. 4B) suggesting that this
reduction was not sufficient to overcome the antibody pressure in
ascitic fluid.
Example 5
Rec IFN.gamma. Can Improve the Effect of H-1PV to Stimulate the
Human Innate Immune System
[0061] In search of clinical relevance of the obtained data, the
previous studies were continued using human PDAC cell lines and
peripheral blood monocytes derived from healthy donors, aiming to
find out whether the latter can be activated more efficiently with
a combination of virus and IFN.gamma.. It was previously reported
that H-1PV infection leads to a limited but significant activation
of human PBMCs as indicated by their TNF.alpha. release. The latter
effect was largely masked in the case when PBMCs were cocultivated
with pancreatic cancer cells irrespective of their infection status
(Grekova S, Aprahamian M, Giese N, Schmitt S, Giese T, Falk C S, et
al. Immune cells participate in the oncosuppressive activity of
parvovirus H-1PV and are activated as a result of their abortive
infection with this agent. Cancer Biol Ther 2011;10:1280-9).
Considering that PDAC cells can express IFN.gamma. receptors first
of all the lethal effect of H-1PV and IFN.gamma. combination on
pancreatic cancer cell was evaluated. IFN.gamma. does not change
H-1PV-induced toxicity on human PDAC cells (SFIG. 2). In a next
step, PBMCs were cocultured with pancreatic cancer cells that had
been previously infected (or not) with H-1PV, and used the release
of TNF.alpha. as a read-out for innate immune cell activation. As
already previously reported, the direct infection of PBMCs with
H-1PV resulted in an increased release of TNF.alpha. at 48 hpi
(FIG. 5, PBMC monoculture) (Grekova S, Aprahamian M, Giese N,
Schmitt S, Giese T, Falk CS, et al. Immune cells participate in the
oncosuppressive activity of parvovirus H-1PV and are activated as a
result of their abortive infection with this agent. Cancer Biol
Ther 2011;10:1280-9). Addition of relatively low dose IFNy (50
UI/ml) to the cultures did not significantly enhance TNF.alpha.
production. The same was the case when PDACs were pre-infected with
H-1PV before coculturing them with the PBMCs. However, in general,
in the presence of IFN.gamma., PBMC cocultures with Panc-1, T3M4,
Capan-1 and especially Colo357 and AsPC-1 produced 100-150 pg/ml
more TNF.gamma., corresponding to a higher level of activation of
innate immune cells. Interestingly, despite the fact that the
fluctuations of TNF.alpha. were not statistically significant, a
tendency could be observed that PDAC cells deriving from metastatic
(lymph node, liver or peritoneal) pancreatic cancer seemed to be
more potent stimulators of PBMCs in the presence of IFNy than the
lines established from primary PDAC tumors. In general, all these
effects support the hypothesis that concomitant application of IFNy
can be beneficial for the anticancer vaccination effect of H-1PV
especially in the treatment of advanced metastatic disease.
Example 6
Conclusion
[0062] The observed reduction in the titers of virus neutralizing
antibodies induced by IFN.gamma. represents a very interesting
phenomenon in the frame of oncolytic virotherapy. It is in
agreement with the changes observed in the II-12/II-10 cytokine
ratio secreted from macrophages pointing to a shift in the Th1/Th2
balance in the peritoneal cavity. Probably, an additional
modification of the IFN.gamma. treatment protocol or its
combination with certain immunosuppressive agents, recently
reported in oncolytic virotherapy may improve the described effect
and reduce the antibodies to levels permitting repeated virus
applications and metastasis transduction (Lun XQ, Jang J H, Tang N,
Deng H, Head R, Bell J C, et al. Efficacy of systemically
administered oncolytic vaccinia virotherapy for malignant gliomas
is enhanced by combination therapy with rapamycin or
cyclophosphamide. Clin Cancer Res 2009;15:2777-88).
[0063] Treatment of peripheral blood mononuclear cells with H-1PV
could prime the release of TNF.alpha., a cytokine that represents
one of the main products secreted upon macrophage activation
possessing also strong antitumor properties. However, coculturing
PBMCs with pancreatic cancer cell lines deriving from different
organ locations caused a generalized increase in TNF.alpha. levels
that seemed to almost completely mask the effect of H-1PV
pre-infection of PDAC cells. IFN.gamma. could serve as an
additional stimulator of TNF.alpha. production mostly in the case
of cocultures between PBMCs and metastatic PDAC cancer lines.
Notably, this effect was most pronounced for AsPC-1, a cell line
deriving from a clinical case of peritoneal metastasis, therefore
giving stronger credibility to the results obtained in animal
experiments with peritoneal carcinosis. In conclusion, the
combination of an oncolytic virus with a powerful imunomodulating
cytokine like IFN.gamma. may represent a promising strategy for
cancer therapy. In view of the forthcoming clinical applications of
H-1PV as an oncolytic agent, a therapeutic protocol involving
co-treatment with the two modalities has potential to improve the
outcome in terminal stage patients with pancreatic cancer.
[0064] The invention is further described by the following numbered
paragraphs: [0065] 1. A combination of a parvovirus and a cytokine
for use in treating pancreatic cancer. [0066] 2. The combination of
compounds according to paragraph 1 for the use according to
paragraph 1 characterized in that the use is for treating a
terminal stage of pancreatic cancer. [0067] 3. The combination of
compounds according to paragraph 1 for the use according to
paragraph 2 characterized in that the use is for treating a
terminal stage of pancreatic cancer characterized by peritoneal
carcinosis. [0068] 4. The combination of compounds according to
paragraph 1 for the use according to any one of paragraphs 1 to 3
characterized in that said cytokine is an interferon. [0069] 5. The
combination of compounds according to paragraph 4 for the use
according to any one of paragraphs 1 to 3 characterized in that
said interferon is IFN-.gamma.. [0070] 6. The combination of
compounds according to any one of paragraphs 1 to 5 for the use
according to any one of paragraphs 1 to 3 characterized in that
said parvovirus is a rodent parvovirus. [0071] 7. The combination
of compounds according to paragraph 6 for the use according to any
one of paragraphs 1 to 3 characterized in that said rodent
parvovirus is LuIII, Mouse minute virus (MMV), Mouse parvovirus
(MPV), Rat minute virus (RMV), Rat parvovirus (RPV), Rat virus (RV)
or H1 (H1-PV). [0072] 8. The combination of compounds according to
any one of paragraphs 1 to 7 for the use according to any one of
paragraphs 1 to 3 characterized in that said parvovirus is
intratumorally administered and the cytokine is intraperitoneally
administered. [0073] 9. The combination of compounds according to
any one of paragraphs 1 to 7 for the use according to any one of
paragraphs 1 to 8 characterized in that the combination of
compounds further comprises an immunosuppressive agent. [0074] 10.
The combination of compounds according to paragraph 9 for the use
according to any one of paragraphs 1 to 8 characterized in that the
immunosuppressive agent is rapamycin or cyclophosphamide.
* * * * *