Note: Descriptions are shown in the official language in which they were submitted.
CA 02435967 2003-07-25
REOVIRUS FOR THE TREATMENT OF LYMPHOID MALIGNANCIES
FIELD OF THE INVENTION
The present invention relates to methods of treating lymphoid malignancies
using reovirus.
REFERENCES
The following publications, patent applications, and patents are cited in this
application:
U.S. Patent No. 6,136,307.
U.S. Patent No. 6,596,268.
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.
Bannerji, R., Byrd, J.C. (2000) Update on the biology of chronic
lymphocytic leukemia. Curr. Opin. Oncol. 12:22-29.
Barbacid, M. (1987) Ras genes. Annu. Rev. Biochem. 56:779-827.
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.
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.
CA 02435967 2003-07-25
Chang, H.W., Uribe, L.H., Jacobs, B.L. (1995) Rescue of vaccinia virus
lacking the E3L gene by mutants of E3L. J. Virol. 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.
Dohner, H., Stilgenbauer, S., Dohner, K., Bentz, M., Lichter, P. (1999)
Chromosome aberrations in B-cell chronic lymphocytic leukemia:
reassessment based on molecular cytogenetic analysis. J. Mol. Med.
77:266-8I .
Drach, J., Kaufinann, H., Urbauer, E., Schreiber, S., Ackermann, J., Huber,
H. (2000) The biology of multiple myelorna. J. Cancer Res. Clin.
Oncol. 126:441-47.
Duncan, R., Horne, D., Strong, J.E., Leone, G., Pon, R.T., Yeung, M.C.,
Lee, P.W. (1991) Conformational and functional analysis of the C-
terminal globular head of the reovirus cell attachment protein. Virology
182:810-19.
Gaidano, G., Pastore, C., Volpe, G. (1995) Molecular pathogenesis of non-
Hodgkin lymphoma: a clinical perspective. Haematologica 80:454-72.
Haig, D.M., McInnes, C.J., Thomson, J., Wood, A., Bunyan, K., Mercer,
A. ( 1998) The orf virus OVZO.OL gene product is involved in
interferon resistance and inhibits an interferon-inducible, double-
stranded RNA-dependent kinase. Immunology 93:335-40.
Hankin, R.C., Hunter, S.V. (1999) Mantle cell lymphoma. Arch. Pathol.
Lab. Med. 123:1182-88.
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.
2
CA 02435967 2003-07-25
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.
Hecht, J.L., Aster, J.C. (2000) Molecular biology of Burkitt's lymphoma.
J. Clin. Oncol. 18:3?07-21.
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 cfc 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.
Kuppers, R. (2002) Molecular biology of Hodgkin's lymphoma. Adv.
Cancer Res. 2002:277-3 I2.
Levitzki, A. (1994) Signal-transduction therapy. A novel approach to
disease management. Eur. J. Biochem. 226:1-13.
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.
Mahmoudi, M., Motoo, Y., Vela, G.R., Bollon, A.P., Osther, K. (1989)
Expression of c-myc and c-Ha-ras oncogenes in human lymphobIastoid
cells (Namalva). Cell. Mol. Biol. 35:75-80.
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.
Motoo, Y., Mahmoudi, M., Osther, K., Bolton, A.P. (1986) Oncogene
expression in human hepatoma cells PLClPRF/5. Biochem. Biophys.
Res. Commun. 135:262-68.
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.
3
CA 02435967 2003-07-25
Nedergaard, T., Guldberg, P., Ralfkiaer, 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.
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.
Neri, A., Murphy, J.P., Cro, L., Ferrero, D., Tarella, C., Baldini, L., Dalla-
Favera, R. (1989) Ras oncogene mutation in multiple myeloma. J. Exp.
Med. 170:1715-25.
Nibert, M.L., Schiff, L.A., and Fields, B.N., Reoviruses and their
replication in Fields Virology, 3rd Edition, Lippencott-Raven Press,
1995, pp. 1557-96.
Ong, S.T. and Le Beau, M.M. (1998) Chromosomal abnormalities and
molecular genetics of non-Hodgkin's lymphoma. Semin. Oncol.
25:447-60.
Pasqualucci, L., Neumeister, P., Goossens, T., Nanjangud, G., Chaganti,
R.S., Kuppers, R., Dalla-Favera, R. (2001) Hypermutation of multiple
proto-oncogenes in B-cell diffuse large-cell lymphomas. Nature
412:341-46.
