U.S. patent application number 10/044955 was filed with the patent office on 2003-03-06 for treatment of neoplasms with viruses.
This patent application is currently assigned to Pro-Virus, Inc.. Invention is credited to Groene, William S., Lorence, Robert M., Rabin, Harvey, Roberts, Michael S., von Borstel, Reid W..
Application Number | 20030044384 10/044955 |
Document ID | / |
Family ID | 26864553 |
Filed Date | 2003-03-06 |
United States Patent
Application |
20030044384 |
Kind Code |
A1 |
Roberts, Michael S. ; et
al. |
March 6, 2003 |
Treatment of neoplasms with viruses
Abstract
The subject invention relates to viruses that are able to
replicate and thereby kill neoplastic cells with a deficiency in
the IFN-mediated antiviral response, and their use in treating
neoplastic disease including cancer and large tumors. RNA and DNA
viruses are useful in this regard. The invention also relates to
methods for the selection, design, purification and use of such
viruses for cancer therapy.
Inventors: |
Roberts, Michael S.;
(Walkersville, MD) ; Lorence, Robert M.;
(Bethesda, MD) ; Groene, William S.; (New Market,
MD) ; Rabin, Harvey; (Rockville, MD) ; von
Borstel, Reid W.; (Potomac, MD) |
Correspondence
Address: |
NIXON & VANDERHYE P.C.
8th Floor
1100 North Glebe Road
Arlington
VA
22201-4714
US
|
Assignee: |
Pro-Virus, Inc.
|
Family ID: |
26864553 |
Appl. No.: |
10/044955 |
Filed: |
January 15, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10044955 |
Jan 15, 2002 |
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09168883 |
Oct 9, 1998 |
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09168883 |
Oct 9, 1998 |
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08948244 |
Oct 9, 1997 |
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Current U.S.
Class: |
424/93.2 ;
435/235.1; 435/456 |
Current CPC
Class: |
A61K 35/768 20130101;
A61P 43/00 20180101; C12N 2770/36132 20130101; A61K 39/42 20130101;
C12N 7/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 38/21 20130101; A61K 2300/00 20130101; C12N 2760/18132
20130101; A61K 35/768 20130101; A61K 38/21 20130101; A61K 39/42
20130101 |
Class at
Publication: |
424/93.2 ;
435/456; 435/235.1 |
International
Class: |
A61K 048/00; C12N
007/00; C12N 015/86 |
Claims
What is claimed is:
1. A method of infecting a neoplasm in a mammal with a virus
comprising administering an interferon-sensitive,
replication-competent clonal RNA virus to said mammal.
2. A method of infecting a neoplasm in a mammal with a virus
comprising administering a replication-competent clonal RNA virus
to said mammal wherein said virus has sensitivity to
interferon.
3. A method of treating a neoplasm in a mammal comprising
administering to said mammal a therapeutically effective amount of
an interferon-sensitive, replication-competent clonal RNA
virus.
4. A method as in claim 1 wherein said RNA virus replicates at
least 100-fold less in the presence of interferon compared to in
the absence of interferon.
5. A method as in claim 1 wherein said RNA virus replicates at
least 1000-fold less in the presence of interferon compared to in
the absence of interferon.
6. A method as in claim 1 wherein said administering step is
systemic.
7. A method as in claim 1 wherein said neoplasm is a cancer.
8. A method as in claim 1 wherein said mammal is a human.
9. A method as in claim 1 wherein said clonal virus is plaque
purified.
10. A method as in claim 1 wherein said clonal virus is of
recombinant clonal origin.
11. A method as in claim 1 wherein said RNA virus is a
Paramyxovirus.
12. A method as in claim 11 wherein said Paramyxovirus is purified
to a level of at least 2.times.10.sup.9 PFU per mg of protein.
13. A method as in claim 11 wherein said Paramyxovirus is purified
to a level of at least 1.times.10.sup.10 PFU per mg of protein.
14. A method as in claim 11 wherein said Paramyxovirus is purified
to a level of at least 6.times.10.sup.10 PFU per mg of protein.
15. A method as in claim 11 wherein said Paramyxovirus is purified
to a level in which the particle per PFU ratio is no greater than
5.
16. A method as in claim 11 wherein said Paramyxovirus is purified
to a level in which the particle per PFU ratio is no greater than
3.
17. A method as in claim 11 wherein said Paramyxovirus is purified
to a level in which the particle per PFU ratio is no greater than
1.2.
18. A method as in claim 11 wherein said Paramyxovirus is avian
paramyxovirus type 2.
19. A method as in claim 11 wherein said Paramyxovirus is NDV.
20. A method as in claim 11 wherein said Paramyxovirus is mumps
virus.
21. A method as in claim 11 wherein said Paramyxovirus is human
parainfluenza virus.
22. A method as claim 1 wherein said RNA virus is selected from the
group consisting of a Rhabdoviris, Togavirus, Flavivirus, Reovirus,
Picornavirus, and Coronavirus.
23. A method as in claim 22 wherein said Togavirus is Sindbis
virus.
24. A method as in claim 22 wherein said Reovirus has a
modification at omega 3.
25. A method as in claim 22 wherein said Reovirus has an
attenuating mutation at omega 1.
26. A method as in claim 22 wherein said Reovirus is an attenuated
rotavirus
27. A method as in claim 26 wherein said rotavirus is rotavirus
WC3.
28. A method of infecting a neoplasm in a mammal with a virus
comprising administering an interferon-sensitive,
replication-competent clonal vaccinia virus, having one or more
mutations in one or more genes selected from the group consisting
of K3L, E3L, and B18R, to said mammal.
29. A method of infecting a neoplasm in a mammal with a virus
comprising administering a replication-competent clonal vaccinia
virus, having one or more mutations in one or more genes selected
from the group consisting of K3L, E3L, and B18R, to said mammal
wherein said virus has sensitivity to interferon.
30. A method treating a neoplasm in a mammal comprising
administering to said mammal a therapeutically effective amount of
an interferon-sensitive, replication-competent clonal vaccinia
virus, having one or more mutations in one or more genes selected
from the group consisting of K3L, E3L, and B18R.
31. A method as in claim 30 wherein said mammal is a human.
32. A method as in claim 30 wherein said vaccinia virus is a
vaccinia virus having an attenuating mutation in a gene selected
from the group encoding vaccinia growth factor, thymidine kinase,
thymidylate kinase, DNA ligase, ribonucleotide reductase and
dUTPase.
33. A method of infecting a neoplasm in a mammal with a virus
comprising administering an interferon-sensitive,
replication-competent clonal DNA virus, selected from the group
consisting of Adenoviruses, Parvoviruses, Papovaviruses, and
Iridoviruses, to said mammal.
34. A method of infecting a neoplasm in a mammal with a virus
comprising administering a replication-competent clonal DNA virus,
selected from the group consisting of Adenoviruses, Parvoviruses,
Papovaviruses, and Iridoviruses, to said mammal wherein said virus
has sensitivity to interferon.
35. A method of treating a neoplasm in a mammal comprising
administering to said mammal a therapeutically effective amount of
an interferon-sensitive, clonal DNA virus selected from the group
consisting of Adenoviruses, Parvoviruses, Papovaviruses, and
Iridoviruses.
36. A method as in claim 33 wherein said mammal is a human.
37. A method as in claim 33 wherein said Adenovirus virus has a
modification in the VA1 transcripts causing said Adenovirus to
become interferon-sensitive.
38. A method as in claim 37 wherein said Adenovirus virus is
selected from the group consisting of vaccine strains of Ad-4, Ad-7
and Ad-21.
39. A method of infecting a neoplasm in a mammal with a virus
comprising administering an interferon-sensitive,
replication-competent clonal Herpesvirus to said mammal.
40. A method of infecting a neoplasm in a mammal with a virus
comprising administering a replication-competent clonal Herpesvirus
to said mammal wherein said virus has sensitivity to
interferon.
41. A method treating a neoplasm in a mammal comprising
administering to said mammal a therapeutically effective amount of
an interferon-sensitive, replication-competent clonal
Herpesvirus.
42. A method as in claim 41 wherein said Herpesvirus is a member of
the subfamily Betaherpesvirus or subfamily Gammaherpesvirus.
43. A method as in claim 41 wherein said Herpesvirus is a member of
the subfamily Alphaherpesvirus that is not HSV-1.
44. A method as in claim 41 wherein said mammal is a human.
45. A method as in claim 41 wherein said Herpesvirus is a member of
the subfamily Alphaherpesvirus which has decreased expression of
the (2'-5') An analog.
46. A method as in claim 45 wherein said Herpesvirus is a
Herpesvirus having an attenuating mutation selected from the group
consisting of the genes encoding thymidine kinase, ribonucleotide
reductase, or a deletion in the b'a'c' inverted repeat locus.
47. A method as in claim 45 wherein said Herpesvirus has a
modification in the gamma 34.5 gene.
48. A method as in claim 41 wherein said Herpesvirus has a
modification in the gamma 34.5 gene and an attenuating mutation in
the gene encoding of thymidine kinase, or a deletion in the b'a'c'
inverted repeat locus or functionally analogous loci.
49. A method as in claim 41 wherein said Herpesvirus is a
Herpesvirus having an attenuating mutation in a gene selected from
the group consisting of thymidine kinase, and ribonucleotide
reductase, or a deletion in the b'a'c' inverted repeat locus.
50. A method as in claim 1 wherein said neoplasm is a cancer
selected from the group consisting of lung, colon, prostate, breast
and brain cancer.
51. A method as in claim 1 wherein said neoplasm is a solid
tumor.
52. A method as in claim 50 wherein said brain cancer is a
glioblastoma.
53. A method as in claim 1 wherein said virus contains a gene
encoding interferon to permit the viral expression of
interferon.
54. A method as in claim 1 wherein said virus contains a gene
encoding a pro-drug activating enzyme.
55. A method as in claim 1 further comprising administering IFN,
before, during or after administration of said virus.
56. A method as in claim 55 wherein said interferon is selected
from the group consisting of .alpha.-IFN, .beta.-IFN, .omega.-IFN,
.gamma.-IFN, and synthetic consensus forms of IFN.
57. A method as in claim 1 further comprising administering a
tyrosine kinase inhibitor before, during or after administration of
said virus.
58. A method as in claim 1 further comprising administering a
compound selected from the group of compounds comprising a purine
nucleoside analog, tyrosine kinase inhibitor, cimetidine, and
mitochondrial inhibitor.
59. A method as in claim 1 further comprising administering a
chemotherapeutic agent before, during or after administration of
said virus.
60. A method as in claim 1 further comprising administering a
cytokine before, during or after administration of said virus.
61. A method as in claim 1 further comprising administering an
immunosuppresant before, during or after administration of said
virus.
62. A method as in claim 1 further comprising administering a viral
replication controlling amount of a compound selected from the
group consisting of IFN, ribavirin, acyclovir, and ganciclovir.
63. A method as in claim 1 wherein said administration is
intravenous or intratumoral.
64. A method of infecting a neoplasm which is at least 1 centimeter
in size in a mammal with a virus comprising administering a clonal
virus, selected from the group consisting of (1) RNA viruses; (2)
Hepadenavirus; (3) Parvovirus; (4) Papovavirus; (5) Herpesvirus;
(6) Poxvirus; and (7) Iridovirus, to said mammal.
65. A method of treating a neoplasm in a mammal, comprising
administering to said mammal a therapeutically effective amount of
a clonal virus selected from the group consisting of (1) RNA virus;
(2) Hepadenavirus; (3) Parvovirus; (4) Papovavirus; (5)
Herpesvirus; (6) Poxvirus; and (7) Iridovirus, wherein said
neoplasm is at least 1 centimeter in size.
66. A method as in claim 64, wherein said neoplasm is at least 300
mm.sup.3 in volume.
67. A method as in claim 64, wherein said RNA virus is a
Paramyxovirus.
68. A method as in claim 67, wherein said Paramyxovirus is NDV.
69. A method as in claim 64, wherein said mammal is a human.
70. A method as in claim 64, wherein said administration is
intravenous or intratumoral.
71. A method as in claim 64, wherein said paramyxovirus is purified
to a level of at least 2.times.10.sup.9 PFU per mg protein.
72. A method as in claim 68, wherein said NDV is mesogenic.
73. A method as in claim 65 wherein said neoplasm is cancerous.
74. A method of treating a tumor in a mammal, comprising
administering to said mammal a therapeutically effective amount of
an RNA virus cytocidal to said tumor, wherein said mammal has a
tumor burden comprising at least 1.5% of the total body weight of
said mammal.
75. A method as in claim 74, wherein said tumor does not respond to
chemotherapy.
76. A method of screening tumor cells or tissue freshly removed
from the patient to determine the sensitivity of said cells or
tissue to killing by a virus comprising subjecting a tissue sample
to a differential cytotoxicity assay using an interferon-sensitive
virus.
77. A method as in claim 76 further comprising the step of
screening said cells or tissue for protein, or mRNA encoding
protein, selected from the group consisting of p68 protein kinase,
C-Myc, C-Myb, ISGF-3, IRF-1, IFN receptor, and p58.
78. A method for identifying a virus with antineoplastic activity
in a mammal comprising: a) using said test virus to infect i) cells
deficient in an interferon-mediated antiviral activity, and ii)
cells competent in an interferon-mediated antiviral activity, and
b) determining whether said test virus kills said cells deficient
in an interferon-mediated antiviral activity preferentially to said
cells competent in interferon-mediated antiviral activity.
79. A method as in claim 78 wherein said cells deficient in an
interferon-mediated antiviral activity are KB human head and neck
carcinoma cells.
80. A method as in claim 78 wherein said cells competent in an
interferon-mediated antiviral activity are human skin
fibroblasts.
81. A method of making viruses for use in antineoplastic therapy
comprising: (a) modifying an existing virus by diminishing or
ablating a viral mechanism for the inactivation of the antiviral
effects of IFN, and optionally (b) creating an attenuating
mutation
82. A method of controlling viral replication in a mammal treated
with a virus selected from the group consisting of RNA viruses,
Adenoviruses, Poxviruses, Iridoviruses, Parvoviruses,
Hepadnaviruses, Varicellaviruses, Betaherpesviruses, and
Gammaherpesviruses comprising administering an antiviral
compound.
83. A method as in claim 82 wherein said antiviral compound is
interferon.
84. A method as in claim 82 wherein said antiviral is selected from
the group consisting of ribavirin, acyclovir, and ganciclovir.
85. A method as in claim 82 wherein said antiviral is a
neutralizing antibody to said virus.
86. A Paramyxovirus purified by ultracentrifugation without
pelleting.
87. A Paramyxovirus purified to a level of at least
2.times.10.sup.9 PFU/mg protein.
88. A Paramyxovirus as in claim 87 wherein said paramyxovirus is
grown in eggs and is substantially free of contaminating egg
proteins.
89. A Paramyxovirus as in claim 87 wherein said paramyxovirus has a
particle per PFU ratio no greater than 5.
90. A Paramyxovirus as in claim 87 wherein said paramyxovirus has a
particle per PFU ratio no greater than 3.
91. A Paramyxovirus as in claim 87 wherein said paramyxovirus has a
particle per PFU ratio no greater than 1.2.
92. A Paramyxovirus purified to a level of at least
1.times.10.sup.10 PFU/mg protein.
93. A Paramyxovirus purified to a level of at least
6.times.10.sup.10 PFU/mg protein.
94. A Paramyxovirus as in claim 87 wherein said virus is
cytocidal.
95. A Paramyxovirus as in claim 87 wherein said Paramyxovirus is
Newcastle disease virus.
96. An NDV as in claim 95 wherein said NDV is cytocidal.
97. An NDV as in claim 95 wherein said NDV is mesogenic.
98. An RNA virus purified to a level of at least 2.times.10.sup.9
PFU/mg protein.
99. An RNA virus as in claim 98 wherein said virus is
replication-competent.
100. A replication-competent cytocidal virus which is
interferon-sensitive and purified to a level of at least
2.times.10.sup.9 PFU/mg protein.
101. A cytocidal virus as in claim 100 wherein said virus is
clonal.
102. A cytocidal DNA virus which is interferon-sensitive and
purified to a level of at least 2.times.10.sup.9 PFU/mg
protein.
103. A cytocidal DNA virus as in claim 102 wherein said virus is a
Poxvirus.
104. A Poxvirus as in claim 103 wherein said Poxvirus is a vaccinia
virus having one or more mutations in one or more genes selected
from the group consisting of K3L, E3L, and B18R.
105. A replication-competent vaccinia virus having a) one or more
mutations in one or more of the K3L, E3L and B18R genes, and b) an
attentuating mutation in one or more of the genes encoding
thymidine kinase, ribonucleotide reductase, vaccinia growth factor,
thymidylate kinase, DNA ligase, dUTPase.
106. A replication-competent vaccinia virus having one or more
mutations in two or more genes selected from the group consisting
of K3L, E3L, and B18R.
107. A Herpesvirus having a modification in the expression of the
(2'-5') A analog.
108. A Reovirus having a mutation at omega 3 and purified to a
level of at least 2.times.10.sup.9 PFU/mg protein
109. A Reovirus having mutations at omega 1 and omega 3.
110. A method of purifying an RNA virus comprising the steps of: a)
generating a clonal virus, and b) purifying said clonal virus by
ultracentrifugation without pelleting.
111. A method as in claim 110 wherein said RNA virus is
replication-competent.
112. A method of purifying a Paramyxovirus comprising purifying
said virus by ultracentrifugation without pelleting.
113. A method as in claim 112 wherein said purifying step
additionally comprises prior to said ultracentrifugation: a) plaque
purifying to generate a clonal virus, b) inoculating eggs with said
clonal virus, c) incubating said eggs, d) chilling said eggs, e)
harvesting allantoic fluid from said eggs and, f) removing cell
debris from said allantoic fluid.
114. A method as in claim 112 wherein said Paramyxovirus virus is
NDV.
115. A method of infecting a neoplasm in a mammal with a virus
comprising administering an interferon-sensitive,
replication-competent RNA virus to said mammal.
116. A method as in claim 1 wherein said virus is selected from the
group consisting of the Newcastle disease virus strain MK107,
Newcastle disease virus strain NJRoakin, Sindbis virus, and
Vesicular stomatitis virus.
117. A method of infecting a neoplasm in a mammal with a virus
comprising administering a clonal virus selected from the group
consisting of the Newcastle disease virus strain MK107, Newcastle
disease virus strain NJRoakin, Sindbis virus, and Vesicular
stomatitis virus.
118. A method as in claim 1 or claim 28 or claim 33 wherein said
virus is administered as more than one dose.
119. A method as in claim 118 wherein the first dose is a
desensitizing dose.
120. A method as in claim 119 wherein said first dose is
administered intravenously and a subsequent dose administered
intravenously.
121. A method as in claim 119 wherein said first dose is
administered intravenously and a subsequent dose administered
intraperitoneally.
122. A method as in claim 119 wherein said first dose is
administered intravenously and a subsequent dose administered
intra-arterially.
123. A method of treating a neoplasm in a mammal comprising
subjecting a sample from said mammal to an immunoassay to detect
the amount of virus receptor present, and if said receptor is
present, administering an interferon-sensitive, replication
competent clonal virus, which bind said receptor, to said
mammal.
