AU2002325406B2 - Treatment of Neoplasms with Viruses - Google Patents

Description

TREATMENT OF NEOPLASMS WITH VIRUSES Field of the Invention 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 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 i tumor burden. (See Hillard Stanley, Cancer Treat. Reports, Vol. 61, No. 1, Jan/Feb 1977, p.29- 36, Tannock, Cancer Research, 42, 4921-4926, Dec. 1982).

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).

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 cancer patients intentionally infected with live mumps virus, partial tumor reression was noted in 79 cases. Se Asada (1994) Caer, 34, 1907-1928. While the side effects of these viruses were temporary, serious sequela of infection with these human pathogens is of major concern.

Viruses are categorized as follows [see Murphy A and Kingsbury DW, 1990, In: Virolo., 2 d Edition (Ed. Fields, Raven Press, New York, pp 9-35]: Dividing Characteristics RNA viruses ssRNA, positive-sense, rO nonsegmented, nonenveloped, ssRNA, positive-sense, nonsegmented, enveloped, S ssRNA, negative-sense, nonsegmented, enveloped, ssRNA, negative-sense, segmented, enveloped ssRNA, ambisense, segmented, enveloped dsRNA, positive-sense segmented, nonenveloped 2. ssRNA, DNA step in replication, positivesense, nonsegmented, enveloped Virus Family Names Picornaviridae, Calciviridae Togaviridae. Flaviviridae, Coronaviridae Rhabdoviridae, Filoviridae, Paramyxoviridae Orthomvxoviridae Bunvaviridae. Arenaviridae Reoviridae, Birnaviridae Retroviridae DNA viruses ss/dsDNA, nonenveloped Hepadnaviridae ssDNA, nonenveloped Parvoviridae dsDNA. nonenveloped Papovaviridae, Adenoviridae dsDNA, enveloped Herpesviridae, Poxviridae, Iridoviridae Included among the family Herpesviridae (or Herpesviruses), are the subfamilies Alphaherpesvirus (including Genus Varicellavirus and Genus Simpexvirus), Betaherpesvirus, and Gammaherpesvirus.

Newcastle disease virus is a member of the Paramyxoviridae (or Paramyxoviruses).

110 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 HTLV-1, Hepatitis Paramyxoviruses are not known to be carcinogenic.

Temporary regression of tumors has been reported in a small number of patients exposed to NDV, See, Csatary, L.K. (1971) Lan.t, 2, 825. Csatary noted the regression of a gastrointestinal cancer in a chicken farmer during an epidemic of Newcastle disease in his .0 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 rumor. Since the mechanism of rumoricidal activity was thought to be immunologic, no work was carried out to address direct tumor cytotoxicity of the virus. Instead, efforts focused upon the immunomodulating effects of NDV.

See, for example, Murray, Cassel, Torbin, Olkowski, Moore, M.E.

(1977) Cancer, 40, 680; Cassel, Murray, Phillips, H.S. (1983) Cancer, 52, 856; Bohle, Schlag, PJ., Liebrich, Hohenberger, Manasterski, Miller, and Schirrmacher, V. (1990) Cancer, 66, 1517-1523.

The selection of a specific virus for tumor regression was based on serendipity or trial and error 3 in the above citations. Only recently, have rational, mechanism-based approaches for virus use 4 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, JR. 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, WW, 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, et al, 1994 Cancer Res., 54:3963-3966.

International Patent Publication No. WO 94/25627, hereby incorporated by reference in its entirety, discloses the use of NDV or other Paramyxovirus in the treatment of cancer.

Viral IFN transgene expression 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.

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.

In Zhang et al. ((1996) Proc. Natl. Acad. Sci., USA 93:4513-4518), a recombinant adenovirus expressing a human interferon consensus synthetic) gene was used to treat human breast cancer (and other) xenografts in nude mice. The authors concluded 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 al. (1996) teach the use of an interferon-resistant adenovirus in the treatment of tumors.

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.

[R:\LIBZZ]04705.doc: rr 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 rechallenged with tumor cells, the mice immunized with virally modified cells had statistical improvement in the disease-free survival time.

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.

Restifo et al. ((1992) J Exp. Med., 175:1423-1431), used IFN gamma-expressing retroviral io 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 Is also less rumorigenic in mice as compared to the non-transduced parent line, and resulted in activation of a tumor-specific CTL response in vivo.

Use of Therapeutic Doses of Interferon as an Adjuvant to Viral Cancer Therapy 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.

In Kirchner et al. ((1995) World J. Uro., 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 x 10' to 2.2 x 10' PFU/kg. This was a local therapy, as opposed to a systemic approach, with the goal of inducing an anti-tumor immune response.

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 -4those that did not. The authors attributed efficacy of IFN to the induction of CD8-positive T cells in those animals.

Arroyo et al. ((1990) Cancer Immunol. Immunother., 31:305-311) used a mouse model of colon cancer to test the effect ofIFN 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 IFN was used in these studies to augment the ability of the cancer cells to be recognized by the immune system.

>O Objects of the Invention It is an object of the invention to provide viruses for the treatment of diseases including cancer.

It is a further object of the invention to provide viruses for the treatment of neoplastic diseases including cancer.

It is a further object of the invention to provide a means by which candidate viruses are selected i S and/or screened for use in the therapy of neoplastic diseases.

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.

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.

It is a still further object of this invention to provide guidance in the management of viral therapy.

It is an object of the invention to provide a method for treating large tumors.

It is a further object of the invention to provide purified virus and methods for obtaining same.

7 Summary of the Invention The present invention aims to achieve at least one of the stated objects.

There is described herein 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.

There is also described herein a method of infecting a neoplasm in a mammal with a virus comprising systemically administering an interferon-sensitive, replicationcompetent clonal virus to the mammal.

There is also described herein 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.

There is also described herein 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 Bl8R, to the mammal.

There is also described herein 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 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.

There is also described herein 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(l) RNA viruses; Hepadenavirus; Parvovirus; Papovavirus; Herpesvirus; Poxvirus; and Iridovirus, to the mammal.

There is also described herein 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 RNA viruses; Hepadenavirus; (3) Parvovirus; Papovavirus; Herpesvirus; Poxvirus; and Iridovirus. wherein the neoplasm is at least 1 centimeter in size.

There is also described herein a method of treating a tumor in a mammal, comprising administering to the mammal a therapeutically effective amount of an RNA [R:\LIBZZ]04406doc:mrr virus cytocidal to the tumor, wherein the mammal has a tumor burden comprising at least 1.5% of the total body weight.

There is also described herein 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.

There is also described herein a method for identifying a virus with antineoplastic activity in a mammal comprising a) using the test virus to infect i) cells deficient in IFNmediated antiviral activity, and ii) cells competent in IFN-mediated antiviral activity, and to 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.

There is also described herein 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.

There is also described herein 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, Hepadenaviruses, Varicellaviruses, Betaherpesviruses, and Ganunmmaherpesviruses comprising administering an antiviral compound.

There is also described herein a method of treating or infecting a neoplasm in a mammal comprising subjecting a sample 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.

There is also described herein a method of infecting a neoplasm in a mammal with a virus comprising systemically administering a desensitizing dose of an interferonsensitive, replication-competent clonal virus to the mammal.

There is also described herein 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.

There is also described herein a method of infecting a neoplasm in a mammal with a virus comprising administering a replication-competent clonal virus selected from the ER:ULBZZ]04406.doc:rr group consisting of the Newcastle disease virus strain MK107, Newcastle disease virus stain NJ Roakin, Sindbis virus, and Vesicular stomatitis virus.

Included in the methods described herein are: i) a Paramyxovirus purified by ultracentrifugation without pelleting; ii) a Paramyxovirus purified to a level of at least 2 x 109 PFU per mg of protein; iii) a Paramyxovirus purified to a level of at least 1 x 10 1 0 PFU per mg of protein; iv) a Paramyxovirus purified to a level of at least 6 x 1010 PFU per mg of to protein; v) an RNA virus purified to a level of at least 2 x 109 PFU per mg of protein; vi) an RNA virus purified to a level of at least I x 1010 PFU per mg of protein; vii) an RNA virus purified to a level of at least 6 x 1010 PFU per mg of protein; viii) a cytocidal DNA virus which is interferon-sensitive and purified to a level of at least 2 x 10 9 PFU/mg protein; ix) a replication-competent vaccinia virus having a) one or more mutations in one or more of the K3L, E3L and Bl 8R 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; 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; xi) a Herpesvirus having a modification in the expression of the analog causing the Herpesvirus to have increased interferon sensitivity; and xii) a Reovirus having an attenuating mutation at omega 3 causing said virus to become interferon-sensitive.

Also described herein are the following methods: so 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 chromatography, and [R:\LIBZZ]4406doc:mrr 22, NOV, 2305 16:12 SPRUSON AND FERGUSON 6'H2615486 NO. 7727 01 o ii) a method of purifying a Paramyxovirus comprising purifying the virus by ultracentrifugation without pelleting. or by tangential flow filtration with or without 0 subsequent gel permeation chromatography.

There is also described herein 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- Isensitive, replication-competent, clonal virus.

Accordingly, in a first embodiment of the invention there is provided a method of Cinfecting a neoplasm in a mammal with a virus comprising administering an interferonlo sensitive, replication-competent clonal RNA virus to said mammal, wherein said RNA virus replicates at least 10 fold less in the presence of interferon compared to in the Cabsence of interferon.

According to a second embodiment of the invention there is provided 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 R.NA virus, where said RNA virus replicates at least 10 fold less in the presence of interferon compared to in the absence of interferon.

According to a third embodiment of the invention there is provided an interferonsensitive, replication-component elonal RNA virus when used for treating a neoplasm in a 2o mammal, wherein said RNA virus replicates at least 10 fold less in the presence of interferon compared to in the absence of interferon.

According to a fourth embodiment of the invention there is provided use of an interferon-sensitive, replication-component clonal DNA virus for the manufacture of a medicament for the treatment of a neoplasm in a mammal, wherein said RNA virus replicates at least 10 fold less in the presence of interferon compared to in the absence of interferon.

Also disclosed herein is 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 RNA viruses; Hepadenavirus; (3) Parvovirus; Papovavirus; Herpesvirus; Poxvirus; and Iridovirus, to said mammal.

Also disclosed herein is a method of treating a neoplasm in a mammal, comprising administering to said mammal a therapeutically effective amount of a clona virus selected from the group consisting of COMS ID No: SBMI-02015948 Received by IP Australia: Time 16:23 Date 2005-11-22 lOa- RNA virus; Hepadenavirus; Parvovirus; Papovavirus; Herpesvirus; Poxvirus; and Iridovirus, wherein said neoplasm is at least 1 centimeter in size.

fRA I !k7Z1O44O6.doc:THR 22, NOV. 2305 16:12 SPRUSON AND FERGUSON 6:2£2615486 NO. 7727 F, 11 11 o Also disclosed herein is a clonal virus selected from the group consisting of (1) RNA virus; Hepadenavirus; Parvovirus; Papovavirus; Herpesvirus; (6)

O

z Poxvirus; and Iridovirus, when used for treating a neoplasm in a mammal, wherein C,1 said neoplasm is at least 1 centimeter in size.

