Note: Descriptions are shown in the official language in which they were submitted.
CA 02436196 2003-07-25
ONCOLYTIC VIRUS FOR PURGING CELLULAR COMPOSITIONS OF CELLS
OF LYMPHOID MALIGNANCIES
FIELD OF THE INVENTION
The present invention relates to a method of selectively removing neoplastic
cells from a mixed cellular composition. Also provided are compositions
prepared
according to this method, and kits comprising viruses useful for practicing
the
invention.
REFERENCES
The following publications, patent applications, and patents are
cited in this application:
U.S. Patent No. 6,596,268.
WO 94/18992, published September l, 1994.
WO 94/25627, published November 10, 1994.
Ahuja, H.G., Foti, A., Bar-Eli, M., Cline, M.J. (1990) The pattern of
mutational involvement of RAS genes in human hematologic
malignancies determined by DNA amplification and direct sequencing.
Blood 75:1684-90.
Bensinger, W.I. (1998) Should we purge? Bone Marrow Transplant. 21:113-
115.
Bischoff, J.R., Kirn, D.H., Williams, A., Heise, C., Horn, S., Muna, M., Ng,
L., Nye, J.A., Sampson-Johannes, A., Fattaey, A., McCormick, F.
(1996) An adenovirus mutant that replicates selectively in p53-
deficient human tumor cells. Science. 274:373-76.
Bos, J. (1989) Ras oncogenes in human cancer: a review. Cancer Res.
49:4682-89.
Brooks, G.F. et al., eds. (1998) Jawetz, Melnick & Adelberg's Medical
Microbiology. New York: McGraw-Hill.
CA 02436196 2003-07-25
Chang, H.W., Jacobs, B.L. (1993) Identification of a conserved motif that is
necessary for binding of the vaccinia virus E3L gene products to
double-stranded RNA. Virology 194:537-47.
Chang, H.W., Uribe, L.H., Jacobs, B.L. (1995) Rescue ofvaccinia virus
lacking the E3L gene by mutants of E3L. .1. Yirol. 69:6605-08.
Chang, H.W., Watson, J.C., Jacobs, B.L. (1992) The E3L gene of vaccinia
virus encodes an inhibitor of the interferon-induced, double-stranded
RNA-dependent protein kinase. Proc. Natl Acad. Sci. U.S.A. 89:4825-
29.
Chaubert, P., Benhattar, J., Saraga, E., Costa, J. (1994) K-ras mutations and
p53 alterations in neoplastic and nonneoplastic lesions associated with
longstanding ulcerative colitis. Am. J. Path. 144:767-775.
Clark, H.M., Yano, T., Sander, C., Jaffe, E.S., Raffeld, M. (1996) Mutation of
the ras genes is a rare genetic event in the histologic transformation of
follicular lymphoma. Leukemia 10:844-47.
Fueyo, J., Gomez-Manzano, C., Alemany, R., Lee, P.S., McDonnell, T.J.,
Mitlianga, P., Shi, Y.X., Levin, V.A., Yung, W.K., Kyritsis, A.P.
(2000) A mutant oncolytic adenovirus targeting the Rb pathway
produces anti-glioma effect in vivo. Oncogene. 19:2-12.
He, B., Gross, M., Roizman, B. (1997) The gamma(1)34.5 protein of herpes
simplex virus 1 complexes with protein phosphatase 1 alpha to
dephosphorylate the alphasubunit of the eukaryotic translation
initiation factor 2 and preclude the shutoff of protein synthesis by
double-stranded RNA-activated protein kinase. Proc. Natl Acad. Sci.
U.S.A. 94:843-48.
Hulkkonen, J., Vilpo, L., Hurme, M., Vilpo, J. (2002) Surface antigen
expression in chronic lymphocytic leukemia: clustering analysis,
interrelationships and effects of chromosomal abnormalities. Leukemia
16:178-85.
Jaffe, E.S. et al. eds. World Health Organization Classification of Tumours
Pathology & Genetics. (2001) Tumours ofHaematopoietic and
Lymphoid Tissues. Geneva: World Health Organization).
Kawagishi-Kobayashi, M., Silverman, J.B., Ung, T.L., Dever, T.E. (1997)
Regulation of the protein kinase PKR by the vaccinia virus
pseudosubstrate inhibitor K3L is dependent on residues conserved
between the K3L protein and the PKR substrate eIF2alpha. Mol. Cell.
Biol. 17:4146-58.
Mah, D.C. et al. (1990) The N-terminal quarter of reovirus cell attachment
protein sigma 1 possesses intrinsic virion-anchoring function. Virology
179:95-103.
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Mills, N.E., Fishman, C.L., Rom, W.N., Dubin, N., Jacobson, D.R. (1995)
Increased prevalence of K-ras oncogene mutations in lung
adenocarcinoma. Cancer Res. 55:1444-47.
Nakamura, N., Nakamine, H., Tamaru, J., Nakamura, S., Yoshino, T.,
Ohshima, K., Abe, M. (2002) The distinction between Burkitt
lymphoma and diffuse large B-Cell lymphoma with c-myc
rearrangement. Mod. Pathol. 15:771-76.
Nedergaard, T., Guldberg, P., Ralflciaer, E., Zeuthen, J. (1997) A one-step
DGGE scanning method for detection of mutations in the K-, N-, and
H-ras oncogenes: mutations at codons 12, 13 and 61 are rare in B-cell
non-Hodgkin's lymphoma. Int. J. Cancer 71:364-69.
Nemunaitis, J. (1999) Oncolytic viruses. Invest. New Drugs 17:375-386.
Neri, A., Knowles, D.M., Greco, A., McCormick, F., Dalla-Favera, R. (1988)
Analysis of RAS oncogene mutations in human lymphoid
malignancies. Proc. Nat'1 Acad. Sci. U.S.A. 85:9268-72.
Nibert, M.L., Schiff, L.A., and Fields, B.N., Reoviruses and their replication
in Fields Virology, 3'd Edition, Lippencott-Raven Press, 1995, pp.
1 S 57-96.
Nieto, Y. and Shpall, E.J. (1999) Autologous stem-cell transplantation for
solid tumors in adults. Hematol. Oncol. Clin. North Am. 13:939-68.
Romano, P.R., Zhang, F., Tan, S.L., Garcia-Barrio, M.T., Katze, M.G., Dever,
T.E., Hinnebusch, A.G. (1998) Inhibition of double-stranded RNA-
dependent protein kinase PKR by vaccinia virus E3: role of complex
formation and the E3 N-terminal domain. Mol. Cell. Biol. 18:7304-16.
Sharp, T.V., Moonan, F., Romashko, A., Joshi, B., Barber, G.N., Jagus, R.
(1998) The vaccinia virus E3L gene product interacts with both the
regulatory and the substrate binding regions of PKR: implications for
PKR autoregulation. Virology 250:302-15.
Spyridonidis, A., Bernhardt, W., Fetscher, S., Behringer, D., Mertelsmann, R.,
Henschler, R. (1998) Minimal residual disease in autologous
hematopoietic harvests from breast cancer patients. Ann. Oncol. 9:821-
26.
Steenvoorden, A.C., Janssen, J.W., Drexler, H.G., Lyons, J., Tesch, H.,
Binder, T., Jones, D.B., Bartram, C.R. (1988) Ras mutations in
Hodgkin's disease. Leukemia 2:325-26.
Stewart, D.A., et al. (1999) Superior autologous blood stem cell mobilization
from dose-intensive cyclophosphamide, etoposide, cisplatin plus G-
3
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CSF than from less intensive chemotherapy regimens. Bone Marrow
Transplant. 23: 111-117.
Stojdl, D.F., Lichty, B., Knowles, S., Marius, R., Atkins, H., Sonenberg, N.,
Bell, J.C. (2000) Exploiting tumor-specific defects in the interferon
pathway with a previously unknown oncolytic virus. Nat. Med. 6:821-
25.
Turner, D.L. et al. (1992) Site directed mutagenesis of the C-terminal portion
of reovirus protein sigmal :evidence for a conformation-dependent
receptor binding domain. Virology 186:219-27.
Winter, J.N. (1999) High-dose therapy with stem-cell transplantation in the
malignant lymphomas. Oncology (Huntingt.) 13:1635-45.
Yoon, S.S., Nakamura, H., Carroll, N.M., Bode, B.P., Chiocca, E.A., Tanabe,
K.K. (2000) An oncolytic herpes simplex virus type 1 selectively
destroys diffuse liver metastases from colon carcinoma. FASEB J.
14:301-11.
Zorn, U., Dallmann, L, Grosse, J., Kirchner, H., Poliwoda, H., Atzpodien, J.
(1994) Induction of cytokines and cytotoxicity against tumor cells by
Newcastle disease virus. Cancer Biother. 9:225-35.
All of the publications, patent applications, and patents, cited above or
elsewhere in this application, are herein incorporated by reference in their
entirety to
the same extent as if each individual publication, patent application or
patent was
specifically and individually indicated to be incorporated by reference in its
entirety.
BACKGROUND OF THE INVENTION
Cell proliferation is regulated by a combination of growth-promoting and
growth-inhibiting signals. Cells that fails to respond to growth-inhibiting
signals, or
over-respond to growth-promoting signals, may proliferate abnormally rapidly
(referred to as neoplastic development) and may eventually develop into cancer
(a
malignant neoplasm).
Chemotherapy and radiation therapy are current methods of treating cancer,
generally based on the rapid proliferation of cancer cells compared to most
other cells
in the body. Rapidly proliferating cells are more sensitive to drugs that
interfere with
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cell proliferation, such as drugs that interfere with DNA synthesis. In
theory, by
carefully choosing the dosage of chemotherapeutic drugs or ionizing radiation
delivered to the body, one can inhibit cancer cell proliferation without
seriously
damaging normal cells. However, some normal cells, such as hematopoietic stem
cells, also proliferate rapidly. Therefore, any dosage that is harmful to
cancer cells is
likely harmful to hematopoietic stem cells.