124. A method as in claim 123 wherein said virus is Sindbis and
said receptor is the high affinity laminin receptor.
125. A method as in claim 1 or claim 28 or claim 33 wherein said
virus is administered over the course of at least 4 minutes.
126. A method of treating tumor ascites comprising administering an
interferon-sensitive, replication competent clonal virus.
127. A method of reducing pain in a mammal comprising administering
an interferon-sensitive, replication competent clonal virus.
Description
FIELD OF THE INVENTION
[0001] The subject invention relates to viruses that are able to
replicate in and cause the death of neoplastic cells with a
deficiency in the interferon (IFN)-mediated antiviral response. RNA
and DNA viruses are useful in this regard. The invention also
relates to the use of these viruses for the treatment of neoplastic
diseases including cancer and large tumors.
BACKGROUND OF THE INVENTION
[0002] Neoplastic disease which includes cancer is one of the
leading causes of death among human beings. There are over 1.3
million new cases of cancer diagnosed in the United States each
year and 550,000 deaths. Detecting cancer early, before it has
spread to secondary sites in the body, greatly increases a host's
chances of survival. However, early detection of cancer is not
always possible, and even when it is, treatments are
unsatisfactory, especially in cases of highly malignant cancers.
Cancer treatments, including chemotherapy and radiation, are much
less effective in latter stages, especially when neoplastic growths
are large and/or constitute a high tumor burden. (See Hillard
Stanley, Cancer Treat. Reports, Vol. 61, No. 1, Jan/Feb 1977,
p.29-36, Tannock, Cancer Research, 42, 4921-4926, December
1982).
[0003] Tumor regression associated with exposure to various viruses
has been reported. Most of the viruses described are pathogenic in
humans, and include mumps and measles. The effect of other specific
viruses on particular types of cancer cells has also been
described. Smith et al, (1956) Cancer, 9, 1211 (effect of
adenovirus on cervix carcinoma); Holzaepfel et al, (1957) Cancer,
10, 557 (effect of adenovirus on epithelial tumor); Taylor et al,
(1970) J. Natl. Cancer Inst., 44, 515 (effect of bovine
enterovirus-1 on sarcoma-1); Shingu et al, (1991) J. General
Virology, 72, 2031 (effect of bovine enterovirus MZ-468 on F-647a
leukemia cells); Suskind et al, (1957) PSEBM, 94, 309 (effect of
coxsackie B3 virus on HeLa tumor cells); Rukavishnikova et al,
(1976) Acta Virol., 20, 387 (effect of influenza A strain on
ascites tumor).
[0004] The earliest references described partial tumor regression
in patients treated with live attenuated viral vaccine with the aim
to vaccinate them against smallpox or rabies. See DePace, N. G.
(1912) Ginecologia, 9, 82-88; Salmon, P. & Baix (1922) Compt.
Rend. Soc. Biol., 86, 819-820. Partial regression of tumors and
regression of leukemias have also been noted during naturally
occurring measles infections. See Pasquinucci, G. (1971) Lancet,
1,136; Gross, S. (1971) Lancet, 1, 397-398; Bluming, A. Z. and
Ziegler, J. L. (1971) Lancet, 2, 105-106. In one study of 90 cancer
patients intentionally infected with live mumps virus, partial
tumor regression was noted in 79 cases. See Asada (1994) Cancer,
34, 1907-1928. While the side effects of these viruses were
temporary, serious sequela of infection with these human pathogens
is of major concern.
[0005] Viruses are categorized as follows [see Murphy A and
Kingsbury D W, 1990, In: Virology, 2.sup.nd Edition (Ed. Fields, B.
N.), Raven Press, New York, pp 9-35]:
1 Dividing Characteristics Virus Family Names RNA viruses ss RNA,
positive-sense, Picornaviridae, Caliciviridae nonsegmented,
nonenveloped, ssRNA, positive-sense, Togaviridae, Flaviviridae,
nonsegmented, enveloped, Coronaviridae ssRNA, negative-sense,
Rhabodoviridae, Filoviridae, nonsegmented, enveloped,
Paramyxoviridae ssRNA, negative-sense, Orthomyxoviridae segmented,
enveloped ssRNA, ambisense, Bunyaviridae, Arenaviridae segmented,
enveloped dsRNA, positive-sense Reoviridae, Birnaviridae segmented,
nonenveloped ssRNA, DNA step in Retroviridae replication, positive-
sense, nonsegmented, enveloped DNA viruses ss/dsDNA, nonenveloped
ss DNA, nonenveloped dsDNA, nonenveloped dsDNA, enveloped
Hepadnaviridae Parvoviridae Papovaviridae, Adenoviridae
Herpesvirdae, Poxviridae, Iridoviridae
[0006] Included among the family Herpesviridae (or Herpesviruses),
are the subfamilies Alphaherpesvirus (including Genus
Varicellavirus and Genus Simpexvirus), Betaherpesvirus, and
Gamimaherpesvirus.
[0007] Newcastle disease virus ("NDV") is a member of the
Paramyxoviridae (or Paramyxoviruses). The natural hosts for NDV are
chickens and other birds. NDV typically binds to certain molecules
on the surface of animal host cells, fuses with the cell surface,
and injects its genetic material into the host. NDV is a cytocidal
virus. Once inside the cell, the viral genes direct the host cell
to make copies of the virus leading to death of the host cell,
releasing the copies of NDV which infect other cells. Unlike some
viruses, NDV is not known to cause any serious human disease.
Unlike other kinds of viruses (e.g., HTLV-1, Hepatitis B),
Paramyxoviruses are not known to be carcinogenic.
[0008] Temporary regression of tumors has been reported in a small
number of patients exposed to NDV, See Csatary, L. K. (1971)
Lancet, 2, 825. Csatary noted the regression of a gastrointestinal
cancer in a chicken farmer during an epidemic of Newcastle disease
in his chickens. In a similar anecdotal report, Cassel, W. A. and
Garrett, R. E. (1965) Cancer, 18, 863-868, noted regression of
primary cervical cancer, which had spread to the lymph nodes, in a
patient following injection of NDV into the cervical tumor. Since
the mechanism of tumoricidal activity was thought to be
immunologic, no work was carried out to address direct tumor
cytotoxicity of the virus. Instead, efforts focused upon the
immuno-modulating effects of NDV. See for example, Murray, D. R.,
Cassel, W. A., Torbin, A. H., Olkowski, Z. L., & Moore, M. E.
(1977) Cancer, 40, 680; Cassel, W. A., Murray, D. R., &
Phillips, H. S. (1983) Cancer, 52, 856; Bohle, W., Schlag, P J.,
Liebrich, W., Hohenberger, P., Manasterski, M., M ller, P., and
Schirrmacher, V. (1990) Cancer, 66,1517-1523.
[0009] The selection of a specific virus for tumor regression was
based on serendipity or trial and error in the above citations.
Only recently, have rational, mechanism-based approaches for virus
use in cancer treatment been developed using DNA viruses. Examples
of this type of approach are found in the development of
recombinant adenoviral vectors that replicate only in tumors of
specific tissue origin (Rodriguez, R. et al, 1997 Cancer Res.,
57:2559-2563), or those that lack certain key regulatory proteins
(Bischoff, J R., et al, 1996 Science, 274:373-376). Another recent
approach has been the use of a replication-incompetent recombinant
adenoviral vector to restore a critical protein function lost in
some tumor cells (Zhang, W W, et al, 1994 Cancer gene therapy,
1:5-13). Finally, herpes simplex virus has also been engineered to
replicate preferentially in the rapidly dividing cells that
characterize tumors (Mineta, T., et al, 1994 Cancer Res.,
54:3963-3966).
[0010] U.S. application Ser. No. 08/260,536, hereby incorporated by
reference in its entirety, discloses the use of NDV or other
Paramyxovirus in the treatment of cancer.
[0011] Viral IFN Transgene Expression
[0012] One common approach to the treatment of cancer with viral
therapeutics has been the use of virus vectors for the delivery of
certain genes to the tumor mass.
[0013] Recombinant adenovirus, adeno-associated virus, vaccinia
virus and retroviruses have all been modified to express an
interferon gene alone or in combination with other cytokine
genes.
[0014] In Zhang et al. ((1996) Proc. Natl. Acad. Sci. USA
93:4513-4518), a recombinant adenovirus expressing a human
interferon consensus (i.e., synthetic) gene was used to treat human
breast cancer (and other) xenografis in nude mice. The authors
concluded ". . . a combination of viral oncolysis with a virus of
low pathogenicity, itself resistant to the effects of IFN and IFN
gene therapy, might be a fruitful approach to the treatment of a
variety of different tumors, in particular breast cancer." In
contrast to subject invention which relates to interferon-sensitive
viruses, Zhang et at (1996) teach the use of an
interferon-resistant adenovirus in the treatment of tumors.
[0015] In Zhang et al. ((1996) Cancer Gene Ther., 3:31-38),
adeno-associated virus (AAV) expressing consensus IFN was used to
transduce human tumor cells in vitro followed by injection into
nude mice. The transduced tumors either did not form tumors or grew
slower than the non-transduced controls. Also, injection of one
transduced human tumor cell into the tumor mass of another,
non-transduced tumor resulted in a small decrease in size.
[0016] In Peplinski et al. ((1996) Ann. Surg. Oncol., 3:15-23), IFN
gamma (and other cytokines, expressed either alone, or in
combination) were tested in a mouse breast cancer model. Mice were
immunized with tumor cells virally modified with recombinant
vaccinia virus. When re-challenged with tumor cells, the mice
immunized with virally modified cells had statistical improvement
in the disease-free survival time.
[0017] Gastl, et al. ((1992) Cancer Res., 52:6229-6236), used IFN
gamma-expressing retroviral vectors to transduce renal carcinoma
cells in vitro. These cells were shown to produce higher amounts of
a number of proteins important for the function of the immune
system.
[0018] Restifo et al. ((1992) J. Exp. Med., 175:1423-1431), used
IFN gamma-expressing retroviral vector to transduce a murine
sarcoma cell line allowing the tumor cell line to more efficiently
present viral antigens to CD8+T cells. Howard, et al. ((1994) Ann.
NY Acad. Sci., 716:167-187), used IFN gamma-expressing retroviral
vector to transduce murine and human melanoma tumor cells. These
cells were observed to increase the expression of proteins
important to immune function. These cells were also less
tumorigenic in mice as compared to the non-transduced parent line,
and resulted in activation of a tumor-specific CTL response in
vivo.
[0019] Use of Therapeutic Doses of Interferon as an Adjuvant to
Viral Cancer Therapy
[0020] Because of the known immune-enhancing properties of IFN,
several studies have examined the use of IFN protein in combination
with other viral cancer vaccine therapies.
[0021] In Kirchner et al. ((1995) World J. Urol., 13:171-173), 208
patients were immunized with autologous, NDV-modified, and lethally
irradiated renal-cell carcinoma tumor cells, and were co-treated
with low dose IL-2 or IFN alpha. The authors stated that this
treatment regime results in an improvement over the natural course
in patients with locally-advanced renal-cell carcinoma. The dose
was approximately 3.3.times.10.sup.3 to 2.2.times.10.sup.5 PFU/kg.
This was a local therapy, as opposed to a systemic approach, with
the goal of inducing an anti-tumor immune response.
[0022] Tanaka et al. ((1994) J. Immunother. Emphasis Tumor
Immunol., 16:283-293), co-administered IFN alpha with a recombinant
vaccinia virus as a cancer vaccine therapy model in mice. This
study showed a statistical improvement in survivability in mice
receiving IFN as compared to those that did not. The authors
attributed efficacy of IFN to the induction of CD8-positive T cells
in those animals.
[0023] Arroyo et al. ((1990) Cancer Immunol. Immunother.,
31:305-311) used a mouse model of colon cancer to test the effect
of IFN alpha and/or IL-2 co-therapy on the efficacy of a vaccinia
virus colon oncolysate (VCO) cancer treatment. They found that the
triple treatment of VCO+IL-2+IFN was most efficacious in this
murine model. This approach relies on immunization as the mechanism
of anti-tumor activity
[0024] IFN was used in these studies to augment the ability of the
cancer cells to be recognized by the immune system.
OBJECTS OF THE INVENTION
[0025] It is an object of the invention to provide viruses for the
treatment of diseases including cancer.
[0026] It is a further object of the invention to provide viruses
for the treatment of neoplastic diseases including cancer.
[0027] It is a further object of the invention to provide a means
by which candidate viruses are selected and/or screened for use in
the therapy of neoplastic diseases.
[0028] It is a further object of the invention to provide guidance
in the genetic engineering of viruses in order to enhance their
therapeutic utility in the treatment of neoplastic diseases.
[0029] It is a further object of this invention to provide a means
with which to screen potential target cells for viral therapy with
the goal of assessing the sensitivity of the candidate target cells
to viral killing.
[0030] It is a still further object of this invention to provide
guidance in the management of viral therapy.
[0031] It is an object of the invention to provide a method for
treating large tumors.
[0032] It is a further object of the invention to provide purified
virus and methods for obtaining same.
SUMMARY OF THE INVENTION
[0033] This invention relates to a method of infecting a neoplasm
in a mammal with a virus comprising administering an
interferon-sensitive, replication-competent clonal virus, selected
from the group consisting of RNA viruses and the DNA virus families
of Adenovirus, Parvovirus, Papovavirus, Iridovirus, and
Herpesvirus, to the mammal.
[0034] This invention also relates to a method of infecting a
neoplasm in a mammal with a virus comprising systemically
administering an interferon-sensitive, replication-competent clonal
virus to the mammal.
[0035] This invention also relates to a method of treating a
neoplasm including cancer in a mammal comprising administering to
the mammal a therapeutically effective amount of an
interferon-sensitive, replication-competent, clonal virus selected
from the group consisting of RNA viruses, and the DNA virus
families of Adenovirus, Parvovirus, Papovavirus, Iridovirus, and
Herpesvirus.
[0036] This invention also relates to a method of infecting a
neoplasm in a mammal with a virus comprising administering an
interferon-sensitive, replication-competent clonal vaccinia virus,
having one or more mutations in one or more viral genes involved
with blocking interferon's antiviral activity selected from the
group of genes consisting of K3L, E3L and B18R, to the mammal.
[0037] The invention also relates to a method of treating a
neoplasm including cancer in a mammal administering to the mammal a
therapeutically effective amount of an interferon-sensitive,
replication-competent vaccinia virus having one or more mutations
in one or more viral genes involved with blocking interferon's
antiviral activity selected from the group of genes consisting of
K3L, E3L and B18R.
[0038] The invention also relates to a method of infecting a
neoplasm at least 1 cm in size with a virus in a mammal comprising
administering a clonal virus, selected from the group consisting
of(1) RNA viruses; (2) Hepadenavirus; (3) Parvovirus; (4)
Papovavirus; (5) Herpesvirus; (6) Poxvirus; and (7) Iridovirus, to
the mammal.
[0039] The invention also relates to a method of treating a
neoplasm in a mammal, comprising administering to the mammal a
therapeutically effective amount of a clonal virus, selected from
the group consisting of(1) RNA viruses; (2) Hepadenavirus; (3)
Parvovirus; (4) Papovavirus; (5) Herpesvirus; (6) Poxvirus; and (7)
Iridovirus, wherein the neoplasm is at least 1 centimeter in
size.
[0040] The invention also relates to a method of treating a tumor
in a mammal, comprising administering to the mammal a
therapeutically effective amount of an RNA virus cytocidal to the
tumor, wherein the mammal has a tumor burden comprising at least
1.5% of the total body weight
[0041] The invention also relates to a method of screening tumor
cells or tissue freshly removed from the patient to determine the
sensitivity of the cells or tissue to killing by a virus comprising
subjecting the cells or tissue to a differential cytotoxicity assay
using an interferon-sensitive virus.
[0042] The invention also relates to a method for identifying a
virus with antineoplastic activity in a mammal comprising a) using
the test virus to infect i) cells deficient in IFN-mediated
antiviral activity, and ii) cells competent in IFN-mediated
antiviral activity, and b) determining whether the test virus kills
the cells deficient in IFN-mediated antiviral activity
preferentially to the cells competent in interferon-mediated
antiviral activity.
[0043] The invention also relates to a method of making viruses for
use in antineoplastic therapy comprising: a) modifying an existing
virus by diminishing or ablating a viral mechanism for the
inactivation of the antiviral effects of IFN, and optionally b)
creating an attenuating mutation that results in lower virulence
than said existing virus.
[0044] The invention also relates to a method of controlling viral
replication in a mammal treated with a virus selected from the
group consisting of RNA viruses, Adenoviruses, Poxviruses,
Iridoviruses, Parvoviruses, Hepadnaviruses, Varicellaviruses,
Betaherpesviruses, and Gammaherpesviruses comprising administering
an antiviral compound.
[0045] This invention also relates to a method of treating or
infecting a neoplasm in a mammal comprising subjecting a sample
(e.g., serum, tumor cells, tumor tissue, tumor section) from the
mammal to an immunoassay to detect the amount of virus receptor
present to determine if the neoplasm will allow the virus to bind
and cause cytolysis, and if the receptor is present, administering
an interferon-sensitive, replication competent clonal virus, which
binds the receptor, to the mammal.
[0046] The invention also relates to a method of infecting a
neoplasm in a mammal with a virus comprising systemically
administering a desensitizing dose of an interferon-sensitive,
replication-competent clonal virus to the mammal.
[0047] The invention also relates to a method of infecting a
neoplasm in a mammal with a virus comprising administering an
interferon-sensitive, replication-competent clonal virus to the
mammal over a course of at least 4 minutes.
[0048] This invention also relates to a method of infecting a
neoplasm in a mammal with a virus comprising administering a
replication-competent clonal virus selected from the group
consisting of the Newcastle disease virus strain MIK107, Newcastle
disease virus strain NJ Roakin, Sindbis virus, and Vesicular
stomatitis virus.
[0049] Included in the invention are:
[0050] i) a Paramyxovirus purified by ultracentrifugation without
pelleting;
[0051] ii) a Paramyxovirus purified to a level of at least
2.times.10.sup.9 PFU per mg of protein;
[0052] iii) a Paramyxovirus purified to a level of at least
1.times.10.sup.10 PFU per mg of protein;
[0053] iv) a Paramyxovirus purified to a level of at least
6.times.10.sup.10 PFU per mg of protein;
[0054] v) an RNA virus purified to a level of at least
2.times.10.sup.9 PFU per mg of protein;
[0055] vi) an RNA virus purified to a level of at least
1.times.10.sup.10 PFU per mg of protein;
[0056] vii) an RNA virus purified to a level of at least
6.times.10.sup.10 PFU per mg of protein;
[0057] viii) a cytocidal DNA virus which is interferon-sensitive
and purified to a level of at least 2.times.10.sup.9 PFU/mg
protein;
[0058] ix) a replication-competent vaccinia virus having a) one or
more mutations in one or more of the K3L, E3L and B18R genes, and
b) an attenuating mutation in one or more of the genes encoding
thymidine kinase, ribonucleotide reductase, vaccinia growth factor,
thymidylate kinase, DNA ligase, dUTPase;
[0059] x) a replication-competent vaccinia virus having one or more
mutations in two or more genes selected from the group consisting
of K3L, E3L, and B18R
[0060] xi) a Herpesvirus having a modification in the expression of
the (2'-5')A analog causing the Herpesvirus to have increased
interferon sensitivity; and
[0061] xii) a Reovirus having an attenuating mutation at omega 3
causing said virus to become interferon-sensitive.
