U.S. patent application number 11/671498 was filed with the patent office on 2007-06-21 for purging of cells using viruses.
This patent application is currently assigned to WELLSTAT BIOLOGICS CORPORATION. Invention is credited to Harold L. Atkins, John C. Bell, Conrad J. JR. Heilman, Brian D. Lichty, Robert M. Lorence, Michael S. Roberts, David F. Stojdl.
Application Number | 20070141033 11/671498 |
Document ID | / |
Family ID | 22797442 |
Filed Date | 2007-06-21 |
United States Patent
Application |
20070141033 |
Kind Code |
A1 |
Atkins; Harold L. ; et
al. |
June 21, 2007 |
PURGING OF CELLS USING VIRUSES
Abstract
The subject invention relates to viruses that are able to purge
(reduce or eliminate) undesirable cells in a mixture of cells.
Undesirable cells can include neoplastic cells, cells mediating
graft-versus host diseases, and autoimmune cells. The subject
invention also relates to the purging of undesirable cells from
bone marrow or peripheral blood cell harvests in the treatment of
mammals including cancer patients, transplant recipients, and
patients with autoimmune disease.
Inventors: |
Atkins; Harold L.; (Orleans,
ON) ; Bell; John C.; (Ottawa, ON) ; Heilman;
Conrad J. JR.; (Landenberg, PA) ; Lichty; Brian
D.; (Brantford, ON) ; Lorence; Robert M.;
(Bethesda, MD) ; Roberts; Michael S.; (Myersville,
MD) ; Stojdl; David F.; (Ottawa, ON) |
Correspondence
Address: |
LEWIS J. KREISLER
LEGAL DEPARTMENT
930 CLOPPER ROAD
GAITHERSBURG
MD
20878
US
|
Assignee: |
WELLSTAT BIOLOGICS
CORPORATION
Gaithersburg
MD
20878
UNIVERSITY OF OTTAWA
Ottawa
K1N 6N5
|
Family ID: |
22797442 |
Appl. No.: |
11/671498 |
Filed: |
February 6, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10717101 |
Nov 19, 2003 |
7192580 |
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11671498 |
Feb 6, 2007 |
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09888626 |
Jun 26, 2001 |
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10717101 |
Nov 19, 2003 |
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60214014 |
Jun 26, 2000 |
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Current U.S.
Class: |
424/93.6 |
Current CPC
Class: |
C12N 2501/24 20130101;
A61P 35/00 20180101; A61P 25/00 20180101; A61K 35/14 20130101; A61P
37/06 20180101; A61P 37/02 20180101; A61K 35/768 20130101; A61K
35/765 20130101; C12N 5/0093 20130101; A61P 29/00 20180101; A61P
35/02 20180101; A61K 35/28 20130101; A61K 35/766 20130101; A61K
35/765 20130101; A61K 2300/00 20130101; A61K 35/768 20130101; A61K
2300/00 20130101; A61K 35/766 20130101; A61K 2300/00 20130101; A61K
35/28 20130101; A61K 2300/00 20130101; A61K 35/14 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
424/093.6 |
International
Class: |
A61K 35/76 20060101
A61K035/76 |
Claims
1. A method of reducing or eliminating neoplastic cells in an ex
vivo mixture of normal, hematopoeitic cells and neoplastic cells,
wherein the mixture is a suspension, comprising incubating said
mixture with an effective amount of a vesicular stomatitis virus,
and administering an interferon to the mixture before, during or
after incubating with said virus.
2. A method as in claim 1 wherein said neoplastic cells are
leukemia cells.
3. A method as in claim 1 wherein said hematopoeitic cells are
marrow cells.
4. A method as in claim 1 wherein said hematopoeitic cells are
peripheral blood cells.
5. A method as in claim 1 wherein said virus is
replication-competent.
6. A method as in claim 1 wherein said neoplastic cells are
lymphoma cells.
7. A method as in claim 1 further comprising administering a
chemotherapeutic agent to the mixture before, during or after
incubating with said virus.
Description
FIELD OF THE INVENTION
[0001] The subject invention relates to viruses that are able to
purge (reduce or eliminate) undesirable cells in a mixture of
cells. Undesirable cells can include neoplastic cells, cells
mediating graft-versus host diseases, and autoimmune cells. The
subject invention also relates to the purging of undesirable cells
from bone marrow or peripheral blood cell harvests in the treatment
of mammals including cancer patients, transplant recipients, and
patients with autoimmune disease.