Potter, M. (1990) Neoplastic development in B-lymphocytes.
Carcinogenesis 11:1-13.
Romano, P.R., Zhang, F., Tan, S.L., Garcia-Barno, 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.
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.
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.
TweeddaLe, M.E., Lim, B., Jamal, N., Robinson, J., Zalcberg, J.,
Lockwood, G., Minder, M.D., Messner, H.A. (1987) The presence of
4
CA 02435967 2003-07-25
clonogenic cells in high-grade malignant lymphoma: a prognostic
factor. Blood 69:1307-14.
Verwer, B.J., Terstappen, L.W. (1993) Automatic lineage assignment of
acute leukemias by flow cytometry. Cytometry 14:862-75.
Wiessmuller, L. and Wittinghofer, F. (1994) Signal transduction pathways
involving Ras. Cellular Signaling 6:247-267.
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.
STATE OF THE ART
Lymphoid malignancies encompass a heterogeneous group of cell proliferative
disorders resulting from the inappropriate proliferation of lymphoid or
lymphoid
precursor cells. These disorders 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. Classification of these disorders is based on
clinical presentation, cell morphology, and chromosomal abnormalities. An
authoritative classification scheme is published by the World Health
Organization
(Jaffe et al. 2001).
Burkitt's lymphoma (BL) is a B-cell lymphoma, usually caused by Epstein-
Barr virus (EBV). BL is frequently characterized by chromosomal translocations
involving Myc and the ~, or K light-chain immunoglobulin genes (reviewed in
Hecht
et al. 2000).
Hodgkin's lymphoma (HL), or Hodgkin's disease, is a lymphoma
characterized by the presence of mononucleated Hodgkin and multinucleated Reed-
Sternberg cells (HRS), which occur at low frequency in tumor tissues. Hodgkin
and
HRS cells harbor somatically mutated, clonally rearranged immunoglobulin
genes,
appearing to originate from germinal center B cells. Mutation in p53,
IkappaBalpha,
CA 02435967 2003-07-25
NFkappaB, and CD95lFas and deletions at lp, 6q, and 7q have been associated
with
HL (reviewed in, e.g., Kuppers, 2002).
Non-Hodgkin's lymphomas (NHL) are a heterogeneous group of lymphoid
malignancies arising from mature lymphoid cells. NHL is often associated with
activation of proto-oncogenes such as BCL-1, BCL-2, BCL-6, and cMyc, and/or
disruption of tumor suppressor genes such as p53. Other causes of NHL include
virus
infection (e.g., EBV), chronic antigenic stimulation, and dysregulation of
cytokine
networks. Documented chromosomal abnormalities include breaks at 3q27 and/or
9p13 and deletions at 6q25-q27 and 6q21-q23 (Gaidano et al. 1995; Ong et al.
1998).
Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy of
mature B-lymphocytes accounting for about 40-50% of non-Hodgkin's lymphomas.
DLBCL are heterogeneous with respect to chromosomal abnormalities but are
often
associated with translocations affecting 3q27, to which locus maps the BCL6
gene
encoding a putative zinc-finger transcription factor. Dysregulation of BCL-2
and
Myc have also been associated with DLBCL (reviewed in Nakamura et al. 2002).
Follicular lymphoma (FL) is a malignancy of follicular center B-cells
accounting for about 25-40% of non-Hodgkin's lymphomas. FL is usually
associated
with the reciprocal chromosomal translocation, t(14;18)(q32;q21), which causes
deregulated expression of the anti-apoptotic gene BCL2, located on chromosome
18.
About 25-60% of FL transform to more aggressive large cell lymphomas. Other
mutations associated with FL include but are not limited to BCL-2 mutations,
p53
mutations, mutations in the 5' non-coding region (NCR) of BCL-6, and cMyc
rearrangements (Clark et al. 1996).
Mantle-cell lymphoma (MCL) primarily affects seniors and accounts for 2%
to 8% of non-Hodgkin lymphomas. MCL arises 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.
MCL is characterized by a t(11;14) translocation affecting the BCL-1 locus
and/or a
t(I1;I4)(q13;q32) translocation that affects the Cyclin D1 gene. In both
cases, proto-
oncogenes are overexpressed by being placed under the control of an
immunoglobulin
heavy-chain gene enhancer element (Hankin and Hunter, 1999).
6
CA 02435967 2003-07-25
Chronic lymphocytic leukemia (CLL) is a proliferative disorder resulting from
the accumulation of mature lymphocytes in the blood and/or bone marrow.