[0062] Also included in the invention are the following
methods:
[0063] i) a method of purifying an RNA virus comprising the steps
of a) generating a clonal virus; and b) purifying said clonal virus
by ultracentrifugation without pelleting; or c) purifying said
clonal virus by tangential flow filtration with or without
subsequent gel permeation chromotagraphy, and
[0064] ii) a method of purifying a Paramyxovirus comprising
purifying the virus by ultracentrifugation without pelleting, or by
tangential flow filtration with or without subsequent gel
permeation chromotagraphy.
[0065] The invention also relates to a method of treating a disease
in a mammal, in which the diseased cells have defects in an
interferon-mediated antiviral response, comprising administering to
the mammal a therapeutically effective amount of an
interferon-sensitive, replication-competent, clonal virus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0066] FIG. 1 shows the effect of anti-interferon-beta antibody on
viral antigen expression and infectious titer in NHEK (normal human
epithelial keratinocytes) cells.
[0067] FIG. 2 shows the effect of interferon-beta on viral antigen
expression in different cells (normal human skin fibroblasts
CCD922-sk and two types of head and neck carcinoma cells (KB and
Hep2 cells).
[0068] FIG. 3A shows the effect of interferon on viral antigen
expression in CCD922-sk cells, and FIG. 3B shows the effect of
interferon on viral antigen expression in KB cells.
[0069] FIG. 4 shows the survival curves for athymic mice bearing
human ES-2 ovarian carcinoma cells and treated with either saline
or NDV strain PPMK107.
[0070] FIG. 5 shows the interferon responsiveness of a number of
human tumor and normal cell lines.
DETAILED DESCRIPTION OF THE INVENTION
[0071] The present invention relates to the discovery of a novel
mechanism by which viral replication selectively kills neoplastic
cells deficient in an interferon (IFN)-mediated anti-viral
response. This invention also provides methods for selection,
design, purification, and use of viruses for the treatment of
neoplastic diseases including cancer and large tumors. The viruses
of the invention selectively replicate in and kill neoplastic cells
based on the selective deficiency in these cells of an IFN-mediated
anti-viral response. Administration of the appropriate dosage of
virus results in neoplastic cell death, whereas normal cells, which
possess an intact IFN-mediated anti-viral response, limit the
replication of the virus and are not killed.
[0072] Included in the subject of the invention is the use of
paramyxoviruses such as NDV, and other viruses, for use in the
treatment of diseases including neoplastic disease such as cancer.
The invention also teaches screening and engineering of other
viruses suitable for use as therapeutics of neoplastic diseases.
Another embodiment of the invention involves a method of
identifying tumor tissues that are candidates for viral therapy.
Finally, the invention also describes the preparation of highly
purified virus.
[0073] Rationale for the Use of Interferon-sensitive Viruses
Including NDV to Treat Neoplastic Disease
[0074] NDV Demonstrates Selective Killing of Tumor Cells
[0075] Newcastle disease virus causes selective cytotoxic effects
against many human tumor cells with markedly less effects on most
normal human cells. In a differential cytotoxicity assay, human
cancer cells derived from sarcomas, melanomas, breast carcinomas,
ovarian carcinomas, bladder carcinomas, colon carcinoma, prostate
carcinoma, small cell and non-small cell lung carcinomas, and
glioblastomas were discovered o be approximately 3 to 4 orders of
magnitude more sensitive to NDV than many normal human cells [renal
epithelial cells, fibroblasts, keratinocytes, melanocytes, and
endothelial cells (see Example 1)]. The differential cytotoxicity
assay can also be applied to fresh isolates from the patient's
cells or tumor tissue.
[0076] An in vitro assay is used to define the tumoricidal activity
of NDV as described in Example 1. The assay measures the amount of
virus required to kill 50% of the tested cell culture in a five day
time period. Examples 2 and 3 show the results of in vivo
experiments in which virus was administered to athymic mice bearing
human tumor xenografts by either the intratumoral (Example 2) or
intravenous (Example 3) route. These results demonstrate that NDV
can cause regression of a variety of human tumor types in a
standard animal model for the testing of potential chemotherapeutic
agents.
[0077] Evidence that NDV is specifically replicating within the
tumor was demonstrated by immunohistochemical staining for virus
antigen (Example 2). Within 30 minutes of intratumoral virus
injection, the tumor tissue was negative for viral antigen.
However, by day 2 post treatment, intense immunostaining for viral
antigen was seen within the tumor, indicating virus replication
within the tumor. Importantly, virus replication was specific for
the tumor tissue since the neighboring connective tissue and skin
was negative for viral antigen.
[0078] Importantly, efficient replication of NDV is crucial for the
ability of the virus to kill infected cells, as demonstrated in
studies using UV-inactivated non-clonal virus (Lorence, R., et al,
1994 J Natl Cancer Inst, 86: 1228-1233).
[0079] NDV can also cause regression of large tumors after
intratumoral and intravenous administration (Examples 4 through 9).
Intratumoral NDV treatment of large intradermal A375 human melanoma
xenografts (.gtoreq.10 mm in maximal dimension; tumor volume of
.gtoreq.300 mm.sup.3) in athymic mice lead to high rates of tumor
regression (Examples 4 through 8). Intravenous NDV treatment of
large subcutaneous HT1080 human fibrosarcoma xenografts (.gtoreq.10
mm in maximal dimension) in athymic mice lead to complete or
partial tumor regression in five out of six mice (Example 9).
[0080] The Class I Interferon Family of Cytokines are Important
Negative Modulators of Viral Infection
[0081] The class I interferons consist of the IFN.alpha., found
primarily in cells of hematopoietic origin, and IFN.beta. found
primarily in fibroblasts and epithelial cells. [Joklik, W. K. 1990.
Interferons. pp. 383-410. Virology, second edition, edited by B. N.
Fields, D. M. Knipe et al, Raven Press Ltd., New York; and
Sreevalsan, T. 1995. Biological Therapy with Interferon-.alpha. and
.beta.: Preclinical Studies. pp. 347-364. Biologic Therapy of
Cancer, second edition, edited by V. T. DeVita, Jr., S. Hellman,
and S. A. Rosenberg, J. B. Lippincott Company, Philadelphia.] Both
types of IFN function through an apparently common mechanism of
action that includes the degradation of double-stranded RNA
intermediates of viral replication, and the inhibition of cellular
translation through the activity of a protein kinase activated by
double-stranded RNA (Joklik, W. K. 1990. Interferons. pp. 383-410.
Virology. Second Edition, edited by B. N. Fields, D. M. Knipe et
al., Raven Press Ltd., New York; and references therein). Several
viruses (influenza, EBV, SV40, adenovirus, vaccinia) have evolved
mechanisms by which one or more pathways of the IFN system are
inactivated, thus allowing the efficient replication of the virus
(Katze, M. G. 1995. Trends in Microbiol. 3:75-78).
[0082] A Wide Variety of Tumor Cells are Deficient in the Ability
to Limit Viral Infection Through an IFN-dependent Mechanism
[0083] Human cervical carcinoma cells (HeLa) were over
three-hundred-fold less sensitive to the inhibition of vesicular
stomatitis virus replication following pre-treatment with IFN than
a non-transformed fibroblast control cell line (Maheshwari R. K.,
1983. Biochem, Biophys. Res. Comm. 17:161-168). The subject
inventors have discovered that infection of a co-culture of
tumorigenic human head and neck carcinoma cells (KB) and normal
human skin fibroblast cells (CCD922-sk) results in viral
replication initially in both cell types, followed by a limiting of
the infection in the normal cells versus continued replication and
killing of the tumor cells (Example 10). Moreover, although IFN was
being secreted by the normal cells into the culture medium, the
tumor cells were unable to respond to the IFN at the concentrations
being produced to establish an antiviral state. Further evidence
for the role of IFN in the differential sensitivity of tumor cells
versus normal cells to killing by NDV was obtained in two separate
experiments in which normal fibroblast cells (CCD922-sk) or normal
epithelial keratinocyte cells (NHEK) were shown to become more
sensitive to infection with NDV in the presence of neutralizing
antibody to IFN (Examples 11 and 12). Finally, parallel infection
of normal fibroblasts (CCD922-sk) and human tumor cells (KB) in the
presence of IFN revealed that the normal cells were at least
100-fold more sensitive to the antiviral effects of added IFN than
were the tumor cells (Examples 13 and 14). Similar testing of
variety tumor cell lines (total of 9) revealed a clear correlation
in the relative sensitivity of a cell line to killing by NDV and an
inability of the cell line to manifest an interferon-mediated
antiviral response (Example 26).
[0084] Interferon and Cell Growth
[0085] There are several species of interferon (IFN) including
natural and recombinant forms of .alpha.-IFN, .beta.-IFN,
.omega.-IFN, and .gamma.-IFN as well as synthetic consensus forms
(e.g., as described in Zhang et al. (1996) Cancer Gene Therapy,
3:31-38). In addition to the anti-viral activities that lead to its
discovery, IFN is now known to play an important role in the normal
regulation of cell growth and differentiation. IFN is viewed as a
negative growth regulator and several key proteins involved in the
function and regulation of IFN activity have been shown to act as
tumor-suppresser proteins in normal cells (Tanaka et al, 1994 Cell
77:829-839). Moreover, several other proteins known to antagonize
the anti-viral activity of IFN have been shown to have oncogenic
potential when expressed inappropriately (see below, Barber, GN,
1994, Proc. Natl. Acad. Sci. USA 91:4278-4282). Cells derived from
a number of human cancers have been shown to be deleted in the
genes encoding IFN (James, C D, et al, 1991, Cancer Res
51:1684-1688), and partial or complete loss of IFN function has
been observed in human cervical carcinoma (Petricoin, E, et al,
1994 Mol. Cell. Bio., 14:1477-1486), chronic lymphocytic leukemia
(Xu, B., et al, 1994, Blood, 84:1942-1949), and malignant melanoma
cells, (Linge, C., et al, 1995, Cancer Res., 55:4099-4104).
[0086] The IFN-inducible protein kinase (p68) has been shown to be
an important regulator of cellular and viral protein synthesis. A
correlation has emerged that links the expression or activity of
the p68 kinase to the cellular state of differentiation. Thus,
poorly differentiated cells, such as those occurring in many
cancers, are deficient in p68 function (Haines, G. K., et al, 1993
Virchows Arch B Cell Pathol. 63:289-95). Cells that lack p68
activity are generally sensitive to viral mediated killing because
the p68 kinase is an important effector of the IFN-inducible
antiviral state. The antiviral activity of p68 can be antagonized
through a direct interaction with a cellular protein identified as
p58. When cloned and overexpressed in NIH3T3 cells, p58 causes the
cells to exhibit a transformed phenotype and anchorage-independent
growth (Barber G N et al., 1994 Proc Natl Acad Sci USA
91:4278-4282), and a number of human leukemia cell lines have been
shown to overexpress the p58 protein (Korth M J, et al., 1996 Gene
170:181-188). Sensitivity to viral killing in undifferentiated
cells can be reversed through the induction of a more
differentiated phenotype (Kalvakolanu, D V R and Sen, G. C. 1993
Proc Natl Acad Sci USA 90:3167-3171).
[0087] Definitions
[0088] Cells competent in an interferon-mediated antiviral
response. As used herein, the term "cells competent in an
interferon-mediated antiviral response" are cells which respond to
low levels (e.g., 10 units per ml) of exogenous interferon by
significantly reducing (at least 10-fold, more advantageously at
least 100-fold, more advantageously at least 1000-fold, and most
advantageously at least 10,000-fold) the replication of an
interferon-sensitive virus as compared to in the absence of
interferon. The degree of virus replication is determined by
measuring the amount of virus (e.g., infectious virus, viral
antigen, viral nucleic acid). CCD922 normal fibroblasts are cells
competent in an interferon-mediated antiviral response.
[0089] Cells deficient in an interferon-mediated antiviral
response. As used herein, the term "cells deficient in an
interferon-mediated antiviral response" are cells which fail to
meet the criteria listed above for a cell competent in an
interferon-mediated antiviral response, that is, they fail to
respond to low levels (e.g., 10 units per ml) of exogenous
interferon by significantly reducing the replication of an
interferon-sensitive virus as compared to in the absence of
interferon. KB oral carcinoma cells are cells deficient in an
interferon-mediated antiviral response.
[0090] Clonal. Use of the term "clonal" virus is defined hereafter
as virus derived from a single infectious virus particle and for
which individual molecular clones have significant nucleic acid
sequence homology. For example, the sequence homology is such that
at least eight individual molecular clones from the population of
virions have sequence homology greater than 95%, more
advantageously greater than 97%, more advantageously greater than
99%, and most advantageously 100% over 300 contiguous
nucleotides.
[0091] Cytocidal. As used herein, the term "cytocidal" virus refers
to a virus that infects cells resulting in their death.
[0092] Desensitizing Dose. As used herein, the phrase,
"desensitizing dose" refers to the amount of virus required to
lessen the side effects of subsequent doses of the virus.
[0093] Differential Cytotoxicity Assay. As used herein, the phrase
"differential cytotoxicity assay" for screening tumor cells or
tissue using a virus refers to the (a) virus infection of the tumor
cells and one or more control cells or tissue; (b) a determination
of cell survivability or death for each sample (for example, by the
use of a dye indicator of cell viability as in detailed in Example
1) after one or more days of infection; and (c) based on the
results, an estimation of the sensitivity (for example, by IC50
determination as detailed in Example 1) of the sample to the virus
compared to the control(s).
[0094] Infecting a Neoplasm. As used herein, the term "infecting a
neoplasm" refers to the entry of viral nucleic acid into the
neoplastic cells or tissues.
[0095] Interferon-sensitive. As used herein, the phrase
"interferon-sensitive" virus (e.g., NDV) means a virus that
replicates significantly less (at least 10-fold less,
advantageously at least 100-fold less, more advantageously at least
1000-fold less, and most advantageously at least 10,000-fold less),
in the presence of interferon compared to in the absence of
interferon. This is determined by measuring the amount of virus
(e.g., infectious virus, viral antigen, viral nucleic acid)
obtained from cells competent in an interferon-mediated antiviral
response in the presence or absence of low levels of exogenous
interferon (e.g., 10 units per ml).
[0096] Neoplasm and Neoplastic Disease. As used herein, "neoplasm"
means new growth of tissue, including tumors, benign growths (e.g.,
condylomas, papillomas) and malignant growths (e.g., cancer). As
used herein, "neoplastic disease" refers to disease manifested by
the presence of a neoplasm.
[0097] Replication Competent. As used herein, the term
"replication-competent" virus refers to a virus that produces
infectious progeny in neoplastic cells.
[0098] Substantially Free of Contaminating Egg Proteins. The term
"substantially free of contaminating egg proteins" refers to a
level of virus purity in which ovalbumin is not detectable in a
Western blot as performed by one skilled in the art by (1) using
1.7.times.10.sup.9 FU of virus per well (3.3 cm in width) run on an
SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel
electrophoresis) gel (1 mm thick); (2) transferring the viral
proteins from the gel to a nitrocellulose membrane; and (3)
immunostaining for ovalbumin with the use of a rabbit
anti-ovalbumin [Rabbit IgG fraction at a 1:200 dilution of a 4
mg/ml antibody concentration (from Cappel, Inc.) or equivalent
polyclonal antibody].
[0099] Therapeutically effective amount. As used herein, the term
"therapeutically effective amount" when referring to the treatment
of neoplastic disease refers to a quantity of virus which produces
the desired effect, e.g., cessation of neoplastic growth, tumor
regression, improved clinical conditions, or increased
survival.
Compounds of the Invention
[0100] A diverse group of viruses are used to selectively kill
neoplastic cells. Natural or engineered viruses can function as an
antineoplastic agent. These viruses i) infect neoplastic cells
resulting in their death; ii) are replication-competent in the
neoplastic cells; and iii) are limited in killing of normal cells
by the antiviral effects of interferon.
[0101] In an advantageous embodiment of the invention, the viruses
possessing the above three characteristics [(i) they infect
neoplastic cells resulting in their death; (ii) they are
replication-competent in the neoplastic cells; and (iii) they are
limited in killing of normal cells by the antiviral effects of
interferon] also induce interferon.
[0102] In another advantageous embodiment of the invention, the
viruses possessing the above three characteristics also cause
regression of human neoplasms; and/or are not neutralized in the
target human population because of the presence of pre-existing
immunity.
[0103] In another advantageous embodiment, the viruses possessing
the above three characteristics are cytocidal to tumor cells.
[0104] A Paramyxovirus (as used herein "Paramyxovirus" refers to a
member of the Paramyxoviridae) can be used according to the present
invention to treat a neoplasm including a large tumor or a host
having a high tumor burden. The Paramyxoviridae family comprises
three genera: (1) paramyxoviruses; (2) measles-like viruses
(morbilli viruses); and (3) respiratory syncytial viruses
(pneuemoviruses). These viruses contain an RNA genome. Use of
Paramyxoviridae viruses which are cytocidal, especially
paramyxoviruses, e.g., Newcastle disease virus ("NDV") and other
avian paramyxoviruses such as avian paramyxovirus type 2, is an
advantageous method of practicing the invention. Attenuated strains
of these viruses are especially useful for treatment of neoplasms
in accordance with the present invention.
[0105] NDV is an especially advantageous virus according to the
present invention. NDV is categorized into three distinct classes
according to its effects on chickens and chicken embryos. "Low
virulence" strains are referred to as lentogenic and take 90 to 150
hours to kill chicken embryos at the minimum lethal dose (MLD);
"moderate virulence" strains are referred to as mesogenic and take
60 to 90 hours to kill chicken embryos at the MLD; "high virulence"
strains are referred to as velogenic and take 40 to 60 hours to
kill chicken embryos at the MLD. See, e.g., Hanson and Brandly,
1955 (Science, 122:156-157), and Dardiri et al., 1961 (Am. J. Vet.
Res., 918-920). All three classes are useful, advantageously,
mesogenic strains of NDV such as strain MK107, strain NJ Roakin,
and strain Connecticut-70726. (see Examples 21-23). See, e.g.,
Schloer and Hanson, 1968 (J. Virol., 2:40-47) for a listing of
other mesogenic strains.
[0106] For certain purposes, it is desirable to obtain a clonal
virus to ensure or increase the genetic homogeneity of a particular
virus strain and to remove defective interfering particles. Removal
of defective interfering particles by cloning allows for increased
purity in the final product as assessed by the number of total
virus particles per infectious particle (e.g., the number of
particles per PFU).
[0107] Clonal virus can be produced according to any method
available to the skilled worker. For example, plaque purification
is routinely utilized to obtain clonal virus. See, e.g., Maassab et
al., In: Plotkin and Mortimer, eds. Vaccines. Philadelphia: W. B.
Saunders Co., 1994, pages 78-801. Triple plaque purification is
especially desirable, where a plaque is selected at each round of
purification having the desired characteristics, such as a
preferred size, shape, appearance, or representative of the
parental strain. Another means of generating clonal virus is by
recombinant DNA techniques applicable by one skilled in the art.