BACKGROUND OF THE INVENTION
[0002] Ex vivo purging techniques have shown limited success for
autologous bone marrow or stem cell transplantation in patients
with leukemia or other malignancies. One goal of purging bone
marrow or peripheral blood progenitor cells (PBPC) is to remove
neoplastic cells while having little effect on normal stem cells
and hematopoeitic progenitor cells. Transplantation of the purged
marrow occurs after myeloablative therapy such as high dose
chemotherapy or radiation [see for example, Stuart R. K., 1993,
Semin. Oncol. 20(5Suppl 6):40-54); Hammert L. C. and Ball, E. D.,
1997, Curr Opin Hematol 4:423-423; Schneidkraut M. J., et al.,
1996, J Hematother 5:631-646]. Transplantation of any neoplastic
cells with the marrow places the patient at risk for relapse of the
malignancy (see for example, Rummel S A and Van Zant G, 1994, J.
Hematother. 3:213-218; Kvalheim G. et al, 1996, J. Hematother.,
5:427-436). Methods undergoing current study to selectively kill
neoplastic cells include the use of chemotherapeutic agents (such
as 4-hydroperoxycyclophosphamide; see for example, Bird J. M.,
1996, Bone Marrow Transplant, 18:309-313), monoclonal antibodies
(see for example, Hammert, L. C. and Ball E. D., 1997, Blood Rev.,
11:80-90), photodynamic therapy (see for example, Villeneuve L.,
1999, Biotechnol Appl. Biochem. 30:1-17), and viral vectors such as
adenovirus (see for example, Hirai M, et al., 1999, Acta Haematol.,
101:97-105; Marini F. C., et al., 1999, Clin. Cancer Res.,
5:1557-1568). However, recent experiments demonstrated that viable
cancer cells remained in the bone marrow or PBPC after therapeutic
purging leading to relapse of the malignancy. Another major
limitation of current methods of ex vivo purging is the delayed
engraftment due to damage to normal stem cells and/or early
hematopoietic progenitor cells (Rummel S A and Van Zant G. 1994, J.
Hematother. 3:213-218, Damon et al., 1996, Bone Marrow Transplant,
17:93-99). Progenitor cells that are actively proliferating are
consequently very sensitive to killing by most chemotherapeutic
agents including 4-hydroperoxycyclophosphamide. The resultant loss
of early progenitor cells causes a prolonged neutropenia and/or
thrombocytopenia which places the patient at increased risk for
life-threatening infection 1 and/or bleeding. A tumor cytotoxic or
cytolytic agent that spares normal hematopoietic cells is an
important advance in cancer therapy.
[0003] In addition to neoplastic cells, bone marrow or peripheral
blood progenitor cell harvests can include other undesirable cells
such as autoimmune cells in people with arthritis or multiple
sclerosis, for example. Other undesirable cells include those that
mediate graft-versus-host disease (e.g., certain T-lymphocytes) in
allogeneic transplants. Reduction or elimination of such
undesirable cells would be an important in the treatment of cancer
and autoimmune diseases.
[0004] PCT applications by Roberts et al. (WO/99118799 and) relates
to the treatment of neoplasms with viruses.
OBJECTS OF THE INVENTION
[0005] It is an object of the invention to provide viruses for the
reduction or elimination of undesirable cells in mixtures of
desirable and undesirable cells.
[0006] It is a further object of the invention to provide viruses
for the reduction or elimination of neoplastic cells in mixtures of
normal and neoplastic cells.
[0007] It is a further object to provide viruses for the ex vivo
purging of neoplastic cells from normal hematopoetic cells such as
bone marrow or peripheral blood progenitor cells.
[0008] It is a further object to provide viruses for the ex vivo
purging of autoimmune cells from normal cells such as bone marrow
or peripheral blood progenitor cells.
[0009] It is a further object to provide viruses for the ex vivo
purging of cells that mediate graft-versus-host disease from normal
hematopoetic cells such as bone marrow or peripheral blood
progenitor cells.
[0010] It is a further object of the invention to provide a method
of treating disease in a mammal by contacting mixtures of desirable
hematopoietic cells and undesirable cells with a virus and
transplanting such cells into the mammal.
[0011] It is a further object of the invention to provide a method
of treating cancer in a mammal by contacting harvested cells with a
virus and transplanting such purged hematopoietic cells into the
mammal after myeloablative treatment.
