MXPA00003467A - Treatment of neoplasms with viruses - Google Patents

Abstract

The subject invention relates to viruses that are able to replicate and thereby kill neoplastic cells with a deficiency in the IFN-mediated antiviral response, and their use in treating neoplastic disease including cancer and large tumors. RNA and DNA viruses are useful in this regard. The invention also relates to methods for the selection, design, purification and use of such viruses for cancer therapy.

Description

TREATMENT OF NEOPLASMS WITH VIRUSES Field of the Invention The present invention relates to viruses that are capable of replicating in, and causing the death of, neoplastic cells with a deficiency in the antiviral response mediated by interferon (IFN). 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 that includes cancer is one of the leading causes of death among humans. There are more than 1.3 million new cases of cancer diagnosed in the United States of America each year, and 550,000 deaths. The early detection of cancer, before it has spread to secondary sites in the body, greatly increases the chances of survival of a host. However, the early detection of cancer is not always possible, and even when it is, the treatments are unsatisfactory, especially in cases of highly malignant cancers. Treatments for cancer, including chemotherapy and radiation, are much less effective in later stages, especially when neoplastic growths are large and / or constitute a high tumor burden. (See Hillard (Stanley, Cancer Treat., Reports, Volume 61, Number 1, Jan / Feb 1977, pages 29-36, Tannock, Cancer Research, 42, 4921-4926, December, 1982.) Tumor regression has been reported. Associated with exposure to different viruses Most of the viruses described are pathogenic in humans, including mumps and measles, and 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 cervical carcinoma), Holzaepfel et al., (1957) Cancer, 10, 557 (effect of adenovirus on epithelial tumor) Taylor et al., (1970) J. Nati.

Cancer Inst .. 44, 515 (effect of bovine enterovirus-1 in sarcoma-1); Shingu et al., (1991) J. General Viroloqy, 72, 2031 (effect of bovine enterovirus MZ-468 on leukemia cells F-647a); Suskind et al., (1957) PSEBM, 94, 309 (effect of B3 Coxsackie virus on HeLa tumor cells); Rukavishnikova et al., (1976) Acta Virol. , 20, 387 (effect of strain A of influenza on ascites tumor). Previous references described the partial regression of the tumor in patients treated with live attenuated viral vaccine, for the purpose of vaccinating them against smallpox or rabies. See DePace, N.G. (1912) Gynecology, 9, 82-88; Salmon, P. and Baix (1922) Compt. Rend. Soc. Biol., 86, 819-820. Partial regression of tumors and regression of leukemia during naturally occurring measles infections have also been noted. 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 a study of 90 cancer patients intentionally infected with live mumps virus, partial regression of the tumor was noted in 79 cases. See Asada (1994) Cancer, 34, 1907-1928. Although the side effects of these viruses were temporary, the serious sequel to infection with these human pathogens is of major concern. Viruses are categorized as follows [see Murphy A and Kingsbury DW, 1990, In: Virology, 2"Edition (Ed. Fields, B.N.), Raven Press, New York, pages 9-35]: Division Character Names of the Virus Family RNA virus ssRNA, positive sense, Picomaviridae, Calciviridae not segmented, not enveloped, ssRNA, positive sense, Togavi dae, Flaviviridae, not segmented, Coronaviridae enveloped, .RNAs, negative sense, Rhabdoviridae, Filoviridae, not segmented, Paramyxoviridae involved,. SssRNA, negative sense, Orthomyxoviridae, segmented, enveloped ssRNA, ambisense, Bunyaviridae, Arenaviridae, segmented, enveloped. ARNds, positive sense, Reoviridae, Birnaviridae, segmented, non-enveloped .RNAs, DNA passage in Retroviridae, replication, positive sense, non-segmented , wrapped virus. DN ssDNA / da, not enveloped Hepadnaviridae ssDNA, not enveloped Parvoviridae .ADNds, not enveloped Papovaviridae, Adenovi dae dsDNA, enveloped Herpesviridae, Poxviridae, Iridoviridae Included among the family Herpesviridae (or Herpetic Virus), are the subfamilies Alphaherpesvirus (including Genus Varicellavirus and Genus Simpexvirus), Betaherpesvirus, and Gamaherpesvirus. The Newcastle disease virus ("VEN") is a member of the Paramyxoviridae (or Paramyxovirus). The natural guests for the VEN are the chickens and other birds. VEN typically binds to certain molecules on the surface of host cells of the animal, fuses with the cell surface, and injects its genetic material into the host. VEN is a cytocidal virus. Once inside the cell, viral genes direct the host cell to make copies of the virus, leading to death to the host cell, releasing copies of the VEN that infect other cells. Unlike some viruses, it is not known that VEN causes any serious human disease. Unlike other virus classes (eg, HTLV-1, Hepatitis B), Paramyxoviruses are not known to be carcinogenic. Temporary regression of tumors has been reported in a small number of patients exposed to VEN, See, Csatary, L.K. (1971) Lancet, 2, 825. Csatary noted the regression of a gastrointestinal cancer in a poultry farmer during an epidemic of Newcastle disease in his chickens. In a similar anecdotal report, Cassel, W.A. and Garrett, R.E. (1965) Cancer, 18, 863-868, noted the regression of primary cervical cancer, which had spread to lymph nodes in a patient, after injection of VEN into the cervical tumor. Since it was believed that the mechanism of tumoricidal activity was immunological, no work has been done to direct the direct cytotoxicity of the virus tumor. Rather, efforts focused on the immunomodulatory effects of VEN. See, for example, urray, D.R., Cassel, W.A. , Torbin, A.H., Olkowski, Z.L., and Moore, M.E. (1977) Cancer, 40, 680; Cassel, W.A., Murray, D.R. and Phillips, H.S. (1983) Cancer, 52, 856; Bohle, W., Schlag, PJ. , Liebrich, W., Hohenberger, P., Manasterski, M., Miller, P., and Schirrmacher, V. (1990) Cancer, 66, 1517-1523. The selection of a virus specific for the regression of the tumor was based on good luck to find things by chance or in trial methods in the previous appointments. Only recently have rational approaches, based on a mechanism, been developed for the use of viruses in the treatment of cancer, 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 (Rodríguez, R. et al., 1997 Cancer Res., 57: 2559-2563), or those that they lack certain key regulatory proteins (Bischoff, JR, et al., 1996 Science, 274: 373-376). Another recent approach has been the use of the incompetent recombinant adenoviral vector for replication, to restore a critical protein function lost in some tumor cells (Zhang, WW, et al., 1994 Cancer gene therapy, 1: 5-13). Finally, the herpes simplex virus has also been designed to replicate preferentially in the rapidly dividing cells that characterize the tumors (Mineta, T., et al., 1994 Cancer Res., 54: 3963-3966). U.S. Patent Application Serial Number 08 / 260,536, incorporated herein by reference in its entirety, discloses the use of VEN or another Paramyxovirus in the treatment of cancer.

Expression of the viral IFN transgene A common approach for the treatment of cancer with viral therapeutics has been the use of virus vectors for the application of certain genes to the tumor mass. > All recombinant adenoviruses, adeno-associated viruses, vaccinia viruses and retroviruses have been modified to express an interferon gene alone, or in combination with other cytokine genes. In Zhang et al ((1996) Proc. Nati. Acad. Sci., USA 93: 4513-4518), a recombinant adenovirus expressing a consensus (ie, synthetic) human interferon gene was used to treat xenografts of human breast cancer (and others) in hairless mice. The authors concluded "... a combination of viral oncolysis with a low pathogenicity virus, itself resistant to the effects of IFN and IFN gene therapy, may be a successful approach for the treatment of a variety of different tumors, in particular breast cancer. " In contrast to the present invention that 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), an adeno-associated virus (AAV) expressing consensus IFN was used to transduce human tumor cells in vitro, followed by injection into mice hairless. Tumors transduced either did not form tumors, or grew more slowly than non-transduced controls. In addition, the injection of a transduced human tumor cell into the tumor mass of another non-transduced tumor resulted in a small decrease in size. Peplinski et al. ((1996) Ann. Sur. Oncol., 3: 15-23) tested IFN range (and other expressed cytokines either alone or in combination) in a mouse breast cancer model. Mice were immunized with virally modified tumor cells with recombinant vaccinia virus. When they were again assaulted with tumor cells, the mice immunized with virally modified cells had statistical improvement in the time of disease-free survival. Gastl, et al. ((1992) Cancer Res., 52: 6229-6236), used retroviral vectors expressing the IFN range to transduce renal carcinoma cells in vitro. It was shown that these cells 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), they used the retroviral vector that expressed gamma IFN to transduce a murine sarcoma cell line, allowing the tumor cell line to more efficiently display viral antigens for CD8 + T cells. Howard, et al. ((1994) Ann. NY Acad. Sci., 716: 167-187 ), used the retroviral vector that expressed gamma IFN to transduce murine and human melanoma tumor cells. It was observed that these cells increased the expression of proteins important for immune function. These cells were also less tumorigenic in mice, compared to the untransduced parent line, and resulted in the activation of a tumor-specific CTL response in vivo.

Use of Interferon Therapeutic Doses as an Adjuvant for Viral Cancer Therapy Due to the known immunity enhancing properties of IFN, many studies have examined the use of the IFN protein in combination with other viral cancer vaccine therapies. In Kirchner et al ((1995) World J. Urol., 13: 171-173), 208 patients were immunized with tumor cells of renal cell carcinoma, autologous, modified with VEN, and lethally irradiated, and cotrataron with a low dose of IL-2 or alpha IFN. The authors stated that this treatment regimen resulted in an improvement over the natural course in patients with locally advanced renal cell carcinoma. The dose was approximately 3.3 x 103 to 2.2 x 105 PFU / kg. This was a local therapy, as opposed to a systematic approach, with the goal of inducing an antitumor immune response. Tanaka et al. ((1994) J. Immunother, Emphasis Tumor Immunol., 16: 283-293), co-administered alpha IFN with a recombinant vaccinia virus as a model of cancer vaccine therapy in mice. This study showed a statistical improvement in survival in mice that received IFN, compared to those who did not receive it. The authors attributed the efficacy of IFN to the induction of positive CD8-T cells in these animals. Arroyo et al. ((1990) Cancer Immunol. Immunother., 31: 305-311) used a mouse model with colon cancer to test the effect of alpha IFN and / or IL-2 therapy on the efficacy of a virus colon cancer (OCV) cancer treatment. vaccinia They found that the triple treatment of OCV + IL-2 + IFN was the most effective in this murine model. This approach depends on immunization as the mechanism of antitumor activity. IFN was used in these studies to increase the ability of cancer cells to be recognized by the immune system.bJ.

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 and / or classified for use in the therapy of neoplastic diseases. It is a further object of the invention to provide guidance in the genetic design of viruses, in order to improve their therapeutic utility in the treatment of neoplastic diseases. It is a further object of the invention to provide a means by which to classify potential target cells for viral therapy, with the goal of evaluating the sensitivity of target cells for viral death. It is still a further object of the invention to provide guidance in the administration 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 viruses and methods for obtaining same.