Another means of obtaining a clonal virus applies the technique of
limiting dilution (e.g., by adding dilutions of the virus sample to
give an average of one or less infectious virus particles per well
containing a monolayer of a susceptible cell).
[0108] In an advantageous embodiment of the invention, purified
virus is used to treat neoplastic diseases. An advantageous method
for purification of egg derived viruses are as follows (virus is
not pelleted at any step in these methods):
[0109] Purification Method A
[0110] a) generating a clonal virus (e.g., plaque purification)
[0111] b) inoculating eggs with the clonal virus
[0112] c) incubating the eggs
[0113] d) chilling the eggs
[0114] e) harvesting the allantoic fluid from the eggs
[0115] f) removing cell debris from the allantoic fluid
[0116] h) ultracentrifugation of the allantoic fluid without
pelleting (e.g., using a discontinuous sucrose gradient)
[0117] In another embodiment of the invention, additional steps,
added after the removal of the cell debris (from the allantoic
fluid) and before ultracentrifugation, consist of:
[0118] freezing then thawing the allantoic fluid
[0119] removing contaminating material from the virus suspension
(e.g., by means of centrifugation)
[0120] In another embodiment of the invention, ultracentrifugation
is accomplished by means of a continuous flow ultracentrifuge.
[0121] One embodiment of the invention relates to a method of
purifying a replication-competent RNA virus comprising the steps
of
[0122] a) generating a clonal virus, and b) purifying said clonal
virus by ultracentrifugation without pelleting.
[0123] Another embodiment of the invention involves a method of
purifying a paramyxovirus (e.g., NDV) comprising purifying the
virus by ultracentrifugation without pelleting. Optionally, the
purifying step additionally comprises prior to the
ultracentrifugation:
[0124] a)plaque purifying to generate a clonal virus,
[0125] b)inoculating eggs with the clonal virus,
[0126] c)incubating the eggs,
[0127] d)chilling the eggs,
[0128] e)harvesting allantoic fluid from the eggs and,
[0129] f)removing cell debris from the allantoic fluid.
[0130] Another, embodiment of the invention involves a method of
purifying a replication-competent clonal virus from eggs or cell
culture comprising the step of ultracentrifugation without a step
in which the virus is pelleted.
[0131] Another embodiment of the invention involves a method of the
purifying a paramyxovirus (e.g., NDV) comprising purifying the
virus by sequential tangential flow filtration (TFF). Optionally,
the virus can be additionally purified by gel permeation
chromatography, where each of these steps occurs in the presence of
a stabilizing buffer (Example 15):
[0132] a) plaque purifying to generate a clonal virus,
[0133] b)inoculating eggs with the clonal virus,
[0134] c)incubating the eggs,
[0135] d)chilling the eggs,
[0136] e)harvesting allantoic fluid from the eggs and dilution of
allantoic fluid with buffer,
[0137] f)removing cell debris from the allantoic fluid by TFF,
[0138] g)purification of the virus by TFF, and
[0139] h)purification of the virus by gel permeation
chromatography.
[0140] Optionally, the virus obtained from the gel permeation step
can be concentrated using TFF.
[0141] Another embodiment of the invention involves a method of
purifying a replication-competent clonal virus from eggs or cell
culture comprising the step purifying the virus by sequential
tangential flow filtration (TFF), optionally followed by gel
permeation chromatography, optionally followed by TFF to
concentrate the virus.
[0142] Clonal virus
[0143] Use of these methods permits purification of a clonal virus
[including Paramyxovirus (e.g., NDV)] to at least 2.times.10.sup.9
PFU/mg protein, advantageously to at least 3.times.10.sup.9 PFU/mg
protein, more advantageously to at least 5.times.10.sup.9 PFU/mg
protein, more advantageously to at least 1.0.times.10.sup.10 PFU/mg
protein, more advantageously to at least 2.0.times.10.sup.10 PFU/mg
protein, more advantageously to at least 3.times.10.sup.10 PFU/mg
protein, more advantageously to at least 4.times.10.sup.10 PFU/mg
protein, more advantageously to at least 5.times.10.sup.10 PFU/mg
protein, and most advantageously at least 6.times.10.sup.10
PFU/mg.
[0144] Use of these methods permits purification of a clonal virus
[including Paramyxovirus (e.g., NDV)] to level in which the number
of virus particles per PFU is less than 10, more advantageously
less than 5, more advantageously less than 3, more advantageously
less than 2, and most advantageously less than 1.2. (Lower numbers
of virus particles per PFU indicate a higher degree of purity.)
[0145] RNA Viruses
[0146] In another embodiment, these methods permit purification (to
the levels cited above for clonal viruses) of an RNA virus
[including (a) a cytocidal RNA virus; (b) a single-stranded RNA
non-segmented, nonenveloped virus; (c) a single-stranded RNA
segmented, enveloped virus; (d) a double-stranded RNA segmented,
nonenveloped virus; (e) and a single-stranded RNA non-segmented,
enveloped virus (e.g., Paramyxovirus (e.g., NDV) and e.g.,
Retroviruses].
[0147] DNA Viruses
[0148] In another embodiment, these methods permit purification (to
the levels cited above for clonal viruses) of an
interferon-sensitive cytocidal virus selected from the group
consisting of (a) enveloped, double-stranded DNA viruses (including
poxviruses); (b) nonenveloped, single-stranded DNA viruses; and (c)
nonenveloped, double-stranded DNA viruses.
[0149] Egg Derived Viruses
[0150] In another embodiment, these methods permit purification of
egg derived viruses to a level substantially free of contaminating
egg proteins. It is preferred to limit the amount of egg proteins
in virus preparations for human therapeutic use since major egg
proteins like ovalbumin are allergens.
[0151] Viruses useful in the treatment of neoplastic diseases
including cancer are shown in Table 1. These viruses are optionally
screened for naturally occurring variations (certain strains or
isolates) that result in altered IFN production relative to the
parental strain.
[0152] In another embodiment of this invention, candidate viruses,
whether naturally occurring or engineered, are tested for the
ability to provide therapeutic utility in the treatment of
neoplasms. In one embodiment, the amount of candidate virus
required to kill 50% of cells deficient in an interferon-mediated
antiviral response, e.g., KB head and neck carcinoma cells, is
compared to the amount of virus required to kill 50% of a similar
number of cells competent in an interferon-mediated antiviral
response, for example normal skin fibroblasts. The amount of
killing is quantified by any number of means including trypan blue
exclusion or MTT assay (see Example 1). A significant reduction
(e.g., at least 5-fold) in the amount of virus required to kill
cells deficient in an interferon-mediated antiviral response
relative to the amount needed to kill cells competent in an
interferon-mediated antiviral response indicates that the virus
being tested exhibits activity required for therapeutic utility in
the treatment of neoplasms. Other NDV viruses and Sindbis virus are
such natural occurring viruses that display tumor-selective killing
(see Examples 21-23, and 25).
2TABLE 1 Naturally Occurring Viruses for Use in Cancer Therapy
Virus Class Virus Family Virus Example RNA, negative
Paramyxoviridae Newcastle Disease Virus stranded Avian
Paramyxovirus Type 2 Mumps Human Parainfluenza Rhabdoviridae
Vesicular Stomatitus Virus RNA, positive Togaviridae Sindbis Virus
stranded Flaviviridae Yellow Fever Virus (attenuated) Picomaviridae
Rhinovirus Bovine Enterovirus Echovirus Coronaviridae Avian
Infectious Bronchitis Virus Human Coronaviruses
[0153] An understanding of the factors involved in the
establishment of an antiviral state allows for the creation of a
screening assay for tumors that are likely to respond to viral
therapy. In principle, patient derived tumor tissue obtained from
biopsy is screened for the expression of p68 kinase, p58, or other
factors involved in the regulation of an antiviral state or
cellular differentiation. Other factors include, but are not
limited to, interferon response factor-I (IRF-1), interferon
stimulatory gene factor-3 (ISGF-3), c-Myc, c-Myb, and IFN
receptors. In the case of c-Myc, c-Myb or p58, high level
expression indicates that the tumor tissue or cells are treatment
candidates for virus therapy. In the case of p68, IRF-I, ISGF-3,
and IFN receptors, low level expression indicates that the tumor
tissue or cells are treatment candidates for virus therapy.
[0154] In another embodiment of this invention, primary tumor
tissue or cells obtained from patient biopsies are expanded in
culture and tested for sensitivity to killing by a suitable viral
therapy. In one embodiment, the amount of virus required to kill
50% of the tumor tissue culture is compared to the amount required
to kill 50% of a culture of normal cells as described above for the
screening of candidate viruses. An increase of ten-fold or greater
in the sensitivity of the tumor cells relative to normal cells to
killing by the viral agent indicates that the tumor cells are
specifically sensitive to the cytocidal effects of the viral
treatment. In a further embodiment of the invention, the ability of
the targeted tumor cells to respond to endogenously or exogenously
supplied IFN is determined by conducting the above screen in the
presence of IFN (alpha or beta form, using e.g., 10 units per ml,
see Example 27).
[0155] An understanding of the cellular receptors required for
virus attachment or entry allows additional screening for tumors
that have high receptor expression and hence enhanced sensitivity
to the interferon-sensitive virus. This is an additional level
screening for patients that are likely to respond to virus therapy.
Advantageously for therapy with an interferon-sensitive virus, the
patient's tumor is both resistant to interferon and has high
expression of the cellular receptor for the virus. In principle,
patient derived serum, tumor cells, tissues, or tissue sections are
screened by immunoassay or immunostain for the amount of virus
receptor present in the serum or on the tumor cells or tumor
tissue. For example, Sindbis virus utilizes the high affinity
laminin receptor to infect mammalian cells (Wang et al., 1992, J
Virol., 66, 4992-5001). This same receptor is known to be expressed
in higher amounts in many diverse types of metastatic cancer. The
PANC-1 renal cancer cell line, and the colon adenocarcinoma cell
line SW620 are known to express a high level of high affinity
laminin receptor mRNA (Campo et al, 1992, Am J Pathol
141:107301983; Yow et al., (1988) Proc. Natl Acad Sci, 85,
6394-6398) and are highly sensitive to Sindbis virus (Example 25).
In contrast, the rectum adenocarcinoma cell line SW1423 is known to
express very low levels of high affinity lamin receptor mRNA (Yow
et al., (1988) Proc. Natl Acad Sci, 85, 6394-6398), and is more
than 4 orders of magnitude more resistant to killing by
PPSINDBIS-Ar339 than SW620 cells.
[0156] Existing strains of NDV, or other viruses including RNA and
DNA viruses, are screened or engineered for altered IFN responses
(e.g., advantageously increased IFN responses) in normal cells. In
addition to the ability to elicit a strong IFN response, other
viral characteristics are screened for or engineered into the
virus. Viruses with altered receptor specificity (e.g., Sindbis
virus PPSINDBIS-Ar339, see Example 25), or low neurovirulence are
included in the subject invention (e.g., NDV virus PPNJROAKIN, see
Example 24). Advantageously, viruses of the invention have the
capacity to spread through direct cell to cell contact.
[0157] The invention described herein includes a broad group of
viruses (see Table 1) that are useful for treatment of neoplasms in
a manner analogous to the indication for NDV. In addition, viruses
that naturally would not be candidates for use, due to the presence
of a mechanism(s) to inactivate the IFN response in normal cells,
are optionally engineered to circumvent the above restrictions. If
left unmodified, viruses with mechanisms to inactivate the
interferon response would be more toxic to normal cells than
viruses with such mechanism removed. The subject invention provides
(1) the development of a vector that can be easily manipulated; and
(2) the creation of a set of therapeutic viruses. Manipulations
include the addition of an IFN gene to permit the viral expression
of a transgene expressing IFN, or other activators of the IFN
response pathway. Additional permutations include the engineered
expression of pro-drug activating enzymes such as the Herpesvirus
thymidine kinase or cytosine deaminase (Blaese R M et al., 1994.
Eur. J. Cancer 30A: 1190-1193) and the expression of suitable
marker antigen to allow targeting of tumor cells by the immune
system. An additional permutation include the engineered expression
of receptor ligands to target cells with those receptors [e.g.,
expression of receptors to other viruses to target cells infected
with those viruses (see Mebastsion et al., 1997, Cell 90:841-847;
and Schnell M J et al., 1997, Cell 90:849-857].
[0158] Several Newcastle Disease virus strains demonstrate
selective killing of tumor cells. In a differential cytotoxicity
assay using a second strain of mesogenic Newcastle Disease virus,
tumor cells were found to be 3 orders of magnitude more sensitive
than normal cells to killing by the virus (Example 21).
Additionally, when a third mesogenic Newcastle Disease virus strain
was used in a differential cytotoxicity assay, tumor cells were
found to be 80 to 5000-fold more sensitive than normal cells to
killing by the virus (Example 22). Both of these mesogenic
Newcastle Disease virus strains also caused tumor growth regression
following intratumoral administration to athymic mice bearing human
tumor xenografts (Example 23).
[0159] In separate experiments, the safety of three distinct
Newcastle Disease virus strains were studied following
intracerebral inoculation in athymic and immune-competent mice. The
results of this study showed that all three virus strains were well
tolerated in mice with an intact immune system. Intracerebral
inoculation into the brains of athymic mice revealed that one of
the viruses was tolerated significantly better than the other two
(Example 24). These results demonstrate that within a single virus
family important differences in viral properties can occur and be
can be exploited therapeutically for greater efficacy or increased
safety.
[0160] Another means by which increased efficacy and lower toxicity
following treatment with oncolytic viruses can be achieved is
through the use of interferon-sensitive viruses that require
specific cell surface receptors that are preferentially expressed
on tumor cells. Sindbis virus provides an example of this type of
restriction. Sindbis virus infects mammalian cells using the high
affinity laminin receptor (Wang et al, (1992) J. Virol. 66,
4992-5001). When normal and tumor cells were infected with Sindbis
virus in a differential cytotoxicity assay, cells which both were
tumorigenic and expressed the high affinity laminin receptor were
found to be more sensitive to killing by this virus than other
cells (Example 25). Normal keratinocytes express the high affinity
laminin receptor (Hand et al., (1985) Cancer Res., 45, 2713-2719),
but were resistant to killing by Sindbis in this assay.
[0161] Vesicular Stomatitis Virus (VSV) provides evidence of
tumor-selective killing of by enclitic viruses, i.e., an inherent
deficiency in interferon responsiveness in tumor cells renders
these cells sensitive to killing by interferon-sensitive
replication-competent viruses. When VSV was used to infect
non-tumorigenic human WISH cells and tumorigenic HT1080 or KB cells
in the presence of exogenous interferon.
[0162] Below is a list of viruses that when modified to remove
naturally-occurring anti-interferon activities, are useful for
viral cancer therapy (see Table 2). Modified viruses
(advantageously, but not necessarily, attenuated in addition to the
anti-interferon modification, see Table 3) that have had endogenous
anti-interferon activities destroyed or reduced, are useful for
cancer therapy. This list includes, but is not be limited to, the
viruses described below. Because of the similarity between viruses
of a common class, the identified mechanisms for each of the
specific viruses listed below, are also present in other members of
that class of virus as identical or functionally analogous
mechanisms. The broader group of viruses is added in parenthesis.
Viruses, such as those below, that have a functional loss of
anti-interferon activity, through any means, including natural
occurring mutations, as well as engineered deletions or point
mutations, are useful in the methods of the subject invention.
[0163] Viruses that exercise more than one mechanism are optionally
modified to contain mutations in one, some, or all of the
activities. Mutations for some of the described activities are
available in the general scientific community.
[0164] Isolates of naturally occurring or engineered virus that are
slower growing, compared to the growth rate of wild-type virus, are
particularly advantageous because a slower virus growth rate will
allow a cell or population of cells competent in an interferon
response to establish an efficient antiviral state before viral
replication can kill the cell or cell population.
[0165] The disabling of viral anti-interferon activities as a
specific alteration of viral character that results in the
augmentation of the interferon response in an infected cell, but
still allows viral replication in neoplastic cells is included in
the subject invention.
[0166] Table 2 shows existing viruses engineered to remove
anti-interferon activity. Table 3 lists viruses engineered to be
attenuated in virulence.
3TABLE 2 Extant Viruses Engineered to Remove Anti-IFN Activity
Virus Class Virus Family Virus Anti-IFN Activity Reference RNA
Reoviridae reovirus .sigma.3 Imani F and Jacobs B (1988) Proc Natl
Acad Sci USA 85:7887-7891 DNA Poxviridae Vaccinia K3L Beattie E et
al. (1991) Virology 183:419 E3L Beattie E et al. (1996) Virus Genes
12:89-94. B18R Symons JA et al (1995) Cell 81:551-560 Adenoviridae
various VA.sub.1 transcripts Mathews MB and Shenk T (1991) J Virol
64:5657-5662. subtypes Alphaherpesvirinae HSV-1 gamma 34.5 Chou J
et al (1996) Proc Natl Acad Sci USA 92:10516-10520 gene product
[0167]
4TABLE 3 Known Attenuating Mutations in Selected Viruses Virus
Class Virus Family Virus Attenuation Reference RNA Reoviridae
reovirus .sigma.1 Spriggs DR and Fields BN (1982) Nature 297:68-70.
rotavirus bovine strains (WC3) Clark HF (1988) J Infect Dis
158:570-587. DNA Poxviridae Vaccinia vaccinia growth factor Buller
RML et al (1988) Virology 164:182. thymidine kinase Buller RML et
al (1985) Nature 317:813-815. thymidylate kinase Hughes SJ et al
(1991) J Biol Chem 266:20103-20109 DNA ligase Kerr SM et al (1991)
EMBO J 10:4343-4350. ribonucleotide reductase Child SJ et al (1990)
Virology 174:625-629. dUTPase Perkus ME et al (1991) Virology
180:406-410 Adenoviridae various Ad-4, Ad-7, Ad-21 Takafugi ET et
al (1979) J Infect Dis 140:48-53. subtypes Alphaherpesvirinae HSV-1
thymidine kinase Field HJ and Wildy P (1978) J Hyg 81:267-277.
ribonucleotide reductase Goldstein DJ and Weller sk (1988) Virology
166:41-51. gamma 34.5 gene product Chou J et al (1995) Proc Natl
Acad Sci USA 92:10416-10520 b'a'c' inverted repeats Meignier B et
al (1988) J Infect Dis 162:313-322
[0168] Treatment of Neoplasms
[0169] The present invention relates to viral therapy of neoplasms,
especially in animals having cancer. In an advantageous embodiment,
the invention relates to the treatment of tumors which are 1
centimeter (cm) or more in size as measured in the greatest
dimension. As used herein, "a 1 cm tumor" indicates that at least
one dimension of the tumor is 1 cm in length. Such tumors are more
sensitive than expected to viral therapy, often at least as
sensitive to virus, if not more sensitive, than tumors which are
smaller in size. In a more advantageous aspect of the invention,
tumors greater than 1 cm. are treated, e.g., tumors which are 2 cm
or greater, from about 2 cm to about 5 cm, and greater than 5
cm.