C s Also disclosed herein is use of a clonal virus selected from the group consisting of RNA virus; Hepadenavirus; Parvovirus; Papovavirus; Herpesvirus; (6) NO Poxvirus; and Iridovirus, for the manufacture of a medicament for treating a neoplasm in a mammal, wherein said neoplasm is at least I centimeter in size.

Cl Also disclosed herein is a method of treating a tumor in a mammal, comprising

C

C to administering to said mammal a therapeutically effective amount of an RNA virus Scytocidal to said tumor, wherein said mammal has a tumor burden comprising at least of the total body weight of said mammal.

Also disclosed herein is an RNA virus cytocidal to a tumor when used for treating said tumor in a mammal, wherein said mammal has a tumor burden comprising at least is 1.5% of the total body weight of said mammal.

Also disclosed herein is use of an RNA virus cytocidal to a tumor for the manufacture of a medicament for treatment of said tumor in a mammal having a tumor burden comprising at least 1.5% of the total body weight of said mammal.

Also disclosed herein is a method of making viruses for use in antineoplastic therapy comprising: modifying an existing virus by diminishing or ablating a viral mechanism for the inactivation of the antiviral effects of IFN.

Also disclosed herein is a virus for use in antineoplastic therapy when made according to the method of the twelfth embodiment.

Also disclosed herein is a pharmaceutical composition comprising a virus according to the thirteenth embodiment, together with a pharmaceutically acceptable carrier or adjuvant.

Also disclosed herein is 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, [R U3BZZ104406 don TR COMS ID No: SBMI-02015948 Received by IP Australia: Time 16:23 Date 2005-11-22 1 la Hepadenaviruses, Varicellaviruses, Betaherpesviruses, and Gammaherpesviruses comprising administering an antiviral compound.

SAlso disclosed herein is an antiviral compound when used in controlling viral 0 replication in a mammal treated with a virus selected from the group consisting of RNA viruses, Adenoviruses, Poxviruses, Iridoviruses, Parvoviruses, Hepadenaviruses, IN Varicellaviruses, Betaherpesviruses, and Gammaherpesviruses.

Also disclosed herein is the use of an antiviral compound for the manufacture of a medicament for controlling viral replication in a mammal treated with a virus selected N from the group consisting of RNA viruses, Adenoviruses, Poxviruses, Iridoviruses, O 10 Parvoviruses, Hepadenaviruses, Varicellaviruses, Betaherpesviruses, and Gammaherpesviruses.

Also disclosed herein is a Paramyxovirus purified by ultracentrifugation without pelleting.

Also disclosed herein is a Paramyxovirus purified to a level of at least 2 x 10 9 PFU/mg protein.

Also disclosed herein is a Paramyxovirus purified to a level of at least 1 x 1010 PFU/mg protein.

Also disclosed herein is a Paramyxovirus purified to a level of at least 6 x 1010 PFU/mg protein.

Also disclosed herein is an RNA virus purified to a level of at least 2 x 109 PFU/mg protein.

Also disclosed herein is a replication-competent cytocidal virus which is interferonsensitive and purified to a level of at least 2 x 109 PFU/mg protein.

According to a fifteenth embodiment of the invention there is provided 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 binds said receptor, to said mammal.

According to a sixteenth embodiment of the invention there is provided an interferon-sensitive, replication-competent clonal virus when used in a method of treating a neoplasm according to the fifteenth embodiment.

[R:\LIBZZ104406.doc:THR 1 llb O According to a seventeenth embodiment of the invention there is provided use of an .C interferon-sensitive, replication-competent clonal virus for the manufacture of a

(D

Smedicament for treatment of a neoplasm in a mammal, wherein said RNA virus replicates 0 0 at least 10 fold less in the presence of interferon compared to in the absence of interferon.

Also disclosed herein is a method of purifying an RNA virus comprising the steps IN0 of: a) generating a clonal virus, and Sb) purifying said clonal virus by ultracentrifugation without pelleting.

C1 Also disclosed herein is an RNA virus purified by the above method.

Also disclosed herein is a method of purifying a Paramyxovirus comprising purifying said virus by ultracentrifugation without pelleting.

Also disclosed herein is a Paramyxovirus purified by the above method.

Also disclosed herein is a method of infecting a neoplasm in a mammal with a virus comprising administering an interferon-sensitive, replication-competent RNA virus to said mammal.

Also disclosed herein is 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 NJ Roakin, Sindbis virus, and Vesicular stomatitis virus.

According to an eighteenth embodiment of the invention there is provided a clonal virus selected from the group consisting of the Newcastle disease virus strain MK107, Newcastle disease virus strain NJ Roakin, Sindbis virus, and Vesicular stomatitis virus when used for infecting a neoplasm in a mammal, wherein said virus replicates at least fold less in the presence of interferon compared to in the absence of interferon.

According to a nineteenth embodiment of the invention there is provided use of a clonal virus selected from the group consisting of the Newcastle disease virus strain MK107, Newcastle disease virus strain NJ Roakin, Sindbis virus, and Vesicular stomatitis virus for the manufacture of a medicament for infecting a neoplasm in a mammal, wherein said virus replicates at least 10 fold less in the presence of interferon compared to in the absence of interferon.

Brief Description of the Drawings Figure 1 shows the effect of anti-interferon-beta antibody on viral antigen expression and infectious titer in NHEK (normal human epithelial keratinocytes) cells.

[R:\LIBZZ]04406.doc:THR lie Figure 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).

Figure 3A shows the effect of interferon on viral antigen expression in CCD922-sk cells, and Figure 3B shows the effect of interferon on viral antigen expression in KB cells.

Figure 4 shows the survival curves for athymic mice bearing human ES-2 ovarian carcinoma cells and treated with either saline or NDV strain PPMK 107.

Figure 5 shows the interferon responsiveness of a number of human tumor and normal cell lines.

Detailed Description-of the Invention There is also described herein a novel mechanism by which viral replication selectively kills neoplastic cells deficient in an interferon (IFN)-mediated anti-viral response. There is also described herein methods for selection, design, purification, and use of viruses for the treatment of neoplastic diseases including cancer and large tumors.

The viruses described herein 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.

There is also described herein use of paramyxoviruses such as NDV, and other viruses, for use in the treatment of diseases including neoplastic disease such as cancer.

There is also described herein screening and engineering of other viruses suitable for use as therapeutics of neoplastic diseases. There is also described herein a method of identifying tumor tissues that are candidates for viral therapy. Finally, there is also described herein the preparation of highly purified virus.

Rationale for the use of interferon-sensitive viruses including NDV to treat neoplastic disease NDV demonstrates selective killing of tumor cells.

Newcastle disease virus causes selective cytotoxic effects against many human tumor cells with markedly less effects on most normal human cells. In a differential cylotoxicity 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 to be approximately 3 to 4 orders of magnitude more sensitive to NDV than [R:ULIBZZ]04406 dac:nrr lid many normal human cells [renal epithelial cells, fibroblasts, keratinocytes, melanocytes, and endothelial cells (see Example The differential cytotoxicity assay can also be applied to fresh isolates from the patient's cells or tumor tissue.

[R:\LIBZZj406 doc:nur

I

An in vitro assay is used to define the tumoricidal activity ofNDV 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 rumor 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.

Evidence that NDV is specifically replicating within the tumor was demonstrated by immunohistochemical staining for virus antigen (Example Within 30 minutes of intratumoral t0 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.

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, et al, 1994 J NatlCancer nst, 86:1228-1233).

NDV can also cause regression of large tumors after intratumoral and intravenous administration (Examples 4 through Intratumoral NDV treatment of large intradermal A375 human melanoma xenografts (210 mm in maximal dimension; tumor volume of 2300 mm in athymic mice lead to high rates of tumor regression (Examples 4 through Intravenous NDV treatment of large subcutaneous HT1080 human fibrosarcoma xenografts 10 mm in maximal dimension) in athymic mice lead to complete or partial tumor regression in five out of six mice (Example 9).

The class I interferon family of cytokines are important negative modulators of viral 2S infection.

The class I interferons consist of the IFNta, found primarily in cells of hematopoietic origin, and IFNp 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 Ld., New York: and Sreevalsan, T. 1995. Biological Therapy with Interferon-a and P: 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, 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).

A wide variety of tumor cells are deficient in the ability to limit viral infection through an IFN-dependent mechanism.

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 nontransformed fibroblast control cell line (Maheshwari 1983. Biochem, Biophys. Res.

IS 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 Moreover, although IFN was being secreted by the normal cells into the culture medium, 2. 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 2.S 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 100fold 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).

Interferon and Cell Growth There are several species of interferon (IFN) including natural and recombinant forms of a-IFN, P-IFN, o-IFN, and y-IFN as well as synthetic consensus forms as described in Zhang et al.

/13 (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 rowth 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 tumorsuppresser 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, CD, et al, 1991, Cancer Res., 51:1684cO 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. et al, 1994, Blood, 84:1942-1949), and malignant melanoma cells, (Linge, C., et al, 1995. Cancer Res., 55:4099-4104).

The IFN-inducible protein kinase (p68) has been shown to be an important regulator of cellular Ss- 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, 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 .O 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 GN 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 MJ, et al., 1996 Gene 170:181-188).

S Sensitivity to viral killing in undifferentiated cells can be reversed through the induction of a more differentiated phenotype (Kalvakolanu, DVR and Sen. G.C. 1993 Proc Natl Acad Sci USA 90:3167-3171).

Definitions 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 10 units per ml) of exogenous interferon by significantly reducing (at least

/V/

I

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 theamount of virus infectious virus, viral antigen, viral nucleic acid). CCD922 normal fibroblasts are cells competent in an interferon-mediated antiviral response.

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 10 units per ml) of exogenous interferon by significantly reducing lO 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.

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 more advantageously greater than 97%, more advantageously greater than 99%, and most advantageously 100% over 300 contiguous nucleotides.

Cytocidal. As used herein, the term "cytocidal" virus refers to a virus that infects cells resulting in their death.

Desensitizing Doseg 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.

Differential Cvtotoxicitv Assay. As used herein, the phrase "differential cytotoxicity assay" for screening tumor cells or tissue using a virus refers to the virus infection of the tumor cells and one or more control cells or tissue; 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 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).

I

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.

Interferon-sensitive. As used herein, the phrase "interferon-sensitive" virus 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 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 10 units per ml).

%O Neoplasm and Neoplastic Disease. As used herein, "neoplasm" means new growth of tissue, including tumors, benign growths condylomas, papillomas) and malignant growths cancer). As used herein, "neoplastic disease" refers to disease manifested by the presence of a neoplasm.

Replication Competent. As used herein, the term "replication-competent" virus refers to a i" virus that produces infectious progeny in neoplastic cells.