Because it is difficult to find a chemotherapeutic drug or radiation treatment
regimen that selectively kills cancer cells without causing unacceptable
damage to
normal cells, high-dose chemotherapy or radiation therapy, followed by
autologous
hematopoietic progenitor stem cell transplantation, has gained acceptance as a
therapeutic approach for treating many cancers (see, e.g., Winter 1999; Nieto
and
Shpall 1999). According to this approach, a portion of a cancer patient's
hematopoietic stem cells are removed prior to high-dose chemotherapy or
radiation
therapy, which will be lethal to most all rapid-proliferating cells, including
cancer
cells and hematopoietic stem cells. Following chemotherapy or radiation
therapy, the
patient's own hematopoietic stem cells (i.e., an autograft) are returned to
hematopoietic centers in the body, e.g., the bone marrow, in the hope that
they will
repopulate the patient's immune system (these processes may be referred to
herein as
"reintroduction" and "repopulation," respectively).
A serious drawback of this type of therapy is that when the hematopoietic
progenitor stem cells are removed from the patients, they are often
contaminated with
cancer cells, which will be returned to the body along with the hematopoietic
cells.
This complication is especially problematic when the patient has a cancer of
hematopoietic origin, although patients with solid tumors may still harbor
cancer cell-
contaminated hematopoietic stem cells, particularly in the case of solid
tumors that
have metastasized. It is therefore desirable to purge autografts of cancer
cells prior to
reintroduction to the patient.
Several methods have been employed to purge autografts of cancer cells
(Spyridonidis et al. 1998; Bensinger 1998). The autograft can be treated ex
vivo with
chemotherapeutic drugs (or radiation) to kill the contaminating neoplastic
cells.
However, as discussed above, it is difficult to find a therapeutic dose that
selectively
kills cancer cells without causing an unacceptable level of damage to
hematopoietic
CA 02436196 2003-07-25
stem cells. Autografts can also be treated with a toxin conjugated to one or
more
antibodies that recognize antigens specific for cancer cells. However, tumor-
specific
antigens are not always available, depending on the specific form of cancer
involved.
It is also possible to isolate stem cells from other cells in the autograft,
including
cancer cells, based on stem cell-specific surface markers (e.g., CD34) using
flow
cytometry, affinity chromatography, or magnetic bead selection, along with
appropriate antibodies. However, by selecting only certain hematopoietic cells
(e.g.,
CD34+ cells), other hematopoietic stem cells, including less differentiated
CD34- cells
and more differentiated but still immature precursor cells, are also
eliminated, thereby
delaying or preventing the full recovery of the patient's immune system
(Bensinger
1998). In addition, this procedure may result in the loss of about half the
CD34+ cells
while failing to remove all contaminating cancer cells (Spyridonidis et al.
1998).
Therefore, there remains a need for a highly selective and efficient method
for
purging autografts of neoplastic cells.
SUMMARY OF THE INVENTION
The present invention relates to a method for removing cells of a lymphoid
malignancy from a mixed cellular composition using a virus that selectively
replicates
in cells of the lymphoid malignancy. The invention is particularly useful for
purging
mixed cellular compositions comprising hematopoietic stem-cells of cells of
lymphoid malignancies. The purged, virus-treated hematopoietic cell
compositions
may then be reintroduced to the same animal or transplanted into a second
animal,
most likely following chemotherapy or radiation therapy.
Accordingly, the invention provides a method of selectively removing cells of
a lymphoid malignancy from a mixed cellular composition, comprising the steps
of
a) obtaining a mixed cellular population suspected of containing cells of a
lymphoid malignancy from an animal,
b) contacting the mixed cellular composition with at least one virus under
conditions that result in substantial killing of cells of the lymphoid
malignancy, thereby producing a treated cellular composition; and
c) collecting the treated cellular composition so produced.
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In one embodiment of the invention, the mixed cellular composition comprises
hematopoietic stem cells. In a preferred embodiment, the mixed cellular
composition
comprises CD34+ cells. In one embodiment of the invention, the mixed cellular
composition has been harvested from bone marrow. In another embodiment, the
mixed cellular composition has been harvested from blood. In yet another
embodiment, the mixed cellular composition is selected from the group
consisting of
a tissue, an organ, or a portion of a tissue or organ, wherein the
corresponding treated
cellular composition is useful for transplantation. In another embodiment, the
mixed
cellular composition comprises cultured cells, semen, or eggs. In a preferred
embodiment of the invention, steps (a) and (b), above, are performed ex vivo.
In one embodiment of the invention, the method is useful for treating a mixed
cellular population comprising (or suspected of comprising) cells of at least
one
lymphoid malignancy. In a preferred embodiment of the invention, the lymphoid
malignancy is a B-cell lymphoid malignancy. Examples of B-cell malignancies
that
can be treated by the instant invention include but are not limited to
Burkitt's
lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, diffuse large
B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, and mantle-
cell lymphoma. The cells of the lymphoid malignancy may comprise normal ras
genes.
Numerous different viruses can be used to practice the invention. In one
embodiment, the virus or viruses are replication competent. In a preferred
embodiment of the invention, at least one reovirus is used. The reovirus may
be a
human reovirus, a non-human reovirus, a modified reovirus, a recombinant
reovirus,
combinations thereof, or chimeras thereof. In another embodiment of the
invention,
at least one virus is an adenovirus, herpesvirus, vaccinia virus, parapoxvirus
orf virus,
Newcastle disease virus, or vesicular stomatitis virus.
In the case of adenovirus, the E 1 A-coding region may be mutated such that
the
resulting ElA gene product does not bind to Rb. Alternatively or additionally,
the
adenovirus may be mutated in the E 1 B-coding region such that the resulting E
1 B
gene product does not bind to p53. The adenovirus may also be capable of
expressing
a wild type p53 protein. Examples of adenovirus useful for practicing the
instant
invention include but are not limited to Delta24 and ONYX-O1 S.
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In another embodiment of the invention, at least one virus is mutated or
modified such that the virus does not produce a gene product that inhibits
double
stranded RNA kinase (PKR). In another embodiment of the invention, the virus
is an
interferon sensitive virus and the method further comprises a step of adding
interferon
to the mixed cellular composition.
In another embodiment of the invention, virus is removed from the mixed
cellular composition or inactivated in the mixed cellular composition
following
treatment.
In one embodiment of the invention, the mixed cellular composition is stored
either before or after treatment with virus. In a preferred embodiment, the
treated
cellular composition is stored. In a most preferred embodiment, the treated
cellular
composition is stored in liquid nitrogen in a solution comprising DMSO.
In a preferred embodiment of the invention, the method further comprises the
step of reintroducing the treated cellular composition to the animal from
which it was
obtained. In a most preferred embodiment of the invention, such an animal is
treated
with chemotherapeutic drugs or radiation therapy following the harvesting of
the
mixed cellular composition but prior to the reintroduction of the treated
cellular
composition. In one embodiment of the invention, the treated cellular
composition is
reintroduced into the bone marrow of the animal. In another embodiment of the
invention, the treated cellular composition is reintroduced into the blood of
the
animal.
In a variation of this preferred embodiment of the invention, virus is
administered to an animal or patient following the harvesting of a mixed
cellular
composition from the animal or patient but prior to treatment of the animal or
patient
with chemotherapy or radiation therapy. The animal or patient is then allowed
to
produce neutralizing antibodies to the virus before being treated with
chemotherapy
or radiation therapy. The mixed cellular composition is treated with the same
virus as
administered to the animal or patient prior to chemotherapy or radiation
therapy, or a
variant thereof, providing that both viruses are recognized by at least some
of the
same antibodies produced in the animal or patient. When the treated cellular
composition is returned to the animal or patient, the animal or patient will
already
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possess circulating antibodies that will recognize and inactivate virus
present in the
treated cellular composition. In one particular embodiment of this invention,
virus
present in the treated cellular composition is inactivated primarily by these
circulating
antibodies. In another particular embodiment, circulating antibodies remove
only
residual virus that was not completely inactivated or removed from the treated
cellular
composition by other means, prior to reintroduction to the animal or patient.
In another embodiment of the invention, a composition comprising anti-virus
antibodies is administered to the animal or patient prior to, simultaneously
with, or
soon after, the treated cellular composition is reintroduced to the animal or
patient.
These antibodies recognize and inactivate virus present in the treated
cellular
composition though passive immunity.
In yet another embodiment of the invention, the treated cellular composition
is
administered to at least one second animal, wherein the second animal is not
the
animal from which the mixed cellular composition was harvested. In a preferred
embodiment of the invention, the second animal is genetically compatible with
the
animal from which the mixed cellular composition was harvested. In one
embodiment of the invention, immunosuppresants are administered to the second
animal to prevent or minimize rejection of the transplanted cellular
composition.
The invention may be administered to a variety of different animals. In a
preferred embodiment of the invention, the animal is selected from the group
consisting of dogs, cats, sheep, goats, cattle, horses, pigs, humans, and non-
human
primates.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Bar graph showing the % viability of Raji, CA46, Daudi, Ramos,
ST486, and four DLBCL cells (OCY-LY1, OCY-LY2, OCY-LYB, and OCY-LY10)
following challenge with reovirus at an MOI of 20. % viability is based on
trypan
blue exclusion.
Figure 2: Bar graph showing the ability of reovirus to replicate in Raji,
CA46, Daudi, Ramos, ST486, and four DLBCL cells (OCY-LY1, OCY-LY2, OCY-
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LYB, and OCY-LY10). Growth is reported in PFUs at 0 hour (open squares) and 96
hours (filled squares) post infection.
Figure 3: Graphs showing the sizes of lymphoma-derived tumors implanted
in mice following infection with either live (filled symbols) or UV-
inactivated (open
circles) reovirus. Panel A: Raji tumors, intratumoral virus administration.