[0012] It is a further object of the invention to provide a method
of preventing raft-versus-host diseases by in a mammal by
contacting harvested cells with a virus and transplanting such
purged hematopoietic cells into the mammal after myeloablative
treatment.
[0013] It is a further object of the invention to provide a method
of treating autoimmune disease in a mammal by contacting harvested
cells with a virus and transplanting such purged hematopoietic
cells into the mammal after myeloablative treatment
[0014] It is a further object of the invention to provide a method
of treating cancer in a mammal receiving a bone marrow or
peripheral blood stem cell transplant comprising the treatment of
the transplant with a virus.
SUMMARY OF THE INVENTION
[0015] This invention relates to a method of reducing or
eliminating undesirable cells in a mixture of desirable cells and
undesirable cells by contacting the mixture of cells with a
virus.
[0016] This invention also relates to a method of reducing or
eliminating undesirable cells in a mixture of desirable cells and
undesirable cells by contacting the mixture of cells with an RNA
virus.
[0017] This invention also relates to a method of reducing or
eliminating neoplastic cells in an ex vivo mixture of normal
hematopoietic cells and neoplastic cells by contacting the mixture
with a virus, such as an RNA virus.
[0018] This invention also relates to a method for ex vivo purging
of neoplastic cells from a bone marrow or peripheral blood stem
cell harvest by contacting the harvested cells with a virus, such
as an RNA virus.
[0019] This invention also relates to a method for ex vivo purging
of autoimmune cells from a bone marrow or peripheral blood stem
cell harvest by contacting the harvested cells with a virus, such
as an RNA virus.
[0020] This invention also relates to a method for ex vivo purging
of cells that mediate graft-versus-host disease from a population
of bone marrow or peripheral blood stem cells by contacting the
cell population with a virus, such as an RNA virus.
[0021] This invention also relates to a method of treating or
preventing disease such as cancer in a mammal comprising: a)
removing bone marrow or peripheral blood cells from said mammal, b)
contacting said bone marrow or peripheral blood cells ex vivo with
a virus, such as an RNA virus, c) performing myeloablative
treatment on said mammal, and d) transplanting into said mammal the
purged hematopoietic cells of step b.
[0022] This invention also relates to a method of treating cancer
in a mammal receiving a bone marrow or peripheral blood progenitor
cell transplant comprising contacting the harvested cells of the
transplant with a virus, such as an RNA virus, and administering
the purged cells to said mammal.
[0023] This invention also relates to a method of treating
autoimmune disease in a mammal receiving a bone marrow or
peripheral blood progenitor cell transplant comprising contacting
the harvested cells of the transplant with a virus, such as an RNA
virus, and administering the purged cells to said mammal.
[0024] This invention also relates to a method of preventing
graft-versus-host disease in a mammal receiving a bone marrow or
peripheral blood progenitor cell transplant comprising contacting
the harvested cells of the transplant with a virus, such as an RNA
virus, and administering the purged cells to said mammal.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention relates to the discovery of viruses
and the use of viruses for the reduction or elimination of
undesirable cells such as neoplastic cells from a mixture of
desirable cells and undesirable cells. This invention provides
viruses and methods for the purging (reducing or eliminating) of
undesirable cells from normal cells using viruses. Undesirable
cells in hematopoeitic cell transplants that are removed by viruses
in the present invention include neoplastic cells, autoimmune cells
(such as in the case of rheumatoid arthritis or multiple
sclerosis), and cells which mediate graft-versus-host disease.
Treatment of the mammal consists of a) removing bone marrow or
peripheral blood cells from said mammal, b) contacting said bone
marrow or peripheral blood cells ex vivo with a virus, c)
performing myeloablative treatment on said mammal, and d)
transplanting into said mammal the purged hematopoeitic cells of
step b).
METHODS OF THE INVENTION
Purging of Neoplastic Cells
[0026] Incubation of mixtures of normal cells and neoplastic cells
with viruses result in the selective killing of the neoplastic
cells and not the normal cells. Effective means of purging
neoplastic cells from hematopoietic cells can be used in the
treatment of cancer in mammals with autologous bone marrow or
peripheral blood stem cell transplantation.