Approximately 95% of all CLL involves mature B-cells, hence the designation B-
cell
chronic lymphocytic leukemia (B-cell CLL). CLL cells typically overexpress BCL-
2,
e.g., from a t(14:18) translocation that causes the expression of BCL-2
(normally on
chromosome 18) to be controlled by an immunoglobulin enhancer element on
chromosome 14. Other molecular genetic abnormalities include deletions at
13q14
andlor 11 q, deletions and/or translocations involving 17p, trisomy 12, p53
aberrations,
and ectopic expression of cell surface markers (Bannerji and Byrd, 2000;
Dohner et
al. 1999).
Ras, a proto-oncogene associated with a variety of cell proliferative
disorders,
is a monomeric guanine nucleotide binding protein that is activated in
response to
upstream mitogenic signals mediated by tyrosine receptor kinases (RTKs). Ras
is
known to interact with a number of downstream effectors, including members of
the
Raf family (e.g., Rafl, B-raf, and A-raf), members of the RaIGEF family (e.g.,
RaIGDS, RGL1, RGLZ, and Rlf), and phosphotidylinositol-3-kinases (PI-3
kinases)
(e.g., isoforms of protein kinase C, AKT kinase/protein kinase B, p70-S6
kinase, and
RacGEFs). (Ward et al. 2001; de Ruiter et al. 2000; Bos 1998; and references
within).
Activating Ras mutations are present in approximately 30% of all human
tumors (Wiessmuller and Wittinghofer 1994; Barbacid 1987); however, such
mutations occur at different frequencies in different tumor cell types. Fox
example,
Ras mutations are common in pancreatic cancers (80%), thyroid cancers (50%),
and
sporadic colorectal carcinomas (40-50%) (Millis et al. 1995; Chaubert et al.
1994;
Bos 1989) but are less common in certain hematopoietic malignancies.
While some hematopoietic malignancies are associated with Ras mutations
(e.g., 30% of myeloid leukemias and 18% of acute lymphoblastic leukemias), Ras
mutations are virtually undetectable in lymphoid malignancies, for example,
NHL and
CLL (Neri et al. 1988; Millis et al. 1995; Chaubert et al. 1994; Bos 1989).
This latter
observation has been confirmed by a number of independent studies. For
example,
Neri et al. (1988) .screened I78 lymphomas and failed to identify N-Ras
mutations
(codons 12 and 13) in NHL or CLL samples. Similarly, using a PCR-based assay
that
7
CA 02435967 2003-07-25
simultaneously scanned six mutational "hot-spots" in codons 12, 13, and 61 of
K-, N-,
and H-Ras, Nedergaard et al. ( 1997) reported only three Ras mutations in 123
NHL
samples. Abuja et al. (1990) and Steenvoorden et al. (1988) reported the
presence of
no N-Ras mutations in six and 25 HL samples, respectively. And Clark et al.
(1996)
reported the presence of only one N-Ras mutation (codon I2) in 16 samples of
FL.
Accordingly, while Ras mutations appear in some acute myeloid and lymphoid
malignancies, they are rare in chronic lymphoid malignancies (Abuja et al.
1990).
As described in U.S. Patent No. 6,136,307, herein incorporated by reference in
its entirety, reovirus selectively replicates in cells with an activated Ras
pathway.
While reovirus can infect "resistant" cells, virus gene transcription is
associated with
activation (via phosphorylation) of double-stranded RNA-activated protein
kinase
(PKR). Activated PKR subsequently affects phosphorylation of translation
initiation
factor eIF-2a , resulting in the inhibition of viral gene translation.
However, PKR
phosphorylation is reduced or reversed in Ras-transformed cells, relieving the
translation block and allowing virus proteins to be translated. Reovirus
therefore
infects and productively replicates only in "susceptible" cells. Because
reovirus
replication causes cytopathic effects (CPE) resulting in cell death, reovirus
may be
used to selectively kill tumor cells in an animal, wherein the tumor cells
have
activated Ras pathways.
SUMMARY OF THE INVENTION
The present invention pertains to a method of treating a lymphoid malignancy
in an animal, the method comprising administering to cells of the lymphoid
malignancy an amount of reovirus sufficient to cause substantial lysis of the
cells.
In one embodiment of the invention, the lymphoid malignancy is selected
from the group consisting of Burkitt's lymphoma, Hodgkin's lymphoma, chronic
lymphocytic leukemia, and non-Hodgkin's lymphoma. In a particular embodiment
of
the invention, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma,
follicular lymphoma, mantle-cell lymphoma, or small lymphocytic lymphoma.