Summary of the Invention This invention relates to a method for infecting a neoplasm in a mammal with a virus, comprising administering an interferon-responsive clonal virus responsive to replication, selected from the group consisting of RNA viruses, and families of Adenovirus .DNA virus, Parvovirus, Papovavirus, Iridovirus, and Herpesvirus, to the mammal. This invention also relates to a method for infecting a neoplasm in a mammal with a virus, comprising systematically administering a clonal virus sensitive to interferon, competent for replication to the mammal. This invention also relates to a method for treating a neoplasm that includes cancer in a mammal, which comprises administering to the mammal a therapeutically effective amount of a clone virus responsive to interferon, competent for replication, selected from the group consisting of .ARN, and the families of DNA viruses of Adenovirs, Parvovirus, Papovavirus, Iridovirus, and Herpesvirus. This invention also relates to a method for infecting a neoplasm in a mammal with a virus, which comprises administering a clonid vaccinia virus sensitive to interferon, competent for replication, having one or more mutations in one or more viral genes, involved with blocking the antiviral activity of interferon, selected from the group of genes consisting of K3L, E3L and B18R, to the mammal. The invention also relates to a method of treating a neoplasm that includes cancer in a mammal, administering to the mammal a therapeutically effective amount of a replication-competent clone vaccinia virus responsive to one or more mutations in one or more viral genes, involved with blocking the antiviral activity of interferon, selected from the group of genes consisting of K3L, E3L and B18R. The invention also relates to a method for infecting a neoplasm of at least 1 centimeter in size, with a virus in a mammal, comprising administering a clonal virus, selected from the group consisting of (1) .ARN virus; (2) Hepadanovirus; (3) Parvovirus; (4) Papovavirus; (5) Herpesvirus; (6) Poxvirus; and (7) Iridovirus, to the mammal. The invention also relates to a method for treating a neoplasm in a mammal, comprising administering to the mammal a therapeutically effective amount of a clonal virus, selected from the group consisting of (1) RNA virus; (2) Hepadanovirus; (3) Parvovirus; (4) Papovavirus; (5) Herpesvirus; (6) Poxvirus; and (7) Iridovirus, where the neoplasm is at least 1 centimeter in size. The invention also relates to a method for treating a tumor in a mammal, which comprises administering to the mammal a therapeutically effective amount of a cytocidal RNA virus to the tumor., wherein the mammal has a tumor load that comprises at least 1.5 percent of the total body weight. The invention also relates to a method for classifying tumor cells or tissue freshly removed from the patient, to determine the sensitivity of cells or tissue to death by a virus, which comprises subjecting cells or tissue to a differential cytotoxicity assay, using an interferon-sensitive virus. The invention also relates to a method for identifying a virus with antineoplastic activity in a mammal, comprising a) using the test virus to infect i) cells deficient in interferon-mediated antiviral activity, and ii) competent cells in mediated antiviral activity by interferon, and b) determining whether the test virus kills cells deficient in interferon-mediated antiviral activity, preferentially to competent cells in interferon-mediated antiviral activity. The invention also relates to a method for creating a virus for use in antineoplastic therapy, comprising: a) modifying an existing virus by means of decreasing or removing a viral mechanism for inactivation of the antiviral effects of IFN, and optionally b) create an attenuating mutation that results in less virulence than the existing virus. The invention also relates to a method for controlling viral replication in a mammal treated with a virus selected from the group consisting of RNA viruses, Adenoviruses, Poxviruses, Iridoviruses, Parvoviruses, Hepadnaviruses, Varicellaviruses, Betaherpesviruses, and Gamaherpesviruses, which comprises administering an antiviral compound. The invention also relates to a method for treating or infecting a neoplasm in a mammal, comprising subjecting a sample (eg, serum, tumor cells, tumor tissue, tumor section) of the mammal to an immunoassay, to detect the amount of receptors of viruses present, to determine if the neoplasm will allow the virus to bind or cause cytolysis, and if the receptor is present, by administering a clonal virus responsive to interferon, competent for replication, to bind the receptor, to the mammal. The invention also relates to a method for infecting a neoplasm in a mammal with a virus, comprising systematically administering a desensitizing dose of a clonal virus sensitive to interferon, competent for replication, to the mammal. The invention also relates to a method for infecting a neoplasm in a mammal with a virus, comprising administering a clonal virus sensitive to interferon, competent for replication, to the mammal, during a course of at least 4 minutes. The invention also relates to a method for infecting a neoplasm in a mammal with a virus, which comprises administering a clone virus competent for replication, selected from the group consisting of the Newcastle disease virus, strain MK107, virus of the Ne castle disease, NJ Roakin strain, Sindbis virus, and vesicular stomatitis virus. Included in the invention are: i) a Paramyxovirus purified by ultracentrifugation without formation of granules; ii) a purified Paramyxovirus to a level of at least 2 x 109 PFU per milligram of protein; iii) a purified Paramyxovirus to a level of at least 1 x 1010 PFU per milligram of protein; iv) a purified Paramyxovirus to a level of at least 6 x 1010 PFU per milligram of protein; v) an RNA virus purified to a level of at least 2 x 109 PFU per milligram of protein; vi) an RNA virus purified to a level of at least 1 x 1010 PFU per milligram of protein; vii) an RNA virus purified to a level of at least 6 x 1010 PFU per milligram of protein; viii) a cytocidal DNA virus, which is sensitive to interferon, and purified to a level of at least 2 x 109 PFU per milligram of protein; ix) a vaccinia virus competent for replication having a) one or more mutations in one or more of the K3L, E3L, and B18R genes, and b) an attenuating mutation in one or more of the genes encoding the thymidine kinase, ribonucleotide reductase, vaccinia growth factor, thymidylate kinase, DNA ligase, dUTPase; x) a vaccinia virus competent for replication, having one or more mutations in two or more genes selected from the group consisting of K3L, E3L, and B18R; xi) a Herpesvirus that has a modification in the expression of the analogue (2'-5 ') A, causing the Herpesvirus to have increased sensitivity to interferon; and xii) a Reovirus that has an attenuating mutation in omega 3, causing that virus to become sensitive to interferon. Also included in the invention are the following methods: i) a method for purifying an RNA virus comprising the steps of a) generating a clonal virus; and b) purifying that clonal virus by ultracentrifugation without formation of granules; or c) purifying that clonal virus by tangential flow filtration, with or without the subsequent gel permeation chromatography, and ii) a method for purifying a Paramyxovirus, which comprises purifying the virus by ultracentrifugation without granule formation, or by flow filtration. tangential, with or without the subsequent gel permeation chromatography. The invention also relates to a method of treating a disease in a mammal, wherein the diseased cells have defects in an interferon-mediated antiviral response, which comprises administering to the mammal a therapeutically effective amount of a competent interferon-sensitive clonal virus. for replication.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the effect of the anti-beta-interferon antibody on viral antigen expression and infectious titer in QEHN cells (normal human epithelial keratinocytes). Figure 2 shows the effect of beta-interferon 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. Figure 3B shows the effect of interferon on viral antigen expression in KB cells. Figure 4 shows the survival curves for athymic mice containing human ES-2 ovarian carcinoma cells, and treated with either saline or VEN, strain PPMK107. Figure 5 shows the responsiveness of interferon to a number of human tumor and normal cell lines.

Detailed Description of the Invention The present invention relates to the discovery of a novel mechanism by which viral replication selectively kills neoplastic cells deficient in an interferon-mediated antiviral response (IFN). This invention also provides methods for the selection, design, purification and use of viruses for the treatment of neoplastic diseases, including cancer and large tumors. The viruses of the invention selectively replicate in, and kill the neoplastic cells based on the selective deficiency in these cells of an antiviral response mediated by interferon. The administration of the appropriate dose of the virus results in the death of the neoplastic cells, whereas normal cells, which possess an antiviral response mediated by intact interferon, limit the replication of the virus and do not die. Included in the present invention are the use of paramyxoviruses such as VEN, and other viruses, for use in the treatment of diseases, including neoplastic disease such as cancer. The invention also teaches the classification and design of other viruses suitable for use as therapeutics of neoplastic diseases. Another embodiment of the invention involves a method for identifying tumor tissues that are candidates for viral therapy. Finally, the invention also describes the preparation of highly purified viruses.

Rationale for the use of interferon-sensitive viruses, including VEN, to treat neoplastic disease VEN demonstrates the selective elimination of tumor cells. The Newcastle disease virus causes selective cytotoxic effects against many human tumor cells, with markedly fewer effects on the majority of normal human cells. In a differential cytotoxicity assay, human cancer cells derived from sarcomas, melanomas, breast carcinomas, ovarian carcinomas, bladder carcinomas, colon carcinoma, prostate carcinoma, small cell lung carcinomas and non-small cell, and glioblastomas, are approximately 3 to 4 orders of magnitude more sensitive to VEN than many normal human cells [renal epithelial cells, fibroblasts, keratinocytes, melanocytes, and endothelial cells (see Example 1)]. The differential cytotoxicity assay can also be applied to fresh cell isolates or tumor tissue from the patient. An in vitro assay was used to define the tumoricidal activity of VEN, as described in Example 1. The assay measures the amount of virus required to eliminate 50 percent of the cell culture tested over a period of time of five days. Examples 2 and 3 show the results of in vivo experiments, in which the virus was administered to athymic mice having human tumor xenografts, by means of either the intratumoral (Example 2) or intravenous route (Example 3). These results demonstrate that VEN can cause the regression of a variety of human tumor types in a standard animal model for the testing of potential chemotherapeutic agents. Evidence that VEN replicates specifically within the tumor was demonstrated by immunohistochemical staining for the virus antigen (Example 2). Within the next 30 minutes of intratumoral injection of the virus, the tumor tissue was negative for the viral antigen. However, by day 2 after the treatment, intense immunostaining was seen for the viral antigen inside the tumor, indicating the replication of the virus inside the tumor. Importantly, the replication of the virus was specific for the tumor tissue since the surrounding connective tissue and the skin were negative for the viral antigen. Importantly, efficient replication of VEN is crucial to the ability of the virus to kill infected cells, as demonstrated in studies using non-clonal UV inactivated virus (Lorence, R., et al., 1994 J Nati Cancer Inst., 86: 1228 -1233). VEN can also cause regression of large tumors after intratumoral and intravenous administration (Examples 4 to 9). Intratumoral treatment with VEN of large intradermal A375 human melanoma xenografts (= 10 millimeters in maximum dimension, tumor volume = 300 cubic millimeters) in athymic mice, led to high rates of tumor regression (Examples 4 to 8). The intravenous treatment of VEN of large subcutaneous human fibrosarcoma HT1080 xenografts (= 10 millimeters in maximum dimension) in athymic mice, led to complete or partial tumor regression in five out of six mice (Example 9).

The family of interferon class I cytokines are important negative modulators of viral infection. Class I interferons consist of lFN, which is found mainly in cells of hematopoietic origin, and of ßlFN, which is found mainly in fibroblasts and epithelial cells. [Joklik, W.K. 1990. Interferons, pages 383-410. Virology, second edition, edited by B.N. Fields, D.M. Knipe et al., Raven Press Ld. , NY; and Sreevalsan, T. 1995. Biological Therapy with Interferon-a and ß: Preclinical Studies, pages 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 work through a seemingly common mechanism of action, which includes the degradation of the double-stranded RNA intermediates of viral replication, and the inhibition of cell translation through the activity of an activated protein kinase. by double-stranded RNA [Joklik, WK 1990. Interferons, pages 383-410. Virology, second edition, edited by B.N. Fields, D.M. Knipe et al., Raven Press Ld. , NY; and references therein]. Many viruses (influenza, EBV, SV40, adenovirus, vaccinia) have developed mechanisms by which one or more IFN system trajectories are inactivated, thus allowing efficient replication of the virus (Katze, MG 1995, Trends in Microbiol., 3: 75- 78).

A wide variety of tumor cells are deficient in the ability to limit viral infection through a mechanism dependent on IFN. Human cervical carcinoma cells (HeLa) were more than one hundred times less sensitive to the inhibition of vesicular stomatitis virus replication after previous treatment with IFN, than a non-transformed fibroblast control cell line (Maheshwari RK, 1983. Biophys, Res. Comm. 17: 161-168). The present inventors have discovered that infection of a co-culture of tumorigenic human head and neck carcinoma (KB) cells and normal human skin fibroblast cells (CCD922-sk) results in viral replication initially in both cell types, followed by a limitation of the infection in the normal cells, against the continuous replication and the elimination of the tumor cells (Example 10). On the other hand, although the normal cells were secreting IFN in the culture medium, the tumor cells were unable to respond to the IFN at the concentrations that were being produced to establish an antiviral state. Additional evidence was obtained for the role of IFN in the differential sensitivity of tumor cells against normal cells, for elimination by VEN, in two separate experiments in which normal fibroblast cells (CCD922-sk) or cells were shown to of normal epithelial keratinocytes (QEHN) became more sensitive to infection with VEN, in the presence of neutralizing antibodies to IFN (Examples 11 and 12).