[0027] For example, bone marrow or peripheral blood stem cells from
a mammal with a neoplasm is contacted with a virus prior to
transplant to prevent relapse from the neoplasm. Neoplastic cells
that can be purged by the methods of the invention include, but are
not limited to, (1) leukemia, (2) lymphoma, (J) carcinomas such as
breast cancer, lung cancer, colon cancer, prostate cancer, and
pancreatic cancer, (4) sarcomas, and (5) cancers of neuroepithelial
origin such as melanoma and neuoblastoma.
[0028] Diverse viruses such as RNA viruses [such as, but not
limited to vesicular stomatitis virus (VSV), Newcastle disease
virus (NDV), and reovirus] can be used to purge neoplastic cells
from normal hematopoietic cells. Normal hematopoeitc cells are
resistant to infection by many viruses including RNA viruses. As an
example, normal human marrow cells are resistant to infection by
VSV, a rhabdovirus, as determined by both infectious virus
production and by viability. Normal marrow cells from two donors
produced no infectious virus even when infected at a high
multiplicity of infection (e.g., 10 plaque forming units
(pfu)/cell; see Example 1). Infected bone marrow cultures were
indistinguishable from mock-infected cultures in their ability to
form the normal spectrum of hematopoietic cell types following in
vitro culture in methylcellulose. As another example, CD34+
enriched normal human marrow were resistant to infection by NDV, a
virus from another family (paramyxovirus); see Roberts et al.,
WO/9918799.
[0029] Many types of neoplastic cells are highly sensitive to cell
killing by many viruses including RNA viruses. As an example using
VSV, acute myelogenous leukemia cell lines OCI/AML3, OCI/AML4 and
OCI/AML5 were highly susceptible to VSV infection with 0.05
pfu/cell killing 50% of the cells at 24 hours and as little as
0.0003 pfu/cell killing 50% at 48 hours (Example 1). The VSV
Indiana serotype used in this experiment was propagated and
harvested from murine L929 cells. As another example, diverse tumor
cell types were shown to be sensitive to VSV including ovarian
carcinoma, fibrosarcoma, lung carcinoma, melanoma, prostate
carcinoma, and leukemia cells (see Example 3). NDV also kills most
human neoplastic cells (see Roberts et al., WO/9918799). Reovirus
type 3 killed human neoplastic fibrosarcoma cells but not normal
fibroblasts (Example 4).
[0030] The selective elimination of neoplastic cells from a
co-culture with normal hematopoietic cells can be achieved with a
variety of viruses. For example, in co-cultures of leukemic
OCI/AML3 cells with normal bone marrow cells (at a 1:9 ratio and at
a 1:3 ratio) VSV killed all of the leukemia cells while having
little if any effect on the normal hematopoeitic cells (Example 2).
The VSV Indiana serotype used in this experiment was propagated and
harvested from murine L929 cells. These data show the selective
destruction of leukemic cells in a mixed population of normal
marrow and the utility of viruses such as VSV in bone marrow
purging. As another example in selective elimination of neoplastic
cells in a mixed culture of normal cells was shown with NDV.
[0031] NDV strain PPMK107, a triple plaqued purified isolate of the
meosogenic NDV strain MK107, selectively killed human oral
carcinoma cells in a mixed culture with normal fibroblasts (see
Roberts et al., WO/9918799).
Purging of Cells Mediating Graft-Versus-Host Disease.
[0032] Incubation of mixtures of undesirable cells such as T
lymphocytes causing graft-versus-host disease and desirable cells
with viruses result in the selective killing of the undesirable
cells. Effective means of purging such marrow or peripheral blood
cells can be used in the prevention of graft-versus-host disease in
mammals.
Purging of Cells Mediating Autoimmune Disease.
[0033] Incubation of mixtures of autoimmune cells causing
autoimmune disease and desirable cells with viruses result in the
selective killing of the autoimmune cells. Effective means of
purging of such undesirable cells can be used in the treatment of
autoimmune disease such as rheumatoid arthritis and multiple
sclerosis.
Screening of Undesirable Cells.
[0034] Undesirable cells of the present invention can include
neoplastic cells with chromosomal deletions or rearrangements of a
gene, or genes, encoding proteins or modulators of the cellular
interferon response or harbor an otherwise defective interferon
response (Colamonici O R, et al, 1992, Blood, 80:744-749; Heyman M,
et al, 1994, Leukemia, 3:425-434; Billard C, et al, 1986, Blood,
67:821-826). The suspended nature of bone marrow or PBPC
populations allows for the facile use of fluorescence activated
cell sorting (FACS) analysis in the determination of the interferon
responsive state of the cell. Probes for interferon responsiveness
include chromosomal hybridization probes for gene deletions or
rearrangements, and probes, such as antibodies, for the analysis of
cellular receptors and components of signal transduction pathways
involved in the cellular response to interferon.