[0170] The present invention can also be employed to treat hosts
having a high tumor burden. As used herein, the phrase "tumor
burden" refers to the total amount of tumor within the body
expressed as a percentage as body weight. Viral therapy of hosts
having a tumor burden, e.g., from about 1% to about 2% of total
body weight is surprisingly effective, e.g., producing tumor
regression and a reduction in the overall tumor load. This is
especially unexpected since a tumor burden of approximately 2% of
the total body weight (e.g., a 1 kg tumor in a 60 kg human) is
approximately the maximum cancer mass compatible with life. See,
e.g., Cotran et al, In Robbins Pathological Basis of Diseases, 4th
Edition, W B Saunders, 1989, page 252. In the Examples, volumes up
to 397 mm.sup.3 for a melanoma cancer (e.g., A375) in a mouse host
showed complete regression in response to treatment with a
Newcastle disease virus (e.g., a triple-plaque purified virus).
Assuming that for tissue 1000 mm.sup.3 equals 1 gram, a tumor
having a volume of 397 mm.sup.3 comprises approximately 2% of the
total body weight for a 20 gram mouse.
[0171] As shown in Examples 4 to 9 below, tumor regression was
achieved with tumors at least 1 cm in size, while untreated,
control animals began dying from tumor burden within several weeks.
Thus, such diseased animals were successfully treated despite being
within two weeks of death. Thus, in accordance with the present
invention, an animal which is near terminal from its tumor burden
can be treated effectively with viral therapy. Consequently, the
present invention can be used to treat patients who have not
responded to conventional therapy, e.g., chemotherapy such as
methotrexate, 5-fluorouracil, and radiation therapy.
[0172] The efficacy of NDV for the treatment of cancer following
administration through the intraperitoneal route has also been
examined. Using an ascites prevention model of ovarian cancer,
intraperitoneal injection of NDV in mice harboring ES-2 human
ovarian tumors resulted in increased survival compared to mice
treated with saline (Example 16). When ES-2 cells were used in an
ovarian cancer tumor model with treatment initiated once ascites
formed, ascites fluid production was markedly decreased in
virus-treated animals compared to saline controls (Example 17).
[0173] In another embodiment of the invention, the administration
of virus results in 1) the relief of tumor related symptoms, such
as but not limited to deceased rate of ascites fluid production,
relief of pain, and relief of obstructive disease, and 2) the
prolongation of life.
[0174] Twenty-three patients have received the plaque purified NDV
isolate by the intravenous route (Example 20). Treatment responses
include the regression of a palpable tumor, the stabilization of
disease in 47% of patients and a reduction in pain medication.
[0175] Administration and Formulation
[0176] In one embodiment of the invention, tumor cells or tissue
are screened in vitro to determine those patients with tumors
sensitive to the virus. Tumor cells removed from the patient (by
methods such as fine needle aspiration for solid tumors or by
paracentesis for ovarian ascites tumors) are grown in vitro and
incubated with virus. In this embodiment of the invention, patients
are selected for therapy if the virus has a high activity against
their tumor cells.
[0177] In an advantageous embodiment of the invention, the amount
of virus administered results in regression of the tumor or tumors.
As used herein, the term "regression" means that the tumor shrinks,
e.g., in size, mass, or volume. Shrinkage in tumor size is
demonstrated by various methods, including physical examination,
chest film or other x-ray, sonography, CT scan, MRI, or a
radionucleotide scanning procedure.
[0178] Various types of neoplasms including cancers are treatable
in accordance with the invention. The viruses of the present
invention are useful to treat a variety of cancers, including but
not limited to lung carcinoma, breast carcinoma, prostate
carcinoma, colon adenocarcinoma, cervical carcinoma, endometrial
carcinoma, ovarian carcinoma, bladder carcinoma, Wilm's tumor,
fibrosarcoma, osteosarcoma, melanoma, synovial sarcoma,
neuroblastoma, lymphoma, leukemia, brain cancer including
glioblastoma, neuroendocrine carcinoma, renal carcinoma, head and
neck carcinoma, stomach carcinoma, esophageal carcinoma, vulvular
carcinoma, sarcoma, skin cancer, thyroid pancreatic cancer, and
mesothelioma. The viruses of the present invention are also useful
to treat a variety of benign tumors, including but not limited to
condylomas, papillomas, meningiomas, and adenomas.
[0179] A therapeutically effective amount of virus is administered
to a host having a neoplasm. It is understood by those skilled in
the art that the dose of virus administered will vary depending on
the virus selected, type of neoplasm, the extent of neoplastic cell
growth or metastasis, the biological site or body compartment of
the neoplasm(s), the strain of virus, the route of administration,
the schedule of administration, the mode of administration, and the
identity of any other drugs or treatment being administered to the
mammal, such as radiation, chemotherapy, or surgical treatment.
These parameters are defined through maximum tolerated dose
determination in animal models and scaling to human dosage as a
function of relative body surface area or body mass. It is also
understood that under certain circumstances, more than one dose of
the virus is given. The optimal interval between such multiple
doses of the virus can be determined empirically and is within the
skill of the art. NDV is generally administered from about
3.times.10.sup.6 to about 5.times.10.sup.12 PFU of virus. For local
administration (e.g., directly into a tumor), total amounts of from
about 3.times.10.sup.6 to about 5.times.10.sup.10 PFU of virus are
typically used. For systemic administration, amounts of from about
1.times.10.sup.8 to about 4.times.10.sup.11 PFU of virus per square
meter of body surface area are used. For intravenous
administration, dosing schedules of once per week, two times per
week and three times per week are used. A virus in accordance with
the present invention, optionally with a chemotherapeutic agent,
can be administered by various routes, e.g., enteral, parenteral,
oral, nasal, rectal, intrathecal, intervenous (e.g., using a
catheter), subcutaneous, intratumor (e.g., directly into its tissue
or into vessels which perfuse it), peritumoral, local, sublingual,
buccal, topical, intramuscular, by inhalation, percutaneous,
vaginal, intra-arterial, intra-cranial, intradermal, epidural,
systemically, topical, intraperitoneal, intrapleural, etc. For lung
tumors, a bronchial route (e.g., bronchial administration) can be
used. Endoscopic injections of gastrointestinal tumors, as well as
suppository treatments of rectal tumors are also used where
appropriate.
[0180] Murine toxicity studies with NDV have indicated that the
acute toxicity following intravenous virus administration is likely
to be caused by cytokine mediated reactions. Cytokine responses to
repeated stimuli are known to be desensitized, or down-regulated,
following the initial induction event (Takahashi et al., (1991)
Cancer Res. 51, 2366-2372). Mice intravenously injected with a
desensitizing dose of virus were able to tolerate approximately
10-fold more virus on a second dose than mice receiving vehicle
alone for the first injection (Example 18).
[0181] The rate of virus administration by the intravenous route
can significantly affect toxicity.
[0182] Two groups of athymic mice were intravenously treated with
identical doses of NDV which was administered either slowly (0.2 ml
over 4 minutes) or rapidly (0.2 ml over 30 seconds). Comparison of
the maximal weight lose in each group revealed 50% less weight loss
in the group receiving slow injection versus a rapid injection
(Example 19).
[0183] In one cohort of a clinical trial, patients received three
injections of the plaque purified NDV isolate over the course of
one week (Example 20). Under these conditions, a desensitizing
effect of the initial dose lessened the toxicity associated with
the second and third doses. These data parallel those obtained with
the animal studies shown in Example 18. One concern related to the
use of oncolytic viruses in the treatment of cancer is the
potential inhibitory effect the humoral immune response can exert
on the therapy. In the clinical study, patients displaying stable
disease after 1 month are eligible for a second course of treatment
which then is administered in the presence of neutralizing
antibodies to NDV. Nevertheless, infectious virus could be found in
patient urine seven days after dosing for the second course,
providing evidence that administration of high doses of virus can
overcome the effect of neutralizing antibodies and establish an
infection within the patient.
[0184] In an advantageous embodiment of the invention, a
desensitizing dose is given before higher subsequent doses. For
desensitization, virus doses of 1.times.10.sup.8 to
2.4.times.10.sup.10 PFU/m.sup.2 are used. After desensitization,
additional virus doses of 3.times.10.sup.8 to 4.times.10.sup.12
PFU/m.sup.2 are used. The time frame between doses, including the
time frame between desensitizing dose and the next dose, is 1 to 14
days, advantageously 1 to 7 days. The desensitizing dose can be
administered by various routes, e.g., intravenous, enteral,
parenteral, oral, nasal, rectal, intrathecal, intervenous,
subcutaneous, intratumor, peritumoral, local, sublingual, buccal,
topical, intramuscular, by inhalation, percutaenous, vaginal,
intra-arterial, intracranial, intradermal, epidural, sytemically,
topical, intraperitoneal, intrapleural, endoscopic, intrabronchial,
etc. The subsequent doses can be administered by the same route as
the desensitizing dose or by another route, e.g., intravenous,
enteral, parenteral, oral, nasal, rectal, intrathecal, intervenous,
subcutaneous, intratumor, peritumoral, local, sublingual, buccal,
topical, intramuscular, by inhalation, percutaenous, vaginal,
intra-arterial, intracranial, intradermal, epidural, sytemically,
topical, intraperitoneal, intrapleural, endoscopic, intrabronchial,
etc.
[0185] Optionally, more than one route of administration can be
used in either a sequential or concurrent mode. Routes for either
concurrent or sequential administration include but are not limited
to intravenous, enteral, parenteral, oral, nasal, rectal,
intrathecal, intervenous, subcutaneous, intratumor, peritumoral,
local, sublingual, buccal, topical, intramuscular, by inhalation,
percutaenous, vaginal, intra-arterial, intracranial, intradermal,
epidural, sytemically, topical, intraperitoneal, intrapleural,
endoscopic, intrabronchial, etc. An example would be the
administration of a intravenous desensitizing dose followed by an
intraperitoneal dose.
[0186] In another advantageous embodiment of the invention, the
virus is administered by slow infusion including using an
intravenous pump or slow injection over the course of 4 minutes to
24 hours.
[0187] A virus, and optionally one or more chemotherapeutic agents,
is administered by a single injection, by multiple injections, or
continuously. The virus is administered before, at the same time,
or after the administration of chemotherapeutic agents (such as but
not limited to: busulfan, cyclophosphamide, methotrexate,
cytarabine, bleomycin, cisplatin, doxorubicin, melphalan,
mercaptopurine, vinblastine, 5-fluorouracil, taxol, and retinoic
acid). Viral therapy in accordance with the present invention is
optionally combined with other treatments, including, surgery,
radiation, chemotherapy (see, e.g., Current Medical Diagnosis and
Treatment, Ed. Tierney et al, Appleton & Lange, 1997,
especially pages 78-94), and biological therapy. The virus is
administered before, at the same time, or after the administration
of biological agents such as (1) other oncolytic agents [such as
but not limited to: adenoviruses with one of its genes under
transcriptional control of a prostate cell specific response
element (see Rodrigues, R. et al, 1997, Cancer Res, 57:2559-2563;
adenoviruses which do not encode a Elb polypeptide capable of
binding p53 (see Bischoff, J. R., et al, 1996, Science
274:373-376); a herpes simplex virus that is incapable of
expressing 99 functional gamma 34.5 gene product (see Mineta, T. et
al, 1995, Nature Medicine, 1:938-943)]; (2) cytokines (such as but
not limited to: colony stimulating factors such as GM-CSF; tumor
necrosis factor, and interleukins such as IL-I, IL-2, IL-6 and
IL-10); (3) viral vectors [such as but not limited to adenovirus
encoding p53 (see Zhang, W W et al, 1994, Cancer Gene Therapy.
1:5-13)]; and (4) cancer vaccines.
[0188] In one embodiment of the invention, therapy consists of the
serial treatment with antigenically distinct viruses which are
cytotoxic and tumor selective via the IFN mechanism. This
embodiment allows viral therapy over an extended period without
immunological interference.
[0189] Another embodiment involves the treatment of patients with
IFN (e.g. .alpha.IFN, .beta.IFN or .gamma.IFN) prior to, concurrent
with, or following administration of NDV (or other virus). The IFN
is selected from the group class I (alpha, beta and omega) and
class II (gamma), and recombinant version and analogs thereof as
discussed in, for example, Sreevalsoun, T., 1995 (In: Biologic
Therapy of Cancer, second edition, edited by V. T. DeVita, Jr., S.
Hellman, and S. A. Rosenberg, J. B. Lippincott Company,
Philadelphia, pp347-364). Normal cells respond to the IFN
pre-treatment with an augmented IFN response to viral infection
affording even greater safety to these cells. Tumor cells deficient
in the IFN signaling pathway remain sensitive to killing by the
virus. This allows even higher doses of viral therapy to be used.
The IFN is administered in accordance with standard clinical
guidelines for doses and regimens known to be effective for
treating viral infections. In another embodiment of the invention,
other drugs, known to affect the IFN response pathway are also
optionally used to increase the sensitivity of tumor cells, or
increase the resistance of normal cells to the cytocidal effects of
viral infection. This class of drugs includes, but is not limited
to tyrosine kinase inhibitors, cimetidine, and mitochondrial
inhibitors. Hypoxia and hyperthermia are also known to modulate
interferon responsiveness.
[0190] In another embodiment of the invention, immunosuppressants
such as cyclosporin A, azathiaprime, and leflunomide, various
corticosteroid preparations, and anti-CD-40 ligand antibodies (Foy,
T. M., et al., 1993, J. Exp. Med. 178:1567-1575) are administered
with the virus. Alternatively, an immunostimulatory compound, e.g.,
lipopeptides, can be administered with the virus.
[0191] An independent mechanism by which the amount of interferon
produced in response to viral infection is increased through the
use of nucleosides (Machida, H., 1979. Microbiol. Immunol.
23:643-650), nucleoside precursors, or drugs that increase the
cellular concentration of one or more nucleosides, are optionally
used as an adjunct to viral therapy.
[0192] Certain purine nucleoside analogs, e.g.,
2-chlorodeoxyadenosine and 2'-deoxycoformycin, reduce interferon
production in vivo. Such compounds are used to further effect
differences in interferon sensitivities of tumor cells versus
normal cells and are optionally used as an adjunct to viral
therapy.
[0193] In one aspect, an effective amount of virus can be
subdivided into smaller dose units and injected at the same time
into different locations of the same tumor. For continuous
administration, the desired agent(s) is administered via an
implanted minipump or it is impregnated into a desired polymer and
then transplanted into a desired location (e.g., directly into the
tumor) for slow or delayed release.
[0194] A virus of the present invention is formulated as a
pharmaceutical preparation by bringing it into a suitable dose
form, together with at least one excipient or auxiliary, and, if
desired, with one or more further active compounds. The
preparations are utilized in both human and veterinary medicine.
Suitable excipients include, e.g., organic and inorganic substances
which are appropriate for enteral or parenteral administration,
e.g., water, saline, tissue culture media, buffers, lysine,
citrate, glycerol triacetate and other fatty acid glycerides,
gelatin, soya lecithin, carbohydrates such as, mannitol, sucrose,
lactose or starch, magnesium stearate, talc, cellulose or protein
carriers, or a combination of the preceding compounds, such as
mannitol/lysine, or mannitol/lysine/sucrose. The preparations are
sterilized and/or contain additives, such as preservatives or
stabilizers. For parenteral administration, e.g., systemic or local
injection, a virus preparation is formulated, e.g., as an aqueous
suspension or emulsion.
[0195] The invention also relates to a method of treating a disease
in a mammal, in which the diseased cells have defects in an
interferon-mediated antiviral response, comprising administering to
the mammal a therapeutically effective amount of an
interferon-sensitive, replication-competent, clonal virus. For
example, cells infected with many viruses like hepatitis B that
disable the interferon response are susceptible to the viruses of
this invention. There is evidence that human immunodeficiency virus
(HIV) disables the interferon response. The interferon-sensitive
viruses of this invention are useful in treating such chronic virus
infections such as those due to hepatitis B, hepatitis C, HIV,
Epstein-Barr virus, human papilloma virus, and herpes virus.
[0196] Unless indicated otherwise herein, details and conditions of
viral therapy of this invention are in accordance with U.S.
application Ser. No. 08/260,536 whose disclosure is incorporated
herein by reference in its entirety. The entire disclosure of all
applications, patents and publications, cited above and in the
figures are hereby incorporated by reference.
[0197] The following examples are illustrative, but not limiting of
the methods and compositions of the present invention. Other
suitable modifications and adaptations of a variety of conditions
and parameters normally encountered in clinical therapy which are
obvious to those skilled in the art are within the spirit and scope
of this invention.
EXAMPLE 1
[0198] PPMK107, (a Triple Plaque Purified Isolate of the NDV Strain
MK107) Demonstrates a Selective Cytotoxic Activity toward many
Human Cancer Cells Compared to Normal Human Cells
[0199] Human tumor cells and normal cells were grown to
approximately 80% confluence in 24 well tissue culture dishes.
Growth medium was removed and PPMK107 was added in 10 fold
dilutions ranging from 10.sup.6 plaque forming units (PFU)/well to
10.sup.-1 PFU/well. Controls wells with no virus added were
included on each plate. Virus was adsorbed for 90 minutes on a
rocking platform at 37.degree. C. At the end of the incubation
period, the viral dilutions were removed and replaced by 1 ml of
growth medium. Plates were then incubated for 5 days at 37.degree.
C. in 5% C02, then assessed qualitatively for the amount of
cytopathic effect (CPE). Cytotoxicity was quantified by using a
colorimetric MTT (2-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium bromide) assay (Cell Titer 96, catalog #G4000, Promega
Corporation, Madison Wis. 53711) monitored at 570 nm, that detects
mitochondrial enzyme activity (Mosman, T., 1983, J. Immunol.
Methods 65:55). The viability in the virus treated wells was
expressed as a percent of the activity in untreated control wells.
The data was plotted graphically as PFU/well vs. viability as a
percent of control. The IC50 was calculated as the amount of virus
in PFU/well causing a 50% reduction in the amount of viable
cells.
[0200] The results are given in Tables 4, 5 and 6. PPMMK107
demonstrated a high degree of cytotoxic activity against a diverse
set of human cancer cells with 30 out of 39 malignant lines having
an IC50 value less than 1000 compared to the relative insensitivity
of normal human cell types. The majority of human cancer cells had
IC50 values that were 2 to 3 orders of magnitude lower than most
normal human cell types.
5TABLE 4 Summary of Cytotoxicity Assay Results Tumor Type CELL LINE
IC.sub.50 (PFU/well) FIBROSARCOMA HT 1080 2 MELANOMA SKMEL2 8
SKMEL3 2 SKMEL5 4 A375 37 MALME-3M 778 HT144 28 BREAST CARCINOMA
SKBR3 10 MDA-MB-468 44 ZR75-1 78 OVARIAN CARCINOMA SW626 4 PA-1 4
ES-2 13 SKOV-3 24 OVCAR3 34 LUNG CARCINOMA H-1299 26 (Large Cell,
Low Passage) GLIOBLASTOMA U87MG 25 U373MG 765 U138 38 A172 207
BLADDER CARCINOMA HT1197 3 UM-UC-3 54 HT1376 422 NEUROBLASTOMA
IMR32 41 CERVICAL CARCINOMA HeLa 4 PROSTATE CARCINOMA DU-145 31 PC3
3.1 .times. 10.sup.3 COLON CARCINOMA SW620 55 HT29 >1.0 .times.