Panel B:
Daudi tumors, intratumoral virus administration. Panel C: Raji tumors,
intravenous
virus injection.
Figure 4: Bar graphs showing cell viability following reovirus infection.
Panels A-C show the purging effects of reovirus on mixed cellular compositions
comprising apheresis product and tumor cells selected from the group
consisting of
MCF7, MDA MB 468 and SKBR3 cells. Panel D shows a control experiment in
which CD34+ cells were infected with reovirus.
Figure 5: Bar graph showing the number of colonies of different cell types
originating from CD34+ cells at various times following reovirus infection.
NV: no
virus. LV: live virus. G: granulocytes. E: erythroids. GEMM: granulocyte
erythroid macrophage megakaryocyte.
DETAILED DESCRIPTION OF THE INVENTION
For the purposes of the following description, the following terms have been
given the following meanings unless otherwise indicated.
Definitions
Activated Ras pathway: A Ras pathway that has become activated (i.e., the
constitutive level of signaling through the pathway has increased compared to
that of
equivalent normal cells) by way of Ras gene structural mutation, elevated
level of Ras
gene expression, increased stability of the Ras gene message, or any mutation
or other
mechanism which leads to the activation of Ras or a factor or factors
downstream or
upstream from Ras in the Ras pathway.
Adenovirus: Double-stranded DNA (dsDNA) viruses of the family
Adenoviridae. In humans, adenoviruses can replicate and cause disease in the
eye and
CA 02436196 2003-07-25
in the respiratory, gastrointestinal, and urinary tracts. About one-third of
the 47
known human serotypes are responsible for most human diseases associated with
adenovirus (Brooks et al. 1998).
Apheresis: A method of isolating specific fractions of whole blood wherein
whole blood is withdrawn from an animal or donor and separated into various
components. The desired components are then retained while the remainder is
returned to the animal or donor.
Burkitt's lymphoma (BL): A B-cell lymphoma, usually caused by Epstein-
Barr virus (EBV).
Cellular composition suspected of containing neoplastic cells: A cellular
composition that may contain neoplastic cells. For example, any autograft
obtained
from a patient, clinical test subject, or experimental animal known to have or
suspected of having a neoplastic disorder. Cells that have been growing in
culture for
a considerable amount of time may also give rise to neoplastic cells as a
result of
spontaneous mutations.
Cellular composition: Any cells or mixture of cells obtained from an animal.
Cellular compositions include but are not limited to bone marrow, tissue from
the
spleen, lymph nodes, Peyer's patches, thymus, tonsils, fetal liver, liver,
bursa of
Fabricus (in Avian species), mucosa-associated lymphoid tissues (MALT),
spleen,
whole blood, and other mixtures of animals cells from various tissues or
organs, as
well as fractions and/or combinations thereof.
Chronic lymphocytic leukemia (CLL): A proliferative disorder resulting from
the accumulation of mature lymphocytes in the blood andlor bone marrow.
Contacting cells with reovirus: Providing reovirus to cells of an animal such
that the virus and cells are in sufficient proximity to allow virus adsorption
to the cell
surface.
Diffuse large B-cell lymphoma (DLBCL): An aggressive malignancy of
mature B-lymphocytes accounting for about 40-50% of non-Hodgkin's lymphomas.
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Ex vivo: Outside the body. As use herein, ex vivo refers to a process that is
performed under cell culture conditions using a cellular composition obtained
from an
animal. Following manipulation of the mixed cellular composition (e.g.,
treatment
with one or more viruses), the treated cellular composition may be
reintroduced to the
same animal or administered to at least one second animal.
Follicular lymphoma (FL): A malignancy of follicular center B-cells
accounting for about 25-40% of non-Hodgkin's lymphomas.
Genetically compatible: As used herein, "genetically compatible" refers to
animals that express substantially identical cell surface antigens (or major
histocompatability markers (MHC markers)) on their cell surfaces.
Hematopoietic stem/progenitor cells: Undifferentiated and/or partially
differentiated cells cable of differentiating into a variety of different
hematopoietic
lineages, including myeloid and lymphoid cells. The presence of CD34 is often
used
as a marker for hematopoietic stem/progenitor cells (i.e., CD34+ cells),
although more
primitive, less differentiated, hematopoietic precursor cells may actually
lack CD34
(i.e., CD34- cells).
Herpesvirus: Members of the family Herpesviridae, including but not limited
to herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2),
cytomegalovirus
(CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), Kaposi's
sarcoma-
associated virus (KSHV), and other numbered herpesviruses (e.g., HHV-6, 7,
etc.) of
humans, pigs, cattle, horses, chickens, turkeys, and other animals. HSV-1 and
HSV-2
are the best studied herpesviruses. HSV gene Y134.5 encodes the gene product
infected-cell protein 34.5 (ICP34.5) that can prevent the antiviral effects
exerted by
PKR. ICP34.5 has a unique mechanism of preventing PKR activity by interacting
with protein phosphatase 1 and redirecting its activity to dephosphorylate eIF-
2a (He
et al. 1997). In cells infected with either wild-type or the genetically
engineered virus
from which the y,34.5 genes were deleted, eIF-2oc is phosphorylated and
protein
synthesis is turned off in cells infected with y~ 34.5-minus virus. It would
be expected
that the Y134.5 minus virus would be replication competent in cells with an
activated
Ras pathway in which the activity of ICP34.5 would be redundant.
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Hodgkin's lymphoma (HL) (or Hodgkin's disease): A lymphoma
characterized by the presence of mononucleated Hodgkin and multinucleated Reed-
Sternberg cells (HRS), which occur at low frequency in tumor tissues.
Interferon sensitive virus: A virus that does not replicate in or kill normal
cells
in the presence of interferon. As defined below, a normal cell is a cell that
is not
neoplastic. To test whether a virus is interferon sensitive, a culture of
normal cells
may be incubated with the virus in the presence of varying concentrations of
interferon, and the survival rate of the cells is determined according to well-
known
methods in the art. A virus is interferon sensitive if less than 20%,
preferably less
than 10%, of the normal cells is killed at a high concentration of interferon
(e.g., 100
units per ml).
Intramedullary: Within or pertaining to the bone marrow of an animal.
Lymphoid cell lines: Cell lines derived from lymphoid cells or their
precursors.
Lymphoid malignancies: As used herein, this term refers broadly to a
heterogeneous group of diseases, disorders, or conditions resulting from the
rapid
proliferation of lymphoid or lymphoid precursor cells. Examples of lymphoid
malignancies include but are not limited to Burkitt's lymphoma, Hodgkin's
lymphoma, chronic lymphocytic leukemia, and non-Hodgkin's lymphomas such as
diffuse large B-cell lymphoma, follicular lymphoma, mantle-cell lymphoma and
small
lymphocytic lymphoma. Current classification is based on numerous factors,
including clinical presentation, cell morphology, and chromosomal
abnormalities. As
used herein, "lymphoid malignancies" is synonymous with "lymphoid neoplasms"
and "lymphoid neoplasias" based on the classification scheme published by the
World
Health Organization (Jaffe et al. 2001 ).
Mantle-cell lymphoma (MCL): A lymphoma arising from naive pre-germinal
center cells of either the primary follicle or the mantle regions of secondary
follicles.
The disease is often associated with gastrointestinal tract lymphomatous
polyposis or
leukemia, primarily affects seniors, and accounts for 2% to 8% of non-Hodgkin
lymphomas.
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Mixed cellular (or cell) composition: A cellular composition comprising at
least two kinds of cells. Typically, the mixed cellular composition comprises
both
normal and neoplastic cells. It is preferable that most of the cells in the
cellular
composition are dividing cells and a virus selectively replicates in the
neoplastic cells.
Mutated (or modified) adenovirus: As used herein, mutated adenoviruses are
those in which the gene product (or products) that prevent the activation of
PKR are
lacking, inhibited, or mutated such that PKR activation is not blocked.
Adenovirus
encodes several gene products that counter antiviral host defense mechanisms.
The
virus-associated RNA (VAI RNA or VA RNAI) of the adenovirus are small,
structured RNAs that accumulate in high concentrations in the cytoplasm at
late time
after adenovirus infection. These VAI RNA bind to the double stranded RNA
(dsRNA) binding motifs of PKR and block the dsRNA-dependent activation of PKR
by autophosphorylation. Thus, PKR is not able to function and the virus can
replicate
within the cell. The overproduction of virons eventually leads to cell death.
In a
mutated or modified adenovirus, the VAI RNA's are preferably not transcribed.
Such
mutated or modified adenovirus would not be able to replicate in normal cells
that do
not have an activated Ras-pathway; however, it would be able to infect and
replicate
in cells having an activated Ras-pathway.
Mutated (or modified) HSV: As used herein, mutated or modified HSV are
those herpesviruses in which the gene product (or products) which prevent the
activation of PKR are lacking, inhibited or mutated such that PKR activation
is not
blocked. Preferably, the HSV gene y134.5 is not transcribed. Such mutated or
modified HSV would not be able to replicate in normal cells that do not have
an
activated Ras-pathway, however, it would be able to infect and replicate in
cells
having an activated Ras-pathway.
Mutated (or modified) parapoxvirus orf: A parapoxvirus orf virus in which the
gene product or products that prevent activation of PKR are lacking,
inhibited, or
mutated such that PKR activation is not blocked. Preferably, the gene OV20.OL
is not
transcribed. Such mutated or modified parapoxvirus orf viruses would not be
able to
replicate in normal cells that do not have an activated Ras-pathway; however,
they
would be able to infect and replicate in cells having an activated Ras-
pathway.
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Mutated (or modified) vaccinia virus: As used herein, these terms refer to
vaccinia virus variants in which the gene product or products that prevent the
activation of PKR are lacking, inhibited or mutated, such that PKR activation
is not
blocked. Preferably, the E3L gene and/or the K3L gene is not transcribed. Such
mutated or modified vaccinia viruses are unable to replicate in normal cells
that do
not have activated ras pathways; however, they are able to infect and
replicate in cells
having activated ras pathways.