COMPOUNDS OF THE INVENTION
[0035] The viruses of the present invention are capable of
distinguishing undesirable cells such as neoplastic cells from
desirable cells such as normal hematopoeitic cells. RNA viruses of
this present invention include, but are not limited to (1)
single-stranded viruses including those of negative-sense RNA
viruses and positive-sense RNA viruses, and (2) double-strand RNA
viruses. Single strand, negative-sense RNA viruses of the invention
include, but are not limited to, those non-segmented virus families
such as rhabdoviruses [(for example, vesicular stomatitis virus
(VSV)] and paramyxoviruses [for example, Newcastle disease virus
(NDV) and human parainfluenza virus type 3]. Single strand,
positive-sense RNA viruses of the present invention include, but
are not limited to picornaviruses [for example, rhinovirus], and
togaviruses [for example, Sindbis virus]. Double-strand RNA virus
families of the invention include reoviruses. Replication-competent
and replication-incompetent RNA viruses are included in the
invention.
[0036] Included in the present invention are the
"interferon-sensitive viruses" described in Roberts et al.,
WO/9918799, which is herein incorporated by reference in its
entirety. These viruses selectively replicate and kill neoplastic
cells based on the selective deficiency in these cells of an
IFN-mediated antiviral response. In addition to RNA viruses,
included among the "interferon-sensitive viruses" are VA1-mutants
of adenovirus, a DNA virus.
[0037] The rhabdovirus family consist of closely related enveloped,
non-segmented negative-sense, RNA viruses and include the following
genera that infect animals: (1) Vesiculovirus genus (e.g, vesicular
stomatitis virus, VSV); (2) the Lyssavirus genus (e.g, rabies
virus); and (3) the Ephemerovirus genus [Dietzschold B et al.,
1996. Rhabdoviruses. In: Fields Virology, 3.sup.rd Edition, (eds.
Fields B. N., et al.), pp 1137-1159]. In an especially advantageous
embodiment according to the present invention, the rhabdovirus is
vesicular stomatitis virus (VSV). Several serologically distinct
VSV strains have been identified along with a multitude of
characterized mutants. The natural hosts of VSV include insects,
rodents and domestic farm animals. In general, very few North
American people have come in contact with the virus with most human
infections occurring in laboratory personnel and farmers. In
humans, infections are either asymptomatic or manifested as
flu-like symptoms. VSV strains include, but are not limited to,
Indiana, New Jersey, Priy, Coccal and Chandipura. While the
examples disclosed herein relates to VSV Indiana, it is to be
understood that one of skill in the art, by following the methods
outlined in this document, will be readily be able to screen other
VSV strains and derivatives of VSV including mutants of VSV that
selectively kill neoplastic cells.
[0038] The paramyxovirus family of non-segmented negative-sense RNA
viruses comprises three genera: (I) paramyxoviruses including
Newcastle disease virus (NDV); (2) measles-like viruses (morbilli
viruses); and (3) respiratory syncytial viruses (pneuviruses). NDV
is an advantageous virus according to the present invention. NDV is
categorized into three distinct classes according to the effects on
chickens and chicken embryos. "Low virulence" strains are referred
to as lentogenic and take 90 to 150 hours to kill chicken embryos
at the minimum lethal dose (MLD); "moderate virulence" strains are
referred to as mesogenic and take 60 to 90 hours to kill chicken
embryos at the MLD; "high virulence" strains are referred to as
velogenic and take 40 to 60 hours to kill chicken embryos at the
MLD. See, e.g., Hanson and Bradley, 1955 (Science, 122:156-157),
and Diardiri et al., 1961 (Am J Vet Res 9:918-920). All three
classes are useful, including, advantageously, mesogenic strains of
NDV such as MK107.
[0039] In an especially advantageous embodiment according to the
present invention, the double-strand RNA virus is reovirus. In a
further advantageous embodiment according to the present invention,
the reovirus is reovirus type 3.