Finally, the parallel infection of normal fibroblasts (CCD922-sk) and human tumor cells (KB), in the presence of IFN, revealed that normal cells were at least 100 times more sensitive to the antiviral effects of IFN added than were tumor cells (Examples 13 and 14).

The similar test of various tumor cell lines (a total of 9) revealed a clear correlation in the relative sensitivity of a cell line to elimination by VEN, and an inability of the cell line to manifest an antiviral response mediated by interferon (Example 26) .

Interferon and Cell Growth There are many species of interferon (IFN) including the natural and recombinant forms of -IFN, β-IFN,? -IFN, and? -IFN, as well as synthetic consensus forms (for example, as described in Zhang). and collaborators, (1996) Cancer Gene Therapy, 3: 31-38). In addition to the antiviral activities that led to its discovery, it is now known that IFN plays an important role in the normal regulation of cell growth and differentiation. IFN is seen as a negative growth regulator, and it has been shown that many key proteins involved in the function and regulation of IFN activity act as tumor suppressor proteins in normal cells (Tanaka, et al., 1994 Cell 77: 829 -839). On the other hand, many other proteins known to antagonize the antiviral activity of IFN have been shown to have oncogenic potential when expressed inappropriately (see below, Barber, GN, 1994, Proc. Nati, Acad. Sci. USA 91: 4278 -4282). It has been shown that cells derived from many human cancers are eliminated in the genes encoding IFN (James, CD, et al., 1991, Cancer Res., 51: 1684-1688), and partial or complete loss has been observed. of the role of IFN in human cervical carcinoma (Petricoin, E, et al., 1994 Mol. Cell. Bio., 14: 1477-1486), chronic lymphocytic leukemia (Xu, B., et al., 1994, Blood, 84 : 1942-1949), and malignant melanoma cells (Linge, C, et al., 1995, Cancer Res., 55: 4099-1404). It has been shown that IFN-inducible protein kinase (p68) is an important regulator of cellular and viral protein synthesis. A correlation has arisen that links the expression or activity of the p68 kinase to the cell differentiation state. In this way, poorly differentiated cells, such as those that occur in many cancers, are deficient in the p68 function (Haines, G.K., et al., 1993 Virchows Arch B Cell Pathol., 63: 289-95). Cells lacking p68 activity are generally sensitive to virus mediated clearance, because p68 kinase is a major effector of the antiviral state inducible by IFN. 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 Nati Acad Sci USA 91: 4278-4282), and a number of cell lines of human leukemia have been shown to overexpress the p58 protein (Korth MJ, et al., 1996 Gene 170: 181-188). The sensitivity to viral elimination in undifferentiated cells can be reversed through the induction of a more differentiated phenotype (Kalvakolanu, DVR and Sen, G.C. 1993 Proc Nati Acad Sci USA 90: 3167-3171).

Definitions Competent cells in an antiviral response mediated by interferon. As used herein, the term "cells competent in an interferon-mediated antiviral response" are cells that respond to low levels (eg, 10 units per milliliter) of exogenous interferon, by means of significantly reducing (at least 10 times , more conveniently at least 100 times, more conveniently at least 1000 times, and more convenient at least 10,000 times) the replication of an interferon-sensitive virus, compared to the absence of interferon. The degree of replication of the virus is determined by measuring the amount of virus (eg, infectious virus, viral antigen, viral nucleic acid). Normal CCD922 fibroblasts are competent cells in an antiviral response mediated by interferon. Cells deficient in an antiviral response mediated by interfßrón. As used herein, the term "cells deficient in an interferon-mediated antiviral response" are cells that fail to meet the criteria listed above for a competent cell in an interferon-mediated antiviral response., that is, they do not manage to respond to low levels (for example, 10 units per milliliter) of exogenous interferon, by means of significantly reducing the replication of a virus sensitive to interferon, in comparison with the absence of interferon. KB oral carcinoma cells are cells deficient in an antiviral response mediated by interferon. Clonal The use of the term "clonal" virus is hereinafter defined as a virus derived from a single infectious virus particle, and for which individual molecular clones have significant nucleic acid sequence homology. For example, sequence homology is such that at least eight individual molecular clones of the virion population have sequence homology greater than 95 percent, more conveniently greater than 97 percent, more conveniently greater than 99 percent, and more conveniently 100 percent over 300 contiguous nucleotides. Cytocidal As used herein, the term "cytocidal" virus refers to a virus that infects cells resulting in death. Desensitizing dose. As used herein, the phrase "desensitizing dose" refers to the amount of virus required to decrease the side effects of subsequent doses of the virus. Differential Cytotoxicity Test. As used herein, the phrase "differential cytotoxicity assay" for classifying tumor cells or tissue, using a virus, refers to (a) virus infection of tumor cells and one or more control cells or tissues.; (b) a determination of cell survival or death for each sample (for example, by using a cell viability dye indicator, as detailed in Example 1) after one or more days of infection; and (c) based on the results, an estimate of the sensitivity (e.g., by the IC50 determination as detailed in Example 1) of the sample to the virus, as compared to the control (s). Infecting a Neoplasm. As used herein, the term "infecting a neoplasm" refers to the entry of viral nucleic acid into neoplastic cells or tissue. Interferon sensitive. As used herein, the phrase "interferon-sensitive" virus (e.g., VEN) means a virus that replicates significantly less (at least 10 times less, conveniently at least 100 times less, more conveniently at least 1000 times less, and more conveniently at least 10,000 times less), in the presence of interferon, compared to the absence of interferon. This is determined by measuring the amount of virus (eg, infectious virus, viral antigen, viral nucleic acid) obtained from competent cells in an interferon-mediated antiviral response, in the presence or absence of low levels of exogenous interferon (eg. example, 10 units per milliliter). Neoplasm and Neoplastic Disease. As used herein, "neoplasm" means the new growth of tissue, including tumors, benign growths (e.g., condylomata, papillomas) and malignant growths (e.g., cancer). As used herein, "neoplastic disease" refers to the disease manifested by the presence of a neoplasm. Competent for Replica. As used herein, the term "replication competent" refers to a virus that produces infectious progeny in neoplastic cells. Egg Protein Free Sustancialmentß Pollutants The term "substantially free of contaminating egg proteins" refers to a level of virus purity in which ovalbumin can not be detected in a Western blot, as determined by one skilled in the art by means of (1) using 1.7 x 109 Virus PFU per well (3.3 centimeters wide) run on an SDS-PAGE gel (sodium dodecylsulfate polyacrylamide gel electrophoresis) (1 millimeter thick), (2) transfer the viral proteins from the gel to a nitrocellulose membrane; and (3) immunostaining ovalbumin with the use of a rabbit antiovalbumin [Rabbit IgG Fraction at a dilution of 1: 200 of an antibody concentration of 4 milligrams / milliliter (from Cappel, Inc.) or an equivalent polyclonal antibody] The Therapeutically Effective Amount As used herein, the term "therapeutically effective amount", when referring to the treatment of neoplastic disease, refers to a quantity of virus that produces the desired effect, for example, cessation of neoplastic growth, tumor regression, improved clinical conditions, or increased survival.

Compounds of the Invention A diverse group of viruses was used to selectively remove neoplastic cells. Natural or designed viruses can function as an antineoplastic agent.

These viruses i) infect the neoplastic cells resulting in their death; ii) are competent for replication in neoplastic cells; and iii) they are limited to eliminate normal cells by the antiviral effects of interferon. In a convenient embodiment of the invention, viruses having the above three characteristics [i) these infect the neoplastic cells resulting in their death; ii) these are competent for replication in neoplastic cells; and iii) these are limited to eliminate normal cells by the antiviral effects of interferon] they also induce interferon. In another preferred embodiment of the invention, viruses possess the three aforementioned characteristics also cause regression of the human neoplasm; and / or are not neutralized in the target human population due to the presence of pre-existing immunity. In another convenient embodiment, viruses possessing the above three characteristics are cytocidal for the tumor cells. A Paramyxovirus (as used herein "Paramyxovirus" refers to a member of the Paramyxoviridae) according to the present invention can be used to treat a neoplasm, including a large tumor or a host having a high tumor burden. The family Paramyxoviridae comprises three genera: (1) paramyxoviruses; (2) measles-like viruses (morbillia virus); and (3) respiratory syncytial virus (pneumovirus). These viruses contain an RNA genome. The use of Paramyxoviridae viruses that are cytocidal, especially paramyxoviruses, for example, Newcastle disease virus ("VEN") and other avian paramyxoviruses such as poultry paramyxovirus type 2, is a convenient method for practicing the invention. Attenuated strains of these viruses are especially useful for the treatment of neoplasms in accordance with the present invention. VEN is a particularly convenient virus according to the present invention. VEN is categorized into three distinct classes, in accordance with its effects on chickens and chicken embryos. The "low virulence" strains are referred to as lentogenic, and take 90 to 150 hours to kill chicken embryos at the minimum lethal dose (MLD); reference is made to strains of "moderate virulence" as mesogenic and take 60 to 90 hours to kill chicken embryos at the minimum lethal dose; The "high virulence" strains are referred to as velogenic and take 40 to 60 hours to kill chicken embryos at the minimum lethal dose. See, for example, Hanson and Brandly, 1955 (Science, 122: 156-157), and Dardiri et al, 1961 (Am. J. Vet. Res., 918-920): The three classes are convenient, conveniently, the mesogenic strains of VEN such as strain MK107, strain NJ Roakin, and strain Connecticut -70726 (see examples 21-23). See, for example, Schloer and Hanson, 1968 (J. 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 interference particles. The removal of defective interference particles by cloning allows an increased purity in the final product, as assessed by the number of total virus particles per infectious particle (e.g., the number of particles per PFU). The clonal virus can be produced in accordance with any method available to the skilled worker. For example, plaque purification is routinely used to obtain the clonal virus. See, for example, Maassab et al., In: Plotkin and Mortimer, editors, Vaccines Philadelphia: W.B. Saunders Co., 1994, pages 78-801. Especially desirable is triple plate purification, wherein a plate is selected in each round of purification, having the desired characteristics, such as a preferred size, shape, appearance, or that is representative of the parent strain. Another means of generating the clonal virus is by means of recombinant DNA techniques, which can be applied by one skilled in the art. Another means of obtaining a clonal virus applies the technique of limiting the dilution (for example, by adding dilutions of the virus sample to give an average of one or fewer infectious virus particles per well, containing a monolayer of a cell susceptible). In a convenient embodiment of the invention, the purified virus is used to treat neoplastic diseases. A convenient method for the purification of egg-derived viruses is as follows (viruses are not formed into granules at any step in these methods): Purification Method A a) generate a clonal virus (eg, plaque purification) b) inoculate the eggs with the clonal virus c) cover the eggs d) cool the eggs e) harvest the allantoic fluid from the eggs f) remove the waste from the allantoic fluid g) ultracentrifugation of the allantoic fluid without formation of granules (for example, using a discontinuous sucrose gradient). In another embodiment of the invention, the additional steps, added after the removal of the cell waste (from the allantoic fluid), and before the ultracentrifugation, consist of: • freezing and then thawing the allantoic fluid, • removing the contaminating material from the suspension of the virus (for example, by means of centrifugation). In another embodiment of the invention, ultracentrifugation is obtained by means of a continuous flow ultracentrifuge. One embodiment of the invention relates to a method for purifying an RNA virus competent for replication, comprising the steps of: a) generating a clonal virus, and b) purifying that clonal virus by ultracentrifugation without granule formation. Another embodiment of the invention involves a method for purifying a paramyxovirus (e.g., VEN), which comprises purifying the virus by ultracentrifugation without formation of granules. Optionally, the purification step additionally comprises, before ultracentrifugation: a) purifying the plate to generate a clonal virus, b) inoculating the eggs with the clonal virus, c) concealing the eggs, d) cooling the eggs, e) harvesting the allantoic fluid of the eggs and, f) remove the waste from the cell of the allantoic fluid. Another embodiment of the invention involves a method for purifying a clonal virus competent for egg replication or cell culture, comprising the ultracentrifugation step without a step in which the virus is formed into granules. Another embodiment of the invention involves a method for the purification of a paramyxovirus (e.g., VEN), which comprises the purification of the virus by sequential tangential flow filtration (FFT). Optionally, the virus can be further purified by gel permeation chromatography, where each of these steps occurs, in the presence of a stabilizing pH regulator (Example 15): a) purify the plate to generate a clonal virus, b ) inoculate the eggs with the clonal virus, c) cover the eggs, d) refrigerate the eggs, e) harvest the allantoic fluid from the eggs, and dilution of the allantoic fluid with pH regulator, f) remove the cellular waste from the allantoic fluid by FFT, g) purify the virus by FFT, and h) purify the virus by gel permeation chromatography. Optionally, the virus obtained from the gel permeation step can be concentrated using FFT. Another embodiment of the invention involves a method for purifying a clonal virus competent for egg replication or cell culture, comprising the step of purifying the virus by sequential tangential flow filtration (FFT), optionally followed by gel permeation chromatography, optionally followed by FFT to concentrate the virus.