Neoplasm: As used herein, neoplasm is used to describe benign or malignant
tumors. As used herein, malignant tumors may include various forms of cancer.
Malignant neoplasms/tumors can be broadly classified into three major types.
Malignant neoplasms arising from epithelial structures are called carcinomas;
malignant neoplasms that originate from connective tissues such as muscle,
cartilage,
fat or bone are called sarcomas; and malignant tumors arising from
hematopoietic
cells, are called leukemias and lymphomas. Other neoplasms include but are not
limited to neurofibromatosis.
Neoplastic cells: Cells that proliferate aberrantly with respect to their
normal
counterparts and typically do not respond to growth inhibition signals. A new
growth
comprising neoplastic cells is a neoplasm or tumor. A neoplasm is an abnormal
tissue
growth, generally forming a distinct mass, which grows more rapidly than
normal
cells. Neoplasms may show partial or total lack of structural organization and
functional coordination with normal tissue. As used herein, a neoplasm is
intended to
encompass hematopoietic neoplasms as well as solid neoplasms. Also as used
herein,
the term "neoplastic cells," or substantially similar terms, refer to cells
with
proliferative disorders.
Non-Hodgkin's lymphomas (NHL): A heterogeneous group of lymphoid
malignancies arising from mature lymphoid cells.
Normal cell: A cell that that is not neoplastic, responds to growth
stimulatory
and inhibitory signals in a manner typical for the particular cell type, does
not cause
tumors in animals, and is not believed to harbor chromosomal abnormalities
that
would result in a neoplastic phenotype.
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Normal hematopoietic stem/progenitor cells: Hematopoietic stem/progenitor
cells that are not associated with a cell proliferative disorder or neoplasm
and are not
believed to harbor chromosomal abnormalities that would cause such growth
phenotypes.
Normal lymphocytes: Lymphocytes that are not associated with a transformed
or malignant growth phenotypes and not believed to harbor chromosomal
abnormalities that would cause such growth phenotypes.
Normal ras gene: A gene encoding a normal, i.e., non-transforming form, of
ras.
Parapoxvirus orf A poxvirus that induces acute cutaneous lesions in different
mammalian species, including humans. Parapoxvirus orf viruses naturally infect
sheep, goats, and humans through broken or damaged skin, replicates in
regenerating
epidermal cells, and induces pustular lesions that turn to scabs. The
parapoxvirus orf
viruses encode the gene OV20.OL that is involved in blocking PKR activity
(Haig et
al. 1998).
Pluripotency: Potential to differentiate into different cell types. As used
herein, pluripotency refers to cells that have broad "differentiation
potential" with
respect to the type of cells that may arise from the undifferentiated cells.
Ras-activated neoplastic cells or ras-mediated neoplastic cells: Cells that
proliferate at an abnormally high rate due to, at least in part, activation of
the ras
pathway. The ras pathway may be activated by way of ras gene structural
mutation,
elevated level of ras gene expression, elevated stability of the ras gene
message, or
any mutation or other mechanism which leads to the activation of ras or a
factor or
factors downstream or upstream from ras in the ras pathway, thereby increasing
the
ras pathway activity. For example, activation of EGF receptor, PDGF receptor
or Sos
results in activation of the ras pathway. Ras-mediated neoplastic cells
include, but are
not limited to, ras-mediated cancer cells, which are cells proliferating in a
malignant
manner due to activation of the ras pathway.
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Reovirus: Any virus in the family Reoviridae. The name reovirus (respiratory
and enteric orphan virus) is a descriptive acronym suggesting that these
viruses,
although not associated with any known disease state in humans, can be
isolated from
both the respiratory and enteric tracts (Sabin 1959).
Replication competent virus: A virus that is capable of replicating in at
least
one cell type.
Resistance of cells to viral infection: Infection of the cells with the
particular
virus or viruses does not result in significant viral production or yield.
SCID/NOD mice: Nonobese diabetic (NOD) mice with severe combined
immunodeficiency (SCID). SCID/NOD mice lack functional T and B-lymphocytes.
SCID/NOD mice are useful for growing palpable tumor masses, derived from
implantated exogenous tumor cells, for subsequent challenge with therapeutic
agents.
Solid lymphoma: A lymphoid malignancy characterized by the formation of a
discrete mass of predominantly malignant cells (i.e., cells of the lymphoid
malignancy) at one or more loci in an animal. Solid lymphomas may remain
localized in an animal or may metastasize, resulting in the formation of
additional
malignant cell masses or resulting in a circulating lymphoid malignancy.
Substantial lysis: As used herein, substantial lysis refers to a decrease in
viability, e.g., through lysis, of cells of a lymphoid malignancy. Lysis can
be
determined by a viable cell count of the treated cells, and the extent of
decrease can
be determined by comparing the number of viable cells in the treated cells to
that in
the untreated cells, or by comparing the viable cell count before and after
reovirus
treatment. Lysis can also be inferred from a reduction in the size of a solid
lymphoma
in terms of either (or both) mass or volume. The decrease in viability is at
least about
10%, preferably at least SO%, and most preferably at least 75% of the
proliferating
cells. The percentage of lysis can be determined for tumor cells by measuring
the
reduction in the size of the tumor in the mammal or the lysis of the tumor
cells in
vitro. Substantial lysis also includes the complete elimination of cells of a
lymphoid
malignancy from an animal or from a mixed cellular composition.
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Transplant recipient: An animal, including but not limited to dogs, cats,
sheep,
goats, cattle, horses, pigs, humans, and non-human primates, that receives a
transplant
of one or more cellular compositions. Preferably the recipient is a human, and
more
preferably the recipient is a human who is receiving a transplant in the
treatment of
cancer.
Vaccinia virus: Viruses of the orthopoxvirus genus that infect humans and
produce localized lesions (Brooks et al. 1998). Vaccinia virus encodes two
genes that
play a role in the down regulation of PKR activity through two entirely
different
mechanisms. E3L gene encodes two proteins of 20 and 25 kDa that are expressed
early in infection and have dsRNA binding activity that can inhibit PKR
activity.
Deletion or disruption of the E3L gene creates permissive viral replication in
cells
having an activated Ras pathway. The K3L gene of vaccinia virus encodes pK3, a
pseudosubstrate of PKR.
Viral oncolysate: A composition prepared by treating tumor cells with an
oncolytic virus in vitro, which composition is subsequently administered to a
tumor
patient with the same kind of tumor in order to induce immunity in the tumor
patient
against this tumor. As such, viral oncolysates are essentially virus-modified
cancer
cell membranes.
The Invention:
The present invention is directed to a method for selectively removing
neoplastic cells from a mixed cellular composition, for example an autograft,
by using
a virus that selectively replicates in neoplastic cells. The instant invention
is based, in
part, on Applicant's discovery that reovirus is capable of replicating in
cells of
lymphoid malignancies despite the relative infrequency of activating Ras
mutations in
lymphoid malignancies, compared to malignancies derived from other cell types
(Neri
et al. 1988; Steenvoorden et al. 1998; Ahuja et al. 1990; Clark et al. 1996;
and
Nedergaard et al. 1997).
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The conclusion that reovirus is able to replicate in cells of lymphoid
malignancies is based on in vitro cell culture data as well as in vivo animal
model
data. In a first set of experiments, a panel of nine lymphoid cell lines was
assembled
for challenge with reovirus. The panel comprised two BL cell lines (Raji and
Daudi)
in which the Epstein-Barr virus (EBV) was detected (i.e., EBV+ cell lines),
three BL
cell lines (CA46, Ramos, and ST486) in which EBV was not detected (i.e., EBV-
cell
lines), and four DLBCL cell lines (OCY-LY1, OCY-LY2, OCY-LY8, and OCY-
LY10). Reovirus replicated in six of the nine lymphoma-derived cell lines,
specifically Raji, CA46, and all four DLBCL cells (Figure 1). Reovirus was
unable to
replicate in only three of these cell lines (Daudi, Ramos, and ST486). This
experiment is described in more detail in Example 1.
In a second experiment, cell suspensions were prepared from 27 lymphoid
tumor biopsy specimens for challenge with reovirus. Of the 27 specimens, 15
were
associated with a clinical diagnosis of CLL, and 12 with a clinical diagnosis
of NHL.
The NHL specimens could be further divided into BL (1); DLBCL (2); small
lymphocytic leukemia (SLL) (2); FL, grade I (1); FL, grade II (4); FL, grade
III (1);
and MCL (1). Three suspensions each of normal primary blood mononuclear cells
(PBMC) and normal bone marrow, CD34+-hematopoietic stem/progenitor cells were
included as negative controls.
Reovirus was able to replicate in all 15 CLL cell suspensions, the BL cell
suspension, both DLBCL cell suspensions, one of the SLL cell suspensions, the
MCL
cell suspension, and the FL, grade I, cell suspension. Reovirus did not
replicate in S
of the 6 FL cells suspensions, one of the SLL cell suspensions, or, as
expected, any of
the six negative control cell suspensions comprising normal PBMC or
hematopoietic
stem/progenitor cells (Figure 2). In total, reovirus was able to replicate in
21 of the 27
cell suspensions prepared from lymphoid tumor biopsy specimens. This
experiment is
described in more detail in Example 2.
In vivo data using a SCID/NOD mouse xenograft model provided direct
evidence that reovirus was effective in reducing the growth of a BL tumor in
an
animal. In this experiment, mice were injected with either the reovirus-
susceptible
Raji cells or the reovirus-resistant, Daudi cells, from above.