8
CA 02435967 2003-07-25
In another embodiment of the invention, the cells of the lymphoid malignancy
comprise an activated Ras pathway. In a particular embodiment of the
invention, the
cells of the lymphoid malignancy comprise a normal Ras gene, with the Ras
pathway
being activated in a manner that does not require an activating Ras mutation.
In one embodiment of the invention, reovirus is administered to hematopoietic
cells in vivo. In preferred embodiments of the invention, reovirus is
administered by a
route selected from the group consisting of intramedullar, intravascular,
intrathecal,
intravenous, intramuscular, subcutaneous, intraperitoneal, topical, oral,
rectal, vaginal,
nasal, and intratumoral routes. In another embodiment of the invention,
reovirus is
administered to a cellular composition ex vivo. Reovirus may also be
administered by
any combination of in vivo routes, alone or in addition to ex vivo
administration.
In one embodiment of the invention, more than one type of reovirus is
administered. In another embodiment of the invention, more than one strain of
reovirus is administered. In yet another embodiment of the invention, one or
more
recombinant reoviruses are administered.
In one embodiment of the invention, the reovirus is administered in a single
dose. In another embodiment of the invention, the reovirus is administered in
more
than one dose.
In one embodiment of the invention, the animal to which the reovirus is
administered is a mammal. In a preferred embodiment of the invention, the
mammal
is selected from the group consisting of dogs, cats, sheep, goats, cattle,
horses, pigs,
humans, and non-human primates.
The invention also provides a method for selectively killing cells of a
lymphoid malignancy in a cellular composition suspected of containing such
cells.
The method includes administering reovirus to cells of a cellular composition
under
conditions that result in substantial lysis of the cells of the lymphoid
malignancy.
In one embodiment of the invention, the cellular composition to which
reovirus is administered or provided is selected from the group consisting of
hematopoietic tissue and transplant tissue. In a preferred embodiment of the
invention, the hematopoietic tissue is selected from the group consisting of
bone
9
CA 02435967 2003-07-25
marrow, spleen, mucosa-associated lymphoid tissues (MALT), lymph nodes,
Peyer's
patches, thymus, tonsils, and fetal liver.
In one embodiment of the invention reovirus is administered to cells of the
cellular composition by a route selected from the group consisting of
intramedullar,
intravascular, intrathecal, intravenous, intramuscular, subcutaneous,
intraperitoneal,
topical, oral, rectal, vaginal, nasal, and intratumoral routes. In another
embodiment of
the invention cells of a cellular composition are removed from an animal,
contacted
with reovirus ex vivo, then returned to an animal.
Another embodiment of the invention, comprises the additional step of
inactivating the reovirus after contacting the cellular composition with
reovirus under
conditions which results in substantial lysis of the cells of the lymphoid
malignancy.
In one embodiment of the invention, reovirus is removed by subjecting the
reovirus-
treated cellular composition to anti-reovirus antibodies or a combination of
anti-
reovirus antibodies and complement to inactivate the reovirus. Alternatively
or
additionally, immobilized anti-reovirus antibodies specific for reovirus
surface
epitopes are used to remove the reovirus particles from cellular composition.
In one
embodiment of the invention, anti-reovirus antibodies are bound to a column
over
which the treated cellular composition is passed. In another embodiment, anti-
reovirus antibodies are bound to beads or other physical structures that can
be mixed
with the cellular composition then recovered by filtration or centrifugation.
The
reovirus-depleted cellular composition is then returned to the donor animal or
provided to another animal.
Alternatively, antireovirus antibodies are administered in vivo to
hematopoietic tissues treated with reovirus. In one embodiment of the
invention, the
anti-reovirus antibodies are provided to the tissue or tissues that were
treated with
reovirus. In another embodiment of the invention, the anti-reovirus antibodies
are
provided or administered systemically.
CA 02435967 2003-07-25
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-LY8, and OCY-LY10}
following challenge with reovirus type 3 at a 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-
LY8, and OCY-LY10). Growth is reported in PFUs at 0 hour (open squares) and 96
hours (filled squares) post infection.
Figure 3: Bar graphs showing the size of lymphoma-derived tumors
implanted in mice following infection with either live (filled symbols) or UV-
inactivated (open circles) reovirus. (A) Raji tumors, intratumoral virus
administration. (B) Daudi tumors, intratumoral virus administration. (C) Raji
tumors,
intravenous virus injection.