Clonal viruses The use of these methods allows the purification of a clonal virus [including Paramyxovirus (eg, VEN)] at least 2 x 109 PFU / milligram of protein, convenient at least 3 x 109 PFU / milligram protein , more conveniently at least 5 x 109 PFU / milligram of protein, more conveniently at least 1.0 x 1010 PFU / milligram of protein, more conveniently at least 2.0 x 1010 PFU / milligram of protein, more conveniently at least 3 x 1010 PFU / milligram of protein, more conveniently at least 4 x 1010 PFU / milligram of protein, more conveniently at least 5 x 1010 PFU / milligram of protein, and more conveniently at least 6 x 1010 PFU / milligram. The use of these methods allows the purification of a clonal virus [including Paramyxovirus (eg, VEN)] at a level at which the number of virus particles per UFP is less than 10, more conveniently less than 5, more conveniently less than 3, more conveniently less than 2, and more conveniently less than 1.2. (The lower numbers of virus particles by UFP indicate a higher degree of purity).

RNA virus In another embodiment, these methods allow the purification (at the levels cited above for the clonal viruses) of an RNA virus [including (a) a cytocidal RNA virus; (b) a non-enveloped, unsegmented, single-stranded RNA virus; (c) a wrapped, segmented, single chain RNA virus; (d) a non-enveloped, double-stranded RNA, segmented virus; (e) and a non-segmented, single-stranded RNA virus (e.g., Paramyxovirus (e.g., VEN) and, e.g., Retrovirus].

DNA Virus In another embodiment, these methods allow the purification (at the levels cited above for the clonal viruses) of an interferon-sensitive cytocidal virus, selected from the group consisting of (a) double-stranded DNA viruses, wrapped (including poxviruses); (b) single stranded DNA viruses, not enveloped; and (c) double stranded DNA viruses, not enveloped.

Egg-derived viruses In another embodiment, these methods allow the purification of virus derived from eggs at a level substantially free of contaminating egg proteins. It is preferred to limit the amount of egg proteins in the virus preparations for human therapeutic use, since the larger egg proteins such as ovalbumin are allergens. As shown in Table 1, viruses are useful in the treatment of neoplastic diseases, including cancer. These viruses are optionally classified for naturally occurring variations (certain strains or isolates) that result in the production of altered IFN relative to the parent strain.

Table 1 Naturally Occurring Viruses for Use in Cancer Therapy In another embodiment of this invention, candidate viruses, either naturally present or designed, are tested for their ability to provide therapeutic utility in the treatment of neoplasms. In one embodiment, the amount of candidate virus that is required to eliminate 50 percent of the cells deficient in an interferon-mediated antiviral response, eg, head and neck carcinoma KB cells, is compared to the amount of virus required to remove 50 percent of a similar number of competent cells in an interferon-mediated antiviral response, eg, normal skin fibroblasts. The amount of removal is quantified by any number of media including the trypan blue exclusion or MTT assay (see Example 1) . A significant reduction (for example, of at least 5 times) in the amount of virus required to eliminate cells deficient in an interferon-mediated antiviral response, relative to the amount needed to kill competent cells in an interferon-mediated antiviral response, indicates that the virus being tested exhibits the activity required for therapeutic utility in the treatment of neoplasms. Other VEN viruses and Sindbis viruses are such naturally occurring viruses that show selective tumor clearance (see Examples 21-23, and 25). An understanding of the factors involved in establishing an antiviral state allows the creation of a screening assay for tumors that are likely to respond to viral therapy. In principle, the tumor tissue derived from the patient, obtained from the biopsy, is classified by the expression of p68 kinase, p58, or other factors involved in the regulation of an antiviral state or cell differentiation. Other factors include, but are not limited to, interferon-response factor-1 (FRI-1), interferon-stimulating gene factor-3 (FGEI-3), c-Myc receptors, c-Myb, and IFN. In the case of c-Myc, c-Myb or p58, the high level of expression indicates that the tissue or tumor cells are candidates for treatment for virus therapy. In the case of the p68, FRI-1, FGEI-3, and IFN receptors, the low level of expression indicates that the tissue or tumor cells are candidates for treatment for virus therapy.

In another embodiment of this invention, the tissue or primary tumor cells, obtained from patient biopsies, are expanded in culture and tested for sensitivity to elimination by appropriate viral therapy. In one embodiment, the amount of virus required to eliminate 50 percent of the tumor tissue culture is compared with the amount required to eliminate 50 percent of a normal cell culture, as described above for the classification of the viruses candidates. An increase of tenfold or greater in the sensitivity of the tumor cells in relation to normal cells, for elimination 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 target tumor cells to respond to IFN supplied endogenously or exogenously by conducting the above classification in the presence of IFN (alpha or beta form, using, for example, 10 units) is determined. per milliliter, see Example 27). An understanding of the cellular receptors that are required for virus binding or entry allows further classification for tumors that have high receptor expression and, consequently, improved sensitivity to the interferon-sensitive virus. This is an additional level classification for patients who are likely to respond to virus therapy. Conveniently, for therapy with an interferon-sensitive virus, the patient's tumor is resistant to interferon, and also has high expression of the cellular receptor for the virus. In principle, serum, tumor cells, tissues or sections of tissue derived from the patient are classified by immunoassay or immunostaining to see the amount of virus receptor present in the serum or in the tumor cells or tumor tissue. For example, the Sindbis virus uses the high-affinity laminin receptor to infect mammalian cells (Wang et al., 1992, J Virol., 66, 4992-5001). It is known that this same receptor is expressed in larger amounts in many different types of metastatic cancer. It is known that the renal cancer cell line PANC-1, and the colon adenocarcinoma cell line SW620, express a high level of laminin receptor of high affinity mRNA (Campo et al., 1992, Am J Pathol 141: 107301983; Yow and collaborators (1988) Proc. Nati, Acad. Sci., 85, 6394-6398) and are highly sensitive to Sindbis virus (Example 25). In contrast, it is known that the rectal adenocarcinoma cell line SW1423 expresses very low levels of high affinity laminin receptor .RNA (Yow et al., (1988) Proc. Nati. Acad. Sci. 85, 6394-6398), and it is more than 4 orders of magnitude more resistant to elimination by PPSINDBIS-Ar339 than SW620 cells.

Existing strains of VEN, or other viruses including RNA and DNA viruses, are classified or designed for altered IFN responses (eg, IFN responses suitably increased) in normal cells. In addition to the ability to produce a strong IFN response, other viral characteristics are classified for, or are designed on the virus. Included in the present invention are viruses with altered receptor specificity (e.g., Sindbis virus PPSINDBIS-Ar339, see Example 25), or low neurovirulence (e.g., VEN PPNJROAKIN virus, see Example 24). Conveniently, the viruses of the invention have the ability to be disseminated through direct cell-to-cell contact. The invention described herein includes a broad group of viruses (see Table 1) which are useful for the treatment of neoplasms in a manner analogous to the indication for VEN. In addition, viruses that would naturally not be candidates for use, due to the presence of a mechanism (s) to inactivate the IFN response in normal cells, are optionally designed to circumvent the above restrictions. If left unchanged, viruses with mechanisms to inactivate the response to interferon would be more toxic to normal cells than viruses with that mechanism removed. The present invention allows (1) the development of a vector that can be easily manipulated; and (2) the creation of a set of therapeutic viruses. Manipulations include the addition of an IFN gene to allow viral expression of a transgene expressing IFN, or other activators of the IFN response pathway. Additional permutations include the engineered expression of prodrug activating enzymes such as thymidine kinase or cytosine deaminase from Herpesvirus (Blaese RM et al., 1994, Eur. J. Cancer 30A: 1190-1193) and expression of appropriate marker antigen for allow the targeting of tumor cells by the immune system. An additional permutation includes the designed expression of receptor ligands to target cells with those receptors [e.g., the 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). Many strains of Newcastle Disease virus demonstrate the selective elimination of tumor cells. In a differential cytotoxicity assay, using a second strain of the mesogenic Newcastle Disease virus, it was found that the tumor cells are 3 orders of magnitude more sensitive than the normal cells to elimination by the virus (Example 21). Additionally, when a third strain of the mesogenic Newcastle Disease virus was used, in a differential cytotoxicity assay, it was found that the tumor cells are 80 to 5000 times more sensitive than the normal cells to elimination by the virus (Example 22). Both strains of the mesogenic Newcastle Disease virus also caused regression of tumor growth, after intratumoral administration to athymic mice that had human tumor xenografts (Example 23). In separate experiments, the safety of three different strains of Newcastle Disease virus after intracerebral inoculation in athymic and immunocompetent mice was studied. The results of this study showed that all three strains of the virus were well tolerated in mice with an intact immune system. Intracerebral inoculation within the brains of athymic mice revealed that one of the viruses was tolerated significantly better than the other two (Example 24). These results demonstrate that important differences in viral properties can occur within a single family of viruses, and can be used therapeutically for increased efficacy or increased safety. Another means by which increased efficacy and lower toxicity can be achieved after treatment with oncolytic viruses, is through the use of interferon-sensitive viruses, which require specific cellular surface receptors that are preferentially expressed in tumor cells. The 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, it was found that the cells were both turaorigenic, as they expressed the high affinity laminin receptor were more sensitive to elimination by this virus than the 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 elimination by the Sindbis virus in this assay. The Vesicular Stomatitis Virus (VSV) provides evidence of selective elimination by the tumor by means of oncolytic viruses, that is, an inherent deficiency in the responsiveness to interferon in the tumor cells produces these cells sensitive to elimination by competent viruses for replication, sensitive to interferon, when VSV was used to infect human WISH cells not your native and HT1080 cells or tumorigenic KB in the presence of exogenous interferon. Then there is a list of viruses that, when modified to remove naturally occurring anti-interferon activities, are useful for viral cancer therapy (see Table 2). The modified viruses (conveniently, but not necessarily, attenuated in addition to the anti-interferon modification, see Table 3) that had their destroyed or reduced endogenous anti-interferon activities, are useful for cancer therapy. This list includes, but is not limited to, the viruses described below. Due to the similarity between viruses of a common class, the mechanisms identified for each of the specific viruses listed below are also present in other members of that virus class as identical or functionally analogous mechanisms. The largest group of viruses is added in parentheses. Viruses, such as those that follow, which have a functional loss of anti-interferon activity, by any means, including naturally occurring mutations, as well as designed deletions or dot mutations, are useful in the methods of the present invention. invention. Viruses that exert more than one mechanism are optionally modified to contain mutations in one, some, or all activities. Mutations for some of the activities described are available in the general scientific community. Isolates of naturally occurring or engineered viruses that are slower growing, compared to the growth rate of wild type viruses, are particularly convenient because a slower virus growth rate will allow a cell or cell population competent in a response to interferon, establish an efficient antiviral state, before the viral replication can eliminate the cell or the cell population. Included in the present invention is the inhibition of viral anti-interferon activities as a specific alteration of the viral character which results in an increased response to interferon in an infected cell, but which still allows viral replication in the neoplastic cells. Table 2 shows the existing viruses designed to remove anti-interferon activity. Table 3 lists viruses designed to be attenuated in virulence.