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Following the establishment of palpable tumor masses, mice were treated with
either live reovirus or LTV-inactivated virus. The administration of live
reovirus to
mice with Raji tumors resulted in an approximately ten-fold reduction in tumor
size
compared to mice receiving UV-inactivated reovirus (Figure 3). These results
showed that reovirus could be used to treat tumors arising from lymphoid
malignancies in an animal. Consistent with the results of the above in vitro
experiment, reovirus was not effective in treating the mice with Daudi tumors.
This
experiment is described in more detail in Example 3.
While the ras genotypes of all nine lymphoid tumor cell lines and all 27
lymphoma biopsy specimens used in the above experiments have not been formally
characterized, the low frequency of ras mutations in lymphoid cells (Neri et
al. 1988;
Mills et al. 1995; Chaubert et al. 1994; Bos 1989; Ahuja et al. 1990)
forecloses the
possibility that 27 out of 36 (75%) lymphoma cell lines tested could harbor
Ras
mutations. Accordingly, the replication of reovirus in the above lymphoid
tumor cell
lines and biopsy specimens cannot be explained by the presence of ras
mutations in
these cells. Rather, the ability of reovirus to replicate in 27 out of 36
lymphoid
malignancy cell types in which ras mutations are rare indicates that reovirus
susceptibility in lymphoid cells is not merely a function of whether ras
mutations are
present in the lymphoid cell type.
It is also known that reovirus can selectively purge neoplastic cells from a
mixed population of cells comprising CD34+ cells, with minimal effect on the
viability and pluripotency of the CD34+ cells. Experiments supporting these
conclusions are described in Examples 4 and 5.
In the experiment described in Example 4, cells of one of three neoplastic
cell
lines (i.e., MCF7, SKBR3, and MDA MB 468), known to be susceptible to reovirus
infection (data not shown), were mixed with an apheresis product comprising
CD34+
cells. The mixed cellular populations were then infected by reovirus and
counted
daily to determine the extent of cell death and the types of cells killed by
reovirus. As
shown in Figures 4A-4D, reovirus infection resulted in the selective killing
of the
neoplastic cells with minimal effect on CD34+ cells. These data indicate that
reovirus
may be effective in purging a cell population of neoplastic cells without
affecting the
viability of the normal cells.
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To determine whether reovirus infection affected the pluripotency of the
CD34+ cells, CD34+ cells were allowed to differentiate in culture after being
exposed
to reovirus. These cell cultures were compared to control cell cultures
comprising
control cells that had not been exposed to virus). As shown Figure 5, CD34+
cells
gave rise to similar numbers of granulocutes (G), erythrocytes (E), or
granulocyte
erythroid macrophage megakaryocytes (GEMM) regardless of whether they were
exposed to reovirus. Therefore, reovirus treatment did not change the
differentiation
potential of CD34+ cells.
These findings suggest that reovirus could be used to purge mixed cellular
compositions of cells of lymphoid malignancies even when the cells of the
lymphoid
malignancies do not harbor ras mutations. Accordingly, the present invention
provides the treatment of mixed cellular compositions comprising or suspected
of
comprising cells of a lymphoid malignancy in an animal, comprising the step of
administering to the mixed cellular compositions animal an amount of reovirus
sufficient to kill the cells of the lymphoid malignancy.
The amount of reovirus required for killing cells of a lymphoid malignancy in
a mixed cellular composition depends on numerous factors, including but not
limited
to the type or strain of virus administered; the volume and cell density of
the mixed
cellular composition; the sensitivity of the cells of the lymphoid malignancy
to virus
infection, and the number of cells of the lymphoid malignancy present in the
sample.
However, because the virus will replicate selectively in cells of the lymphoid
malignancy, releasing progeny virus with the same specificity, the initial
amount of
reovirus that should be administered to a mixed cellular composition may
encompass
a wide range. Administration of an excessive number of virus particles is
unlikely to
cause toxic effects because of the blockage of virus translation in non-
permissive
cells. Administration of a less than optimal number of virus particles is
likely to
increase the time required to kill the cells of the lymphoid malignancy
because
additional rounds of virus replication will be required to generate sufficient
virus
particles to infect the cells of the lymphoid malignancy.
Accordingly, a feature of the invention is the wide range of virus particle
dosages effective in purging mixed cellular compositions of cells of lymphoid
malignancies. In one embodiment of the invention, an effective amount of
reovirus is
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from about 1.0 plaque forming unit (PFU)/kilogram (kg) sample weight (i.e.,
the
sample weight of the mixed cellular composition) to about 1015 PFU/kg sample
weight, more preferably from about 102 PFU/kg sample weight to about 1013
PFU/kg
sample weight. The treatment can be administered to a variety of animals,
including
but not limited to dogs, cats, sheep, goats, cattle, horses, pigs, humans, and
non-human primates.
In one embodiment of the invention, the mixed cellular population is a
hematopoietic stem cell-comprising autograft that is obtained from an animal
or
patient prior to the administration of a chemotherapeutic or radiotherapeutic
regimen.
The autograft is treated with virus prior to transplantation to remove
contaminating
neoplastic cells. This "cleaned up" or "purged" autograft is returned to the
animal or
patient following high-dose chemotherapy/radiation therapy.
In a preferred embodiment of the invention, the mixed cellular composition is
obtained from a hematopoietic center in the animal known to have or suspect of
having a lymphoid malignancy. In a most preferred embodiment of the invention,
the
mixed cellular composition is obtained from the bone marrow of such an animal.
In
another preferred embodiment of the invention, the mixed cellular composition
is
obtained from the blood of the animal. In another embodiment, the mixed
cellular
composition is an apheresis product.
The invention may be used to purge a mixed cellular composition of cells of a
variety of lymphoid malignancies. In one embodiment, the invention is used to
purge
cells of a B-cell lymphoid malignancy. In another embodiment, the invention is
used
to purge cells of a T-cell lymphoid malignancy. In yet another embodiment of
the
invention, reovirus is used to purge cells of a lymphoid malignancy comprising
both
B-cells and T-cells, in any proportion. The invention may also be used to
purge cells
of a lymphoid malignancy arising from lymphoblasts, prolymphocytes, or other
lymphoid cells at various stages of differentiation and/or maturity.
In one embodiment of the invention, reovirus is used to purge cells of a B-
cell
lymphoid malignancy such as Burkitt's lymphoma (BL). In another embodiment of
the invention, reovirus is used to purge cells of a non-Hodgkin's lymphoma
(NHL),
including but not limited to chronic lymphocytic leukemia (CLL), diffuse large
B-cell
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lymphoma (DLBCL), and follicular lymphoma (FL). In one embodiment of the
invention, the FL is a type I FL. In another embodiment of the invention,
reovirus is
used to purge cells of small lymphocytic lymphoma (SLL) or mantle-cell
lymphoma
(MCL).
In a preferred embodiment of the invention, hematopoietic progenitor stem
cells can be obtained from the bone marrow of an animal or patient.
Alternatively,
hematopoietic stem cells may be obtained from the peripheral blood of an
animal,
with or without the use of colony stimulating factor priming. In the latter
case,
hematopoietic progenitor stem cells can be obtained from blood as an apheresis
product, which may be stored for a long time before being transplanted. The
present
invention can be applied to stem cell-containing autografts that are harvested
from
any tissue source, including bone marrow and blood.
In addition to hematopoietic stem cells, the present invention can be broadly
applied to remove neoplastic cells from many other cellular compositions. For
example, reovirus can be used as a routine practice to "clean-up" any tissue
or organ
transplant prior to implantation.
Of particular interest will be the use of the claimed methods to clean-up
whole
blood or any portion thereof for a subsequent transfusion. Similarly, since
tissue and
organ transplantation has become increasingly common, it will be beneficial to
clean-
up the transplant tissues or organs to remove cells of lymphoid malignancies
prior to
transplantation. Liver, kidney, heart, cornea, skin graft, pancreatic islet
cells, bone
marrow, or any portions thereof are just a few examples of the tissues or
organs to
which this invention can be applied.
The tissue or organ can be autologous, allogeneic, or xenogeneic. The tissue
or organ may also be derived from a transgenic animal, may be a tissue/organ
that is
developed in vitro from stem cells, or may be expanded ex vivo. The tissue or
organ
to be treated with reovirus can be from an embryonic or adult origin. For
example,
embryonic neuronal cells can be treated before being transplanted into an
Alzheimer's
patient. Similarly, the invention can be used to treat semen or donor eggs ex
vivo.
In all instances of transplantation, a transplant recipient may be given
treatments to stimulate the immune system and/or to reduce the risk of virus
infection.
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This treatment may be performed prior to, contemporaneously with, or after the
transplantation, but is preferably performed prior to the transplantation.
Administering immunostimulants to a patient is particularly desirable when the
treated cellular compositions are autografts, which should not invoke a graft-
versus-
host reaction upon reintroduction to the patient or animal.
Virus may also be administered to a transplant recipient following the
harvesting of a mixed cellular composition but prior to chemotherapy or
radiation
therapy. The transplant recipient is then allowed to produce neutralizing
antibodies to
the virus before being treated with chemotherapy or radiation therapy. The
mixed
cellular composition (in this case, transplant tissue) is treated with the
same virus as
administered to the transplant recipient prior to chemotherapy or radiation
therapy, or
a variant thereof, providing that both viruses are recognized by at least some
of the
same antibodies produced in the transplant recipient. When the transplant
tissue is
given to the transplant recipient, circulating anti-virus antibodies will
recognize and
inactivate virus present in the transplant tissue. In another embodiment of
the
invention, the transplant recipient is administered with a composition
comprising anti-
virus antibodies that will recognize and inactivate virus present in the
transplant
tissues through passive immunity.
Application of the present invention is not limited to autografts and
transplants. Rather, any cellular compositions can be "cleaned up" with
reovirus for
any purpose. Thus, all the examples described above or below are applicable
even if
the tissue or organ is not meant for transplantation.
Cell lines may also be treated routinely to safeguard against spontaneous or
contaminating neoplastic cells. Again, any cell line will be a good candidate
for this
treatment, providing that reovirus, or another suitable virus, selectively
grows in the
neoplastic cells.