[0040] In another advantageous embodiment of the invention, RNA
viruses capable of replicating in neoplasms deficient in the
expression of substilism-related proteases are used. Human
parainfluenze virus type 3 is a virus of this type.
[0041] For certain purposes, it is desirable to obtain a clonal
virus to ensure or increase genetic homogeneity of particular virus
strain and to remove defective interfering particles. Removal of
defective interfering particles allows for increased purity in the
final product as assessed by the number of total virus particles
per infectious particle. Clonal virus can be generated by plaque
purification or by other means as described in Roberts et al.,
WO/9918799.
[0042] In another embodiment of the invention, the virus is
genetically modified, as for example, to increase its selectivity
for neoplastic cells. Methods of genetic manipulation of
rhabdoviruses such as VSV are well established (Roberts A., and J.
K. Rose, Virology, 1998, 247:1-6) making it possible to alter the
genetic properties of the virus.
[0043] Furthermore, standard techniques well known to one of skill
in the art can be used to genetically modify VSV and introduce
desired genes within the VSV genome to produce recombinant VSVs
(e.g., Sambrook et al., 1989, A Laboratory Manual, New York, Cold
Spring Harbor Press). In one embodiment of the invention, the G
protein of VSV can be modified to produce fusions that target
specific sites on tumor cells. In another embodiment of the
invention, the VSV is genetically altered to express one or more
suicide genes capable of metabolizing a prodrug into a toxic
metabolite thereby permitting VSV infected tumor cells to be killed
by administration of a prodrug. VSV engineered to express the
herpes virus thymidine kinase or the cytosine deaminase gene can be
used to convert ganciclovir or 5-FC, respectively, into a toxic
compound. However, it is understood that other suicide genes can
also be employed.
FORMULATION AND ADMINISTRATION
[0044] An advantageous embodiment of the invention relates a kit
for use in the ex vivo purging of undesirable cells from a mixture
of desirable and undesirable cells. The kit includes premeasured
amounts of formulated virus, or viruses, appropriate to treat a
mixture containing a certain number of desirable and undesirable
cells. Advantageous formulations include excipients that stabilize
the virus against loss of infectivity, or boost the viability or
survival of the desirable cells. A more advantageous kit allows for
the contacting of the virus formulation with the target mixture of
cells to occur in an aseptic step without the need for
biocontainment equipment. An example of such a device is a
compartmentalized collection container for the target mixture of
cells that contains the pre-measured virus in a separate
compartment. Creation of a patent pathway between the compartments
allows contact between the virus, the target cell population, and
one or more excipients. Contact with the virus and excipients, if
separate, can occur simultaneously, or sequentially. A further
advantageous embodiment of the invention is a type of
compartmentalized container that maintains the optimum temperature
for virus cell interaction during the time of contact between the
virus, or viruses, and the target mixture of desirable and
undesirable cells. Another advantageous embodiment of the invention
relates to a kit that allows for the separation of the contacted
mixture of cells from the virus, excipients. or both after an
appropriate amount of time. The appropriate amount of contact time
would be known or determined by someone skilled in the art.
[0045] Suitable formulations for viruses of the present invention
include those listed for viruses used in the treatment of neoplasms
(Roberts, et al, 1999, PCT WO9918799). In addition, advantageous
formulations include compounds or biologicals that have one or more
of the following activities on the desirable cells in the mixture
of desirable and undesirable cells: differentiating, proliferating,
sparing, stimulating, protecting, and inducing quiescence. For
example, compounds and biologicals of these types include,
cytokines, peptide regulators of cell cycling, interleukins, growth
factors, energy sources, vitamins and electrolytes. Additional
desirable excipients include cryoprotective compounds.
[0046] An effective amount of virus in the invention is to be used
for the reduction or elimination of the undesirable cells with the
maintenance of the desirable cells. It is understood by those
skilled in the art that the amount of virus to be used for the
reduction or elimination of the undesirable cells will vary
depending upon the virus selected, the type and amount of
undesirable cells, and the type and amount of desirable cells to be
maintained. For example, VSV is used at 0.00001 to 10 plaque
forming units (pfu) per cell, and more advantageously at 0.0003 to
10 pfu per cell.
[0047] In another advantageous embodiment of the invention, the
virus is used to contact the mixture of neoplastic cells and normal
cells which are treated before, during or after contact with
interferon. Interferon allows for enhanced protection of normal
cells (see Example 3 and see Roberts et al., WO/9918799). The
interferon (IFN) is selected from the group--class I (alpha, beta
and omega) and class U (gamma), and recombinant versions and
analogs thereof as discussed in, for example, Sreevalsoun, T., 1995
(In: Biologic Therapy of Cancer, second edition, edited by V. T.