Table 2 Existing Viruses Designed to Remove Anti-IFN Activity Table 3 Known Attenuation Mutations in Selected Viruses Treatment of Neoplasms The present invention relates to the viral therapy of neoplasms, especially in animals that have cancer. In a convenient embodiment, the invention relates to the treatment of tumors that are 1 centimeter (cm) or more in size, as measured in the largest dimension. As used herein "a 1 centimeter tumor" indicates that at least one dimension of the tumor is 1 centimeter in length. These tumors are more sensitive than expected to viral therapy, often at least as sensitive to the virus, if not more sensitive, than tumors that are smaller in size. In a more convenient aspect of the invention, tumors greater than 1 centimeter are treated, for example, tumors that are 2 centimeters or more, from about 2 centimeters to about 5 centimeters, and greater than 5 centimeters. The present invention can also be used to treat hosts having a high tumor burden. As used herein, the phrase "tumor burden" refers to the total amount of tumors within the body, expressed as a percentage of body weight. Viral therapy of hosts having a tumor burden, for example, from about 1 percent to about 2 percent of the total body weight is surprisingly effective, for example, by causing tumor regression and a reduction in tumor burden. global This is especially unexpected since a tumor burden of approximately 2 percent of the total body weight (eg, a 1-kilogram tumor in a 60-kilogram human) is approximately the maximum mass of life-supporting cancer. See, for example, Cotran et al., In Robbins Pathological Basis of Diseases, 4th Edition, WB Saunders, 1989, page 252. In the Examples, volumes of up to 397 cubic millimeters for a melanoma cancer (eg, A375) in a mouse host showed a complete regression in response to treatment with a Newcastle disease virus (eg, a purified triple plaque virus). Assuming that for the tissue 1000 cubic millimeters is equal to 1 gram, a tumor having a volume of 397 cubic millimeters comprises approximately 2 percent of the total body weight for a 20 gram mouse. As shown in Examples 4 to 9 below, regression of the tumor was achieved in tumors at least 1 cm in size, while untreated control animals began to die from the tumor burden, within several weeks. In this way, those sick animals were successfully treated regardless of whether they were within two weeks to die. Therefore, in accordance with the present invention, an animal that is almost terminal to its tumor load can be effectively treated with viral therapy. Accordingly, the present invention can be used to treat patients who have not responded to conventional therapy, for example, chemotherapy such as with methotrexate, 5-fluorouracil, and radiation therapy. The efficacy of VEN for the treatment of cancer after administration through the intraperitoneal route has also been examined. Using an ovarian cancer ascites prevention model, intraperitoneal injection of VEN in mice harboring human ovarian tumors ES-2 resulted in increased survival compared to mice treated with saline (Example 16). When ES-2 cells were used in an ovarian cancer tumor model, with treatment initiated once the ascites was formed, the production of ascites fluid in the animals treated with the virus markedly decreased, compared to the saline controls ( Example 17). In another embodiment of the invention, administration of the virus resulted in 1) relief of tumor-related symptoms., such as, but not limited to, a decreased rate of ascites fluid production, pain relief, and relief of obstructive disease, and 2) prolongation of life. Twenty-three patients received the VEN isolate purified from plaque by the intravenous route (Example 20). Responses to treatment include regression of a palpable tumor, stabilization of the disease in 47 percent of patients, and a reduction in pain medications.

Administration and Formulation In one embodiment of the invention, tumor cells or tissue were classified in vi tro, to determine those patients with tumors sensitive to the virus. The tumor cells removed from the patient (by methods such as fine-needle aspiration for solid tumors, or by paracentesis for ascitic ovarian tumors) are grown in vi tro and covered with the virus. In this embodiment of the invention, patients are selected for therapy if the virus has high activity against their tumor cells. In a convenient embodiment of the invention, the amount of virus administered resulted in the regression of the tumor or tumors. As used herein, the term "regression" means that the tumor shrinks, for example, in size, mass or volume. Shrinkage in tumor size is demonstrated by different methods, including physical examination, chest film, or other X-rays, sonography, CT scanning, MRI, or a radionucleotide scanning procedure. Different types of neoplasms, including cancers, are treatable in accordance with the invention. The viruses of the present invention are useful for treating a variety of cancers, including but not limited to lung carcinoma, be 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, cancer of skin, pancreatic thyroid cancer, and mesothelioma. The viruses of the present invention are also useful for treating a variety of benign tumors, including, but not limited to, condylomas, papillomas, meningiomas, and adenomas. A therapeutically effective amount of the virus is administered to a host having a neoplasm. Those skilled in the art understand that the dose of virus administered varies depending on the selected virus, the type of neoplasm, the extent of neoplastic cell growth or metastasis, the biological site or the body compartment of the neoplasm (s), the strain of the virus , the route of administration, the administration program, 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 the determination of maximum tolerated dose in animal models, and escalation to the human dose as a function of body surface area or relative body mass. It is also understood that under certain circumstances, more than one dose of the virus is given. The optimal interval between these multiple doses of the virus can be determined empirically, and is within the experience of the technique. The VEN is generally administered from about 3 x 10 6 to about 5 x 10 12 PFU of the virus. For local administration (e.g., directly within a tumor), total amounts of from about 3 x 10 6 to about 5 x 10 10 PFU of the virus are typically used. For routine administration, amounts of from about 1 x 108 to about 4 x 10 11 PFU of the virus are used per square meter of body surface area. For intravenous administration, dosing schedules are used once a week, twice a week and three times a week. A virus according to the present invention, optionally with a chemotherapeutic agent, can be administered by means of different routes, for example, enteric, parenteral, oral, nasal, rectal, intrathecal, intravenous (for example, using a catheter), subcutaneous , intratumoral (for example, directly inside your tissue or in vessels that irrigate it), oral, local, sublingual, oral, topical, intramuscular, inhalation, percutaneous, vaginal, intra-arterial, intracranial, intradermal, epidural, systematically, topical, intraperitoneal, intrapleural, and so on. For lung tumors, a bronchial route (eg, bronchial administration) can be used. Endoscopic injections of gastrointestinal tumors are also used, as well as rectal tumor suppository treatments, where appropriate. Studies of murine toxicity with VEN have indicated that it is possible that acute toxicity after intravenous administration of the virus is caused by cytokine-mediated reactions. It is known that cytokine responses to repeated stimuli are desensitized, or downregulated, after the initial induction event (Takahashi et al. (1991) Cancer Res., 51, 2366-2372). Mice injected intravenously with a virus desensitizing dose were able to tolerate approximately 10 times more virus in a second dose, than mice that received the vehicle only for the first injection (Example 18). The speed of administration of the virus by the intravenous route can significantly affect the toxicity. Two groups of athymic mice were treated intravenously with identical doses of VEN, which was administered either slowly (0.2 milliliters for 4 minutes) or quickly (0.2 milliliters for 30 seconds). The comparison of the maximum weight loss in each group revealed 50 percent less weight loss in the group that received the slow injection, versus a quick injection (Example 19). In one group of a clinical trial, patients received three injections of the plaque-purified VEN isolate during the course of a week (Example 20). Under these conditions, a desensitizing effect of the initial dose decreased the toxicity associated with the second and third doses. These data are parallel to those obtained with the animal studies shown in Example 18. A concern related to the use of oncolytic viruses in the treatment of cancer, is the potential inhibitory effect that the humoral immune response can exert on the therapy . In the clinical study, patients who showed stable disease after 1 month were eligible for a second course of treatment that was then administered in the presence of neutralizing antibodies to VEN. However, the infectious virus could be found in the patient's urine seven days after dosing for the second year, providing evidence that the administration of high doses of the virus can overcome the effect of neutralizing the antibodies, and establish an infection within the patient. of the patient. In a convenient embodiment of the invention, a desensitizing dose is given before subsequent higher doses. For desensitization, virus doses of 1 x 108 to 2.4 x 1010 PFU / square meter are used. After desensitization, additional virus doses of 3xl08 PFU / m2 were used. The time frame between doses, including the time frame between the desensitizing dose and the next dose, is from 1 to 14 days, conveniently from 1 to 7. days. The desensitizing dose can be administered by means of different routes, for example, intravenous, enteric, parenteral, oral, nasal, rectal, intrathecal, intravenous, subcutaneous, intratumoral, peritumoral, local, sublingual, buccal, topical, intramuscular, by inhalation, percutaneous, vaginal, intra-arterial, intracranial, intradermal, epidural, systematically, topical, intraperitoneal, intrapleural, endoscopic, intrabronchial, and so on. Subsequent doses may be administered by the same route as the desensitizing dose, or by another route, for example, intravenous, enteric, parenteral, oral, nasal, rectal, intrathecal, intravenous, subcutaneous, intratumoral, peritumoral, local, sublingual, buccal. , topical, intramuscular, by inhalation, percutaneous, vaginal, intra-arterial, intracranial, intradermal, epidural, systematically, topical, intraperitoneal, intrapleural, endoscopic, intrabronchial, etcetera. Optionally, more than one administration route can be used in a sequential or concurrent mode. Routes for administration either concurrent or sequential include but are not limited to intravenous, enteral, parenteral, oral, nasal, rectal, intrathecal, intravenous, subcutaneous, intratumoral, peritumoral, local, sublingual, buccal, topical, intramuscular, inhalation , percutaneous, vaginal, intra-arterial, intracranial, intradermal, epidural, systematically, topical, intraperitoneal, intrapleural, endoscopic, intrabronchial, and so on. An example would be the administration of an intravenous desensitizing dose followed by an ij-n-peritoneal dose. In another convenient embodiment of the invention, the virus is administered by means of slow infusion, including the use of an intravenous pump or slow injection during the course of 4 minutes to 24 hours. A virus, and optionally one or more chemotherapeutic agents, are administered by means of a single injection, by multiple injections, or continuously. The virus is administered before, at the same time, or after administration of the chemotherapeutic agents (such as, but not limited to: bisulfan, cyclophosphamide, methotrexate, cytarabine, bleomycin, cisplatin, doxorubicin, melphalan, mercaptopurine, vinblastine, fluoroacyl, taxol, and retinoic acid). Viral therapy according to the present invention is optionally combined with other treatments, including, surgery, radiation, chemotherapy (see, for example, Current Medical Diagnosis and Treatment, Ed. Tierney et al., Appleton &; Lange, 1997, especially pages 78-94), and biological therapy. The virus is administered before, at the same time, or after administration of biological agents such as (1) other oncolytic agents [such as, but not limited to: adenovirus with one of its genes under the transcriptional control of a response element specific to the prostate cells (see Rodríguez, R. et al., 1997, Cancer Res., 57: 2559-2563); adenoviruses that do not encode an Elb polypeptide capable of binding p53 (see Bischoff, J.R. et al., 1996, Science 274: 373-376); a herpes simplex virus that is unable to express a 34.5 functional range gene product (see Mineta, T. et al., 1995, Nature Medicine, 1: 938-943)]; (2) cytokines (such as, but not limited to: colony stimulating factors such as GM-CSF, tumor necrosis factor, and interleukins such as IL-1, IL-2, IL-6 and IL-10); (3) viral vectors [such as, but not limited to, adenovirus encoding p53 (see Zhang, WW et al., 1994, Cancer Gene Therapy, 1: 5-13)]; and (4) cancer vaccines. In one embodiment of the invention, the therapy consists of the serial treatment with antigenically distinct viruses, which are cytotoxic and selective to the tumor, by means of the IFN mechanism. This modality allows viral therapy for an extended period, without immunological interference. Another modality involves the treatment of patients with IFN (for example, lFN, ßlFN or? IFN) before, concurrent with, or after administration of the VEN (or other virus). IFN is selected from the class I group (alpha, beta and omega) and class II (gamma), and the recombinant version and analogs thereof, as described in, for example, Sreevalsoun, T., 1995 (In: Biologic Therapy of Cancer, second edition, edited by VT DeVita, Jr., S. Hellman and SA Rosenberg, JB Lippincott Company, Philadelphia, pages 347-364). Normal cells respond to previous treatment with IFN, with an increased response of IFN to viral infection, providing even greater safety to these cells. The tumor cells deficient in the path of IFN signaling remain sensitive to elimination by the virus. This allows even higher doses of viral therapy to be used. IFN is administered in accordance with standard clinical guidelines for the doses and regimens known to be effective in treating viral infections. In another embodiment of the invention, other drugs, which are known to affect the IFN response pathway, are also optionally used to increase the sensitivity of the tumor cells, or increase the resistance of normal cells to the cytocidal effects of the viral infection. This class of drugs includes, but is not limited to inhibitors of tyrosine kinase, cimetidine, and mitochondrial inhibitors. It is also known that hypoxia and hyperthermia modulate the responsiveness of interferon. In another embodiment of the invention, immunosuppressants such as cyclosporin A, azathiaprim, and leflunomide, different corticosteroid preparations, and anti-CD-40 ligand antibodies are administered (Foy, TM, et al., 1993, J Exp Med. 178: 1567-1575) with the virus. Alternatively, an immunostimulatory compound, for example, lipopeptides, can be administered with the virus. Optionally, an independent mechanism is used by means of which the amount of interferon produced in response to viral infection is increased, through the use of nucleotides (Machida, H., 1979, Microbiol, Imol. 23: 643-650), nucleoside precursors, or drugs that increase the cellular concentration of one or more nucleosides, as an adjunct to viral therapy. Certain purine nucleoside analogs, for example, 2-chlorodeoxyadenosine and 2'-deoxyco- moricin, reduce interferon production in vivo. These compounds are used to further effect differences in the interferon sensitivities of tumor cells against normal cells, and are optionally used as an adjunct to viral therapy. In one aspect, an effective amount of virus can be subdivided into small dose units, and injected at the same time in different locations of the same tumor. For continuous administration, the desired agent (s) is administered by means of an implanted minipump, or impregnated in a desired polymer, and then transplanted into the desired location. (for example, directly inside the tumor) for slow or delayed release. A virus of the present invention is formulated as a pharmaceutical preparation by means of bringing it into a suitable dosage form, together with at least one excipient or auxiliary and, if desired, with one or more active compounds. The preparations are used in both human and veterinary medicine. Suitable excipients include, for example, organic and inorganic substances which are suitable for enteral or parenteral administration, for example, water, saline, tissue culture medium, pH regulators, lysine, citrate, glycerol triacetate and other acid glycerides. fatty acids, gelatin, soy lecithin, carbohydrates such as mannitol, sucrose, lactose or starch, magnesium stearate, talc, cellulose or protein carriers, or a combination of the above compounds, such as mannitol / lysine, or mannitol / lysine / saccharose. The preparations are sterilized and / or contain additives, such as preservatives or stabilizers. For parenteral administration, eg, systematic or local injection, a virus preparation is formulated, for example, as an aqueous suspension or emulsion. The invention also relates to a method of treating a disease in a mammal, wherein the diseased cells have defects in an interferon-mediated antiviral response, which comprises administering to the mammal a therapeutically effective amount of a clonal virus, sensitive to interferon, competent for reply. For example, cells infected with many viruses such as hepatitis B, which disable the interferon response, are susceptible to the viruses of this invention. There is evidence that the human immunodeficiency virus (HIV) disables the response to interferon. The interferon-sensitive viruses of this invention are useful for treating those chronic virus infections, such as those due to hepatitis B, hepatitis C, HIV, * Epstein-Barr virus, human papilloma virus, and herpes virus. Unless otherwise indicated herein, the details and conditions of the viral therapy of this invention are in accordance with United States of America Patent Application Serial Number 08 / 260,536, the disclosure of which is incorporated herein by reference. to the present as a reference in its entirety. The full description of all the applications, patents and publications, cited above, and in the figures, are incorporated herein by reference. The following examples are illustrative, but not limiting, of the methods and compositions of the present invention. Other modifications and suitable adaptations of a variety of conditions and parameters normally encountered in clinical therapy, which are obvious to those skilled in the art, are within the spirit and scope of this invention.