DETAILED DESCRIPTION OF THE INVENTION
Definitions:
Cellular composition: Any 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, and bursa of
Fabricus (in
Avian species), mucosa-associated lymphoid tissues (MALT), whole blood, and
fractions thereof.
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.
Hematopoietic stem/progenitor cells: Partially differentiated cells cable of
differentiating into a variety of different hematopoietic lineages, including
lymphoid
cells. The presence of CD34 is often used as a marker for hematopoietic
stemlprogenitor cells (i.e., CD34+ cells), although more primitive, less
differentiated,
hematopoietic precursor cells may actually lack CD34 (i.e., CD34- cells).
11
CA 02435967 2003-07-25
Intramedullary: Within the bone marrow.
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. The 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).
Normal hematopoietic stem/progenitor cells: Hematopoietic stemlprogenitor
cells not associated with a transformed or malignant growth phenotypes and not
believed to harbor chromosomal abnormalities that would cause such growth
phenotypes.
Normal lymphocytes: Lymphocytes 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.
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.
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).
12
CA 02435967 2003-07-25
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 a particular location 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.
The Invention:
The instant invention is based, in part, on Applicant's discovery that
reovirus is
capable of replicating in tumor 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).
The conclusion that reovirus is able to replicate in lymphoid tumor cells 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
13
CA 02435967 2003-07-25
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-LYB, and OCY-LY10).
Reovirus replicated in six of the nine lymphoma-derived cell lines,
specifically Raji,
CA46, and all four DLBCL cells. Reovirus was unable to replicate in only three
of
these cell lines (Daudi, Ramos, and ST486).
In a second experiment, cell suspensions were prepared from 27 lymphoid
tumor biopsy specimens for challenge with reovirus. Of the 27 specimens, 1 S
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 5
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. In total, reovirus was able to replicate in 21 of the
27 cell
suspensions prepared from lymphoid tumor biopsy specimens (see, Example 2 and
Table 1, below).
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 (see also,
Example 3,
below).
Following the establishment of palpable tumor masses, mice were treated with
either live reovirus or UV-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 LTV-inactivated reovirus. These results showed that
14
CA 02435967 2003-07-25
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.
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.
These findings suggest that reovirus could be used to treat lymphoid
malignancies in an animal even when the cells of the lymphoid malignancies do
not
harbor Ras mutations. Accordingly, the present invention provides the
treatment of a
lymphoid malignancy in an animal, comprising the step of administering to the
animal
an amount of reovirus sufficient to kill the cells of the lymphoid malignancy.
In one
embodiment of the invention, treatment results in a reduction in the size of a
solid
lymphoma or a reduction in the number of cells of a circulating lymphoma
(e.g., a
leukemia).
As used herein, the size of a solid lymphoma refers to the mass or volume of a
solid lymphoma. In one embodiment of the invention, reduction in the size of a
lymphoma is achieved when the lymphoma is less than about 50% of its original
volume following reovirus treatment. In a preferred embodiment of the
invention
reduction in the size of a lymphoma is achieved when the lymphoma is less than
about
25% of its original volume following reovirus treatment. In a most preferred
embodiment, reduction in the size of a lymphoma is achieved when the lymphoma
is
less than about 10% of its original volume following reovirus treatment. In
another
embodiment of the invention, treatment results in the complete elimination of
a solid
lymphoma from an animal.
CA 02435967 2003-07-25
In the case of a circulating lymphoma, or circulating cells from a solid
lymphoma that has metastasized, treatment results in a reduction in the number
of
circulating lymphoma cells. In one embodiment of the invention, treatment
results in
at least a 50% decrease in the number of circulating lymphoma cells. In a
preferred
embodiment of the invention, treatment results in at least a 75% decrease in
the
number of circulating lymphoma cells. In a most preferred embodiment,
treatment
results in at least a 90% decrease in the number of circulating lymphoma
cells. In
another embodiment of the invention, treatment results in the complete
elimination of
circulating lymphoma cells from an animal.
The amount of reovirus required for treatment of a lymphoid malignancy
depends on the body mass, age, gender, and physical condition of the animal;
the type
or strain of reovirus administered; the route or combination of routes of
administration; the severity and characteristics of the lymphoid malignancy,
the
seventy of the patient's symptoms; the rate of virus replication in
susceptible cells,
and other factors. In addition, because reovirus replicates selectively in
cells of the
lymphoid malignancy, releasing progeny virus with the same specificity, the
initial
amount of reovirus that is administered to an animal may encompass a wide
range.