DeVita, Jr., et al., J. B. Lippincott Company, Philadelphia, pp
347-364).
[0048] In another advantageous embodiment of the invention, the
virus is used to contact the mixture of neoplastic cells and normal
cells which are treated with a chemotherapeutic agent before during
or after contact with said virus. The chemotherapeutic agent is
used to further reduce the viability of the neoplastic cells.
[0049] The following examples are illustrative, but not limiting of
the methods and compositions of the present invention. Other
suitable modifications and adaptations of a variety of conditions
and parameters normally encountered in clinical therapy which are
obvious to those skilled in the art are within the spirit and scope
of this invention.
EXAMPLE 1
Selective Killing of Leukemia Cells and Not Normal Marrow Cells by
Vesicular Stomatitis Virus as Determined in Separate Cell
Cultures
[0050] The VSV Indiana serotype was plaque purified on mouse L929
cells. An individual plaque was used to infect a monolayer of L929
cells and 18 hours later the supernatant harvested and subjected to
centrifugation at 6,000.times.g for ten minutes. The clarified
supernatant was then filtered through a 0.2 micron filter
(Millipore) and then titered on L cells and stored at -80.degree.
C. in aliquots. Individual aliquots of virus was used only once.
Normal bone marrow cultures from two separate healthy donors were
resistant to infection by this Indiana serotype of vesicular
stomatitis virus (VSV). Normal bone marrow cells produced no
infectious VSV particles, even when infected at a multiplicity of
infection of 10 pfu/cell. Moreover, the infected bone marrow
cultures were indistinguishable from mock-infected cultures in
their ability to form the normal spectrum of hematopoietic cell
types following in vitro culture in methylcellulose. In contrast,
AML cell lines OCI/AML3, OCI/AML4 and OCI/AML5 were highly
susceptible to VSV infection with 0.05 pfu/cell killing 50% of the
cells at 24 hours and as little as 0.0003 pfu/cell killing 50% at
48 hours.
EXAMPLE 2
Selective Killing of Leukemia Cells by Vesicular Stomatitis Virus
in Mixed Cultures Containing Normal Marrow Cells
[0051] The VSV Indiana serotype used in this example was prepared
as indicated in Example 1 In co-cultures of leukemic OCI/AML3 cells
mixed with normal bone marrow cells (1:9 ratio) VSV had selective
oncolytic properties. In this experiment (Table 1), co-cultures
were infected with VSV at a multiplicity of infection of 1 plaque
forming unit (pfu)/cell or 5 pfu/cell for 24 hours and then plated
in methylcellulose with or without growth factors. In the presence
of growth factors, both normal marrow and tumor cells grew while
only OCI/AML3 cells formed colonies in the absence of growth
factors. Colony counts were performed after 14 days (Table 1) and
demonstrated a complete ablation of growth factor-independent
leukemic cells and sparing of normal bone marrow progenitors.
Identical results were observed when a 1:3 mixture of OCI/AML3
cells and normal marrow were used. This data shows the selective
destruction of leukemic cells in a mixed population of normal
marrow and the utility of VSV in ex vivo bone marrow purging.
TABLE-US-00001 TABLE 1 Selective killing of acute myelogenous
leukemia (AML) cells co-cultured with normal bone marrow. Colonies
per dish (receiving 10.sup.4 cells) observed two weeks after VSV
infection are tubulated below for neoplastic cells (leukemia) and
normal hematopoeitic cells (neutrophil, mixed, and monocyte).
Multiplicity of Infection Colony Type 0.0 1.0 5.0 Leukemic 172 0 0*
Neutrophil 12 7 5 Mixed 6 3 4 Monocyte 10 7 5 *No leukemic colonies
were detected on the growth factor minus dishes even when 10.sup.5
cells were plated per dish.