Example 1 PPMK107, (a purified isolate of triple VEN plate, strain MK107) demonstrates a selective cytotoxic activity towards many human cancer cells, as compared to normal human cells. Tumor cells and human normal cells were cultured at approximately 80 percent confluence in 24-well tissue culture boxes. The growth medium was removed and PPMK107 was added in 10-fold dilutions ranging from plaque forming units (PFU) / well to 10"1 PFU / well Control wells were included without virus added in each plate. Viruses were absorbed for 90 minutes on an oscillating platform at 37 ° C. At the end of the incubation period, viral dilutions were removed and replaced with 1 milliliter of growth medium, and then the plates were incubated for 5 days at 37 ° C. ° C in C02 at 5 percent, then qualitatively evaluated to see the amount of cytopathic effect (ECP). Cytotoxicity was quantified by the use of a MTT colorimetric assay (2- [4,5-dimethylthiazole-2-bromide] il] -2,5-diphenyltetrazolium) (Cell Title 96, catalog # G4000, Promega Corporation, Madison Wl 53711), monitored at 570 nm, which detects the mitochondrial activity of the enzyme (Mosman, T., 1983, J. Immunol Methods 65:55.) Viability in wells t Virus-infected rats were expressed as a percentage of the activity in untreated control wells. The data were graphically plotted as PFU / well versus viability as a percentage of control. IC50 was calculated as the amount of virus in PFU / well, causing a 50% reduction in the number of viable cells. The results are given in Tables 4, 5, and 6. PPMK107 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 of less than 1000, compared with the relative insensitivity of normal human cell types. Most human cancer cells had IC50 values that were 2 to 3 orders of magnitude lower than most normal human cell types.

Table 4. Summary of Cytotoxicity Test Results TUMOR TYPE CELLULAR LINE IC ,,, (PFU / well) FIBROSARCOMA HT1080 2 MELANOMA SKMEL2 8 SKMEL3 2 NCI-H345 1.2 X 10.6 CA CELL SMALL, PROSTATE NCI-H660 1.0 X 10.5 LEUKEMIA (AML) K562 5.4 X 10" Table 5. Summary of Cytotoxicity Test Results Using Normal Human Cells TYPE OF CELL CELL IC. "(UFP / DOZO NHEK keratinocytes 9.0 x 106 Fibroblasts CCD-922 1.4 x 10.5 NHDF 8.1 x 103 HPAEC 5.2 x 10"Endothelial Cells Renal Cells RPTEC 2.7 x 10" NHEM 5.1 x 10"Melanocytes NHA astrocytes 3.8 x lO3 Table 6. Summary of Cytotoxicity Test Results Using Normal Human Cells that Proliferate Fast 1 Human breast epithelial cells (CEPH) had a high rate of proliferation after stimulation with bovine pituitary extract and human epidermal growth factor. In marked contrast, normal breast epithelial cells almost always have a very low degree of proliferation in adult women with cancer.

Example 2 Use of PPMK107 for the Intratumoral Treatment of Human Tumor Xenografts (<10 millimeters and> 5 millimeters) in Athymic Mice. Athymic mice were injected intradermally with 10 million human tumor cells. After tumors reached a size range between 5 and 10 millimeters, a single injection of PPMK107 (at a dose of 3 x 108 PFU) or saline was given. Almost all tumor types exhibited a complete or partial regression rate of 50 percent to 100 percent (see Table 7) in mice treated with PPMK107. The only exception was the case of the U87MG experiment (experiment I): Although only one of the 9 tumors treated with PPMK107 had complete regression, two more tumors treated with virus showed a regression of 32 percent and 20 percent, and two more tumors treated with virus had a slower growth than the 8 tumors treated with saline control. Tumor regression was virtually null in tumors treated with saline control: In all of these experiments (A to I, listed in Table 7) only one of the 73 control tumors showed regression. These results indicate that different types of tumors showed responses to treatment with intratumoral PPMK107. To examine the virus replication inside the tumor, immunohistochemical staining for viral antigens was performed (using a monoclonal antibody against the VEN protein), using the subcutaneous HT1080 fibrosarcoma model. Within the next 30 minutes of the tumor injection of 3 x 108 PFU of PPMK107, the tumor tissue was negative for the viral antigen. However, by day 2 after treatment, an intense immunotease was seen for the viral antigen inside the tumor, indicating the replication of the virus inside the tumor. Importantly, the replication of the virus was specific for the tumor tissue, since the surrounding connective tissue and the skin were negative for the viral antigen.

The Example 3 Use of PPMK107 for the Intravenous Treatment of Human Tumor Xenografts (<8.5 millimeters and> 5.5 millimeters) in Athymic Mice. Athymic mice were injected intradermally with million human HT1080 fibrosarcoma cells. After the tumors reached a size range between 5 and 8 millimeters, an intravenous injection (s) of PPMK107 or saline solution was made. As shown in Table 8, at the highest level of virus dose (1 x 109 PFU) a complete regression of the tumor was seen in all seven mice. Individual injections of 3 x 10a and 6 x 107 resulted in regression rates of more than 90 percent. Although a single intravenous injection of 3 x 108 gave only a 55 percent regression rate of the tumor, three intravenous injections at this dose level produced a 100 percent response rate. Mice treated with intravenous saline did not exhibit any evidence of tumor regression. These results indicate that HT1080 subcutaneous tumors are very responsive to intravenous treatment with PPMK107.