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 using 1.7 x 10 9 PFU of virus per well (3.3 cm in width) run on an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel (1 mm thick); transferring the viral proteins from the gel to a nitrocellulose membrane; and immunostaining for ovalbumin with the use of a rabbit antiovalbumin [Rabbit IgG fraction at a 1:200 dilution of a 4 mgiml antibody concentration (from Cappel, Inc.) or equivalent polyclonal antibody].

Therapeutically effective amount. As used herein, the term "therapeutically effective amount" 2. when referring to the treatment of neoplastic disease refers to a quantity of virus which produces the desired effect, cessation of neoplastic growth, tumor regression, improved clinical conditions, or increased survival.

.Compounds of the Invention A diverse group of viruses are used to selectively kill neoplastic cells. Natural or engineered viruses can function as an antinebplastic agent. These viruses 1) infect neoplastic cells resulting in their death; ii) are replication-competent in the neoplastic cells; and iii) are limited in killing s of normal cells by the antiviral effects of interferon.

In an advantageous embodiment of the invention, the viruses possessing the above three characteristics they infect neoplastic cells resulting in their death; (ii) they are replicationcompetent in the neoplastic cells; and (iii) they are limited in killing of normal cells by the antiviral effects of interferon] also induce interferon.

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.

In another advantageous embodiment, the viruses possessing the above three characteristics are cytocidal to tumor cells, IS 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; measles-like viruses (morbilli viruses); and respiratory syncyrial viruses (pneumoviruses). These viruses contain an RNA genome. Use of Paramyxoviridae viruses which are cytocidal, especially paramyxoviruses, 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.

NDV is an especially advantageous virus according to the present invention. NDV is 2S 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, 0 Hanson and Brandly, 1955 (Science, 122:156-157), and Dardiri et al., 1961 (Am. J. Vet.

/7

I

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 Virol., 2:40-47) for a listing of other mesogenic strains.

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 the number of particles per PFU).

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, 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 CS recombinant DNA techniques applicable by one skilled in the art. Another means of obtaining a clonal virus applies the technique of limiting dilution 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).

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): Purification Method A a) generating a clonal virus plaque purification) b) inoculating eggs with the clonal virus 3S c) incubating the eggs d) chilling the eggs e) harvesting the allantoic fluid from the eggs f) removing cell debris from the allantoic fluid g) ultracentrifugation of the allantoic fluid without pelleting using a discontinuous sucrose gradient) /f 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: freezing then thawing the allantoic fluid, removing contaminating material from the virus suspension by means of centrifugation) In another embodiment of the invention, ultracentrifugation is accomplished by means of a continuous flow ultracentrifuge.

One embodiment of the invention relates to a method of purifying a replication-competent RNA virus comprising the steps of: a) generating a clonal virus, and b) purifying said clonal virus by ultracentrifugation without pelleting.

Another embodiment of the invention involves a method of purifying a paramyxovirus NDV) comprising purifying the virus by ultracentrifugation without pelleting.

Optionally, the purifying step additionally comprises prior to the ultracentrifugation: S" a) plaque purifying to generate a clonal virus, b) inoculating eggs with the clonal virus, c) incubating the eggs, d) chilling the eggs, e) harvesting allantoic fluid from the eggs and, f) removing cell debris from the allantoic fluid.

Another embodiment of the invention involves a method of purifying a replicationcompetent clonal virus from eggs or cell culture comprising the step of ultracentrifugation without a step in which the virus is pelleted.

Another embodiment of the invention involves a method of the purifying a 2" paramyxovirus 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 a) plaque purifying to generate a clonal virus, /9

I

b) inoculating eggs with the clonal virus, c) incubating the eggs.

d) chilling the eggs, e) harvesting allantoic fluid from the eggs and dilution of allantoic fluid with buffer, f) removing cell debris from the allantoic fluid by TFF, g) purification of the virus by TFF, and h) purification of the virus by gel permeation chromatography.

Optionally, the virus obtained from the gel permeation step can be concentrated using TFF.

1o 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.

Clonal virus (S Use of these methods permits purification of a clonal virus [including Paramyxovirus NDV)] to at least 2 x 10 9 PFU/mg protein, advantageously to at least 3 x 10 9 PFU/mg protein, more advantageously to at least 5 x 10 9 PFU/mg protein, more advantageously to at least 1.0 x 10'° PFU/mg protein, more advantageously to at least x 1010 PFU/mg protein, more advantageously to at least 3 x 101 PFU/mg protein, more advantageously to at least 4 x 1010 PFU/mg protein, more advantageously to at least 5 x 1010 PFU/mg protein, and most advantageously at least 6 x 10' 0 PFU/mg.

Use of these methods permits purification of a clonal virus [including Paramyxovirus NDV)] to level in which the number of virus particles per PFU is less than 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 2 C per PFU indicate a higher degree of purity.) RNA viruses In another embodiment, these methods permit purification (to the levels cited above for clonal viruses) of an RNA virus [including a cytocidal RNA virus; a singleo2tA stranded RNA non-segmented, nonenveloped virus: a single-stranded RNA segmented, enveloped virus: a double-stranded RNA segmented, nonenveloped virus; and a single-stranded RNA non-segmented, enveloped virus Paramyxovirus NDV) and Retroviruses].

s DNA viruses 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 enveloped, double-stranded DNA viruses (including poxviruses); (b) nonenveloped, single-stranded DNA viruses; and nonenveloped, double-stranded ,0 DNA viruses.

Egg derived viruses 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.

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.

a./ Table 1 Naturally Occurring Viruses for Use in Cancer Therapy Virus Class____ Virus Family 1Virus Example RNA. negative stranded Pararnyxovinidas Newcastle Disease Virus Avan Pramxovirus Type 2 S- -Human Parainfluenza Rhabdovindae Vesicular Stomnaitis Virus RNA, positive stranded Togavcridae Sindbis Virus iYellow Fever Virus (attenuated) [Rhinovirus I Bovine Enterovirus I Echovirus Coronaviridae fAvian Infectious Bronchitis Virus lHuman Coronaries &Zc:- 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, 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 interferonmediated 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 A significant reduction 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 i C 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 An understanding of the factors involved in the establishment of an antiviral state allows for the Is 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 ofc-Myc, c- Myb or p 5 8, high level expression indicates that the tumor tissue or cells are treatment candidates for virus therapy. In the case of p 6 8, IRF- I, ISGF-3, and IFN receptors, low level expression indicates that the tumor tissue or cells are treatment candidates for virus therapy.

In another embodiment of this invention, primary rumor tissue or cells obtained from patient biopsies are expanded in culture and tested for sensitivity to killing by a suitable viral therapy.

aS 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 10 units per ml, see Example 27).

i^ 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 iS 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 orders of magnitude more resistant to killing by PPSINDBIS-Ar339 than SW620 cells.

Existing strains of NDV, or other viruses including RNA and DNA viruses, are screened or engineered for altered IFN responses 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 Sindbis virus PPSINDBIS-Ar339, see Example 25), or low neurovirulence are included in the subject invention NDV virus PPNJROAKIN, see Example 24). Advantageously, viruses of the invention have the capacity to spread through direct cell to cell contact.

S 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 3C would be more toxic to normal cells than viruses with such mechanism removed. The subject invention provides the development of a vector that can be easily manipulated; and 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

,S.L

response pathway. Additional permutations include the engineered expression of pro-drug activating enzymes such as the Herpesvirus thymidine kinase or cytosine deaminase (Blaese RM 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 expression of receptors to other viruses to target cells infected with those viruses (see Mebastsion et al., 1997, Cell 90:841-847; and Schnell MJ et al., 1997, Cell 90:849-857].

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, Io 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).

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 Oc 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.

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 0 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.

Vesicular Stomatitis Virus (VSV) provides evidence of tumor-selective killing of by oncolytic viruses, 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 HT 1080 or KB cells in the presence of exogenous interferon.

Below is a list of viruses that when modified to remove naturally-occurring anti-interferon activities, are useful- for viral cancer therapy (see Table 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 to 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.

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.

9, 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.

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.

Table 2 shows existing viruses engineered to remove anti-interferon activity.

Table 3 lists viruses engineered to be attenuated in virulence.

4,

I

Table 2 Extant Viruses Engineered to Remove Anti-IFN Activity o7

I

Table 3 Known Attenuating Mutations in Selected Viruses Virus Virus Family Virus Attenuation Reference class RNA IReovmridae rearus at ISpriggs DR and Fields N (182) Nature 297:68-70.

rotavins [bovine strains (WC3) Clark HF (1988)4 Infect Dis 158:570-587.

DNA Poxviridac V&lFvaccin growth factor DuIe RML et S (1988) Virology 164:182.

hymidhnke kinase f1ulier RML et at (1985) Nature 317:813-815.

thymidylate kinase Hughes S4 et a (1991)J Bio Chern 266i200320109 DNA ligase Kerr SM et a1(1991) EMBO J 10:4343-4350.

ribonucleonde reauctase Child SJ et at (1990) Virology 174:525-629.

dUTPase Perkus ME et al (1991) Virology 180:401-410.

denoviridam various Ad-4, Ad-21 Takatugi FT et al (1979)J infect Dis 140:48-53.

Atphanierpesvirnae HSV-1 mymidine kinase Field Hi and Wildy P(1978)J Hyg 81:267-277.

ndbonuceotide reductase Goldstein 0J and Weter S (1988) Virology 166:41-51.

9amnma 34.5 gene product Chou J et al (1995) Proc Nat] Acad Sci USA 92:10516-10520.

b'a'c inverted repeats Meignier B at a (1988)J Infect Dis 162:313-322.

Treatment of Neoplasms 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, rumors greater than 1 cm. are treated, tumors which are 2 cm or greater, from about 2 cm to about 5 cm, and greater than 5 cm.

\o 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 trumor within the body expressed as a percentage as body weight. Viral therapy of hosts having a tumor burden. from about 1% to about 2% of total body weight is surprisingly effective. producing tumor regression and a reduction in the overall tumor load. This is especially unexpected since a tumor burden of IS approximately 2% of the total body weight a 1 kg tumor in a 60 kg human) is approximately the maximum cancer mass compatible with life. See, Cotran et al., In Robbins Pathological Basis of Diseases, 4th Edition, WB Saunders, 1989, page 252. In the Examples, volumes up to 397 mm 3 for a melanoma cancer A375) in a mouse host showed complete regression in response to treatment with a Newcastle disease virus a triple-plaque no purified virus). Assuming that for tissue 1000 mm 3 equals 1 gram, a tumor having a volume of 397 mm 3 comprises approximately 2% of the total body weight for a 20 gram mouse.

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, chemotherapy such as methotrexate, 5-fluorouracil, and radiation therapy.

The efficacy of NDV for the treatment of cancer following administration through the 0 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 -t9

I

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).

^S In another embodiment of the invention, the administration of virus results in I) 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.

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 ic of disease in 47% of patients and a reduction in pain medication.

Administration and Formulation 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 ls tumors) are grown in vitro and incubated with viius. In this embodiment of the invention, patients are selected for therapy if the virus has a high activity against their tumor cells.