10.sup.6 HEAD-AND-NECK KB 4 CARCINOMA A253 >2.7 .times. 10.sup.3
FaDu >2.9 .times. 10.sup.3 Hep-2 >1.5 .times. 10.sup.4
NEUROEPITHELIOMA SK-N-MC 20 SMALL CELL CA, LUNG DMS-114 48 DMS-153
1.1 .times. 10.sup.5 NCl-H345 1.2 .times. 10.sup.6 SMALL CELL CA,
NCl-H660 1.0 .times. 10.sup.5 PROSTATE LEUKEMIA (AML) K562 5.4
.times. 10.sup.4
[0201]
6TABLE 5 Summary of Cytotoxicity Assay Results Using Normal Human
Cells CELL TYPE CELL IC.sub.50 (PFU/well) Keratinocytes NHEK 9.0
.times. 10.sup.6 Fibroblasts CCD-922 1.4 .times. 10.sup.5 NHDF 8.1
.times. 10.sup.3 Endothelial Cells HPAEC 5.2 .times. 10.sup.4 Renal
Cells RPTEC 2.7 .times. 10.sup.4 Melanocytes NHEM 5.1 .times.
10.sup.4 Astrocytes NHA 3.8 .times. 10.sup.3
[0202]
7TABLE 6 Summary of Cytotoxicity Assay Results Using Rapidly
Proliferating Normal Human Cells RATE OF PROLIFERATION CELL TYPE IN
VIVO IN VITRO IC.sub.50 (PFU/well) Bone Marrow Cells Moderate to
High 6.2 .times. 10.sup.3 CD34+ Enriched to 50% High Breast
Epithelial Cells Very Low.sup.1 High.sup.1 30 .sup.1Human breast
epithelial cells tested (HMEC) had a high rate of proliferation
after stimulation with bovine pituitary extract and human epidermal
growth factor. In marked contrast, normal breast epithelial cells
almost always have a very low degree of proliferation in adult
women with cancer.
EXAMPLE 2
[0203] Use of PPMK107 for the Intratumoral Treatment of Human Tumor
Xenografts (<10 mm and >5 mm) in Athymic Mice
[0204] Athymic mice were injected intradermally with 10 million
human tumor cells. After tumors reached a size range from between 5
and 10 mm, a single injection of PPMK107 (at a dose of
3.times.10.sup.8 PFU) or saline was given. Almost all tumor types
exhibited a rate of complete or partial regression of 50% to 100%
(see Table 7) in mice treated with PPMK107. The one exception is
the case of the U87MG experiment (experiment I): Although only one
of 9 tumors treated with PPMK107 completely regressed, two more
virus-treated tumors showed regression of 32% and 20% and two more
virus-treated tumors had slower growth than all 8 tumors treated
with saline control. Tumor regression was virtually absent in the
saline control treated tumors: In all of these experiments (A
through I listed in Table 7) only one of 73 control tumors showed
regression. These results indicate that diverse tumor types showed
responses to intratumoral PPMK107 treatment.
[0205] To examine virus replication within the tumor,
immunohistochemical staining for viral antigen (using a monoclonal
antibody against the NDV P protein) was performed using the
subcutaneous HT1080 fibrosarcoma model. Within 30 minutes of
intratumoral injection of 3.times.10.sup.8 PFU of PPMK107, the
tumor tissue was negative for viral antigen. However, by day 2 post
treatment, intense immunostaining for viral antigen was seen within
the tumor, indicating virus replication within the tumor.
Importantly, virus replication was specific for the tumor tissue
since the neighboring connective tissue and skin was negative for
viral antigen.
EXAMPLE 3
[0206] Use of PPMK107 for the Intravenous Treatment of Human Tumor
Xenografts (<8.5 mm and >5.5 mm) in Athymic Mice
[0207] Athymic mice were injected intradermally with 10 million
human HT1080 fibrosarcoma cells. After tumors reached a size range
from between 5 and 8 mm, a intravenous injection(s) of PPMK107 or
saline were made. As shown in Table 8, at the highest virus dose
level (1.times.10.sup.9 PFU) complete tumor regression was seen in
all seven mice. Single injections of 3.times.10.sup.8 and
6.times.10.sup.7 resulted in regression rates of over 90%. While a
single IV injection of 3.times.10.sup.8 only a 55% rate of tumor
regression, three IV injections at this dose level yielded a 100%
rate of response. Mice treated with IV saline exhibited no evidence
of tumor regression. These results indicate that subcutaneous
HT1080 tumors are very responsive to IV treatment with PPMK107.
8TABLE 7 PPMK107 intratumoral treatment of subcutaneous human tumor
xenografts (<10 mm and <5 mm) in athymic mice Complete
Complete + Tumor Tumor Type Expt # Dose N Regression partial
Regression HT1080 Fibroscarcoma A 3.00E+08 12 11 11 B 3.00E+08 9 8
8 C 3.00E+08 8 8 8 PA-1 Ovarian Carcinoma D 3.00E+08 9 9 9 KB Oral
Carcinoma E 3.00E+08 12 7 10 SKMEL5 Melanoma F 3.00E+08 8 5 7 A375
Melanoma G 3.00E+08 8 5 7 H 3.00E+08 8 1 4 U87MG Glioblastoma I
3.00E+08 9 1 1
[0208]
9TABLE 8 PPMK107 intravenous treatment of subcutaneous human HT1080
fibrosarcoma xenografts (<8.5 mm and <5.5 mm) in athymic mice
Complete Complete + % Re- Dose Schedule N Regression partial
gression 1.00E+09 One Injection 7 7 7 100% 3.00E+08 One Injection
10 9 10 100% 6.00E+07 One Injection 11 10 10 91% 2.00E+07 One
Injection 11 5 6 55% 2.00E+07 Three Injections 7 5 7 100% Every
Other Saline One Injection 10 0 0 0% Saline Three Injections 6 0 0
0% Every Other
EXAMPLE 4
[0209] First Experiment Using PPMK107 for Intratumoral Treatment of
Large A375 Melanoma Xenografts in Athymic Mice
[0210] Athymic mice were injected intradermally with 10 million
A375 human melanoma cells. Ten days later, tumors of various sizes
were treated with a single injection PPMK107 (doses of
3.times.10.sup.8, 9.times.10.sup.8, and 1.5.times.10.sup.9 PFU) or
saline. For those tumor with a single largest dimension of 10 to 11
mm, all nine completely regressed in response to intratumoral
treatment with these doses of PPMK107, while of those tumors with a
single largest dimension of 8 to 9.5 mm, twelve out of 24
completely regressed in response to virus therapy (P<0.008;
Table 9, section A). No tumor regression was seen in any mouse
treated with saline.
[0211] These same tumors when sorted by tumor volume also indicated
a high percentage of complete regression in those of larger tumor
volume. In response to these doses PPMK107, complete regression
occurred in 14 out of 17 tumors with volumes >300 mm.sup.3
(range of 304 to 397 mm.sup.3) and in 7 out of 16 tumors with
volumes <300 mm.sup.3 (range of 144 to 295; P <0.023; Table
9, section B).
[0212] These results indicate that tumors at least 1 cm in length
or 300 mm.sup.3 in volume were at least as sensitive, if not more
sensitive, to intratumoral PPMK107 treatment than smaller
tumors.
EXAMPLE 5
[0213] Second Experiment Using PPMK107 for Intratumoral Treatment
of Large A375 Melanoma Xenografts in Athymic Mice
[0214] Tumors were established as in Example 4 ten days after tumor
cell inoculation. Treatment consisted of various doses of PPMK107
(3.times.10.sup.6 PFU 3.times.10.sup.7, 3.times.10.sup.8, and
1.5.times.10.sup.9) or saline, For tumors 10 to 11.5 mm in single
largest dimension, complete or partial (at least 50%) regression
occurred in all 28 tumors treated with PPMK107 using these doses in
contrast to no regression in any of the saline-treated mice (Table
10, section A).
[0215] When these same tumors were sorted by tumor volume, all 26
tumors greater than 300 mm.sup.3 (range: 309 to 525 mm.sup.3)
regressed completely or partially (at least 50%) in response to
PPMK107 in contrast to none of the saline treated mice (Table 10,
section B).
[0216] These results confirm that tumors at least 1 cm in length or
300 mm.sup.3 in volume are sensitive to intratumoral PPMK107
treatment.
10TABLE 9 Intratumoral PPMK107 Treatment of Intradermal A375
Melanoma Xenografts Tumor Dimension: 8 to 9.5 mm Tumor Dimension:
10 to 11 mm Complete Complete Treatment Dosage N Regression % N
Regression % A. Tumors Sorted Based on the Single Largest Dimension
PPMK107 1.5 .times. 10.sup.9 8 2 25% 3 3 100% PPMK107 9.0 .times.
10.sup.8 8 7 88% 3 3 100% PPMK107 3.0 .times. 10.sup.8 8 3 38% 3 3
100% Total 24 12 50% 9 9 100% a Saline 6 0 0% 3 0 0% Tumor Volume:
<300 mm.sup.3 Tumor Volume: >300 mm.sup.3 Complete Complete
Treatment Dosage N Regression % N Regression % B. Tumors Sorted
Based on the Tumor Volume PPMK107 1.5 .times. 10.sup.9 6 2 33% 5 3
60% PPMK107 9.0 .times. 10.sup.8 4 3 75% 7 7 100% PPMK107 3.0
.times. 10.sup.8 6 2 33% 5 4 80% Total 16 7 44% 17 14 82% b Saline
8 0 0% 1 0 0% a- P < 0.008 for complete regression in the
PPMK107 10-11 mm group vs. the PPMK107 8-9.5 mm treatment group b-
P < 0.023 for complete regression in the PPMK107-treated >300
mm.sup.3 group vs. the PPMK107-treated <300 mm.sup.3group
[0217]
11TABLE 10 Intratumoral PPMK107 Treatment of Intradermal A375
Melanoma Xenografts Regressions Complete + Treatment Dose N
Complete % Partial % A. Tumors 10 to 11.5 mm (Sorted Based on the
Single Largest Dimension) 1.5 .times. 10.sup.9 7 7 100% 7 100% 3.0
.times. 10.sup.8 7 6 86% 7 100% 3.0 .times. 10.sup.7 7 5 71% 7 100%
3.0 .times. 10.sup.6 7 5 71% 7 100% All PPMK107 28 23 82% 28 100%
Groups Saline 6 0 0% 0 0% B. Tumors >300 mm.sup.3 (Sorted Based
on the Tumor Volume) 1.5 .times. 10.sup.9 7 7 100% 7 100% 3.0
.times. 10.sup.8 7 6 86% 7 100% 3.0 .times. 10.sup.7 6 4 67% 6 100%
3.0 .times. 10.sup.6 6 4 67% 6 100% All PPMK107 26 21 81% 26 100%
Groups Saline 5 0 0% 0 0%
EXAMPLE 6
[0218] Third Experiment Using PPMK107 for Intratumoral Treatment of
Large A375 Melanoma Xenografts in Athymic Mice
[0219] Tumors were established as in Example 4 nineteen days after
tumor cell inoculation. Intratumoral treatment consisted of various
doses of PPMK107 (3.times.10.sup.8, 3.times.10.sup.6,
3.times.10.sup.5, 3.times.10.sup.4, 3.times.10.sup.3,
3.times.10.sup.2 PFU) or saline. For tumors 12.5 to 14 mm in single
largest dimension (volume range: 632 to 787 mm.sup.3; average
volume 698 mm.sup.3), tumor regressions of at least 50% occurred in
two out of three mice treated with 3.times.10.sup.8 PFU in contrast
to no regression in both saline-treated mice (Table 11). Using the
same dose of PPMK107 (3.times.10.sup.8 PFU) to treat tumors with a
single largest dimension of 10 to 12 mm (volume range: 320 to 600
mm.sup.3; average volume: 411 mm.sup.3), seven of 8 mice exhibited
regression of at least 25% (P<0.001 for regression of at least
25% compared to the saline treated mice which exhibited no
regressions, Table 11). Regressions of at least 25% for tumors of
length 10 to 12 mm tumors were also seen in mice treated with
3.times.10.sup.6 PFU, 3.times.10.sup.5 PFU, 3.times.10.sup.4 PFU,
and 3.times.10.sup.3 PFU, but not for mice treated with
3.times.10.sup.2 PFU or saline (Table 11).
[0220] These results confirm that tumors at least 1 cm in length or
300 mm.sup.3 in volume are sensitive to intratumoral PPMK107
treatment.
EXAMPLE 7
[0221] Fourth Experiment Using PPMK107 for Intratumoral Treatment
of Large A375 Melanoma Xenografts in Athymic Mice
[0222] Tumors of largest dimension 10 to 12 mm were established as
in Example 4 thirteen days after tumor cell inoculation.
Intratumoral treatment consisted of a single injection of
3.times.10.sup.8 PFU of PPMK107 or saline. Volumes of those tumors
treated with PPMK107 ranged from 295 to 600 mm.sup.3 (average tumor
volume of 437 mm.sup.3). Groups of mice in each treatment group
were euthanized on days 0, 2, 3, 4, 7, and 14 for tumor histology.
For those mice observed for a minimum of 4 days, eleven out to 12
mice treated with PPMK107 exhibited regression of at least 25%
compared to none of 8 in the saline group (P<0.0001, Table 12).
At 2 days after PPMK107 treatment, two tumors already exhibited
signs of regression but the degree of regression was less than
25%.
12TABLE 11 3.sup.rd Experiment Using PPMK107 for the Intratumoral
Treatment of A375 Melanoma Xenografts (at least 10 mm in size)
Regressions Total # of % Treatment N Volume Range Avg Volume
Complete Partial.sup.a >25% & <50%.sup.b
Regressions.sup.c Regressions.sup.c Size: 12.5 to 4 mm 3.0E+08 3
632 to 787 698 1 1 0 2 67% Saline 2 717 to 860 788 0 0 0 0 0% Size:
10 to 12 mm 3.0E+08 8 320 to 600 411 0 3 4 7 88% 3.0E+06 8 425 to
662 502 0 0 2 2 25% 3.0E+05 8 245 to 600 421 0 0 1 1 13% 3.0E+04 8
336 to 600 477 0 0 1 1 13% 3.0E+03 8 281 to 542 349 2 0 0 2 25%
3.0E+02 8 281 to 662 372 0 0 0 0 0% Saline 8 379 to 666 518 0 0 0 0
0% .sup.aPartial Regression is defined as regression less than 100%
and equal to or greater than 50%. .sup.b"Regression >25% &
<50%" is defined as tumor regression greater than 25% and less
than 50%. .sup.cIncludes all tumor regression that is greater than
25% d - P < 0.001 for Regression greater than 25% in the 3E+08
group vs the saline group.
[0223]
13TABLE 12 4.sup.th Experiment Using PPMK107 for the Intratumoral
Treatment of A375 Melanoma Xenografts (at least 10 mm in size) Day
Euthanized Regressions Total # of % Treatment Post Treatment N
Complete Partial.sup.a >25% & <50%.sup.b
Regressions.sup.c Regressions.sup.c Tumor Size: 10 to 12 mm 3.0E+08
14 Days 3 0 2 1 3 100% 3.0E+08 7 Days 3 0 2 1 3 100% 3.0E+08 4 Days
3 0 2 1 3 100% 3.0E+08 3 Days 3 0 0 2 3 67% 3.0E+08 All PPMK107
Groups 12 0 6 5 11 92% d,e Saline 14 Days 2 0 0 0 0 0% Saline 7
Days 2 0 0 0 0 0% Saline 4 Days 2 0 0 0 0 0% Saline 3 Days 2 0 0 0
0 0% Saline All Saline Groups 8 0 0 0 0 0% .sup.aPartial Regression
is defined as regression less than 100% and equal to or greater
than 50%. .sup.b"Regression >25% & <50%" is defined as
tumor regression greater than 25% and less than 50%. .sup.cIncludes
Regression that is at least 25% d - P < 0.03 for Complete or
Partial Regression in the PPMK107 group of 12 mice vs the saline
group of 8 mice. e - P < 0.0001 for Regression at least 25% in
the PPMK107 group of 12 mice vs the saline group of 8 mice.
EXAMPLE 8
[0224] Fifth Experiment Using PPMK107 for Intratumoral Treatment of
Large A375 Melanoma Xenografts in Athymic Mice
[0225] Tumors of largest dimension 10 to 12 mm were established as
in Example 4 twenty days after tumor cell inoculation. Intratumoral
treatment consisted of a single injection of 3.times.10.sup.8 PFU
of PPMK107 or saline. Volumes of those tumors treated with PPMK107
ranged from 361 to 756 mm.sup.3 (average tumor volume of 551
mm.sup.3). Nine out of 10 mice treated with PPMK107 exhibited a
regression of at least 25% compared to none of 10 in the saline
group (P<0.0001, Table 13).
EXAMPLE 9
[0226] First Experiment Using PPMK107 for Intravenous Treatment of
Large HT1080 Fibrosarcoma Xenografts
[0227] Athymic mice were injected subcutaneously with 10 million
HT1080 human fibrosarcoma cells. Six days later, tumors were
treated with a single injection PPMK107(at a dose of
1.5.times.10.sup.9 PFU) or saline. For those tumors 10 to 11 mm in
single largest dimension, five out of six tumors completely or
partially regressed in response to a single intravenous injection
of PPMK107 compared to none of the saline treated tumors (Table 14,
P<0.025). These results indicate that tumors at least 1 cm in
length are sensitive to intravenous PPMK 107 treatment.
14TABLE 13 5.sup.th Experiment Using PV701 for the Intratumoral
Treatment of A375 Melanoma Xenografts (at least 10 mm in size)
Regressions Total # of % Treatment N Complete Partial >25% &
<50%.sup.b Regressions.sup.c Regressions.sup.c Size: 10 to 12 mm
3.0E+08 10 0 4 5 9 90% d,e Saline 10 0 0 0 0 0% a - Partial
Regression is defined as regression less than 100% and equal to or
greater than 50%. .sup.b"Regression >25% & <50%" is
defined as tumor regression greater than 25% and less than 50%.
.sup.cIncludes all tumor regression that is greater than 25% d - P
< 0.05 for Complete or Partial Regression in the PV701 group of
vs the saline group. e - P < 0.0001 for all tumor regression at
least 25% in the PV701 group vs the saline group.
[0228]
15TABLE 14 Intravenous Treatment of Subcutaneous HT1080 Human
Fibrosarcoma Xenografts in Athymic Mice Complete + Treatment Dose N
Complete % Partial % Size: 10 to 11 mm PPMK107 1.5E+09 6 4 67% 5
83% a Saline 4 0 0 0 0 a- P < 0.25 (by Fisher's exact test) for
complete or partial regression (at least 50% regression) in the
PPMK107 treated group compared to the saline group.
EXAMPLE 10
[0229] Specific Clearing of PPMK107 Infection from Normal but Not
Tumor Cells
[0230] In order to examine the mechanism of tumor-specific killing
by NDV strain PPMK107, representative tumor cells were chosen based
on the following criteria: a) ability to form tumors as xenografts
in athymic mice; b) the tumor xenografts are specifically killed in
vivo following administration of PPMK107; c) the tumors cells
exhibit killing by PPMK107 in vitro at virus concentrations that
are several logs below the concentration to kill resistant, normal
cells; and d) tumor cells must be easily distinguished from the
normal cells when present as a co-culture. Xenograft tumors
comprised of KB head and neck carcinoma cells exhibit 83% complete
or partial regression in response to a single intratumoral
injection of PPMK107, are more than four logs more sensitive to
killing by PPMK107 in vitro than are normal primary skin
fibroblasts (CCD922-sk), and are easily distinguished from
CCD922-sk cells when present as a co-culture.