Example 4 First Experiment Using PPMK107 for the Intratumoral Treatment of A375 Large Melanoma Xenografts in Athymic Mice Athymic mice were injected intradermally with 10 million human A375 melanoma cells. Ten days later, tumors of different sizes were treated with a single injection of PPMK107 (dose of 3 x 108, 9 x 108, and 1.5 x 109 PFU) or saline. For those tumors with a single larger dimension of 10 to 11 millimeters, all nine showed regression in response to intratumoral treatment with these doses of PPKM107, whereas of those tumors with a single larger dimension of 8 to 9.5 millimeters, twelve of 24 they showed complete regression in response to the virus therapy (P <0.008, Table 9, section A). No regression of the tumor was seen in any mouse treated with saline. When the same tumors were classified by tumor volume they also indicated a high percentage of complete regression in those with the highest tumor volume. In response to these doses of PPMK107, complete regression occurred in 14 of 17 tumors with volumes > 300 cubic millimeters (range of 304 to 397 cubic millimeters), and in 7 of 16 tumors with volumes < 300 cubic millimeters (range from 144 to 295; P <0.023; Table 9, section B). These results indicate that tumors of at least 1 centimeter in length or 300 cubic millimeters in volume were at least as sensitive, if not more sensitive, to treatment with intratumoral PPMK107 than smaller tumors. EXAMPLE 5 Second Experiment Using PPMK107 for the Intratumoral Treatment of Xenoin Melanoma Agars Large A375 in Athymic Mice Tumors were established as in Example 4 ten days after the inoculation of tumor cells. The treatment consisted of different doses of PPMK107 (3 x 106 PFU, 3 x 107 PFU, 3 x 108 PFU, and 1.5 x 109 PFU) or saline. For tumors of 10 to 11.5 millimeters in a single larger dimension, complete or partial regression (at least 50 percent) occurred in all 28 tumors treated with PPMK107 using these doses, in contrast to no regression in any of the mice treated with saline (Table 10, section A). When these same tumors were classified by tumor volume, all 26 tumors larger than 300 cubic millimeters (range: 309 to 525 cubic millimeters) showed complete or partial regression (at least 50 percent), in response to PPMK107, in contrast to none of the mice treated with the saline solution (Table 10, section B). These results confirm that tumors of at least 1 centimeter in length, or 300 cubic millimeters in volume, are sensitive to intratumoral treatment with PPMK107. fifteen «• 15 • EXAMPLE 6 Third Experiment Using PPMK107 for the Intratumoral Treatment of Large A375 Melanoma Xenografts in Athymic Mice Tumors were established as in Example 4, nineteen days after the inoculation of tumor cells. Intratumoral treatment consisted of different doses of PPMK107 (3 x 108, 3 x 106, 3 x 105, and 3 x 104, 3 x 103, 3 x 102 PFU) or saline. For tumors from 12.5 to 14 millimeters in a single larger dimension (volume range: 632 to 787 cubic millimeters; average volume of 698 cubic millimeters), tumor regressions of at least 50 percent occurred in two out of three mice treated with 3 x 108 PFU, in contrast to no regression in both mice treated with saline (Table 11). Using the same dose of PPMK107 (3 x 108 PFU) to treat tumors with a single larger dimension of 10 to 12 millimeters (volume range: 320 to 600 cubic millimeters, average volume: 411 cubic millimeters), seven of 8 mice exhibited regression of at least 25 percent (P <0.001 for a regression of at least 25 percent, compared to mice treated with saline, which did not show any regression, Table 11). Regressions of at least 25 percent were also seen for tumors with lengths of 10 to 12 millimeters in mice treated with 3 x 106 PFU, 3 x 105 PFU, 3 x 10 * PFU, and 3 x 103 PFU, but not for mice treated with 3 x 102 PFU or saline (Table 11). The results confirm that tumors of at least 1 centimeter in length, or 300 cubic millimeters in volume are sensitive to intratumoral treatment with PPMK107. EXAMPLE 7 Fourth Experiment Using PPMK107 for the Intratumoral Treatment of Large A375 Melanoma Xenografts in Athymic Mice Larger tumors of 10 to 12 millimeters were established as in Example 4, thirteen days after the inoculation of tumor cells. Intratumoral treatment consisted of a single injection of 3 x 108 PFU of PPMK107 or saline solution. The volumes of those tumors treated with PPMK107 ranged from 295 to 600 cubic millimeters (average tumor volume of 437 cubic millimeters). Euthanasia was performed in the groups of mice in each treatment group on days 0, 2, 3, 4, 7, and 14 to make the histology of the tumor. For those mice observed for a minimum of 4 days, eleven out of 12 mice treated with PPMK107 exhibited regression of at least 25 percent, compared with none of 8 mice in the saline group (P <0.0001, Table 12). Two days after treatment with PPMK107, two tumors already showed signs of regression, but the degree of regression was less than 25 percent. fifteen twenty • fifteen twenty EXAMPLE 8 Fifth Experiment Using PPMK107 for the Intratumoral Treatment of Large A375 Melanoma Xenografts in Athymic Mice Larger tumors were established from 10 to 12 millimeters as in Example 4, twenty days after the inoculation of tumor cells. Intratumoral treatment consisted of a single injection of 3 x 108 PFU of PPMK107 or saline solution. The volumes of those tumors treated with PPMK107 ranged from 361 to 756 cubic millimeters (average tumor volume of 551 cubic millimeters). Nine out of 10 mice treated with PPMK107 exhibited a regression of at least 25 percent, compared to none of the 10 in the saline group (P < 0.0001, Table 13).

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 of PPMK107 (at a dose of 1.5 x 109 PFU) or saline. For those tumors of 10 to 11 millimeters in a single larger dimension, five of six tumors showed complete or partial regression in response to a single intravenous injection of PPMK107, compared with none of the saline treated tumors (Table 14, P <0.025). These results indicate that tumors of at least 1 centimeter in length are sensitive to intravenous treatment with PPMK107.

EXAMPLE 10 Specific Clearance of Infection by PPMK107 of Normal but Non-Tumor Cells In order to examine the mechanism of tumor-specific elimination by the VEN, strain PPMK107, representative tumor cells were chosen based on the following criteria: ability to form tumors as xenografts in athymic mice; b) tumor xenografts are specifically removed in vivo, after administration of PPMK107; c) the tumor cells exhibit the elimination by the PPMK107 in vitro, at virus concentrations that are many records below the concentration to eliminate normal, resistant cells; and d) tumor cells should be easily distinguished from normal cells when present as a co-culture. Xenograft tumors comprising KB head and neck carcinoma cells exhibited 83 percent complete or partial regression, in response to a single intratumoral injection of PPMK107, more than two records are sensitive to elimination by PPMK107 in vitro what are normal primary skin fibroblasts (CCD922-sk), and are easily distinguished from CCD922-sk cells, when present as a co-culture. In accordance with the above, the cocultures of KB and CCD922-sk cells were infected at a multiplicity of infection (mdi, the proportion of virus added per cell) of 0.0005, and the course of the infection was followed for 5 days by immunohistochemical staining for a viral antigen (VEN P protein). Infection of normal cells reached the peak at 2 days with little or no apparent cell death, as determined by visual inspection of the cell monolayer. On the third day after infection, the amount of viral expression in the normal cells decreased significantly, while the infection of the tumor cells was clearly evident. The amount of viral antigen virtually disappeared in normal cells on days 4 and 5, while infection in the tumor cells progressed rapidly through the tumor cell population, resulting in the destruction of most of the tumor cells present. in the cocultivation. Therefore, normal cells were infected and easily cleared the infection in a manner consistent with the antiviral effects of IFN. The tumor cells were unable to establish an antiviral state in response, and were eliminated by the non-abolished viral growth, despite the presence of physiologically effective concentrations of IFN, secreted in the medium by normal cells.

EXAMPLE 11 Demonstration that Interferon is an Important Component of Viral Clearance in Normal CCD922-sk Cells The hypothesis that interferon was mediating the ability of CCD922-sk cells to clear infection of PPMK107 was tested. Polyclonal nelizing antibodies were added daily to human or-interferon or human ß-interferon, used alone or in combination, to cultures of CCD922-sk cells infected with PPMK107, at a mdi of 0.0005, and the progress of the infection was followed for three days. days. The amount of viral antigen present in the cells was increased in proportion to the concentration of nelizing antibody, with the effect that the anti-β-interferon antibody is more marked than the anti-interferon antibody; consistent with reports that fibroblasts predominantly produce the beta form of interferon. The ability to make normally insensitive cells more susceptible to infection with PPMK107 through the addition of the nelizing antibody to interferon, supports the hypothesis that a key difference between the sensitivity of normal and tumor cells to eliminate PPMK107 lies in the ability of normal cells, but not of tumor cells, to establish an antiviral response mediated by interferon.

Example 12 Demonstration of ß-Interferon as an Important Component of Viral Clearance in Other Normal Cells In this experiment, it was determined that another normal cell (QEHN, normal human epithelial cells), known to be very resistant to elimination by PPMK107, it became more sensitive through the addition of polyclonal anti-β-interferon antibody to a culture of infected cells. The QEHN cells (normal human epithelial keratinocytes) were infected at a mdi of either 0.0005 or 0.05, and antibody was added daily for five days. In cultures infected at low mdi (0.0005), the antibody-dependent increase in viral antigen expression was clear at five days after infection, but was less clear earlier in the experiment. The addition of antibodies to cultures infected with PPMK107 at a mdi of 0.05 resulted in a marked increase in the viral antigen on days 4 and 5 after infection. On days 2 and 3 after infection, the addition of nelizing antibody resulted in less accumulation of viral antigen (Figure 1).

The culture supernatants of the samples with high mdi were also titrated, to see the amount of infectious virus present by plaque assay in human HT1080 fibrosarcoma tumor cells: the standard assay system in our laboratory. The results of this analysis showed that five days after infection there was a 19-fold increase in the amount of infectious virus in the cultures treated with antibodies, in relation to controls falsely treated (Figure 1). These results suggest a general mechanism by which normal elimination cells are protected by PPMK107, through a mechanism related to interferon.

Example 13 Comparison of the Effect of β-interferon on Infection by PPMK107 on Tumor and Normal Cells A comparison of the effect of exogenously added β-interferon on infection of normal (CCD922-sk) and tumoral cells of sensitivity to high PPMK107 ( KB) or intermediate (HEp2). Separate cultures of the three cells were treated with β-interferon at 20, 200, or 2000 units / milliliter, 1 day before infection and 2 days after infection at a mdi of 0.0005. At 3 days after infection, the low level of viral antigen expression present in normal cells was eliminated in all the doses of interferon used. Conversely, the addition of interferon to highly sensitive KB tumor cells at concentrations of 2 or 200 units / milliliter decreased the relative levels of viral antigen expression twice, with complete suppression at 1000 units / milliliter of interferon. Moderately sensitive HEp-2 cells responded to exogenous interferon by clearing viral antigen expression at all doses of interferon used (Figure 2). The pattern of sensitivity in KB and CCD922-sk cells to the antiviral effects of exogenously added β-interferon was inversely proportional to the sensitivity of these cells to elimination by PPMK107. The ability of HEp-2 cells to respond to the effects of interferon indicates that these cells are able to efficiently utilize the interferon concentrations used in this experiment. Similarly, the response of KB cells to high doses of interferon suggests that the inability to establish an interferon-mediated antiviral response is not the result of an absolute defect in the path of interferon, but rather a relative insensitivity in comparison with normal cells.

EXAMPLE 14 Effect of Low ß-Interferon Concentrations on Normal and Tumor Cell Infection by PPMK107 In this experiment, normal (CCD922-sk) and tumoral (KB) cells were treated with low concentrations of β-interferon (0.2, 2, and 20 units / milliliter) 1 day before and two days after infection with PPMK107 at a mdi of 0.05. Under these conditions, the normal cells experienced a dose-dependent decrease in the amount of viral antigen, while the relative levels of the viral antigen in the tumor cells was not affected by the addition of the exogenous interferon (Figure 3).

Example 15 Purification of PPMK107 Method A PPMK107 was derived from 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 ° C for 46 hours, the eggs were cooled, and then the allantoic fluid was harvested. Cells and cell waste were removed from the allantoic fluid by centrifugation at 1750 x g for 30 minutes. The clarified allantoic fluid (supernatant containing the virus) was then layered on a gradient of discontinuous sucrose at 20 percent / 55 percent) and centrifuged at approximately 100,000 x g for 30 minutes. The purified virus was harvested from the interface at 20 percent / 55 percent and dialyzed against the saline solution to remove the sucrose.

Method B In another convenient mode, the clarified allantoic fluid was frozen at -70 ° C. After thawing, the fluid was kept at 4 ° C overnight, and then the contaminating material was removed from the virus suspension, by means of of centrifugation (1750 xg for 30 minutes). This material was further processed using the discontinuous sucrose gradient in the ultracentrifuge as above.

Method C In another convenient embodiment, ultracentrifugation was performed in the discontinuous sucrose gradient by means of a continuous flow ultracentrifuge.

Method D In another convenient embodiment, the allantoic fluid harvested was diluted with a pH regulator containing 5 percent mannitol and 1.0 percent 1-lysine, pH 8.0 (pH regulator ML), and clarified and exchanged with ML pH regulator by means of tangential flow filtration (FFT), through filters with a nominal pore size of 0.45 μ. The permeate containing the clarified virus was collected in pH regulator ML, and the virus was purified by tangential flow filtration, through filters with a nominal cut-off of 300,000 daltons in the ML pH regulator. The purified virus was collected, concentrated in the ML pH regulator as the retentate of this step, and diluted again with the ML pH regulator, before being applied to a gel permeation column of Sephacryl S500 (Pharmacia) equilibrated with pH regulator. ML. Fractions containing the purified virus were collected, pooled, and reconcentrated by tangential flow filtration, through filters with a nominal cut-off of 300,000 daltons with ML pH regulator.