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, in size, mass, or volume. Shrinkage in tumor size is demonstrated by various :2 methods, including physical examination, chest film or other x-ray, sonography, CT scan, MRI, or a radionucleotide scanning procedure.

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 'a

I

invention are also useful to treat a variety of benign rumors, including but not limited to condylomas, papillomas. meningiomas. and adenomas.

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 x 106 to about 5 x 10 12 PFU of virus. For local administration directly into a S" tumor), total amounts of from about 3 x 106 to about 5 x 10 1 0 PFU of virus are typically used.

For systemic administration, amounts of from about 1 x 10 s to about 4 x 10"1 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 a0 various routes, enteral, parenteral, oral. nasal, rectal, intrathecal, intravenous using a catheter), subcutaneous, intratumor 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 bronchial administration) can be used.

2.S Endoscopic injections of gastrointestinal rumors, as well as suppository treatments of rectal tumors are also used where appropriate.

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 Sinduction 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).

s/ The rate of virus administration by the intravenous route can significantly affect toxicity. 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).

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 iC 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 tS evidence that administration of high doses of virus can overcome the effect of neutralizing antibodies and establish an infection within the patient.

In an advantageous embodiment of the invention, a desensitizing dose is given before higher subsequent doses. For desensitization, virus doses of 1 x 10' to 2.4 x 1010 PFU/m 2 are used.

After desensitization, additional virus doses of 3 x I0' to 4 x 1012 PFU/m 2 are used. The time frame between doses, including the time frame between desensitizing dose and the next dose, is I to 14 days, advantageously 1 to 7 days. The desensitizing dose can be administered by various routes, intravenous, enteral, parenteral, oral, nasal, rectal, intrathecal, intravenous, subcutaneous, intratumor, peritumoral, local, sublingual, buccal, topical, intramuscular, by inhalation, percutaenous, vaginal, intra-arterial, intracranial, intradermal, epidural, systemically, 2.S 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, intravenous, subcutaneous, intratumor, peritumoral, local, sublingual, buccal, topical, intramuscular, by inhalation, percutaenous, vaginal, intra-arterial, intracranial, intradermal, epidural, systemically, topical, intraperitoneal, intrapleural, endoscopic, intrabronchial, etc.

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 .d6 limited to intravenous. enteral, parenteral, oral. nasal, rectal, intrathecal. intravenous, subcutaneous, intrarumor, peritumoral, local. sublingual, buccal, topical, intramuscular, by inhalation, percutaenous, vaginal, intra-arterial, intracranial, intradermal, epidural, systemically, topical, intraperitoneal, intrapieural, endoscopic, intrabronchial, etc. An example would be the S administration of a intravenous desensitizing dose followed by an intraperitoneal dose.

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.

A virus, and optionally one or more chemotherapeutic agents, is administered by a single 0O 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, Current Medical Diagnosis and Treatment, Ed. Tiemey 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 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 Rodriques, R. et al, 1997, Cancer Res, 57:2559-2563; adenoviruses which do not encode a Elb polypeptide capable of binding p53 (see Bischoff. et al, 1996, Science 274:373-376); a herpes simplex virus that is incapable of expressing a functional gamma 34.5 gene product (see Mineta, T. et al, 1995, Nature Medicine, 1:938-943)]; cytokines (such as but not limited to: colony stimulating factors such as GM-CSF; tumor necrosis factor, and interleukins such as IL- -2S 1, IL-2, IL-6 and IL-10); viral vectors (such as but not limited to adenovirus encoding p53 (see Zhang, WW et al, 1994, Cancer Gene Therapv, and cancer vaccines.

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.

2% Another embodiment involves the treatment of patients with IFN aIFN, 3IFN or yIFN) 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 A3 version and analogs thereof as discussed in, for example, Sreevalsoun. 1995 (In: Biologic Therapy ofCancer, second edition, edited by V.T. DeVita, Jr., S. Hellman, and S.A. Rosenberg, J.B. Lippincott Company, Philadelphia, pp3 4 7-364). Normal cells respond to the IFN pretreatment 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.

In another embodiment of the invention, immunosuppressants such as cyclosporin

A.

azathiaprime, and leflunomide, various corticosteroid preparations, and anti-CD-40 ligand %1 antibodies (Foy, et al., 1993, J. Exp. Med. 178:1567-1575) are administered with the virus. Alternatively, an immunostimulatory compound, lipopeptides, can be administered with the virus.

An independent mechanism by which the amount of interferon produced in response to viral infection is increased through the use ofnucleosides (Machida, 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.

Certain purine nucleoside analogs, 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.

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 directly 3 into the tumor) for slow or delayed release.

-a' 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, 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 0to preparations are sterilized and/or contain additives, such as preservatives or stabilizers.

For parenteral administration, systemic or local injection, a virus preparation is formulated, as an aqueous suspension or emulsion.

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 is administering to the mammal a therapeutically effective amount of an interferonsensitive, 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, Epstcin-Barr virus, human papilloma virus, and herpes virus.

Unless indicated otherwise herein, details and conditions of viral therapy of this invention are in accordance with International Patent Publication No. WO 94/25627 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.

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 within the spirit and scope of this invention.

(R:\LUBZZI047 doc: r Example I PPMKI07, (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.

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 106 plaque forming units (PFU)/well to 10- 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 37C. At the end of the incubation period, the viral dilutions were removed and replaced by I ml of growth medium. Plates were then incubated for 5 days at 37°C in O C02, then assessed qualitatively for the amount ofcytopathic 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 WI 53711) monitored at 570 nm, that detects mitochondrial enzyme activity (Mosman, 1983, J.

Immunol. Methods 65:55). The viability in the virus treated wells was expressed as a percent of IS 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.

The results are given in Tables 4, 5 and 6. PPMMK 107 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.

1L

I

Table 4. Summary of Cytotoxicity Assay Results TUMOR TYPE

FIBROSARCOMA

MELANOMA

BREAST CARCINOMA OVARIAN CARCINOMA LUNG CARCINOMA (Large Cell, Low Passage)

GLIOBLASTOMA

BLADDER CARCINOMA

NEUROBLASTOMA

CERVICAL CARCINOMA PROSTATE CARCINOMA COLON CARCINOMA

HEAD-AND-NECK

CARCINOMA

NEUROEPITHELIOMA

SMALL CELL CA, LUNG SMALL CELL CA,

PROSTATE

LEUKEMIA (AML) CELL LINE HT1080 SKMEL2 SKMEL3

SKMELS

A375 MALME-3M HT144 SKBR3 MDA-MB-468 ZR75-1 SW626 PA-1 ES-2 SKOV-3 OVCAR3 H-1299 U87MG U373MG U138 A172 HT1197 UM-UC-3 HT1376 IC, (PFU/well) 2 8 2 4 37 778 28 44 78 4 4 13 24 34 26 765 38 207 3 54 422 IMR32 HeLa DU-145 PC3 SW620 HT29

KB

A253 FaDu Hep-2

SK-N-MC

DMS-114 DMS-153 NCI-H345 NCI-H660 41 4 31 3.1 x I:0 x 4 2.7 x 2.9 x 1.5 x 48 1.1 x 1.2 x 1.0 x 5.4 x K562 Table 5.

Summary of Cytotoxicity Assay Results Using Normal Human Cells Table 6. Summary of Cytotoxicity Assay Results Using Rapidly Proliferating Normal Human Cells RATE OF PROLIFERATION CELL TYPE /N vivo N VMRO IC, (PFU/well) Bone Marrow Cells Moderate to High 6.2 x CD34 Enriched to 50% igh 6.210 Breast Epithelial Cells Very Low High 3 Human breast epithial cells tested (HMEC) had a high rare of proliferaton after stimulation with bovine pituitry act ad human pidera growth factor. In marked contras, nornma breast pithelal cells almost alwas have a y low degree of arked Proliferaon in adult women with cancer. very low dgr of

MMMMMM

Example 2 Use of PPMK107 for the Intratumoral Treatment of Human Tumor Xenografts (<10 mm and >5 mm) in Athymic Mice.

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 PPMK 07 (at a dose of 3 x 10 PFU) or saline was given. Almost all rumor 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 Although only one of 9 tumors treated with PPMK107 completely regressed, two more virus-treated tumors showed regression of 32% and S 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.

IS 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 ofintratumoral injection of 3 x PFU of PPMK 107, 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.

Use of PPMK107 for the Intravenous Treatment of Human Tumor Xenografts mm and >5.5 mm) in Athymic Mice.

"2 Athymic mice were injected intradermally with 10 million human HTI1080 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 (I x 10 9 PFU) complete tumor regression was seen in all seven mice. Single injections of 3 x 10' and 6 x 7 resulted in regression rates of over 90%. While a single IV injection of 3 x 10 gave only a .49 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 rumor regression. These results indicate that subcutaneous HT1080 tumors are very responsive to IV treatment with PPMK107.

Table 7. PPMK107 intratumoral treatment of subcutaneoui human tumnor xenografts (<10 Mm and 5 m) in athymic mice Tumor Tumor Type Expnt Dose N Rcmegeion pet HT1080 Fibrosarcoma A 3.OOE+oa 1 2 Rersso C 3.002+081 8 PA-i Ovarian Carcinoma D 2±9SfE+o8 929 KB Crai Carcinoma 2 -3.OOE+08 12 7 SKMELS Melanoma F 3.OOE+.08I a. 5 7 A3-75 Melanoma G 3.002+08 857 3.00E+o8 8 1 4 U8S7MG IGlioblastoma 1 1 3.000E+0 8 L. 41I Table 8. PPMK107 Intravenous treatment of subcutaneous human 8T1080 fibrosarcoma .xenografts 8.5 'mm and 5.5 mm) In athymic mice.

omplete C~ompete.+ Dose Schedule N ession partal Regression Regression 1.OOEi09 One Intecton 7 7 100% 3.00E+08 One Injection 10 9 10 100% 6.OOE+O7 One Inlection 11 10 10 91% 2.OOE+07 One lecion 11 5 6 2.00E+07 Three Infectons 7 5 7 100% Every Other Day ____Saline One Injection 10. 0 0 0% Salie Three Injections 6 0 0 Every Other Day Example 4 First Experiment Using PPMKI07 for Intratumoral Treatment of Large A375 Melanoma Xenografts in Athymic Mice Athymic mice were injected intradermally with 10 million A375 human melanoma cells. Ten days later, rumors of various sizes were treated with a single injection PPMK107 (doses of 3 x 9 x 10 and 1.5 x 10 9 PFU) or saline. For those tumor with a single largest dimension of to 11 mm, all nine completely regressed in response to intratumoral treatment with these doses of PPMKI07, 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 No tumor regression was seen in any mouse treated with saline.

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 PPMK 107, complete regression occurred in 14 out of 17 tumors with volumes >300 mm 3 (range of 304 to 397 mm 3 and in 7 out of 16 tumors with volumes <300 mm 3 (range of 144 to 295; P <0.023; Table 9, section B).

These results indicate that tumors at least 1 cm in length or 300 mm 3 in volume were at least as sensitive, if not more sensitive, to intratumoral PPMK107 treatment than smaller tumors.