[0231] Accordingly, co-cultures of KB and CCD922-sk cells were
infected at a multiplicity of infection (m.o.i., the ratio of virus
added per cell) of 0.0005 and the course of the infection followed
for 5 days by immunohistochemical staining for a viral antigen (NDV
P protein). Infection of normal cells peaked at 2 days with little
or no apparent cell death as determined by visual inspection of the
cell monolayer. On the third day post-infection the amount of viral
expression in the normal cells decreased significantly, while
infection of the tumor cells was clearly apparent. The amount of
viral antigen virtually disappeared in the normal cells on days 4
and 5, while the infection in the tumor cells progressed rapidly
through the tumor cell population resulting in destruction of the
majority of the tumor cells present in the co-culture.
[0232] Thus, normal cells were infected and easily cleared the
infection in a manner consistent with the anti-viral effects of
IFN. The tumor cells were unable to establish an anti-viral state
in response and were killed by the unabated viral growth, despite
the presence of physiologically effective concentrations of IFN
secreted into the media by the normal cells.
EXAMPLE 11
[0233] Demonstration that Interferon is an Important Component of
Viral Clearing in Normal CCD922-sk Cells
[0234] The hypothesis that interferon was mediating the ability of
CCD922-sk cells to clear the infection of PPMK107 was tested.
Polyclonal neutralizing antibodies to human interferon-a or human
interferon-.beta., used alone or in combination, were added daily
to cultures of CCD922-sk cells infected with PPMK107 at an moi of
0.0005 and the progress of the infection followed for three days.
The amount of viral antigen present in the cells increased in
proportion to the concentration of neutralizing antibody, with the
effect of the anti-interferon-.beta. antibody being more marked
than that of the anti-interferon-.alpha. antibody; consistent with
reports that fibroblasts produce predominantly the beta form of
interferon.
[0235] The ability to make the normally insensitive cells more
susceptible to infection with PPMK107 through the addition of
neutralizing antibody to interferon supports the hypothesis that a
key difference between the sensitivity of normal and tumor cells to
killing by PPMK107 lies in the ability of normal cells, but not
tumor cells, to establish an interferon-mediated anti-viral
response.
EXAMPLE 12
[0236] Demonstration that Interferon-.beta. is an Important
Component of Viral Clearing in Other Normal Cells
[0237] In this experiment, it was determined that another normal
cell (NHEK, normal human epithelial cells) known to be quite
resistant to killing by PPMK107, was made more sensitive through
the addition of polyclonal anti-interferon-.beta. antibody to a
culture of infected cells. NHEK (normal human epithelial
keratinocyte) cells were infected at an moi of either 0.0005 or
0.05 and had antibody added daily over five days.
[0238] In the cultures infected at the low moi (0.0005), antibody
dependent augmentation of viral antigen expression was clear at
five days post-infection, but was less clear earlier in the
experiment. Antibody addition to cultures infected with PPMK107 at
an moi of 0.05 resulted in a marked increase in viral antigen at 4
and 5 days post-infection. At 2 and 3 days post-infection the
addition of neutralizing antibody resulted in less accumulation of
viral antigen (FIG. 1).
[0239] The culture supernatants from the high moi samples were also
titrated for the amount of infectious virus present by plaque assay
on human HT1080 fibrosarcoma tumor cells; the standard assay system
in our laboratory. Results from this analysis demonstrated that at
five days post-infection there was 19-fold increase in the amount
of infectious virus in the antibody-treated cultures relative to
mock-treated controls (FIG. 1).
[0240] These results suggest a general mechanism by which normal
cells are protected from killing by PPMK107 through an
interferon-related mechanism.
EXAMPLE 13
[0241] Comparison of the Effect of Interferon-.beta. on PPMK107
Infection in Tumor and Normal Cells
[0242] A comparison of the effect of exogenously added
interferon-.beta. on the infection of normal (CCD922-sk) and tumor
cells of high (KB) or intermediate (HEp2) sensitivity PPMK107 was
performed. Separate cultures of the three cells were treated with
interferon-.beta. at 20, 200, or 2000 units/ml 1 day pre- and 2
days post-infection at an moi of 0.0005.
[0243] At 3 days post-infection the low level of viral antigen
expression present in the normal cells was eliminated at all doses
of interferon used. Conversely, the addition of interferon to the
highly sensitive KB tumor cells at concentrations of 2 or 200
units/ml decreased relative levels of viral antigen expression
2-fold, with complete suppression at 1000 units/ml interferon. The
intermediately sensitive HEp-2 cells responded to the exogenous
interferon by clearing viral antigen expression at all of the
interferon doses used (FIG. 2).
[0244] The pattern of sensitivity in the KB and CCD922-sk cells to
the anti-viral effects of exogenously added interferon-.beta. was
inversely proportional to the sensitivity of these cells to killing
by PPMK107. The ability of the HEp-2 cells to respond to the
effects of interferon indicates that these cells are able to
efficiently utilize the concentrations of interferon used in this
experiment. Similarly, the response of the KB cells to the high
doses of interferon suggests that the inability to establish an
interferon-mediated anti-viral response does not result from an
absolute defect in the interferon pathway, but rather a relative
insensitivity compared to normal cells.
EXAMPLE 14
[0245] Effect of Low Concentrations of Interferon-.beta. on the
Infection of Normal and Tumor Cells by PPMK107
[0246] In this experiment normal (CCD922-sk) and tumor (KB) cells
were treated with low concentrations of interferon-.beta. (0.2, 2,
and 20 units/ml) 1 day before and 2 days post-infection with
PPMK107 at an moi of 0.05.
[0247] Under these conditions the normal cells experienced a
dose-dependent decrease in the amount of viral antigen, while the
relative levels of viral antigen in the tumor cells was unaffected
by the addition of exogenous interferon (FIG. 3).
EXAMPLE 15
[0248] PPMK107 Purification
[0249] Method A
[0250] PPMK107 was derived from the mesogenic Newcastle disease
virus strain Mass-MK107 by triple plaque purification.
Approximately 1000 PFUs (plaque forming units) of live PPMK107 were
inoculated into the allantoic fluid cavity of each 10 day old
embryonated chicken egg. After incubation at 36.degree. C. for 46
hours, the eggs were chilled and then the allantoic fluid was
harvested. Cells and cell debris were removed from the allantoic
fluid by centrifugation at 1750.times.g for 30 minutes. The
clarified allantoic fluid (supernatant containing virus) was then
layered over a 20%/55% discontinuous sucrose gradient) and
centrifuged at approximately 100,000.times.g for 30 minutes. The
purified virus was harvested from the 20%/55% interface and
dialyzed against saline to remove the sucrose.
[0251] Method B
[0252] In another advantageous embodiment, the clarified allantoic
fluid was frozen at -70.degree. C. After thawing, the fluid was
maintained at 1 to 4 C overnight and then the contaminating
material was removed from the virus suspension by means of
centrifugation (1750.times.g for 30 minutes). This material was
further processed using the discontinuous sucrose gradient on the
ultracentrifuge as above.
[0253] Method C
[0254] In another advantageous embodiment, ultracentrifugation on
the discontinuous sucrose gradient was accomplished by means of a
continuous flow ultracentrifuge.
[0255] Method D
[0256] In another advantageous embodiment, harvested allantoic
fluid is diluted with a buffer containing 5% mannitol and 1.0%
1-lysine, pH 8.0 (ML buffer) and is clarified and exchanged with ML
buffer by tangential flow filtration (TFF) through filters with a
nominal pore size of 0.45.mu.. The permeate containing the
clarified virus in ML buffer is collected and virus is purified by
TFF through filters with a nominal cut-off of 300,000 daltons in ML
buffer. The concentrated, purified virus in ML buffer is collected
as the retentate from this step and is again diluted with ML buffer
before being applied to a Sephacryl S500 (Pharmacia) gel permeation
column equilibrated with ML buffer. Fractions containing purified
virus are collected, pooled and can be reconcentrated by TFF
through filters with a nominal cut-off of 300,000 daltons with ML
buffer.
[0257] Results
[0258] *Clonal Virus
[0259] After generation of PPMK107 by plaque purification, eight
individual molecular clones from the population of virions were
found to have an identical sequence (e.g. a homology of 100%) of
over 300 contiguous nucleotides within the fusion protein gene of
NDV. PPMK107 is a clonal virus with a high degree of genetic
homogeneity.
[0260] PFU/mg Protein
[0261] One quantitative means of measuring purity is by
determination of a PFU/mg protein. Higher values indicate a greater
level of purity. Using Method A, PFU/mg values of at least
4.8.times.10.sup.10 were achieved (see Table 15). Using Method C,
PFU/mg protein values of at least 2.0.times.10.sup.10 were
achieved. For a mesogenic strain of NDV, a literature value for
this measurement of purity has not been found. The best estimate
for a mesogenic strain of NDV is the virus preparation (NDV
Mass/MK107, lot RU2, prepared as in Faaberg K S and Peeples, M D,
1988, J. Virol. 62:586; and Bratt, M A and Rubin, H. 1967, Virology
33:598-608). This RU2 lot was found to have a PFU/mg of
1.3.times.10.sup.9 PFU/mg of protein. The purity values achieved by
Method A are approximately 40 times better than what the Peeples
method achieved (see Table 15).
[0262] *Particle per PFU Ratio
[0263] Another quantitative means of measuring purity is by
determination of a ratio of particles per PFU. Lower values
indicate a greater level of purity. Particle counts were done by
electron microscopy using standard methods. Using either Method A
or Method B, particles per PFU values near one were achieved (Table
15).
16TABLE 15 Virus Purity Virus Preparation PFU per Particle Method
Virus Lot # mg protein per PFU Preferred Method A PPMK107 L2 4.8
.times. 10.sup.10 0.80 L4 6.9 .times. 10.sup.10 NT.sup.a L5 6.6
.times. 10.sup.10 NT L6 7.7 .times. 10.sup.10 0.55 L7 6.1 .times.
10.sup.10 NT Preferred Method C PPMK107 D004 2.0 .times. 10.sup.10
0.32 D005 4.5 .times. 10.sup.10 0.52 D010 4.4 .times. 10.sup.10 NT
Preferred Method D PPMK107 RD2 5.6 .times. 10.sup.10 NT RD3 5.0
.times. 10.sup.10 NT .sup.aNT, Not Tested
[0264] Virus preparations using Methods A and C also permitted
purification of NDV to a level substantially free of contaminating
egg proteins. For the PPMK107 lot 7 preparation using Method A,
ovalbumin, was not detectable in a Western blot using (1)
1.7.times.10.sup.9 PFU of purified virus per well (3.3 cm in width)
run on an SDS-PAGE (sodium dodecyl 10 sulfate-polyacrylamide gel
electrophoresis) gel (1 mm thick); (2) a nitrocellulose membrane
for transfer; and (3 rabbit anti-ovalbumin (Cappel rabbit IgG
fraction at a 1:200 dilution of a 4 mg/ml antibody concentration).
For PPMK107 preparations using Method D and analyzed by SDS-PAGE
followed by silver staining, no band corresponding to ovalbumin was
observed.
EXAMPLE 16
[0265] Use of PPMK107 To Prevent Deaths from ES-2 Ovarian Carcinoma
Ascites in Athymic Mice
[0266] In this experiment, all of the athymic mice (female, NCR
nu/nu, 8 weeks old) were given an intraperitoneal injection of
10.sup.6 ES-2 cells. Seven days later before ascites had developed,
they were treated intraperitoneally with saline or PPMK107 (at
1.times.10.sup.9 PFU). As shown in FIG. 4, there was a markedly
improved survival in the animals treated with PPMK107 compared to
saline. The majority of the mice in the saline treated group had
developed ascites by seven days post-treatment and by day 38, all
of these animals had died. In marked contrast, 92% of the mice
treated with PPMK107 were still alive by day 38 and 25% of these
animals were long term survivors (>120 day survival).
EXAMPLE 17
[0267] PPMK107 Treatment of ES-2 Ovarian Carcinoma in Athymic Mice
When Ascites is Present
[0268] In this experiment, all of the athymic mice (female, NCR
nu/nu, 8 weeks old) were given an intraperitoneal injection of
10.sup.6 ES-2 cells. Fourteen days later when the majority of mice
had developed ascites, the mice without ascites were excluded and
the mice with ascites were randomized into 7 intraperitoneal
treatment groups (PPMK107-one treatment on day 0; PPMK107-two
treatments for the first week; PPMK107-one treatment each week;
PPMK107-two treatments each week; saline-one treatment on day 0;
saline-two treatments for the first week; saline-two treatments
each week). A dose of 1.times.10.sup.9 PFU/mouse was used for each
virus treatment. All of the mice before the first treatment and any
additional treatments were drained of the ascites fluid. Day 0
refers to the day of first treatment.
[0269] The degree of ascites for each mouse was quantified and
noted as follows:
17 Ascites Score Degree of Ascites 1.0 Animal appears normal-little
or no ascites present 2.0 Abdomen slightly distended; animal is
capable of normal functions 3.0 Abdomen distended; animal is
slow-moving, hunched with a staggered gait. 4.0 Abdomen completely
distended; animal moribund 5.0 Death after ascites development
[0270] As shown in Table 16, all of the saline-treated animals had
more advanced ascites than the PPMK107-treated animals on both days
7 and 10. On day 7 post initial treatment, each the saline group
had average ascites scores above 3.5 while all of the
PPMK107-treated groups had average ascites scores at 3.0 or below.
Similarly on day 10 post initial treatment, each the saline group
had average ascites scores above 4.5 while all of the
PPMK107-treated groups had average ascites scores at 4.1 or below.
These results indicate that ascites fluid production was markedly
decreased in virus-treated animals compared to saline controls.
18TABLE 16 PPMK107 Treatment of ES-2 Ovarian Carcinoma in Athymic
Mice When Ascites is Present. Average Average Ascites # of Ascites
Score, Score, Treatment Mice Day 7 Day 10 Saline x1 12 4.3 4.7
Saline x2 12 3.7 4.6 Saline x2 each wk 12 4.3 4.8 PPMK107 x1 17 3.0
4.1 PPMK107 x2 17 2.3 3.6 PPMK107 x1 each wk 17 2.6 2.6 PPMK107 x2
each wk 17 2.2 3.6
EXAMPLE 18
[0271] Use of a Desensitizing Dose of PPMK107 to Reduce the
Lethality of a Subsequent Dose of PPMK107
[0272] C57BL/6 mice (seven weeks old) were injected intravenously
on day 0 with either saline or a desensitizing dose of PPMK107
(3.times.10.sup.8 PFU/mouse). Two days later each set of mice were
further subdivided into groups for intravenous dosing with saline
or PPMK107 (at doses of Ix 10.sup.9, 2.5.times.10.sup.9,
5.times.10.sup.9, and 1.times.10.sup.10 PFU/mouse). As shown in
Table 17, when saline was used to pretreat the mice, deaths were
recorded in the mice subsequently dosed with 2.5.times.10.sup.9,
5.times.10.sup.9, and 1.times.10.sup.10 PFU. The doses of
5.times.10.sup.9 and 1.times.10.sup.10 PFU were 100% lethal to the
mice pretreated with saline. In contrast, no deaths were seen in
any group of mice given a desensitizing dose of PPMK107 on day 0
followed by PPMK107 injection two days later at dose levels up to
1.times.10.sup.10 PFU. These data indicate that PPMK107 can be used
to prevent the lethality of subsequent dosing with this same agent.
Furthermore, the maximal tolerated dose of PPMK107 can be raised by
an approximate order of magnitude when using this virus as a
desensitizing agent.
19TABLE 17 Use of a Desensitizing Dose of PPMK107 to Reduce the
Lethality of a Subsequent Dose of PPMK107. Dose on # of # of %
Group Injection on Day 0 Day 2 Mice Deaths Lethality 1 Saline
Saline 8 0 0 2 Saline PPMK107, 8 0 0 1.0E+09 3 Saline PPMK107, 8 3
38 2.5E+09 4 Saline PPMK107, 8 8 100 5.0E+09 S Saline PPMK107, 8 8
100 1.0E+10 6 PPMK107, 3E+08 Saline 8 0 0 7 PPMK107, 3E+08 PPMK107,
8 0 0 1.0E+09 8 PPMK107, 3E+08 PPMK107, 8 0 0 2.5E+09 9 PPMK107,
3E+08 PPMK107, 8 0 0 5.0E+09 10 PPMK107, 3E+08 PPMK107, 8 0 0
1.0E+10
EXAMPLE 19
[0273] Slower Intravenous Injection Rate Reduces the Toxicity of
PPMK107
[0274] Twenty two athymic mice (8 weeks old) were anesthetized with
a combination of ketamine/xylazine and placed into a restrainer to
help inhibit their movement during the injection process to allow
for either a slow or rapid injection of PPMK107. For the slow
injection group, 0.2 mL of 4.times.10.sup.9 PFU of PPMK107 in
saline was injected intravenously over a 4 minute period with 0.01
mL given every 10 to 15 seconds. The rapid injection group received
the same dose and volume but over a 30 second period. As shown in
Table 18, the animals receiving their dose of PPMK107 over 4
minutes had half as much maximal weight loss (recorded on day 2
after dosing) as the animals receiving the same IV dose over 30
seconds. These results indicate that PPMK107 has less toxicity and
is safer for intravenous administration when injected at such
slower rates.
20TABLE 18 Slower IV Injection of PPMK107 Results in Reduced
Toxicity. Length of Time That Dose was # of Maximal Percent Group
Administered Mice Weight Loss Rapid Injection of 30 seconds 11 12%
4E+09 Slow Injection of 4 minutes 11 6% 4E+09
EXAMPLE 20
[0275] Use of PPMK107 in the Treatment of Patients with Advanced
Cancer.
[0276] PPMK107 has been tested in a phase 1 clinical trial in the
U.S.A. by the intravenous route. Twenty-three patients with
advanced solid tumors, no longer amenable to established therapies,
have been treated with PPMK107. Seventeen of these patients have
received a single dose for the initial treatment course. Six other
patients are receiving three doses per week for one week for the
initial treatment course. The sizes of each patient's tumors were
followed once per month. Patients with at least stable disease
(less than 25% increase and less than 50% decrease in the sum of
the products of all measurable tumors in the absence of any new
lesions) were eligible for additional treatment courses each
month.
[0277] Regression of a Palpable Tumor
[0278] A 68 year old female with colon carcinoma had a palpable
abdominal tumor among her widespread metastases. After a single IV
treatment with PPMK107, this patient experienced a 91% regression
of this single abdominal wall tumor over the course of two weeks
(Table 19). Measurements of the tumor one day after dosing
(3.75.times.3 cm) were similar to the baseline measurements of
4.times.3 cm. However, by day 7 post dosing, the tumor had
decreased in size to 2.times.2 cm and continued to decrease in size
to 1.5.times.1.5 cm by day 14 after PPMK107 dosing. Previous to
PPMK107 treatment, this tumor mass had been rapidly growing with a
1065% increase in tumor volume in the two weeks before PPMK107
dosing. This patient went off study because of increased growth of
the tumor elsewhere.