Administration of an excessive number of virus particles is unlikely to cause
toxic
effects because of the blockage of reovirus 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 treating lymphoid malignancies. In one embodiment of the
invention, an effective amount of reovirus is from about 1.0 plaque forming
unit
(PFU)lkilogram (kg) body weight to about 1015 PFUIkg body weight, more
preferably
from about 102 PFU/kg body weight to about 10'3 PFU/kg body 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.
16
CA 02435967 2003-07-25
The reovirus may be administered in a single dose or in multiple doses. In one
embodiment of the invention, the reovirus is administered to the lymphoid
malignancy by an intratumoral route. In another embodiment, the reovirus is
administered to the bone marrow by an intramedullary route. In another
embodiment,
the reovirus is administered intravenously. In yet another embodiment, the
reovirus is
administered by an intravascular, intrathecal, intramuscular, subcutaneous,
intraperitoneal, topical, oral, rectal, vaginal, nasal, or intratumoral route,
alone or in
any combination with other routes discussed herein.
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 reovirus (Jones), or
serotype 3
reovirus (bearing or Abney) is most preferable. The reovirus may also be a
field
isolate, laboratory strain, or engineered reovirus.
The reovirus may be a recombinant reovirus resulting from the
recombination/reassortment of genomic segments from two or more genetically
distinct reoviruses. RecombinationJreassortment 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 reassorhnent 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
17
CA 02435967 2003-07-25
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
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 COS 1 cells will result in the incorporation of the mutated protein into
the reovirus
virion particle (Turner et ad. 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
18
CA 02435967 2003-07-25
One virus that was particularly useful for selectively killing cells with an
activated Ras pathway was 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.
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 55 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-015 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 adenovirusese will also replicate in the
cells of
lymphoid malignancies. Accordingly, attenuated or modified adenoviruses may be
used to practice the instant invention.
19
CA 02435967 2003-07-25
Infected-cell protein 34.5 (ICP34.S) of both type 1 and type 2 herpes simplex
viruses (HSV) can also prevent the antiviral effects exerted by PKR. ICP34.S
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.S
thereby allows herpesvirus to prevent or reverse PKR activation. Herpesviruses
that
lack or are unable to express ICP34.S cannot replicate in cells with activated
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, it is reasonable
to predict
that ICP34.S-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 2S 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
CA 02435967 2003-07-25
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-2a 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.
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.
The invention may be used to treat a variety of lymphoid malignancies
providing reovirus is able to replicate in the cells of the lymphoid
malignancy. In one
embodiment of the invention, reovirus is used to treat a B-cell lymphoid
malignancy.
In another embodiment of the invention, reovirus is used to treat a T-cell
lymphoid
malignancy. In yet another embodiment of the invention, reovirus is used to
treat a
lymphoid malignancy comprising both B-cells and T-cells, in any proportion.
The
invention may also be used to treat 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 treat a B-cell
lymphoid malignancy such as Burkitt's lymphoma (BL). In another embodiment of
the invention, reovirus is used to treat a non-Hodgkin's lymphoma (NHL),
including
but not limited to chronic lymphocytic leukemia (CLL), diffuse large B-cell
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
21
CA 02435967 2003-07-25
used to treat small lymphocytic lymphoma (SLL) or mantle-cell lymphoma (MCL).
In yet another embodiment of the invention, reovirus is used to treat
precursor B-
lymphoblastic leukemia (also called precursor B-cell acute lymphoblastic
leukemia),
prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone B-
cell lymphoma, hairy cell leukemia, plasma cell myeloma/plasmacytoma,
extranodal
marginal zone B-cell lymphoma of MALT type, nodal marginal zone B-cell
lymphoma, mediastinal large B-cell lymphoma, or primary effusion lymphoma.
In another embodiment of the invention, reovirus is used to treat a B-cell
lymphoma such as Hodgkin's lymphoma (HL), including but not limited to nodular
lymphocyte-predominant Hodgkin's lymphoma in addition to classical Hodgkin's
lymphoma, comprising nodular sclerosis Hodgkin's lymphoma (grades 1 and 2),
lymphocyte-rich classical Hodgkin's lymphoma, mixed cellularity Hodgkin's
lymphoma, and lymphocyte depletion Hodgkin's lymphoma.