Numerous viruses may be used to practice the invention. In one embodiment
of the invention, the virus is a reovirus. The reovirus used to practice the
invention
may be any type or strain of reovirus with a tropism for the target lymphoid
malignancies in the animal to be treated. For example, to treat a human
patient, a
human reovirus, including but not limited to serotype 1 reovirus (Lang) ,
serotype 2
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reovirus (Jones), or serotype 3 reovirus (bearing or Abney) is most
preferable. The
reovirus may also be a field isolate, laboratory strain, chimera, recombinant,
or
otherwise engineered reovirus, or a reovirus comprising any combination of
these
viruses.
Such a recombinant reovirus may result from the recombination/reassortment
of genomic segments from two or more genetically distinct reoviruses.
Recombination/reassortment of reovirus genomic segments may occur in nature
following infection of a host organism with at least two genetically distinct
reoviruses. Recombinant virions can also be generated in cell culture, for
example, by
co-infection of permissive host cells with genetically distinct reoviruses
(Nibert et al.
1995).
Accordingly, the invention contemplates the use of recombinant reovirus
resulting from reassortment of genome segments from two or more genetically
distinct reoviruses, including but not limited to, human reovirus, such as
type 1 (e.g.,
strain Lang), type 2 (e.g., strain Jones), and type 3 (e.g., strain bearing or
strain
Abney), non-human mammalian reoviruses, or avian reovirus. The invention
further
contemplates the use of recombinant reoviruses resulting from reassortment of
genome segments from two or more genetically distinct reoviruses wherein at
least
one parental virus is genetically engineered, comprises one or more chemically
synthesized genomic segment, has been treated with chemical or physical
mutagens,
or is itself the result of a recombination event. The invention further
contemplates the
use of recombinant reovirus that have undergone recombination in the presence
of
chemical mutagens, including but not limited to dimethyl sulfate and ethidium
bromide, or physical mutagens, including but not limited to ultraviolet light
and other
forms of radiation.
The invention further contemplates the use of recombinant viruses that
comprise deletions or duplications in one or more genome segments, that
comprise
additional genetic information as a result of recombination with a host cell
genome, or
that comprise synthetic genes.
The reovirus may be modified by incorporation of mutated surface proteins,
for example, capsid proteins, and, where applicable, membrane proteins. The
proteins
CA 02436196 2003-07-25
may be mutated by substitution, insertion or deletion. Replacement includes
the
insertion of different amino acids in place of the native amino acids.
Insertions
include the insertion of additional amino acid residues into the protein at
one or more
locations. Deletions include deletions of one or more amino acid residues in
the
protein. Such mutations may be generated by methods known in the art. For
example, oligonucleotide site directed mutagenesis of the gene encoding for
one of
the coat proteins could result in the generation of the desired mutant coat
protein.
Expression of the mutated protein in reovirus infected mammalian cells in
vitro such
as COS1 cells will result in the incorporation of the mutated protein into the
reovirus
virion particle (Turner et al. 1992; Duncan et al. 1991; Mah et al. 1990).
The reovirus may comprise more than one reovirus, including but not limited
to, any combination of the reoviruses identified herein. Different reovirus
may be
administered simultaneously or at different times.
While reovirus is discussed as an embodiment of the invention, the invention
is by no means limited to the use of reovirus to kill the cells of lymphoid
malignancies. The use of other modified viruses to selectively kill cells with
activated
Ras pathways has been described in U.S. Patent No. 6,596,268. Representative
types
of modified virus included adenovirus, herpes simplex virus (HSV),
parapoxvirus orf
virus, or vaccinia virus. For reasons that will become apparent, these viruses
may
also be useful for killing cells of lymphoid malignancies
One virus that is particularly useful for selectively killing cells with an
activated Ras pathway is adenovirus. Adenoviruses encode several gene products
that
counter antiviral host defense mechanisms. For example, virus-associated RNA
(VAI
RNA or VA RNAI) refers to small, structured RNAs that accumulate in the
cytoplasm
of infected cells late in the adenovirus replication cycle. VAI RNA binds to
the to the
double stranded RNA (dsRNA) binding motifs of PKR blocking activation by
phosphorylation. With PKR unable to function, adenovirus replicates in the
cell,
causing lysis.
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Some attenuated or modified adenoviruses lack or fail to transcribe VAI RNA.
As a consequence, these viruses are unable to replicate in cells that express
PKR.
However, attenuated or modified adenovirus can replicate in cells with
activated Ras-
pathways, which have reduced PKR activity.
In addition to VAI RNA, a SS kDa cellular p53 inhibitor is encoded by the
E1B region of the adenovirus genome. p55 allows adenovirus to overcome the
replication-inhibitory effect of p53. The ONYX-O1 S adenovirus is deficient
for p55
(Bischoff et al. 1996; WO 94/18992), limiting virus replication to cells that
express
mutated p53. Since p53 mutations often accompany Ras mutations, particularly
in the
later stages of certain cancers, the ONYX-015 adenovirus will replicate in at
least a
subpopulation of cells that harbor activating Ras mutations.
Similarly, the Delta24 adenovirus harbors a 24 base-pair deletion in the ElA-
coding region (Fueyo et al. 2000), responsible for binding to and inhibiting
the
function of the cellular tumor suppressor Rb. Accordingly, Delta 24
replication is
limited to cells in which Rb is inactivated, as is the case in at least a
subset of cancer
cells.
Based on the discovery that reovirus, known to replicate in cells with
activated
Ras pathways, also replicates in the cells of lymphoid malignancies, it
follows that at
least some attenuated or modified adenoviruses will also replicate in the
cells of
lymphoid malignancies. Accordingly, attenuated or modified adenoviruses may be
used to practice the instant invention.
Infected-cell protein 34.5 (ICP34.5) of both type 1 and type 2 herpes simplex
viruses (HSV) can also prevent the antiviral effects exerted by PKR. ICP34.5
causes
cellular protein phosphatase-1 to act on eIF-2a, resulting in
dephosphorylation of eIF-
2a (He 1997), the same protein phosphorylated by PKR. The activity of ICP34.5
thereby allows herpesvirus to prevent or reverse PKR activation. Herpesviruses
that
lack or are unable to express ICP34.5 cannot replicate in cells with activated
(i.e.,
phosphorylated) PKR; however, such attenuated or mutated viruses can replicate
in
cells with activated Ras pathways, in which PKR activity is reduced.
Accordingly,
based on the finding that reovirus can replicate in the cells of lymphoid
malignancies,
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it is reasonable to predict that ICP34.5-deficient herpesviruses can also
replicate in
cells of lymphoid malignancies and therefore be used to practice the instant
invention.
Parapoxvirus orf virus is a poxvirus that induces acute cutaneous lesions in
different mammalian species, including humans. Parapoxvirus orf virus
naturally
infects sheep, goats, and humans through broken or damaged skin, replicates in
regenerating epidermal cells and induces pustular lesions that turn to scabs
(Haig
1998). The virus encodes gene OV20.OL, involved in blocking PKR activity (Haig
1998). Parapoxvirus orf viruses deficient in the expression of OV20.OL cannot
escape the effect of PKR activation. However, such viruses can replicate in
cells that
are deficient in PKR activation, such as cells with activated Ras pathways.
Accordingly, OV20.OL-deficient parapoxvirus orf viruses are also predicted to
replicate selectively in cells of lymphoid malignancies, and are therefore
useful for
practicing the instant invention.
Vaccinia virus is a member of the Orthopoxvirus genus that infects humans,
producing characteristic localized lesions (Brooks 1998). The virus encodes
two
proteins that play a role in down-regulating PKR activity through different
mechanisms.
The E3L gene encodes proteins of 20 and 25 kDa that are expressed early in
infection. The amino terminal region of the E3 proteins interacts with the
carboxy-
terminal region of PKR, preventing function (Chang et al. 1992, 1993, and
1995;
Sharp et al. 1998; and Romano et al. 1998). Deletion or disruption of the E3L
gene
precludes vaccinia virus from replicating in cells with activated PKR,
limiting its
replication to cells with reduced PKR activity, such as cells with an
activated Ras
pathway.
The vaccinia virus K3L gene encodes pK3, a protein possessing a carboxy-
terminal region that is structurally analogous to residues 79-83 of eIF-2a,.
pK3 acts as
an eIF-2a-decoy for PKR, preventing the activation of eIF-2oc and allowing
vaccinia
virus to replicate. Carboxy-terminal mutations or truncations in K3L protein
abolish
its PKR-inhibitory function (Kawagishi-Kobayashi et al. 1997), thereby
limiting the
replication of vaccinia virus to cells with reduced or absent PKR activity,
such as cells
with an activated Ras pathway.
28
CA 02436196 2003-07-25
Attenuated or modified vaccinia viruses are deficient in terms of E3L or pK3
function, and preferably both functions. Such attenuated or modified viruses
are
unable to replicate in normal cells in which PKR is activated. Accordingly,
replication of these viruses is limited to cells having an activated Ras-
pathway, or, as
predicted from the findings related to the instant invention, cells of
lymphoid
malignancies. Accordingly, an attenuated or modified vaccinia virus should be
useful
for practicing the instant invention.
Vesicular stomatitis virus (VSV) selectively kills neoplastic cells in the
presence of interferon. Interferons are circulating factors which bind to cell
surface
receptors which ultimately lead to both an antiviral response and an induction
of
growth inhibitory and/or apoptotic signals in the target cells. Although
interferons
can theoretically be used to inhibit proliferation of tumor cells, this
attempt has not
been very successful because of tumor-specific mutations of members of the
interferon pathway.