Example Second Experiment Using PPMK107 for Intratumoral Treatment of Large A375 '0 Melanoma Xenografts in Athymic Mice.

Tumors were established as in Example 4 ten days after tumor cell inoculation. Treatment consisted of various doses of PPMK107 (3 x 106 PFU, 3 x 107, 3 x 10 s and 1.5 x 109) or saline.

For tumors 10 to 11.5 mm in single largest dimension, complete or partial (at least 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).

43 When these same rumors were sorted by tumor volume, all 26 tumors greater than 300 mm 3 (range: 309 to 525 mm regressed completely or partially (at least 50%) in response to PPMK107 in contrast to none of the saline treated mice (Table 10, section B).

These results confirm that tumors at least 1 cm in length or 300 mm 3 in volume are sensitive to intratumoral PPMK107 treatment.

Table 9: intratumnoral PPMKIo7 Treatment of Intradermat A375 Melanoma Xenagrafts A. T umors Sorted Hasedo the Single Largest DImne lon j ITun' r imension: lo 99 m..r Tu o slon 10 to Complete ___Complete- _____Treatment Dosag N Rgrsson N Regression%- PPMKI07 1.5 x io 9 a2 25% 3 3 100% ____PPMK1O7 9.0 x 108 a_ 7 88% 3, 3 100% PPMK1O7 3.0 x 10' a 3 38% ___Total 24 12 50% 99 1003% a aie 6 0% 3 0 0 B3. Tumors Sorted Based on the Tumor Volume t 1 III___ I"'pra1u"' :_<0Qtsnn Thpgr Voumea, 300l~m I __If Conomplete_ Teatment Dnsacn I M? *,IomL I4 .'j..~w.ooavI I-i.

PPMKC107 -PPMKI07 1 IPPMKI 07 15x i0o 3.0 x t0 8 3 33% 75% Jifigrsslon 7 4 14 S82%

I

Total Siaitno 11161 7 [(7 339II a 0 -70% a. P 0.008 for complete rgresson In the PPMKIO7 10-11 mm roup vs. he PPMK107 -9.5 mm tronlectrou 2

L

p. P 0.023 for cmpleate regression In the PPMKIO7-reated '>300 mme group vs. the PPMKOZ-trealed <300 'm 3 ru

I-

Table 10: Intraturnoral PPMK1O7 Treatment of Intradennal A375 Melanoma nogralts.

A. Tumors 10to 11.5 mm (Sorted Based on the Single Largest Dimension) Reressn Treatment Dose N Complete Complete.+ Partial oXI1 8 7 6 86% 100%_ X~ 10 7,7% 7 100% X10 7 7 571%". -7 100% APPMKlO7GERos 28 2-3 132% 28 100%/ alie 60 0% 0% B. Tumors 300 mm3 (Sorted Based on the Tumor Vlme) Re rsslons Treatment Dose N Complete Complete Partial rx 1og 7 7 100% 7 100% 6 7 678% X.17 6 4 6% 6 .0r rlog 6 4 67% 100%__ APPMX107 Grups 2t 215 81 01 001.% ,14/ Example 6 Third Experiment Using PPMK107 for Intratumoral Treatment of Large A375 Melanoma Xenografts in Athymic Mice.

Tumors were established as in Example 4 nineteen days after tumor cell inoculation.

Intratumoral treatment consisted of various doses of PPMK107 (3 x 10 8 3 x 10', 3 x 10 s 3 x 104, 3 x 10 3 3 x I02 PFU) or saline. For tumors 12.5 to 14 mm in single largest dimension (volume range: 632 to 787 mm 3 average volume 698 nmm 3 tumor regressions of at least occurred in two out of three mice treated with 3 x I 0 PFU in contrast to no regression in both saline-treated mice (Table 11). Using the same dose of PPMK107 (3 x 10 8 PFU) to treat tumors 1O with a single largest dimension of 10 to 12 mm (volume range: 320 to 600 mm 3 average volume: 411 mm3), 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 x 106 PFU, 3 x 10 s PFU, 3 x 10 PFU, and 3 x 103 PFU, but not for mice treated Is with 3 x 102 PFU or saline (Table 11).

These results confirm that tumors at least 1 cm in length or 300 mm 3 in volume are sensitive to intratumoral PPMK107 treatment.

Example 7 Fourth Experiment Using PPMK107 for Intratumoral Treatment of Large A375 Melanoma Xenografts in Athymic Mice.

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 x 108 PFU of PPMKI07 or saline. Volumes of those tumors treated with PPMK107 ranged from 295 to 600 mm 3 (average tumor volume of 437 Groups of mice in each treatment group were 2S" 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 -17 PPMK107 treatment, two tumors already exhibited signs of regression but the degree of regression was less than Table 11: 3td Experiment U I M~7 ofeInrtmra ra of A375 elanoma xestf mm-In slze Size: 1 ,5 a 14--M ns 2tal Of Treatment N Volume Range Avg Volum Complte Parial a 25% .4BO% Regressonso Regressons c E +08 a132t 787 698 1 -1 0 2 670/ Salino 2 717 Wo 86078 l lo size, 10 to2 m e rresslott Toal L of Treatment N Volume Ran JAvg Volume Eomplte Patial 25% -c50%b IRegressins Regresslonso E +08 320 to 600 411 4 a~t E +06 8 425, to 662 '0 2 2 E +05 8 245 to 600 4 4 2 1, 0 0 1 13% E +03 8 281t )542 4 9 130/c225 E +02 8 281 to 662 872, 0 0 0 0%oo S 2 no8 379 666 18 0 0 0 0( 0% ffh a- artlai RegresslnI efnda regression less than 100% ai i al to or gieater than b Regression -C50%" Is deflne surrgress lon gratrthan 25% and less than Bo%.

c ncldes all tumor regression that Is greater 1'rai 215134 d P 0.001 for Regression greater than 25% In the 3 E+08 gro'vsth anegup Table 12. 4th Experiment Using PPMK1O7 for the Intratumoral Treatment of A375 Melanoma Xenogtt -atleast Size: 10 to 12 Day Euthani zedi eressons Total it of Treatment Post Treatment N Complete, Partial 2 a 2 <c 0% bRegressionse Regressionso E +08 14 Days 3 2 1 3 100% E +08 7lDays 30 2 13 100% 3.OE +08 4 Days 3 0- -2 13- 100% 3.02E+08 3 Days 3 0 0 2 3 67% E +08 All PPMKI07 Groups 12-- 0 65 1 92% do Saline 2 0 o 0 Saie Yas20 0 0 0 0% Saline. 4fDays 2 0o 0 0 0 0 Saline 3flays 2 0 0 0 0 0% Saline All Sains Groups 8 0 0 0 0 0% o-Partial Regression Is defined as regression less than 100% and equal to or greater than S0%.

b "Regression So0% Is defined as tumor regreslon, g reater than 25% and less than 50%. o lndiudes Regression that Is at least 25%. 1 d P <0b.03 for Cornplets or Partial l egresslon In the PPMKIo07 group of 12 mice vs the saline group of 8 mIce.

e P 0.0001 for Regression at least 25% In the PPMKI07 groul of 12 mice vs Example 8 Fifth Experiment Using PPMKI07 for Intratumoral Treatment of Large A375 Melanoma Xenografts in Athymic Mice.

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 x 10 s PFU of PPMK107 or saline. Volumes of those tumors treated with PPMKI07 ranged from 361 to 756 mm 3 (average tumor volume of 551 mm 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).

OC Example 9 First Experiment Using PPMK107 for Intravenous Treatment of Large HT1080 Fibrosarcoma Xenografts.

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 x 109 1' 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 I cm in length are sensitive to intravenous PPMK 107 treatment.

I

Table 14: Intravenous Treatment of Subcutaneous HT1080 Human Fibrasarcoma Xenografts in Athyric Mice Size: 10 to 11 mm Regressions Treatment Dose N Complete Complete Partial PPMK107 1.5E0-9 6 4 67% s83% a Saline 1 41 0 0 0101 1 a- P c 0.025 Fishers exact test) for complate or gartlal regression (at least 50% reression) In the PPMK1 07 teated aro comoared to the saline UI Exampe Specific Clearing of PPMK107 Infection from Normal but Not Tumor Cells.

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 S 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 t0 partial regression in response to a single intratumoral injection of PPMK 107, 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.

Accordingly, co-cultures of KB and CCD922-sk cells were infected at a multiplicity of infection the ratio of virus added per cell) of 0.0005 and the course of the infection followed for 'S 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.

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 ofIFN secreted into the media by the normal cells.

c-7 Example 11 Demonstration that Interferon is an Important Component of Viral Clearing in Normal CCD922-sk Cells.

The hypothesis that interferon was mediating the ability of CCD922-sk cells to clear the infection of PPMKI07 was tested. Polyclonal neutralizing antibodies to human interferon-a or human interferon-p, used alone or in combination, were added daily to cultures of CCD922-sk cells infected with PPMK 107 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-p antibody being IC more marked than that of the anti-interferon-a antibody; consistent with reports that fibroblasts produce predominantly the beta form of interferon.

The ability to make the normally insensitive cells more susceptible to infection with PPMKI07 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 Demonstration that Interferon- is an Important Component of Viral Clearing in Other Normal Cells.

.2c 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- p 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.

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 PPMK 107 at an moi of 0.05 resulted in SiCy 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).

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 antibodytreated cultures relative to mock-treated controls (Fig. 1).

These results suggest a general mechanism by which normal cells are protected from killing by PPMKI07 through an interferon-related mechanism.

SExample 13 Comparison of the Effect of Interferon- on PPMK107 Infection in Tumor and Normal Cells.

A comparison of the effect of exogenously added interferon-p on the infection of normal (CCD922-sk) and rumor cells of high (KB) or intermediate (HEp2) sensitivity PPMK107 was IS performed. Separate cultures of the three cells were treated with interferon-p at 20, 200, or 2000 units/ml 1 day pre- and 2 days post-infection at an moi of 0.0005.

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/mI decreased relative levels of viral U3 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).

The pattern of sensitivity in the KB and CCD922-sk cells to the anti-viral effects of exogenously added interferon-p was inversely proportional to the sensitivity of these cells to killing by 2S PPMKI07. 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 <56to 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 Effect of Low Concentrations of Interferon-p on the Infection of Normal and Tumor Cells S by PPMK107.

In this experiment normal (CCD922-sk) and tumor (KB) cells were treated with low concentrations of interferon-P 2, and 20 units/mi) 1 day before and 2 days post-infection with PPMK107 at an moi of 0.05.

Under these conditions the normal cells experienced a dose-dependent decrease in the amount of tO viral antigen, while the relative levels of viral antigen in the tumor cells was unaffected by the addition of exogenous interferon (Fig. 3).

Example PPMK107 Purification Method A 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 0 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 2a x 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 x g for 30 minutes. The purified virus was harvested from the 20%/55% interface and dialyzed against saline to remove the sucrose.