EXAMPLE 3
VSV Selectively Grows in and Kills Neoplastic Cells Compared to
Normal Cells as Determined in Separate Cell Cultures
[0052] A variety of normal and transformed cell lines were either
untreated or pre-treated with 100 units of IFN-alpha, infected with
VSV Indiana at an MOI of 0.1 pfu/ml and incubated for 18 hours at
37.degree. C. (Table 2). Culture media from each sample was titred
for VSV production. Pre-treatment of the normal cell cultures with
interferon reduced viral production to <1000 infectious viral
particles per ml., while tumor cell lines continued to produce
copious amounts of virus particles (10.sup.5-10.sup.8 plaque
forming units per ml.). In tumor cells, a more rapid and fulminant
growth of VSV was observed than in primary normal cell cultures of
fibroblastic or epithelial origin. The differences between the
various cell types was reflected not only in production of virus
particles, but also in the cytopathic effect (cpe) observed at the
microscopic level. TABLE-US-00002 TABLE 2 Virus yield of VSV after
overnight infection of various cell lines either untreated or
treated with IFN Viral Titre (pfu/ml) Cell Line Untreated
IFN-.alpha. OSF7 (primary normal human fibroblast) 1 .times.
10.sup.6 <10 OSF12 (primary normal human fibroblast) 2 .times.
10.sup.5 <10 OSF16 (primary normal human fibroblast) 1 .times.
10.sup.5 <10 PrEC (primary normal human prostate epithelium) 8
.times. 10.sup.6 <10 HOSE (primary normal human ovarian surface
1 .times. 10.sup.7 <1000 epithelium) A2780 (human ovarian
carcinoma) 2 .times. 10.sup.8 1 .times. 10.sup.7 OVCA 420 (human
ovarian carcinoma) 1 .times. 10.sup.8 3 .times. 10.sup.6 C13 (human
ovarian carcinoma) 1 .times. 10.sup.8 1 .times. 10.sup.5 LC80
(human lung carcinoma) 2 .times. 10.sup.9 6 .times. 10.sup.7
SK-MEL3 (human melanoma) 1 .times. 10.sup.9 1 .times. 10.sup.9
LNCAP (human prostate carcinoma) 4 .times. 10.sup.9 5 .times.
10.sup.9 HCT116 (human colon carcinoma) 1 .times. 10.sup.9 2
.times. 10.sup.9 293T (HEK cells transformed with T antigen and 1
.times. 10.sup.8 8 .times. 10.sup.7 Ad virus E1A)
EXAMPLE 4
Selective Killing of Neoplastic Cells and Not Normal Fibroblast
Cells by Reovirus Type 3 in Separate Cell Cultures
[0053] Human tumor cells (HT1080 fibrosarcoma) and normal cells
(CCD922sk, normal human skin fibroblasts) were grown to
approximately 80% confluence in 24 well tissue culture dishes.
Growth medium was removed and PPVR-824, a plaque purified clone of
human reovirus type III, Dearing strain, was added at 1E+6 plaque
forming units (PFU)/well, to 10 PFU/well in 10 fold dilutions (Exp
I) or at 7.2E+7 PFU/well, and 10-fold dilutions ranging from
10.sup.7 l to 100 PFU/well (Exp II). Controls wells with no virus
added were included on each plate. Virus was adsorbed for 90
minutes on a rocking platform at 37.degree. C. At the end of the
incubation period, the viral dilutions were removed and replaced by
1 ml of growth medium. Plates were then incubated for 5 days at
37.sup.0 C in 5% CO.sub.2. Cytotoxicity was quantified by using a
calorimetric MTT (2-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium bromide) assay (Cell Titer 96, catalog #G4000, Promega
Corporation, Madison Wis. 53711) monitored at 570 nm, that detects
mitochondrial enzyme activity (Mosman, T., 1983, J. Immunol.
Methods 65:55). The viability in the virus treated wells was
expressed as a percent of the activity in untreated control wells.
The data was plotted graphically as PFU/well vs. viability as a
percent of control. The IC50 was calculated as the amount of virus
in PFU/well causing a 50% reduction in the amount of viable. The
neoplastic cells were orders of magnitude more sensitive to killing
by PPVR-824 (Table 3). TABLE-US-00003 TABLE 3 Selective Killing of
Neoplastic Cells and Not Normal Fibroblast Cells by Reovirus Type 3
in Separate Cell Cultures Normal Fibroblast Neoplastic Cell
(HT1080), (CCD922sk), IC50 IC50 Expt I >1.0E+06 125 Expt II
>7.2E+07 417
[0054] The foregoing examples are intended as illustrative of the
present invention but not limiting. Numerous variations and
modifications can be effected without departing from the true scope
of the invention.
* * * * *