21TABLE 19 Size of Palpable Abdominal Wall Tumor in Patient #123
(68 year old Female with Metastatic Colon Carcinoma) After a Single
IV PPMK107 Dose of 12 Billion PFU/m.sup.2. % Tumor Tumor Volume
Reduction Time After Dimensions (0.5 .times. L .times. in Tumor
Date Dosing (L .times. W, cm.sup.3) W .times. W, cm.sup.3) Volume
Jul. 23, 1998 Day 0 4 .times. 3 18. -- Jul. 24, 1998 Day 1 3.75
.times. 3 16.9 6% Jul. 30, 1998 Day 7 2 .times. 2 4.0 78% Aug. 6,
1998 Day 14 1.5 .times. 1.5 1.7 91%
[0279] Stabilization of Cancer
[0280] Eight other patients, all of whom previously had tumor
progression with conventional cancer therapies, experienced benefit
in the form of stabilization of their advanced cancer after PPMK107
dosing. These patients with stable disease represent those with
diverse types of cancer including renal cancer, pancreatic cancer,
breast cancer and lung cancer. After three months of PPMK107
treatment, a 67 year old man with advanced and widely metastatic
renal cancer currently had stable disease with no indications of
any new growth and no indication of an increase in tumor size.
There has been a higher rate for stable disease benefit with higher
doses of PPMK107: Two out of 6 patients with stable disease (33% of
patients) at the first two single dose levels (5.9 and 12 billion
PFU per m.sup.2) and 4 out of 5 patients (80% of patients) with
stable disease at the highest single dose level (24 billion PFU per
m.sup.2 (Table 20).
22TABLE 20 Treatment of Patients with Advanced Cancer with PPMK107
# of Dose Patients # of % of Level Treated Patients Patients Types
of Cancer with Stable (Billion at this with with Disease for at
Least One PFU per Dose Stable Stable Month & Length of Stable
in.sup.2) Level Disease Disease Disease 5.9 6 2 33% Renal Cancer-
Ongoing 3 months Lung Cancer- Ongoing 2 months 12 6 2 33%
Pancreatic Cancer- Ongoing 2 months Ovarian Cancer- Ongoing 1 month
24 5 4 80% Breast Cancer- Ongoing 1 month Breast Cancer- Ongoing
month Lung Cancer- Ongoing 1 month Pancreatic Cancer- Ongoing 1
month Total 17 8 47% Noted Above.
[0281] Reduction in Pain Medication
[0282] One patient at the single dose 5.9 billion PFU/m.sup.2 dose
level benefited from PPMK107 treatment in the form of symptomatic
relief of cancer pain as denoted by a reduction in narcotic pain
medication.
[0283] Desensitization
[0284] A clear desensitizing effect from the first dose (at 5.9
billion PFU/m2) is seen on subsequent doses (also at 5.9 billion
PFU/m.sup.2) within the same week. In general, the reported side
effects from second and third doses have been much less. For
example, the first 4 patients in this multidose treatment regimen
(three doses per week for one week) had fever after the first dose
in spite of receiving prophylactic antipyretic treatment with
acetaminophen and ibuprofen. The majority of these patients had no
fever after receiving the second and third doses, even in cases in
which they did not receive antipyretics. This indicates that
administration of the first dose in the three times per week
schedule reduces the toxicity for the second and third doses.
[0285] Dosing Through Neutralizing Antibodies in Serum
[0286] Using the dose range in this phase I study (.gtoreq.5.9
billion PFU/m.sup.2), there is also clear indication that one can
effectively deliver virus to patients even if they have generated
neutralizing antibodies. A 72 year old woman with pancreatic cancer
at the 12 billion PFU/m.sup.2 single dose level has had stable
disease for 2 months since beginning PPMK107 treatment. A second
course (consisting of a single IV dose of PPMK107) was administered
one month after the first dose when the patient had produced
neutralizing antibodies in her serum. Seven days after this second
course, her urine was positive for PPMK107 at a titer of at least
40 PFU per mL. This result indicates that the neutralizing
antibodies to PPMK107 in this patient's serum was not able to
completely inhibit the virus with a second treatment course.
EXAMPLE 21
[0287] Summary of Cytotoxicity Assay Results with Newcastle Disease
Virus PPNJROAKIN
[0288] Human tumor cells and normal cells were grown to
approximately 80% confluence in 24 well tissue culture dishes.
Growth medium was removed and PPNJROAKIN, a plaque purified clone
of the mesogenic Newcastle disease virus strain New Jersey
Roakin-1946, was added in 10 fold dilutions ranging from 10.sup.7
plaque forming units (PFU)/well to 1 PFU/well. Controls wells with
no virus added were included on each plate. Virus was adsorbed for
90 minutes on a rocking platform at 37.degree. C. At the end of the
incubation period, the viral dilutions were removed and replaced by
1 ml of growth medium. Plates were then incubated for 5 days at
37.degree. C. in 5% CO2. Cytotoxicity was quantified by using a
calorimetric MTT (2-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium bromide) assay (Cell Titer 96, catalog #G4000, Promega
Corporation, Madison Wis. 53711) monitored at 570 nm, that detects
mitochondrial enzyme activity (Mosman, T., 1983, J. Immunol.
Methods 65:55). The viability in the virus treated wells was
expressed as a percent of the activity in untreated control wells.
The data was plotted graphically as PFU/well vs. viability as a
percent of control. The IC5O was calculated as the amount of virus
in PFU/well causing a 50% reduction in the amount of viable
cells.
23TABLE 21 Summary of Cytotoxicity Assay Results with PPNJROAKIN.
IC.sub.50 Cell Type Cell Line (PFU/well) Fibrosarcoma HT1080 13.8
Head and Neck KB 2.4 Carcinoma Normal Fibroblast CCD922sk 1.2
.times. 10.sup.4
[0289] These results (Table 21) show that PPNJROAKIN demonstrates
tumor-selective killing of at least two different human tumor cells
(HT1080 and KB) relative to normal skin fibroblasts. The IC50
values for the two tumor cell lines are between 800 and 5000-fold
lower than that for normal cells.
EXAMPLE 22
[0290] Summary of Cytotoxicity Assay Results with Newcastle Disease
Virus PPCONN70726
[0291] Human tumor cells and normal cells were grown to
approximately 80% confluence in 24 well tissue culture dishes.
Growth medium was removed and PPCONN70726, a plaque purified clone
of the mesogenic Newcastle disease virus strain Connecticut
70726-1946, was added in 10 fold dilutions ranging from 10.sup.7
plaque forming units (PFU)/well to 1 PFU/well. Controls wells with
no virus added were included on each plate. Virus was adsorbed for
90 minutes on a rocking platform at 37.degree. C. At the end of the
incubation period, the viral dilutions were removed and replaced by
1 ml of growth medium. Plates were then incubated for 5 days at
37.degree. C. in 5% C02. Cytotoxicity was quantified by using a
colorimetric MTT (2-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium bromide) assay (Cell Titer 96, catalog #G4000, Promega
Corporation, Madison Wis. 53711) monitored at 570 nm, that detects
mitochondrial enzyme activity (Mosman, T., 1983, J. Immunol.
Methods 65:55). The viability in the virus treated wells was
expressed as a percent of the activity in untreated control wells.
The data was plotted graphically as PFU/well vs. viability as a
percent of control. The IC50 was calculated as the amount of virus
in PFU/weIl causing a 50% reduction in the amount of viable
cells.
24TABLE 22 Summary of Cytotoxicity Assay Results with PPCONN70726.
Cell Type Cell Line IC.sub.50 (PFU/well) Head and Neck KB 18.1
Carcinoma Glioblastoma U87MG 12.7 Glioblastoma U373MG 879 Normal
Fibroblast U373MG 7.3 .times. 10.sup.4
[0292] These results (Table 22) show that PPCONN70726 demonstrates
tumor-selective killing of at least three different human tumor
cells (KB, U87MG, and U373MG) relative to normal skin fibroblasts.
The IC50 values for the two tumor cell lines are between 80 and
5000-fold lower than that for normal cells.
EXAMPLE 23
[0293] Intratumoral Treatment of HT1080 Fibrosarcoma Xenografts in
Athymic Mice Using PPMK107, PPNJROAKIN, or PPCONN70726
[0294] In this experiment, athymic mice (female, NCR nu/nu, 5 to 6
weeks old) received a subcutaneous injection of 10.sup.7 HT1080
tumor cells. Four days later when tumors reached a size range of 6
to 8.5 mm, mice were treated intratumorally with saline, PPMK107
(at 1.times.10.sup.8 PFU), PPNJROAK1N (at 1.times.10.sup.8 PFU), or
PPCONN70726 (at 1.times.10.sup.8 PFU). As shown in Table 23, tumor
regression was noted in mice treated with these three viruses
(PPMK107, PPNJROAKIN, and PPCONN70726). After PPMK107 treatment of
12 mice, four experienced complete tumor regression and six
experienced partial regression. After PPNJROAKIN treatment of 12
mice, one mouse experienced complete tumor regression and two
experienced partial regression. After PPCONN70726 treatment of 12
mice, three experienced complete tumor regression and two
experienced partial regression. No tumor regression was noted in
any of the animals treated with saline. These results show that the
three mesogenic strains of NDV can cause tumor regression.
25TABLE 23 Regression of HT1080 Fibrosarcoma Tumors in Athymic Mice
after Treatment with One of Three Viruses (PPMK107, PPNJROAKIN and
PPCONN70726) Each at a Dose of 1 .times. 10.sup.8 PFU. It of
Regression Treatment Mice Partial (PR) Complete (CR) PR + CR (%)
PPMK107 12 6 4 10 (83%) PPNJROAKTN 12 2 1 3 (25%) PPCONN70726 12 2
3 5 (42%) Saline 11 0 0 0 (0%)
EXAMPLE 24
[0295] Effects of PPMK107, PPNJROAKIN, PPCONN70726 After
Intracerebral Injection in Immunodeficient Athymic (nu/nu) and
Immunocompetent Heterozygote (nu/+) Mice
[0296] Fifty-six athymic mice (nu/nu) and 56 immunocompetent
heteroxygote (nu/+) mice were given stereotaxic intracerebral
injections with either saline, PPMK107, PPNJROAKIN, or PPCONN70726.
Eight additional mice of each type were used as untreated controls.
Viruses were used at one of two dose levels (2.times.10.sup.4 or
3.5.times.10.sup.6 PFU/mouse). As shown in Table 24, all of the
heterozygote nu/+ mice treated with each of the three viruses at
the two dose levels survived through day 39 with the exception of
one mouse at the lower PPCONN70726 dose level that was euthanized
for non-neurological symptoms. Athymic nu/nu animals treated with
either PPMK107 or PPCONN70726 had significantly less survival than
the heterozygotes. This was especially true for the highest PPMK107
or PPCONN70726 virus dose of 3.5.times.10.sup.6 PFU/mouse where
only 13% (1 of 8) of the athymic animals in each virus group
survived through day 39. In contrast, there was 75% survival of the
PPNJROAKIN-treated athymic mice at this same dose level
(3.5.times.10.sup.6 PFU/mouse). These data indicate that PPNJROAKIN
is better tolerated in the brains of athymic mice than the other
two virus strains.
26TABLE 24 Survival of Mice Following Intracerebral Injection of
PPMK107, PPCONN70726, and PPNJROAKIN % Survival Intracranial
Injection # of Mice at Day 39 nu/+ Untreated 8 100 nu/+ Saline 8
100 nu/+ PPMK107, 2E+04 8 100 nu/+ PPMK107, 3.5E+06 8 100 nu/+
PPCONN70726, 2E+04 8 88 * nu/+ PPCONN70726, 3.5E+06 8 100 nu/+
PPNJROAKIN, 2E+04 8 100 nu/+ PPNJROAKIN, 3.5E+06 8 100 nu/nu
Untreated 8 100 nu/nu Saline 8 100 nu/nu PPMK107, 2E+04 8 75 nu/nu
PPMK107, 3.5E+06 8 13 nu/nu PPCONN70726, 2E+04 8 75 nu/nu
PPCONN70726, 3.5E+06 8 13 nu/nu PPNJROAKiN, 2E+04 8 100 nu/nu
PPNJROAKIN, 3.5E+06 8 75 * The one non-surviving mouse in this
treatment group was euthanized for non-neurological symptoms.
EXAMPLE 25
[0297] Summary of Cytotoxicity Assay Results with Sindbis Virus
PPSINDBIS-Ar339
[0298] Human tumor cells and normal cells were grown to
approximately 80% confluence in 24 well tissue culture dishes.
Growth medium was removed and PPSINDBIS-Ar339, a plaque purified
clone of Sindbis Ar-339 was added in 10 fold dilutions ranging from
10.sup.7 plaque forming units (PFU)/well to 1 PFU/well. Controls
wells with no virus added were included on each plate. Virus was
adsorbed for 90 minutes on a rocking platform at 37.degree. C. At
the end of the incubation period, the viral dilutions were removed
and replaced by 1 ml of growth medium. Plates were then incubated
for 5 days at 37.degree. C. in 5% C02. Cytotoxicity was quantified
by using a colorimetric MTT (2-[4,5-dimethylthiazol-2-yl]-2,5--
diphenyl tetrazolium bromide) assay (Cell Titer 96, catalog #G4000,
Promega Corporation, Madison Wis. 53711) monitored at 570 nm, that
detects mitochondrial enzyme activity (Mosman, T., 1983, J.
Immunol. Methods 65:55). The viability in the virus treated wells
was expressed as a percent of the activity in untreated control
wells. The data was plotted graphically as PFU/well vs. viability
as a percent of control. The IC50 was calculated as the amount of
virus in PFU/well causing a 50% reduction in the amount of viable
cells.
27TABLE 25 Summary of Cytotoxicity Assay Results with
PPSINDBIS-Ar339 Cell Type Cell Line IC.sub.50 (PFU/well) Pancreatic
Panc-1* 69 Carcinoma Colorectal SW620* 13 Carcinoma Colorectal
SW1463 1.8 .times. 10.sup.5 Carcinoma Non-small cell Lung A427
>1 .times. 10.sup.6 carcinoma Non-small cell Lung A549 5.2
.times. 10.sup.4 carcinoma Renal carcinoma A498 2.4 .times.
10.sup.4 Renal carcinoma Caki-1 3.4 .times. 10.sup.4 Fibrosarcoma
HT1080 7.4 .times. 10.sup.5 Normal Keratinocyte NHEK 2.0 .times.
10.sup.5 Normal Fibroblast CCD922sk 1.6 .times. 10.sup.5 *Cells
known to overexpress the mRNA for the high affinity laminin
receptor.
[0299] The cellular receptor for Sindbis virus on mammalian cells
is the high affinity laminin receptor, that is expressed mainly on
cells of epithelial lineage, but is often overexpressed in many
metastatic cancer cells like the Panc-1 pancreatic carcinoma line,
and the SW620 colon adenonocarcinoma cell line (Campo et al.,
(1992) Am. J. Pathol. 141, 1073-1083; Yow et al., (1988) Proc. Natl
Acad Sci. 85, 6394-6398). In contrast, the rectum adenocarcinoma
cell line SW1423 is known to express very low levels of high
affinity laminin receptor mRNA (Yow et al., (1988) Proc. Natl Acad
Sci, 85, 6394-6398), and is more than 4 order of magnitude more
resistant to killing by PPSINDBIS-Ar339 than SW620 cells. These
results (Table 25) demonstrate that cells that are tumorigenic and
express high levels of the high affinity laminin receptor are more
sensitive to killing by Sindbis Clone PPSINDBIS-Ar339 than other
tumor or normal cells.
EXAMPLE 26
[0300] VSV Killing of Tumorigenic and Non-tumorigenic Cells in the
Presence of Interferon
[0301] In 96 well plates, tumorigenic KB and HT1080 cells
(3.times.10.sup.4 cells per well) and non-tumorigenic WISH cells
(2.5.times.10.sup.4 cells per well) were seeded in the presence of
serially diluted interferon-.alpha. ranging from 2800 to 22
Units/ml and allowed to incubate for 24 hours at 37.degree. C. The
cells were then infected with vesicular stomatitis virus (VSV,
Indiana strain) at an moi of 10. Controls were included for cells
without interferon, and cells without interferon or virus. The
cells were incubated at 37.degree. C. for 24 hours. Cytotoxicity
was quantified by using a colorimetric MTT
(2-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)
assay (Cell Titer 96, catalog #G4000, Promega Corporation, Madison
Wis. 53711) monitored at 570 nm, that detects mitochondrial enzyme
activity (Mosman, T., 1983, J. Immunol. Methods 65:5 5). The
viability in the virus treated wells was expressed as a percent of
the activity in control wells not receiving virus.
28TABLE 26 Comparison of the Cell Killing Activity of VSV in Cells
Treated with Exogenous Interferon. Percent Viable Cells WISH HT1080
KB 100 U/ml IFN 50 6 0 1000 U/ml IFN 95 20 12
[0302] These results (Table 26) demonstrate that VSV is able to
selectively kill tumor cells deficient in interferon responsiveness
(see Example 27). WISH cells (human amnion cells) are a well
established cell line for the use in interferon bioassays because
of their ability to respond efficiently to interferons.
EXAMPLE 27
[0303] Interferon Responsiveness in Cells Sensitive or Resistant to
Killing by PPMK107
[0304] Individual cell lines were grown to near confluence in 96
well microtiter plates and treated with between 5 and 5000 U/ml of
IFN.alpha.A for 24 hours. The cultures were then infected with
PPMK107 at an moi of 1.0 and cultured for an additional 24 hours.
Following chemical fixation, the amount of viral expression was
quantified by immunohistochemistry using a soluble indicator dye.
The amount of virus growth is represented as the percent of P
antigen expressed relative to control cells untreated with
interferon (FIG. 5). In this assay, interferon responsive cells
manifest at least a 50% decrease in the viral antigen in response
to interferon. Cells in FIG. 5 that are sensitive to PPMK107 are
indicated by the solid lines; cells less sensitive are indicated by
the dashed lines.
[0305] The results of this experiment show a strong correlation
between the resistance of the cell line to the antiviral effects of
exogenous interferon and the relative sensitivity of the cell to
killing by PPMK107 (indicated by the IC50 value shown in
parentheses next to the cell line name in the graph legend, see
FIG. 5). For example, following pretreatment with 5 U/ml of
interferon, 6 of 7 (86%) cell lines nonresponsive to interferon are
sensitive to killing by PPMK107; when pretreated with 500 U/ml of
interferon, all (4 of 4) of the nonresponsive cell lines are
sensitive to killing by PPMK107.
[0306] The data above also present an example of a screening assay
to identify candidate cells that are likely to be sensitive to
killing by PPMK107 or other interferon-sensitive viruses. For
example, infected cells expressing significant (e.g., more than 50%
of controls) viral antigen following pretreatment with exogenous
interferon would be considered interferon deficient and thereby
sensitive to viral oncolysis.
[0307] The foregoing is intended as illustrative of the present
invention but not limiting. Numerous variations and modifications
may be effected without departing from the true spirit and scope of
the invention.
* * * * *