However, by disrupting the interferon pathway to avoid growth inhibition
exerted by interferon, tumor cells may simultaneously compromise their anti-
viral
response. Indeed, it has been shown that VSV, an enveloped, negative-strand
RNA
virus, rapidly replicated in and killed a variety of human tumor cell lines in
the
presence of interferon, while normal human primary cell cultures were
apparently
protected by interferon. Intratumoral injection of VSV also reduced tumor
burden in
nude mice bearing subcutaneous human melanoma xenografts (Stojdl et al. 2000).
Accordingly, in another embodiment of the present invention, VSV is used to
remove neoplastic cells from a mixed cellular composition in the presence of
interferon. Moreover, it is contemplated that this aspect of the invention be
applied to
any other interferon-sensitive virus (WO 99/18799), i.e., a virus that does
not replicate
in a normal cell in the presence of interferon. Such a virus may be identified
by
growing a culture of normal cells, contacting the culture with the virus of
interest in
the presence of varying concentrations of interferons, then determining the
percentage
of cell killing after a period of incubation.
It is also possible to take advantage of the fact that some neoplastic cells
express high levels of particular enzymes and construct a virus that is
dependent on
29
CA 02436196 2003-07-25
these enzymes. For example, the enzyme ribonucleotide reductase is abundant in
cells associated with liver metastases but is scarce in normal liver cells. In
a related
experiment, an HSV-1 mutant defective in ribonucleotide reductase expression
(hrR3)
was shown to replicate in colon carcinoma cells but not normal liver cells
(Moon et al.
2000).
In addition to the viruses discussed above, a variety of other viruses have
been
associated with oncolysis, although the underlying mechanisms were not always
apparent. For example, Newcastle disease virus (NDV), particularly the 73-T
strain,
replicates preferentially in malignant cells (Reichard et al. 1992; Zorn et al
1994; Bar-
Eli et al. 1996). NDV reduction of tumor burden after intratumor inoculation
was
also observed in cervical, colorectal, pancreatic, gastric, and renal cancer,
in addition
to melanoma (WO 94/25627; Nemunaitis 1999). Therefore, NDV can be used to
remove neoplastic cells from a mixed cellular composition.
Tumor regression has also been described in tumor patients infected with,
e.g.,
varicella-zoster virus (VZV), hepatitis virus, influenza virus, and measles
virus
(reviewed in Nemunaitis 1999).
According to the methods disclosed herein and techniques well known in the
art, a skilled artisan can test the ability of any of the above viruses, or
other viruses, to
selectively kill cells of a lymphoid malignancy in a particular mixed cellular
composition. If desired, a biopsy specimen (including but not limited to a
bone
marrow specimen or a blood sample) comprising cells of the lymphoid
malignancies
(or the suspected lymphoid malignancies) may be obtained from the patient or
animal.
The biopsy specimen may be harvested in advance of chemotherapy or radiation
treatment and tested with different viruses to determine which viruses
efficiently kill
the cells of the lymphoid malignancy. The Examples, below, describe assays
useful
for performing such tests, although other assays are known in the art and may
be
equally useful for such purposes. Based on results obtained with biopsy
specimens,
one or more viruses can be selected to practice the invention.
As an optional feature of many of the embodiments of the invention described
herein, cellular compositions that have been treated with a virus, or are
intended to be
treated with a virus, are stored before reintroducing the treated cell
compositions to
CA 02436196 2003-07-25
the same animal or transplanting the treated cell composition into at least
one second
animal.
In a preferred embodiment of the invention, the mixed cellular composition to
be treated with virus, or the treated cell composition, is frozen in a
solution containing
DMSO and thawed prior to reintroduction or transplantation. Freezing all or a
portion
of the cellular composition may allow more time to administer chemotherapy or
radiation therapy or may provide a second opportunity to reintroduce or
transplant a
cellular population into a patient or animal if the first attempt fails to
yield a suitable
clinical outcome. In instances wherein the cellular compositions are frozen
after virus
treatment, freezing the compositions in DMSO may have the added advantage of
inactivating the virus prior to transplantation.
In one embodiment of the invention, virus is administered to mixed cellular
compositions in conjunction with standard techniques for purging hematopoietic
mixed cellular compositions, including but not limited to chemotherapy and/or
radiation therapy. Known chemotherapeutic agent include but are not limited to
5-
fluorouracil, mitomycin C, methotrexate, hydroxyurea, cyclophosphamide,
dacarbazine, mitoxantrone, anthracyclins (Epirubicin and Doxurubicin),
antibodies to
receptors, such as herceptin, etopside, pregnasome, platinum compounds such as
carboplatin and cisplatin, taxanes such as taxol and taxotere, hormone
therapies such
as tamoxifen and anti-estrogens, interferons, aromatase inhibitors,
progestational
agents and LHRH analogs. 1n another embodiment of the invention, virus is
administered mixed cellular compositions prior to or in place of a
chemotherapeutic
agent or radiation.
Prior to, contemporaneous with, or following reintroduction or transplantation
of a treated cellular composition to an animal, immunosuppressants and/or
immunoinhibitors may be given to the animal. The use of immunosuppressants
and/or immunoinhibitors is particularly desirable when transplanting treated
cellular
compositions from one animal to another, even if such animals are genetically
compatible. Immunosuppressants and immunoinhibitors known to those of skill in
the art include but are not limited to such agents as cyclosporin, rapamycin,
tacrolimus, mycophenolic acid, azathioprine and their analogs, and the like
(see, e.g.,
31
CA 02436196 2003-07-25
Goodman and Gilman, 7'h Edition, page 1242, the disclosure of which is
incorporated
herein by reference).
Immunoinhibitors also include anti-antivirus antibodies, which are antibodies
directed against anti-virus antibodies. Such anti-antivirus antibodies may be
administered prior to, at the same time, or shortly after the reintroduction
or
transplantation of the virus. Preferably an effective amount of the anti-
antivirus
antibodies are administered in sufficient time to reduce or eliminate an
immune
response to residual live virus or residual virus antigens.
The invention includes pharmaceutical compositions that comprise, as an
active ingredient, one or more of the above identified viruses associated with
pharmaceutically acceptable Garners or excipients. The pharmaceutical
compositions
may also comprise an appropriate immunosuppresant or immunostimulant
associated
with pharmaceutically acceptable Garners or excipients. The pharmaceutical
compositions may be solid, semi-solid, or liquid, in the form of tablets,
pills, powders,
lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions,
syrups, aerosols
(as a solid or in a liquid medium), ointments, gelatin capsules,
suppositories, sterile
injectable solutions, transdermal patches, and sterile packaged powders, where
appropriate.
Examples of suitable excipients include but are not limited to lactose,
dextrose
(glucose), sucrose, sorbitol, mannitol, starches, gum acacia, calcium
phosphate,
alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose,
polyvinylpyrrolidone, cellulose, syrup, methyl cellulose and sterile water.
Pharmaceutical compositions may additionally comprise lubricating agents such
as
talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and
suspending
agents; preserving agents such as methyl- and propylhydroxy-benzoates;
sweetening
agents; and flavoring agents. The pharmaceutical compositions may be
formulated to
provide quick, sustained or delayed release of the active ingredients)
following
administration to the patient. Other suitable formulations for use in the
present
invention can be found in Remington's Pharmaceutical Sciences.
32
CA 02436196 2003-07-25
The virus or the pharmaceutical composition comprising the reovirus may be
packaged into convenient kits providing the necessary materials packaged into
suitable containers. It is contemplated the kits may also include
chemotherapeutic
agents and/or anti-antivirus antibodies. Such kits may comprise a number of
different
viruses, allowing the practitioner to determine which virus is most efficient
in killing
cells of a particular lymphoid malignancy, for example, using a biopsy
specimen as
described above.
The following Examples are offered to illustrate this invention and are not to
be construed in any way as limiting the scope of the present invention.
Examples
In the examples below, the following abbreviations have the following
meanings. Abbreviations not defined have their generally accepted meanings:
pg microgram
pl microliter
pM micromolar
B-cell B-cell chronic lymphocytic
CLL leukemia
BL Burkitt's lymphoma
CLL chronic lymphocytic leukemia
CPE cytopathic effects
DLBCL diffuse large B-cell lymphoma
DMEM Dulbecco's modified Eagle's
medium
DMSO dimethylsulfoxide
DTT dithiothrietol
EBV Epstein-Barr virus
EBV- Epstein-Barn virus not detected
EBV+ Epstein-Barr virus detected
EGF epidermal growth factor
FBS fetal bovine serum
FL follicular lymphoma
GCSF granulocyte colony stimulating
factor
HL Hodgkin's lymphoma
hr hour
M molar
MCL mantle-cell lymphoma
2-ME 2-mercaptoethanol (also
called ~3-ME)
MEM -modif ed Eagle's medium
mg milligram
min minute
ml milliliter
33
CA 02436196 2003-07-25
mM millimolar
mm2 square millimeters
MOI multiplicity of infection
n number of test subjects in a particular
group
NHL non-Hodgkin's lymphoma
C degree Celsius
PAGE polyacrylamide gel electrophoresis
PBMC primary blood mononuclear cells
PBS phosphate buffered saline
PDGF platelet derived growth factor
PFU plaque forming units
PKR double-stranded RNA activated protein
kinase
rpm revolutions per minute
SDS sodium dodecyl sulfate
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel
electrophoresis
SLL small lymphocytic leukemia
UV ultraviolet
Examyle 1: Susceptibility Of Various Lymphoid Cell Lines To Reovirus
Infection.
To determine the susceptibility of lymphoid cells to reovirus infection, a
panel
of lymphoma-derived cells was assembled for challenge with reovirus. The panel
included EBV+ BL cells (Raji and Daudi), EBV- BL cells (CA46, Ramos, and
ST486), and DLBCL cells (OCY-LY1, OCY-LY2, OCY-LYB, and OCY-LY10).
About 106 cells of each type were challenged with reovirus type 3 at a MOI of
20. While no morphological changes were detected in Daudi, Ramos, or ST486
cells
96 hours post-infection, Raji, CA46, and all four lines of DLBCL cells
exhibited CPE.