Method B In another advantageous embodiment, the clarified allantoic fluid was frozen at -70 0 C. After thawing, the fluid was maintained at 1 to 4C overnight and then the contaminating material was removed from the virus suspension by means ofcentrifugation (1750 x g for 30 minutes). This 5-7 material was further processed using the discontinuous sucrose gradient on the ultracentrifuge as above.

Method C In another advantageous embodiment, ultracentrifugation on the discontinuous sucrose gradient was accomplished by means of a continuous flow ultracentrifuge.

Method D 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 O 0.45p. 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.

Results Clonal Virus After generation ofPPMK107 by plaque purification, eight individual molecular clones from theo2 population of virions were found to have an identical sequence 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.

PFU/mg protein 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 x were achieved (see Table 15). Using Method C, PFU/mg protein values of at least 2.0 x 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 MassMK 07, lot RU2, prepared as in Faaberg KS and Peeples, ME, 1988, J Virol 62:586: and Bratt, MA and Rubin, H. 1967, Virology 33:598-608). This RU2 lot was found to have a PFU/mg of 1.3 x 10 PFU/mg of protein. The purity values achieved by Method A are approximately 40 times better than what the Peeples method achieved (see Table Particle per PFU Ratio 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 Table 15. Virus Purity

-I

Virus Preparation Method Virus PPMK107 Lot L2 Preferred Method A PFU per mg protein 4.8 x 10'0 6.9 x 10' 0 6.6 x 100° 7.7 x 10 t 6.1 x 10'° Particle per PFU 0.80 NT a

NT

0.55

NT

Preferred Method C Preferred Method D PPMK107 D004 D005 PPMKI07 RD2 RD3 2.0 x 10'1 4.5 x 10'" 4.4 x 10'° 5.6 x 10'0 5.0 x 10'° 0.32 0.52

NT

NT

NT

a NT, Not Tested Virus preparations using Methods A and C also permitted purification ofNDV to a level IO substantially free of contaminating egg proteins. For the PPMK107 lot 7 preparation using Method A. ovalbumin, was not detectable in a Western blot using 1.7 x 109 PFU of purified virus per well (3.3 cm in width) run on an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel (1 mm thick); a nitrocellulose membrane for transfer; and 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 Use of PPMK107 To Prevent Deaths from ES-2 Ovarian Carcinoma Ascites in Athymic Mice.

In this experiment, all of the athymic mice (female, NCR nu/nu, 8 weeks old) were given an intraperitoneal injection of 106 ES-2 cells. Seven days later before ascites had developed, they 0t were treated intraperitoneally with saline or PPMK 107 (at 1 x 10 9 PFU). As shown in Figure 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 PPMKI07 were still alive by day 38 and 25% of these animals were long term \S survivors (>120 day survival).

Example 17 PPMK107 Treatment of ES-2 Ovarian Carcinoma in Athymic Mice When Ascites is Present.

In this experiment, all of the athymic mice (female, NCR nu/nu, 8 weeks old) were given an intraperitoneaI injection of 10 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; PPMK 107- tvo treatments for the first week; PPMK107- one treatment each week; PPMK107two 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 x 109 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.

I

The degree of ascites for each mouse was quantified and noted as follows: Ascites Degree of Ascites Score Animal appears normal- little or no ascites present Abdomen slightly distended; animal is capable of normal functions Abdomen distended; animal is slow-moving, bunched with a staggered gait.

Abdomen completely distended; animal moribund Death after ascites development 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 PPMKl07-treated groups had S 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 PPMK 107-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.

Table 16. PPMKI07 Treatment of ES-2 Ovarian Carcinoma in Athymic Mice When Ascites is Present.

Treatment of Mice Average Ascites Average Ascites Score, Score, Day 7 Day Saline xl 12 4.3 4.7 Saline x2 12 3.7 4.6 Saline x2 each wk 12 4.3 4.8 PPMK107 x 1 17 3.0 4.1 PPMK107 x 2 17 2.3 3.6 PPMKI07 xl each wk 17 2.6 2.6 PPMKI07 x2 each wk 17 2.2 3.6 Example 18 Use of a Desensitizing Dose of PPMK107 to Reduce the Lethality of a Subsequent Dose of PPMK107.

C57BL/6 mice (seven weeks old) were injected intravenously on day 0 with either saline or a desensitizing dose of PPMK107 (3 x 10 8 PFU/mouse). Two diys later each set of mice were further subdivided into groups for intravenous dosing with saline or PPMKI07 (at doses of I x 109, 2.5 x 10 9 5 x 10', and 1 x 10'0 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 x 109, 5 x and I x 1010 PFU. The doses of 5 x 10 9 and 1 x 1010 PFU were 100% lethal to the mice to 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 x 1010 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 PPMK 107 can be raised by an approximate order of magnitude when using this virus as a desensitizing agent.

6.-2 Table 17. Use of a Desensitizing Dose of PPMKI07 to Reduce the Lethality of a Subsequent Dose of PPMKI07.

Group Injection on Day 0 Dose on Day 2 of of Mice Deaths Lethality I Saline Saline 8 0 0 2 Saline PPMK107, 1.OE+09 8 0 0 3 Saline PPMK 107, 2.5E+09 8 3 38 4 Saline PPMKI07, 5.0E+09 8 8 100 Saline PPMKI07, 1.0E+10 8 8 100 6 PPMK 07, 3E+08 Saline 8 0 0 7 PPMKI07, 3E+08 PPMKI07, 1.0E+09 8 0 0 8 PPMKI07, 3E+08 PPMKI07, 2.5E+09 8 0 0 9 PPMKI07, 3E+08 PPMKI07, 5.0E+09 8 0 0 PPMK107,3E+08 PPMK107, 1.0E+10 8 0 0 Example 19 Slower Intravenous Injection Rate Reduces the Toxicity of PPMKIO7.

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 x 109 PFU of PPMKI07 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 PPMKI07 over 4 minutes had half as much maximal weight loss O1 (recorded on day 2 after dosing) as the animals receiving the same IV dose over 30 seconds.

These results indicate that PPMK 107 has less toxicity and is safer for intravenous administration when injected at such slower rates.

403 Table 18. Slower IV Injection of PPMKI07 Results in Reduced Toxicity.

Group Length of Time of Mice Maximal Percent That Dose was Weight Loss Administered Rapid Injection of 30 seconds 11 12% 4E+09 Slow Injection of 4 minutes Ii1 6% 4E+09 Example Use of PPMK107 in the Treatment of Patients with Advanced Cancer.

PPMK107 has been tested in a phase I 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 PPMKI07. 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 io eligible for additional treatment courses each month.

Regression of a Palpable Tumor A 68 year old female with colon carcinoma had a palpable abdominal tumor among her widespread metastases. After a single IV treatment with PPMK 107, 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 x 3 cm) were similar to the baseline measurements of 4 x 3 cm. However, by day 7 post dosing, the tumor had decreased in size to 2 x 2 cm and continued to decrease in size to 1.5 x 1.5 cm by day 14 after PPMK107 dosing. Previous to PPMKI07 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. s Table 19. Size of Palpable Abdominal Wall Tumor in Patient #123 (68 year old Female with Metastatic Colon Carcinoma) After a Single IV PPNIK1O7 Dose of 12 Billion PFU/mt Tumor Tumor Volume Time After Dimensions (0.5 x L x Wy x Reducion Date Dosing (L x W, cm 3) cm 3 i uo Volume 7/23/98 DayO0 4 x3 18.

7/24/98 Day 1 3.75 x 3 16.9 6% 7/30/98 Day 7 2x240780%0 8/6/98 Day 14 15x.5.791%o Stabilization of Cancer 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 PPMKI 07 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 PPMIK 107 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 to doses of PPMKlO7: Two out of 6 patients with stable disease (33% of patients) at the first two single dose levels (5.9 and 12 billion PPU per r2) and 4 out of 5 patients of patients) with stable disease at the highest single dose level (24 billion PFU per m 2 (Table Table 20. Treatment of Patients with Advanced Cancer with PPMKI07.

Dose of of of Types of Cancer with Stable Disease Level Patients Patients Patients for at Least One Month Length of Stable Disease (Billion Treated with with PFU per at this Stable Stable Dose Disease Disease Level 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 I month 24 5 4 80% Breast Cancer- Ongoing 1 month Breast Cancer- Ongoing 1 month Lung Cancer- Ongoing I month Pancreatic Cancer- Ongoing 1 month Total 17 8 47% Noted Above.

Reduction in Pain Medication One patient at the single dose 5.9 billion PFU/m 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.

Desensitization A clear desensitizing effect from the first dose (at 5.9 billion PFU/m') is seen on subsequent doses (also at 5.9 billion PFU/m 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.

44< Dosing Through Neutralizing Antibodies in Serum Using the dose range in this phase I study billion PFU/m2), 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/Im single dose level has had stable disease for 2 months since beginning PPMK107 treatment. A second course (consisting of asingle 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 Summary of Cytotoxicity Assay Results with Newcastle Disease Virus PPNJROAKIN Human tumor cells and normal cells were grown to approximately 80% confluence in 24 well (S 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' 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°C. At the end of the incubation period, the viral dilutions were removed and replaced by I ml of growth medium. Plates were then incubated for 5 days at 37 0 C in 5% C02.

Cytotoxicity was quantified by using a colorimetric MTT (2-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide) assay (Cell Titer 96, catalog #G4000, Promega Corporation, Madison WI 53711) monitored at 570 nm, that detects mitochondrial enzyme activity (Mosman, 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.

47 Table 21. Summary of Cytotoxicity Assay Results with PPNJROAKIN.

Cell Type Cel Line IC, (PFU/well) Fibrosarcoma HT1080 13.8 Head and Neck KB 2.4 Carcinoma Normal Fibroblast CCD922sk 1.2 x 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 values for the two tumor cell lines are between 800 and 5000-fold lower than that for normal cells.

S Example 22 Summary of Cytotoxicity Assay Results with Newcastle Disease Virus PPCONN70726 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 0 dilutions ranging from 107 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°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°C in 5% C02.

Cytotoxicity was quantified by using a colorimetric MTT (2-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide) assay (Cell Titer 96, catalog #G4000, Promega Corporation, Madison WI 53711) monitored at 570 nm, that detects mitochondrial enzyme activity (Mosman, 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 )0 in PFU/well causing a 50% reduction in the amount of viable cells.

14 Table 22. Summary of Cvtotoxicity Assay Results with PPCONN70726.

Cell Tvye Cell Line IC, (PFU/well) Head and Neck KB 18.1 Carcinoma Glioblastoma U87MG 12.7 Glioblastoma U373MG 879 Normal Fibroblast CCD922sk 7.3 x 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 Intratumoral Treatment of HT1080 Fibrosarcoma Xenografts in Athymic Mice Using PPMK107, PPNJROAKIN, or PPCONN70726.

In this experiment, athymic mice (female, NCR nuinu, 5 to 6 weeks old) received a subcutaneous injection of 107 HT1080 tumor cells. Four days later when tumors reached a size to range of 6 to 8.5 mm, mice were treated intratumorally with saline, PPMK107 (at 1 x 10

S

PFU), PPNJROAKIN (at 1 x 108 PFU), or PPCONN70726 (at 1 x 10' 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 1s 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.