In yet another embodiment of the invention, reovirus is used to treat
predominantly T-cell and natural killer (NK)-cell neoplasms, including but not
limited
to precursor T-cell neoplasm, precursor T-lymphoblastic lymphoma/leukemia
(also
called precursor T-cell acute lymphoblastic leukemia), T-cell prolymphocytic
leukemia, T-cell granular lymphocytic leukemia, aggressive NK-cell leukemia,
adult
T-cell lymphoma/leukemia (e.g., caused by HTLV-1), extranodal NK/T-cell
lymphoma (nasal type), enteropathy-type T-cell lymphoma, hepatosplenic gamma-
delta T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis
fungoides/Sezary syndrome, anaplastic large-cell lymphoma, T/null cell
(primary
cutaneous type), anaplastic large-cell lymphoma (T/null cell, primary systemic
type),
angioimmunoblastic T-cell lymphoma, and peripheral T-cell lymphomas that are
not
otherwise characterized.
In yet another embodiment of the invention, reovirus is used to treat post-
transplant lymphoproliferative disorders (PTLD), including but not limited to
polyclonal PTLD, monoclonal PTLD, reactive plasmacytic hyperplasia, infectious
mononucleosis-like PTLD, and monomorphic PTLD.
22
CA 02435967 2003-07-25
Reovirus can be used to treat any lymphoid malignancy in which cells it is
able to replicate. Moreover, the lymphoid malignancy need not be clinically,
morphologically, or genetically characterized prior to or in order to be
treated with
reovirus. That reovirus is able to replicate in the cells of the malignancy is
sufficient
to practice the invention. One method of determining whether reovirus is able
to
replicate in cells of a lymphoid malignancy is provided in Example 2, below.
Accordingly, lymphoma cells from a biopsy specimen obtained from an
animal with a lymphoid malignancy, or suspected of having a lymphoid
malignancy,
can be grown in culture and infected with reovirus. Reovirus replication can
be
measured using the assays described in the Examples, below, or by other
methods
known in the art, including but not limited to plaque assays and labeled
nucleoside or
nucleotide monophosphate incorporation. The ability of reovirus to replicate
in cells
derived from the biopsy specimen is evidence that administration of reovirus
to an
animal will be effective in treating the malignancy or suspected malignancy.
In another embodiment of the invention, cells of hematopoietic tissues are
removed from an animal contacted with reovirus ex vivo. The treated
hematopoietic
cells are then returned to the animal, preferably to the site from which they
were
harvested.
In another embodiment of the invention, reovirus is used to remove cells of a
lymphoid malignancy from a tissue or organ prior to transplantation. Because
this
method can be applied without regard to the type or age of the transplant or
the
specific nature of the lymphoid malignancy, and because the reovirus is
essentially
harmless to normal cells and tissues, this method can be used as a routine
practice to
"clean up" any transplant before transplantation. Transplanted tissues may be
autologous, allogeneic, or even xenogeneic. Preferably the transplanted
tissues are
autologous.
Reovirus may optionally be removed following treatment of a cellular
composition, such as hematopoietic tissues treated ex vivo or transplant
tissue, by
subjecting the mixture to anti-reovirus antibodies or a combination of anti-
reovirus
antibodies and complement to inactivate the reovirus (e.g., by lysis or capsid
disruption). Alternatively or additionally, anti-reovirus antibodies that
recognize
23
CA 02435967 2003-07-25
epitopes on the surface of reovirus may be used to remove the reovirus
particles by
immobilizing the antibodies, applying the cellular composition to the
immobilized
antibodies, and collecting the part of the composition that does not bind to
the
antibodies. The cellular composition is then transplanted into an animal or
returned to
the animal from which it was derived.
Alternatively, antireovirus antibodies are administered to an animal in vivo,
following administration of reovirus and, preferably, after substantial lysis
of cells of
a lymphoid malignancy has occurred. According to this embodiment of the
invention,
the antibodies are administered by the same route as the reovirus they are
administered to inactivate, by a separate route of administration, or any
combination
thereof.
Reovirus may also be used to treat more than one lymphoid malignancy in an
animal, including but not limited to any combination of the diseases,
disorders, and
conditions identified herein, or any combination of lymphoid malignancies that
have
not been characterized but in which reovirus is able to replicate.
In one embodiment of the invention, reovirus is administered in conjunction
with surgery or removal of at least a portion of the lymphoid malignancy. The
reovirus may be administered in conjunction with or in addition to radiation
therapy
and/or known anticancer compounds or chemotherapeutic agents. Such agents,
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. In
another embodiment of the invention, reovirus is administered prior to or in
place of
radiation therapy and/or chemotherapeutic agent.