Cells with CPE were determined to have 40-70% reduced viability, based on
trypan
blue exclusion straining (Figure 1).
Virus replication in infected cells was also assayed by pulse labeling
infected
cultures with [35S]-methionine for six hours, followed by immunoprecipitating
the
labeled extracts with a rabbit polyclonal antireovirus type 3 antibody. The
immune
complexes were then analyzed by SDS-PAGE, and the results visualized by
autoradiography. Reovirus proteins were observed in Raji, CA46, and all DLBCL
cells but not in Daudi, Ramos, or ST486 cells, consistent with the pattern of
CPE
observed in the cells.
34
CA 02436196 2003-07-25
The results of the above experiments are summarized in Table l, below.
These results demonstrate that reovirus was able to replicate in six out of
nine
lymphoid malignancy-derived cell lines, with CPE corresponding to the presence
of
virus proteins. The presence of virus protein further indicates that virus
translation in
Raji, CA46, and the DLBCL cells is not blocked by PKR activation.
Table 1: Susceptibility of lymphoid cell lines to reovirus
Cell line Cell type Reovirus replication
Raji Burkitt's lymphoma, EBV+Yes
Daudi Burkitt's lymphoma, EBV'~No
CA46 Burkitt's lymphoma, EBV-Yes
Ramos Burkitt's lymphoma, EBV'No
ST486 Burkitt's lymphoma, EBV-No
OCY-LY1 Diffuse large B-cell Yes
lymphoma
OCY-LY2 Diffuse large B-cell Yes
lymphoma
OCY-LY8 Diffuse large B-cell Yes
lymphoma
OCY-LY 10 Diffuse large B-cell Yes
lymphoma
Example 2: Reovirus Infection Of Primary Lymphoma Cells.
A total of 27 lymphoid tumor biopsy specimens were obtained for the
preparation of cell suspensions for reovirus challenge. The specimens included
peripheral blood, bone marrow, lymph nodes, or other tissues. In the case of
solid
biopsy specimens, the tumor masses were disrupted to obtain cell suspensions.
Of the 27 biopsy specimens, 15 were associated with a clinical diagnosis of
CLL. The remaining 12 samples were associated with a clinical diagnosis of NHL
and could be further divided into BL (1); DLBCL (2); SLL (2); FL, grade I (1);
FL,
grade II (4); FL, grade III (1); and MCL (1). PBMC (n=3) and CD34+-
hematopoietic
stem/progenitor cells (n=3) from normal individuals were used as negative
controls.
CA 02436196 2003-07-25
About 106 cells from each sample were infected with reovirus at a MOI of 20
then pulse labeled, immunoprecipitated, and resolved by SDS-PAGE, as described
in
Example 1. Reovirus failed to replicate in the control PBMC and CD34+ cells
but
appeared to replicate in 15/15 CLL samples and 6/12 NHL samples (i.e., 1/1 BL;
2/2
DLBCL; 1/2 SLL; 1/1 FL, grade I; 0/4 FL, grade II; 0/1 FL, grade III; and 1/1
MCL).
The results are shown in Figure 2 and summarized in Table 2, below.
Table 2: Susceptibility of lymphoid biopsy specimens to reovirus
Disease Cell type Total SusceptibleResistant
specimensspecimens specimens
CLL Chronic lymphocytic 1 S 0
leukemia 15
NHL Burkitt's lymphoma 1 1 0
NHL 2 2 0
Diffuse largeB-cell
lymphoma
NHL Small lymphocytic lymphoma2 1 1
NHL Follicular lymphoma, 1 1 0
grade I
NHL Follicular lymphoma, 4 0 4
grade II
NHL Follicular lymphoma, 0 0 1
grade III
NHL Mantle-cell lymphoma 1 1 0
The CLL cells, as well as the BL cells, were also analyzed by flow cytometry,
before and after infection, to identify the population of cells killed by
reovirus
infection. CLL cells are characterized by expression of the CDS and CD20 cell-
surface markers (i.e., the cells are CDS+/CD20+) (Hulkkonen et al. 2002). BL
cells
are characterized by expression of the CD10 and CD20 cell-surface markers
(i.e., the
cells are CD10+/CD20+) (Nakamura et al. 2002). Before infection and at
approximately 96 hours post-infection, cells were washed with PBS then
incubated
with CD10, CDS, and CD20-specific antibodies in the presence of 7-amino-
actinomycin D, for 15 minutes at room temperature, in the dark. The cells were
then
washed and resuspended in PBS.
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CA 02436196 2003-07-25
Flow cytometry of CLL cell populations before and after infection revealed
significant reductions in CDS+/CD20+ cells, but not other cells, as a result
of reovirus
infection, indicating that CLL cells were selectively killed as a result of
reovirus
infection. Similarly, flow cytometry a BL cell population before and after
infection
revealed a significant reduction in CD10+/CD20+ cells, but not other cells, as
a result
of reovirus infection, indicating that BL cells were selectively killed as a
result of
reovirus infection.
These results show that reovirus is able to replicate in cells of CLL and NHL
lymphoid malignancies, taken directly from biopsy specimens, and that
malignant
cells are selectively killed as a result of infection.
Example 3: Efficacy Of Reovirus Treatment On Lymphoid Tumors In A
Xenograft Model.
A marine xenograft model was used to evaluate the ability of reovirus to treat
lymphoma-derived tumors in vivo. About 10' Raji or Daudi cells in about 100 pl
PBS
were administered by subcutaneous injection in the hind flank of 6-8-week old
SCID/NOD mice. Once palpable tumor masses were established, animals received
either live or UV-inactivated reovirus by either intratumoral or intravenous
injection
(day 0).
Animals receiving intratumoral reovirus were injected with approximately 10'
PFU of live (n=8) or UV-inactivated (n=7) reovirus in 50 p,l PBS, delivered to
the
tumor masses. Tumors size was measured every other day for 30 days or until
animals showed excess tumor burden.
Animals receiving intravenous reovirus were injected with either 10' (n=7) or
Sx 10' (n=7) PFU reovirus, or no reovirus (n=7) in 100 p,l saline solution,
delivered
into the tail vain. Tumors size was measured every other day for 20 days or
until
animals showed excess tumor burden. The results are shown in Figure 3.
The growth of Raji-derived tumors was reduced at least 10-fold (in terms of
tumor area, expressed in mm2) by intratumoral administration of live reovirus.
UV-
inactivated reovirus had no effect on tumor size (Figure 3A). Daudi tumors
were
resistant to reovirus treatment (Figure 3B). The growth of Raji-derived tumors
was
37
CA 02436196 2003-07-25
reduced about a 5-fold following intravenous administration of live reovirus
at either
of the concentrations tested. UV-inactivated reovirus had no effect on tumor
size
(Figure 3C).
Hematoxylin and eosin staining of paraffin-embedded sections prepared at day
20 days following live or UV-inactivated reovirus administration confirmed the
killing of Raji tumor cells in animals treated with live reovirus.
Immunohistochemical staining using an antireovirus polyclonal antibody and
avidin-
biotin horseradish peroxidase color-development system (Vector, Burlingame,
CA)
confirmed the presence of reovirus proteins in residual tumor cells,
confirming virus
replication (data not shown).
These results show that reovirus was able to infect and kill human Burkitt's
Lymphoma cells (Raji) in vivo following either intratumoral or intravenous
administration.
Example 4: Reovirus selectively removed cancer cells from a mixed cellular
composition
MCF7 (ATCC number HTB-22), SKBR3 (ATCC number HTB-30), and
MDA MB 468 (ATCC number HTB 132) are known to be susceptible to reovirus
infection (i.e., these cells are killed by reovirus). Cells of each type were
mixed with
apheresis product and subjected to reovirus infection to investigate if
reovirus can
selectively remove neoplastic cells from the mixed cellular composition.
Apheresis
product was prepared according to a procedure previously described (Stewart et
al.
1999; Duggan et al. 2000).
When admixtures of apheresis product (90%) and, e.g., MCF7 (10%) were
treated with reovirus and tested daily for cell count and viability, there was
a 100-fold
depletion in the numbers of cytokeratin-positive MCF7 cells while the CD34+
stem
cells remained intact and viable. Figures 4A-4C show the purging effect of
reovirus
to mixtures of apheresis product with MCF7, SKBR3 or MDA MB 468 cells.
However, in a control experiment, infection of CD34+ cells with reovirus did
not
significantly alter the number of viable cells in culture (Figure 4D). Taken
together,
38
CA 02436196 2003-07-25
these results suggest that reovirus selectively kills cancer cells while not
significantly
affecting the viability of CD34+ stem cells.
Example 5: Reovirus treatment neither inhibited cell proliferation nor altered
differentiation potential of CD34+ cells
While the number of CD34+ cells was unaffected by reovirus infection (data
not shown), there remained the question whether reovirus changed the potential
of
CD34+ stem cells to differentiate into all the hematopoietic lineages in the
appropriate
proportion. To investigate this possibility, CD34+ cells were incubated with
reovirus
for 2, 24, 48 or 72 hours, respectively. The reovirus was then removed and the
cells
were diluted and cultured in fresh media for 14 days to allow colonies to
form. Each
colony was examined to determine if it belongs to the granulocyte, erythroid,
or
granulocyte erythroid macrophage megakaryocyte lineage. As shown in Figure S,
stem cells treated with live virus (LV) yielded similar numbers of
granulocutes (G),
erythrocytes (E) or granulocyte erythroid macrophage megakaryocytes (GEMM) as
the no virus (NV) control. Therefore, reovirus treatment did not change the
differentiation potential of CD34+ cells.
While this invention has been particularly shown and described with
references to preferred embodiments thereof, it will be understood by those
skilled in
the art that various changes in form and details may be made therein without
departing from the spirit and scope of the invention as defined by the
appended claims
39