Table 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 I x 10' PFU.

Regression Treatment of Mice Partial (PR) Complete (CR) PR CR PPMKI07 12 6 4 10 (83%) PPNJROAKIN 12 2 1 3 PPCONN70726 12 2 3 5 (42%) Saline 11 0 0 0 0%) Example 24 Effects of PPMKI07, PPNJROAKIN, PPCONN70726 after Intracerebral Injection in Immunodeficient Athymic (nu/nu) and Immunocompetent Heterozygote Mice.

Fifty-six athymic mice (nu/nu) and 56 immunocompetent heterozygote 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 x 10' or 3.5 x 106 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 x 106 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 x 106 PFU/mouse). These data indicate that PPNJROAKIN is better tolerated in the brains ofathymic mice than the other two virus strains.

I

Table 24. Survival of Mice Following Intracerebral Injection of PPMK107, PPCONN70726, and PPNJROAKIN.

Intracranial Injection of Mice Survival 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 nu/nu PPMK 107, 3.5E+06 8 13 nu/nu PPCONN70726, 2E+04 8 nu/nu PPCONN70726, 3.5E+06 8 13 nu/nu PPNJROAKIN, 2E+04 8 100 nu/nu PPNJROAKIN, 3.5E+06 8 The one non-surviving mouse in this treatment group was euthanized for non-neurological symptoms.

Example Summary of Cytotoxicity Assay Results with Sindbis Virus PPSINDBIS-Ar339 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 107 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 0 C. At the end of the incubation period, the viral dilutions were removed and replaced by I ml of growth medium. Plates were then incubated for 5 days at 37C in 5% C02. Cytotoxicity was quantified by using a colorimetric MTT (2-[4,5-dimethylthiazol-2-yI]-2,5-diphenyl tetrazolium bromide) assay (Cell Titer 96, catalog #G4000, Promega Corporation, Madison WI 53711) monitored at 570 nm, that detects mitochondrial enzyme activity (Mosman, 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 was calculated as the amount of virus in PFU/well causing a 50% reduction in the amount of viable cells.

Table 25. Summary of Cytotoxicity Assay Results with PPSINDBIS-Ar339.

Cell Type Cell Line Ce PFU/well Pancreatic Carcinoma Panc- 1* 69 Colorectal Carcinoma SW620* 13 Colorectal Carcinoma SW1463 1.8 Non-small cell Lung carcinoma A427 >1 x Non-small cell Lung A549 5.2 x carcinoma Renal carcinoma A498 2.4 x Renal carcinoma Caki-1 3.4 x Fibrosarcoma HT1080 7.4 x 105 Normal Keratinocyte NHEK 2.0 x 105 Normal Fibroblast CCD922sk 1.6 x 105 Cells known to overexpress the mRNA for the high affinity laminin receptor.

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. Nat! 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 demonstrate that cells that are tumorigenic and express high levels of the high affinity laminin ,7a--

I

receptor are more sensitive to killing by Sindbis Clone PPSINDBIS-Ar33 9 than other rumor or normal cells.

Example 26 VSV Killing of Tumorigenic and Non-Tumorigenic Cells in the Presence of Interferon.

In 96 well plates, tumorigenic KB and HT1080 cells (3 x 104 cells per well) and nontumorigenic WISH cells (2.5 x 10' cells per well) were seeded in the presence ofserially diluted interferon-a ranging from 2800 to 22 Units/ml and allowed to incubate for 24 hours at 37°C.

The cells were then infected with vesicular stomatitis virus (VSV, Indiana strain) at an moi of Controls were included for cells without interferon, and cells without interferon or virus.

The cells were incubated at 370 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 WI 53711) monitored at 570 nm. that detects mitochondrial enzyme activity (Mosman, 1983, J. Immunol. Methods 65:55). The viability in the virus treated wells was expressed as a percent of the activity in control wells not receiving IS virus.

Table 26. Comparison of the Cell Killing Activity of VSV in Cells Treated with Exogenous Interferon.

Percent Viable Cells WISH HT1080

KB

100 U/mi IFN 50 6 0 l000 U/mlIFN 95 20 12 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.

73

I

Example 27 Interferon Responsiveness in Cells Sensitive or Resistant to Killing by PPMK107.

Individual cell lines were grown to near confluence in 96 well microtiter plates and treated with between 5 and 5000 U/ml of IFNcrA 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 (Figure In this assay, interferon responsive cells manifest at least a 50% decrease in the viral antigen in response to interferon.

I0 Cells in Figure 5 that are sensitive to PPMK107 are indicated by the solid lines; cells less sensitive are indicated by the dashed lines.

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 I 5 graph legend, see Figure For example, following pretreatment with 5 U/ml of interferon, 6 of 7 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. 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 more than 50% of controls) viral antigen following pretreatment with exogenous interferon would be considered interferon deficient and thereby sensitive to viral oncolysis.

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 S3 the invention.

74

Claims (71)