In another embodiment of the invention, a method is provided for reducing the
growth of metastastic tumors in a mammal comprising administering an effective
amount of a reovirus to the mammal.
24
CA 02435967 2003-07-25
The reovirus may be administered to immunocompetent mammals in
conjunction with the administration of immunosuppressants and/or
immunoinhibitors.
Such immunosuppressants and immunoinhibitors are known to those of skill in
the art
and include such agents as cyclosporin, rapamycin, tacrolimus, mycophenolic
acid,
azathioprine and their analogs, and the like. Other agents are known to have
immunosuppressant properties as well (see, e.g., Goodman and Gilman, 7~'
Edition,
page 1242, the disclosure of which is incorporated herein by reference).
Immunoinhibitors include anti-antireovirus antibodies, which are antibodies
directed against anti-reovirus antibodies. Such anti-antireovirus antibodies
may be
administered prior to, at the same time, or shortly after the administration
of the
reovirus. Preferably an effective amount of the anti-antireovirus antibodies
are
administered in sufficient time to reduce or eliminate an immune response by
the
mammal to the administered reovirus.
The invention includes pharmaceutical compositions that comprise, as an
active ingredient, one or more of the reoviruses associated with
pharmaceutically
acceptable carriers or excipients. The pharmaceutical compositions may also
comprise an appropriate immunosuppresant, associated with pharmaceutically
acceptable carriers 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
CA 02435967 2003-07-25
administration to the patient. Other suitable formulations for use in the
present
invention can be found in Remington's Pharmaceutical Sciences.
The reovirus 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-antireovirus antibody.
In order to further illustrate the present invention and advantages thereof,
the
following specific examples are given but are not meant to limit the scope of
the
claims in any way.
EXAMPLES
In the examples below, the following abbreviations have the following
meanings. Abbreviations not defined have their generally accepted meanings:
~1 microliter
B-cell CLL B-cell chronic lymphocytic leukemia
BL Burkitt's lymphoma
CLL chronic lymphocytic leukemia
CPE cytopathic effects
DLBCL diffuse large B-cell lymphoma
EBV Epstein-Barr virus
EBV- Epstein-Barr virus not detected
EBV+ Epstein-Barr virus detected
FL follicular lymphoma
HL Hodgkin's lymphoma
MCL mantle-cell lymphoma
mm2 square millimeters
MOI multiplicity of infection
n number of test subjects in
a particular group
NHL non-Hodgkin's lymphoma
PBMC primary blood mononuclear cells
PBS phosphate-buffered saline
PFU plaque forming units
SDS-PAGE sodium dodecyl sulfate polyacrylamide
gel
electrophoresis
SLL small lymphocytic leukemia
UV ultraviolet
26
CA 02435967 2003-07-25
Example l: 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.
The results of the above experiments are summarized in Table 1, 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
27
CA 02435967 2003-07-25
ST486 Burkitt's lymphoma, No
EBV-
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-LY10 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.
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 I. Reovirus failed to replicate in the control PBMC and CD34+ cells
but
appeared to replicate in 15/IS CLL samples and 6/I2 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.
28
CA 02435967 2003-07-25
Table 2: Susceptibility of lymphoid biopsy specimens to reovirus
Disease Cell type Total SusceptibleResistant
s ecimens s ecimenss ecimens
CLL Chronic lymphocytic 15 15 0
leukemia
NHL Burkitt's lym homa 1 1 0
NHL Diffuse largeB-cell 2 2 0
lymphoma
NHL Small lym hoc is 1 2 1 1
homa
NHL Follicular lym homa, 1 1 0
grade I
NHL Follicular lymphoma, 4 0 4
grade II
NHL Follicular lym homa, 0 0 1
ade III
NHL Mantle-celllyrnphoma 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 1 S minutes at room temperature, in the dark. The cells
were then
washed and resuspended in PBS.
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 CD 10+/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.
29
CA 02435967 2003-07-25
Examule 3: Efficacy Of Reovirus Treatment On Lymphoid Tumors In A
Xenograft Model.
A murine xenograft model was used to evaluate the ability of reovirus to treat
lymphoma-derived tumors in vivo. About 107 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 107
PFU of live (n=8) or UV-inactivated (n=7) reovirus in 50 pl 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 107 (n=7) or
5x107 (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
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)
CA 02435967 2003-07-25
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
admini stration.
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.
31