1. 2. A method of treating a neoplasm in a mammal comprising administering to IDsaid mammal a therapeutically effective amount of an interferon-sensitive, replication- competent clonal RNA virus, where said RNA virus replicates at least 10 fold less in the C presence of interferon compared to in the absence of interferon.
2. 3. The method according to claim 1 or 2, wherein said RNA virus replicates at least 100-fold less in the presence of interferon compared to in the absence of interferon.
3. 4. The method according to claim 1 or 2, wherein said RNA virus replicates at least 1000-fold less in the presence of interferon compared to in the absence of interferon. The method according to any one of claims 1 to 4, wherein said administering step is systemic.
4. 6. The method according to any one of claims 1 to 5, wherein said neoplasm is a cancer.
5. 7. The method according to any one of claims 1 to 6, wherein said mammal is a human.
6. 8. The method according to any one of claims 1 to 7, wherein said clonal virus is plaque purified.
7. 9. The method according to any one of claims 1 to 7, wherein said clonal virus is of recombinant clonal origin. The method according to any one of claims 1 to 9, wherein said RNA virus is a Paramyxovirus.
8. 11. The method according to claim 10, wherein said Paramyxovirus is purified to a level of at least 2 x 10 9 PFU per mg of protein.
9. 12. The method according to claim 10, wherein said Paramyxovirus is purified to a level of at least 1 x 1010 PFU per mg of protein.
10. 13. The method according to claim 10, wherein said Paramyxovirus is purified to a level of at least 6 x 1010 PFU per mg of protein.
11. 14. The method according to claim 10, wherein said Paramyxovirus is purified to a level in which the particle per PFU ratio is no greater than [R:\LIBVV]501 959dl speci.doc:THR purified to a
12. 16. The method according to claim 10, wherein said Paramyxovirus is level in which the particle per PFU ratio is no greater than 3. The method according to claim 10, wherein said Paramyxovirus is purified to a level in which the particle per PFU ratio is no greater than 1.2.
13. 17. The method according to any one of claims 1C Paramyxovirus is avian paramyxovirus type 2.
14. 18. The method according to any one of claims 1C Paramyxovirus is NDV.
15. 19. The method according to any one of claims 1C Paramyxovirus is mumps virus. The method according to any one of claims 1C Paramyxovirus is human parainfluenza virus. 0 to 16, wherein said Sto 16, wherein said Sto 16, wherein said Sto 16, wherein said
16. 21. The method according to any one of claims 1 to 20, wherein said neoplasm is a cancer selected from the group consisting of lung, colon, prostate, breast and brain cancer.
17. 22. The method according to any one of claims 1 to 20, wherein said neoplasm is a solid tumor.
18. 23. The method according to claim 21, wherein said brain cancer is a glioblastoma.
19. 24. The method according to claim 1, wherein said virus contains a gene encoding interferon to permit the viral expression of interferon. The method according to claim 1, wherein said virus contains a gene encoding a pro-drug activating enzyme.
20. 26. The method according to claim 1, further comprising administering IFN before, during or after administration of said virus.
21. 27. The method according to claim 26, wherein said interferon is selected from the group consisting of ca-IFN, p-IFN, co-IFN, y-IFN, and synthetic consensus forms of IFN.
22. 28. The method according to claim 1, further comprising administering a tyrosine kinase inhibitor before, during or after administration of said virus.
23. 29. The method according to 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. The method according to claim 1, further comprising administering a chemotherapeutic agent before, during or after administration of said virus. [R:\LIBVV]501959dl_speci.doc:THR 77
24. 31. The method according to claim 1, further comprising administering a r, cytokine before, during or after administration of said virus.
25. 32. The method according to claim 1, further comprising administering an Simmunosuppressant before, during or after administration of said virus. 00 N 5 33. The method according to claim 1, further comprising administering a viral replication controlling amount of a compound selected from the group consisting of IFN, Iribavirin, acyclovir, and ganciclovir. S34. The method according to any one of claims 1 to 33, wherein said administration is intravenous or intratumoral.
26. 35. The method according to any one of claims 1 to 34, wherein said virus is Sadministered as more than one dose.
27. 36. The method according to claim 35, wherein the first dose is a desensitizing dose.
28. 37. The method according to claim 36, wherein said first dose is administered intravenously and a subsequent dose administered intravenously.
29. 38. The method according to claim 36, wherein said first dose is administered intravenously and a subsequent dose administered intraperitoneally.
30. 39. The method according to claim 36, wherein said first dose is administered intravenously and a subsequent dose administered intra-arterially.
31. 40. The method according to any one of claims 1 to 39, wherein said virus is administered over the course of at least 4 minutes.
32. 41. The method according to any one of claims 1 to 39, wherein said virus is selected from the group consisting of the Newcastle disease virus strain MK107, Newcastle disease virus strain NJ Roakin, Sindbis virus, and vesicular stomatitis virus.
33. 42. An interferon-sensitive, replication-component clonal RNA virus when used for treating a neoplasm in a mammal, wherein said RNA virus replicates at least fold less in the presence of interferon compared to in the absence of interferon.
34. 43. The RNA virus according to claim 42, wherein said RNA virus replicates at least 100-fold less in the presence of interferon compared to in the absence of interferon.
35. 44. The RNA virus according to claim 42, wherein said RNA virus replicates at least 1000-fold less in the presence of interferon compared to in the absence of interferon. The RNA virus according to any one of claims 42 to 44, wherein said neoplasm is a cancer. [R:\LIBVV]501959d Ispeci.doc:THR 78 S46. The RNA virus according to any one of claims 42 to 45, wherein said CN1 mammal is a human.
36. 47. The RNA virus according to any one of claims 42 to 46, wherein said Sclonal virus is plaque purified. 00 N 5 48. The RNA virus according to any one of claims 42 to 47, wherein said clonal virus is of recombinant clonal origin. O 49. The RNA virus according to any one of claims 42 to 48, wherein said SRNA virus is a Paramyxovirus.
37. 50. The RNA virus according to claim 49, wherein said Paramyxovirus is purified to a level of at least 2 x 109 PFU per mg of protein.
38. 51. The RNA virus according to claim 49, wherein said Paramyxovirus is purified to a level of at least 1 x 1010 PFU per mg of protein.
39. 52. The RNA virus according to claim 49, wherein said Paramyxovirus is purified to a level of at least 6 x 1010 PFU per mg of protein.
40. 53. The RNA virus according to claim 49, wherein said Paramyxovirus is purified to a level in which the particle per PFU ratio is no greater than
41. 54. The RNA virus according to 49, wherein said Paramyxovirus is purified to a level in which the particle per PFU ratio is no greater than 3. The RNA virus according to claim 49, wherein said Paramyxovirus is purified to a level in which the particle per PFU ratio is no greater than 1.2.
42. 56. The RNA virus according to any one of claims 49 to 55, wherein said Paramyxovirus is avian paramyxovirus type 2.
43. 57. The RNA virus according to any one of claims 49 to 55, wherein said Paramyxovirus is NDV.
44. 58. The RNA virus according to any one of claims 49 to 55, wherein said Paramyxovirus is mumps virus.
45. 59. The RNA virus according to any one of claims 49 to 55, wherein said Paramyxovirus is human parainfluenza virus. The RNA virus according to any one of claims 42 to 59, wherein said neoplasm is a cancer selected from the group consisting of lung, colon, prostate, breast and brain cancer.
46. 61. The RNA virus according to any one of claims 42 to 59, wherein said neoplasm is a solid tumor.
47. 62. The RNA virus according to claim 60, wherein said brain cancer is a glioblastoma. [R:\LIBVV]501959dlspeci.doc:THR 79 S63. The RNA virus according to claim 42, wherein said virus contains a gene C encoding interferon to permit the viral expression of interferon.
48. 64. The RNA virus according to claim 42, wherein said virus contains a gene Sencoding a pro-drug activating enzyme. 00 CN 5 65. The RNA virus according to claim 42, wherein said use further comprises administering IFN, before, during or after administration of said virus. N 66. The RNA virus according to claim 65, wherein said interferon is selected from the group consisting of c-IFN, P-IFN, o-IFN, y-IFN, and synthetic consensus forms of IFN.
49. 67. The RNA virus according to claim 42, wherein said use further comprises C administering a tyrosine kinase inhibitor before, during or after administration of said virus.
50. 68. The RNA virus according to claim 42, wherein said use further comprises administering a compound selected from the group of compounds comprising a purine nucleoside analog, tyrosine kinase inhibitor, cimetidine, and mitochondrial inhibitor.
51. 69. The RNA virus according to claim 42, wherein said use further comprises administering a chemotherapeutic agent before, during or after administration of said virus. The RNA virus according to claim 42, wherein said use further comprises administering a cytokine before, during or after administration of said virus.
52. 71. The RNA virus according to claim 42, wherein said use further comprises administering an immunosuppressant before, during or after administration of said virus.
53. 72. The RNA virus according to claim 42, wherein said use further comprises administering a viral replication controlling amount of a compound selected from the group consisting of IFN, ribavirin, acyclovir, and ganciclovir.
54. 73. The RNA virus according to claim 42, wherein said virus is selected from the group consisting of the Newcastle disease virus strain MK107, Newcastle disease virus strain NJ Roakin, Sindbis virus, and vesicular stomatitis virus.
55. 74. Use of an interferon-sensitive, replication-competent clonal RNA virus for the manufacture of a medicament for the treatment of a neoplasm in a mammal, wherein said RNA virus replicates at least 10 fold less in the presence of interferon compared to in the absence of interferon. The use according to claim 74, wherein said RNA virus replicates at least
56. 100-fold less in the presence of interferon compared to in the absence of interferon. [R:\LIBVV]501959dlspeci.doc:THR O 76. The use according to claim 74, wherein said RNA virus replicates at CN least 1000-fold less in the presence of interferon compared to in the absence of interferon. 77. The use according to any one of claims 74 to 76, wherein said neoplasm is a cancer. 00 C 5 78. The use according to any one of claims 74 to 77, wherein said mammal is a human. 79. The use according to any one of claims 74 to 78, wherein said clonal virus iis plaque purified. 80. The use according to any one of claims 74 to 79, wherein said clonal virus is of recombinant clonal origin. C 81. The use according to any one of claims 74 to 80, wherein said RNA virus is a Paramyxovirus. 82. The use according to claim 81, wherein said Paramyxovirus is purified to a level of at least 2 x 10 9 PFU per mg of protein. 83. The use according to claim 81, wherein said Paramyxovirus is purified to a level of at least 1 x 1010 PFU per mg of protein. 84. The use according to claim 81, wherein said Paramyxovirus is purified to a level of at least 6 x 1010 PFU per mg of protein. The use according to claim 81, wherein said Paramyxovirus is purified to a level in which the particle per PFU ratio is no greater than 86. The use according to claim 81, wherein said Paramyxovirus is purified to a level in which the particle per PFU ratio is no greater than 3. 87. The use according to claim 81, wherein said Paramyxovirus is purified to a level in which the particle per PFU ratio is no greater than 1.2. 88. The use according to claim 81, wherein said Paramyxovirus is avian paramyxovirus type 2. 89. The use according to any one of claims 81 to 88, wherein said Paramyxovirus is NDV. The use according to any one of claims 81 to 88, wherein said Paramyxovirus is mumps virus. 91. The use according to any one of claims 81 to 88, wherein said Paramyxovirus is human parainfluenza virus. 92. The use according to any one of claims 74 to 91, wherein said neoplasm is a cancer selected from the group consisting of lung, colon, prostate, breast and brain cancer. [R:\LIBVV]5019O59dlspeci.doc:THR 22, NOV. 2305 16:13 SPRUSON AND FERGUSON 6:2£2615486 NO. 7727 12 U) 81 93. The use according to any one of claims 74 to 91, wherein said neoplasm is l a solid tumor. S94. The use according to claim 92, wherein said brain cancer is a glioblastoma. Z 95. The use according to claim 74, wherein said virus contains a gene Cl s encoding interferon to permit the viral expression of interferon. 96. The use according to claim 74, wherein said virus contains a gene encoding a pro-drug activating enzyme. S97. The use according to any one of claims 74 to 96, wherein said medicament In is for intravenous or intratumoral administration. S1to 98. A method of treating a neoplasm in a mammal comprising subjecting a C sample from said mammal to an immunoassay to detect the amount of virus receptor 0 present, and if said receptor is present, administering an interferon-sensitive, replication competent clonal virus, which binds said receptor, to said mammaL 99. An interferon-sensitive, replication-competent clonal virus when used in a is method of treating a neoplasm according to claim 98. 100. The method according to any one of claims 1 to 41, wherein said RNA virus is purified by: generating a clonal virus; and purifying said clonal virus by ultracentrifugation without pelleting.
57. 101. The RNA virus according to any one of claims 42 to 73, wherein said RNA virus is purified by: generating a clonal virus; and purifying said clonal virus by ultracentrifugation without pelleting.
58. 102. The use according to any one of claims 74 to 97, wherein said RNA virus is purified by: generating a clonal virus; and purifying said clonal virus by ultracentrifugation without pelleting.
59. 103. The method according to any one of claims 1 to 41, wherein said RNA virus is a Paramyxovirus purified by ultracentrifugation without pelleting.
60. 104. The RNA virus according to any one of claims 42 to 73, wherein said RNA virus is a Paramyxovirus purified by ultracentrifugation without pelleting.
61. 105. The use according to any one of claims 74 to 97, wherein said RNA virus is a Paramyxovirus purified by ultracentrifugation without pelleting.
62. 106. The method according to claim 103, wherein said purification additionally comprises prior to said ultracentrifugation: [R:\LIBVY]501959dlpeci.doc;THR COMS ID No: SBMI-02015948 Received by IP Australia: Time 16:23 Date 2005-11-22 24, NOV. 2305 11:21 SPRUSON FERGUSON NO. 7208 FP, 82 plaque purifying to generate a clonal virus; inoculating eggs with said clonal virus; incubating said eggs; 0 chilling said eggs; Z s harvesting allantoic fluid from said eggs and; S(f) removing cell debris from said allantoic fluid.
63. 107. The KNA virus according to claim 104, wherein said purification IN additionally comprises prior to said ultracentrifugation: 0 plaque purifying to generate a clonal virus; Sto inoculating eggs with said clonal virus; c-, Cf incubating said eggs; c-l S(d) chilling said eggs; 0i harvesting allantoic fluid from said eggs and; removing cell debris from said allantoic fluid. i 108. The use according to claim 105, wherein said purification additionally comprises prior to said ultracentrifugation: plaque purifying to generate a clonal virus; inoculating eggs with said clonal virus; incubating said eggs; chilling said eggs; harvesting allantoic fluid from said eggs and; removing cell debris from said allantoic fluid.
64. 109. The method according to claim 103 or 108 wherein the said Paramyxovirus is NDV.
65. 110. The RNA virus according to claim 104 or 107, wherein said Paramyxovirus is NDV,
66. 111. The use according to claim 105 or 108, wherein said Paramyxovirus is NDV.
67. 112. A method according to claim 1 or 2, wherein said clonal virus is derived from a single infectious virus particle and for which examination of at least eight molecular clones have sequence homology of at least 95% over 300 contiguous nucleotides.
68. 113. A method according to claim 112, wherein said least eight molecular clones have sequence homology of at least 97% over 300 contiguous nucleotides. IR:\LIBVV]501959dl speci.doc:TIHR COMS ID No: SBMI-02036458 Received by IP Australia: Time 11:25 Date 2005-11-24 24. NOV. 2305 11:21 SPRUSON FERGUSON NO. 7208 P. 6 83 i114. A method according to claim 112, wherein said least eight molecular Sclones have sequence homology of at least 99% over 300 contiguous nucleotides.
69. 115. A method according to claim I or 2, wherein the neoplasm is selected from o lung carcinoma, breast carcinoma, prostate carcinoma, colon adenocarcinoma, cervical Z s carcinoma, endometrial carcinoma, ovarian carcinoma, bladder carcinoma, Wilm's tumor, fibrosarcoma, osteosarcoma, melanoma, synovial sarcoma, neuroblastoma, lymphoma, leukaemia, brain cancer including glioblastoma, neuroendocrine carcinoma, renal Scarcinoma, head and neck carcinoma, stomach carcinoma, esophageal carcinoma, Svulvular carcinoma, sarcoma, skin cancer, thyroid pacreatic cancer, and mesothelioma.
70. 116. The method according to claim 18, wherein the NDV is administered systemically in one or more doses of from about 1 x 10 8 to about 4 x 10" PFU per square Smeter of body surface area per dose. 0
71. 117. The use according to claim 89, wherein the NDV is formulated for systemic administration in one or more doses of from about I x 10$ to about 4 x 10" PFU is per square meter of body surface area per dose. Dated 24 November 2005 Pro-Virus, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [R.;LIBVV]501959dl_specci.doc:TH COMS ID No: SBMI-02036458 Received by IP Australia: Time 11:25 Date 2005-11-24