Results * Clonal Virus After generation of PPMK107 by plaque purification, it was found that eight individual molecular clones of the virion population had an identical sequence (eg, 100 percent homology) of more than 300 contiguous nucleotides within the VEN fusion protein gene. PPMK107 is a clonal virus with a high degree of genetic homogeneity. • 4b * PFU / mg of Protein 5 A quantitative means to measure purity is by determining a PFU / milligram of protein. Higher values indicate a higher level of purity. Using the Method A, values of PFU / milligram were obtained at least 4. 8 x 1010 (see Table 15). Using Method C, they were achieved values of PFU / milligram of protein of at least 2.0 x 1010. For a mesogenic strain of VEN, no value has been found in the literature for this measurement of purity. The best estimate for a mesogenic VEN strain is virus preparation (VEN MassMK107, 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). It was found that this lot RU2 has a PFU / milligram of 1.3 x 109 PFU / milligram of protein. The purity values achieved by Method A are Éfc approximately 40 times better than what the method of Peeples (see Table 15). * Proportion of Particles by PFU Another quantitative means to measure purity is by determining a proportion of particles by UFP The lower values indicate a higher level of purity.

The particle counts were made by electron microscopy, using standard methods. Using either Method A or Method B, particle values were obtained by UFP close to one (Table 15).

Table 15. Purity of the Virus UFP by Particles Method of Virus Preparation Virus # Lot of protein ñor UFP Preferred Method A PPMK107 L2 4.8 x lO10 0.80 L4 6.9 x lO10 NPa L5 6.6 x lO10 NP L6 7J l010 0.55 L7 6.1 x lO10 NP Preferred Method C PPMK107 D004 2.0 x 10 '° 0.32 D005 4.5 x 10' ° 0.52 D010 4.4 x 10 '° NP Preferred Method D PPMK107 RD2 5.6 x lO10 NP RD3 5.0 x lO10 NP to NP, Not Tested The virus preparations using Methods A and C also allowed the purification of the VEN to a substantially free level of contaminating egg proteins. For PPMK107, batch 7, the preparation using Method A, ovalbumin was not detectable in a Western blot using (1) 1.7 x 109 PFU of purified virus per well (3.3 centimeters wide) running on an SDS-PAGE gel ( sodium dodecylsulphate polyacrylamide gel electrophoresis) (1 millimeter thick); (2) a nitrocellulose membrane for transfer; and (3) antiovalbumin (Fraction of Cappel's rabbit IgG at a 1: 200 dilution of an antibody concentration of 4 milligrams / milliliter). For preparations of PPMK107 using method D, and analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis, followed by silver staining, no band corresponding to ovalbumin was observed.

Example 16 Use of PPMK107 to Prevent Death by Ovarian Carcinoma Ascites ES-2 in Athymic Mice In this experiment, all nude mice (females, NCR nu / nu, 8 weeks old) were given an intraperitoneal injection of 106 ES-2 cells. Seven days later, before the ascites had developed, they were treated intraperitoneally with saline or PPMK107 (at 1 x 109 PFU). As shown in Figure 4, there was markedly improved survival in the animals treated with PPMK107, compared to the saline. Most of the mice in the saline group had developed ascites seven days after treatment, and by day 38, all of these animals had died. In stark contrast, 92 percent of mice treated with PPMK107 were still alive by day 38, and 25 percent of these animals were long-term survivors (> 120 days of survival).

Example 17 Treatment with Ovarian Carcinoma PPMK107 in Athymic Mice When Ascites Is Present In this experiment, all athymic mice (female, NCR nu / nu, 8 weeks old) were given an intraperitoneal injection of 106 ES-2 cells . Fourteen days later, when the majority of mice had developed ascites, mice without ascites were excluded, and mice with ascites were randomly assigned to 7 intraperitoneal treatment groups (PPMK107- one treatment on day 0).; PPMK107- two treatments for the first week; PPMK107- one treatment every week; PPMK107- two treatments every week; saline solution- a treatment on day 0; saline solution - two treatments for the first week; saline solution- two treatments every week). A dose of 1 x 109 PFU / mouse was used for each virus treatment. All mice were drained before the first treatment and any additional treatments of the ascites fluid. Day 0 refers to the day of the first treatment. The degree of ascites was quantified for each mouse, and scored as follows: As shown in Table 16, all animals treated with saline had more advanced ascites than animals treated with PPMK107 on days 7 as well as . On day 7 after the initial treatment, each of the group treated with saline had average ascites scores over 3.5, while all of the groups treated with PPMK107 had average ascites scores in 3. 0 or less. Similarly, on day 10 after the initial treatment, each of the group treated with saline had average ascites scores above 4.5, while all of the groups treated with PPMK107 had average ascites scores of 4.1 or less. These results indicate that the production of ascites fluid was markedly decreased in the virus-treated animals, compared to controls treated with saline.

Table 16. Treatment with PPMK107 of Ovarian Carcinoma ES-2 in Athymic Mice When Ascites Is Present Example 18 Use of Desensitizing Dose of PPMK107 to Reduce the Lethality of a Subsequent Dose of PPMK107 Six C57BL (seven weeks old) mice were intravenously injected on day 0, either with saline or a desensitizing dose of PPMK107 (3 x 108 UFP / mouse). Two days later, each set of mice was subdivided into groups for intravenous dosing with saline or PPMK107 (at doses of 1 x 109, 2.5 x 109, 5 x 109, and 1 x 1010 PFU / mouse). As shown in Table 17, when saline was used to pretreat the mice, deaths were recorded in the mice dosed subsequently with 2.5 x 109, 5 x 109, and 1 x 1010 PFU. Doses of 5 x 109, and 1 x 1010 PFU were 100 percent lethal for mice previously treated with saline. In contrast, no deaths were seen in any group of mice given a desensitizing dose of PPMK107 on day 0, followed by injection of PPMK107 two days later, at dose levels of up to 1 x 1010 PFU. These data indicate that PPMK107 can be used to avoid the lethality of the subsequent dosing with this same agent. In addition, the maximum tolerated dose of PPMK107 can be raised by an approximate order of magnitude, when this virus is used as a desensitizing agent.

Table 17. Use of Desensitizing Dosage of PPMK107 to Reduce the Lethality of a Subsequent Dosage of PPMK107 Example 19 Slower Intravenous Injection Rate Reduces PPMK107 Toxicity Twenty-two athymic mice (8 weeks old) were anesthetized with a combination of ketamine / xylazine and placed in a restrictor to help inhibit their movement during the injection process , to allow a slow or rapid injection of PPMK107. For the slow injection group, 0.2 milliliters of 4 x 109 PFU of PPMK107 was injected intravenously in saline, for a period of 4 minutes, with 0.01 milliliter given every 10 to 15 seconds. The rapid injection group received the same dose and volume, but for a period of 30 seconds. As shown in Table 18, animals that received their dose of PPMK107 for 4 minutes, had half the maximum weight loss (recorded on day 2 after dosing) than animals that received the same intravenous dose for 30 days. seconds. These results indicate that PPMK107 had less toxicity and is safer for intravenous administration, when injected at those slower speeds. Table 18. Slower Intravenous Injection of PPMK107 Result in Reduced Toxicity Example 20 Use of PPMK107 in the Treatment of Patients with Advanced Cancer PPMK107 has been tested in a phase 1 clinical trial in the United States of America, by means of the intravenous route. Twenty-three patients with advanced solid tumors, no longer willing to receive the results, have been treated with PPMK107. established therapies. Seventeen of these patients have received a single dose for the initial course of treatment. Six other patients are receiving three doses per week for a week for the initial course of treatment. The tumor sizes of each patient have been followed once a month. Patients with at least one stable disease (less than 25 percent increase and less than 50 percent decrease in the sum of the products of all tumors that can be measured in the absence of any new injuries) were eligible for courses of additional treatment every month.

Regression of a Palpable Tumor A 68-year-old woman with colon carcinoma had a palpable abdominal tumor among her extended metastases. After a single intravenous treatment with PPMK107, this patient experienced a 91 percent regression of this single abdominal wall tumor over the course of two weeks (Table 19). Tumor measurements one day after dosing (3.75 x 3 centimeters) were similar to baseline measurements of 4 x 3 centimeters. However, by day 7 after dosing, the tumor had decreased in size to 2 x 2 centimeters, and continued to decrease in size to 1.5 x 1.5 centimeters by day 14 after dosing with PPMK107. Before treatment with PPMK107, this tumor mass had been growing rapidly with a 1065 percent increase in tumor volume in the two weeks before dosing with PPMK107. This patient left the study due to increased tumor growth elsewhere.

Table 19. Size of Palpable Abdominal Wall Tumor in Patient # 123 (68-year-old woman with Metastatic Colon Carcinoma) After a single intravenous dose of PPMK107 of 12 Billion PFU / m2 Cancer Stabilization Another eight patients, all of whom previously had tumor progression with conventional cancer therapies, experienced benefits in the form of stabilizing their advanced cancer after dosing with PPMK107. These patients with stable disease represent those with different types of cancer, including kidney cancer, pancreatic cancer, breast cancer, and lung cancer. After three months of treatment with PPMK107, a 67-year-old man with advanced and largely metastatic kidney cancer, currently had a stable disease with no indication of any new growth, and no indication of an increase in tumor size. There has been a higher rate for the benefit of stable disease with higher doses of PPMK107: Two out of 6 patients with stable disease (33 percent of patients) in the first two individual dose levels (5.9 and 12 billion UFP per square meter), and 4 out of 5 patients (80 percent of patients) with stable disease at the highest individual dose level (24 billion PFU per square meter) (Table 20).

Table 20. Treatment of Patients with Advanced Cancer with Medication Reduction for Pain One patient at a single dose level dose of 5.9 billion PFU / square meter benefited from treatment with PPMK107, in the form of symptomatic relief of cancer pain, as denoted by a reduction in Narcotic medication for pain.

Desensitization A clear desensitizing effect is seen from the first dose (to 5.9 billion PFU / square meter) in the subsequent doses (also to 5.9 billion PFU / square meter) within the same week. In general, side effects reported from the second and third doses have been much lower. For example, the first four patients in this multi-dose treatment regimen (three doses per week for a week) had fever after the first dose, despite. have received prophylactic antipyretic treatment with acetaminophen and ibuprofen. Most 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 the administration of the first dose in the program three times a week reduces the toxicity for the second and third doses.

Dosage through the Neutralization of. Antibodies in the Serum Using the dose range in this phase I study (= 5.9 billion PFU / square meter), there is also clear indication that one can effectively apply the virus to the patients, even if they have generated neutralizing antibodies. A 72-year-old woman with pancreatic cancer, at the individual dose level of 12 billion PFU / square meter he had had a stable disease for 2 months from the start of treatment with PPMK107. A second course (consisting of a single intravenous 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, his urine was positive for PPMK107 at a rate of at least 40 PFU per milliliter. This result indicates that the neutralizing antibodies for PPMK107 in this patient's serum were not able to fully inhibit the virus with a second course of treatment. Example 21 Summary of Cytotoxicity Assay Results with Newcastle Disease Virus PPNJROAKIN Tumor cells and human normal cells were grown to a confluence of about 80 percent in 24-well tissue culture boxes. The growth medium was removed and PPNJROAKIN, a purified clone of Newcastle disease virus plate, New Jersey strain Roakin-1946 mesogenic, was added in 10-fold dilutions varying from 107 plaque forming units (PFU) / well at 1 PFU / well. Control wells without virus added in each plate were included. The virus was absorbed for 90 minutes on an oscillating platform at 37 ° C. At the end of the incubation period, the viral dilutions were removed and replaced with 1 milliliter of growth medium. The plates were then incubated for 5 days at 37 ° C in 5 percent C02. Toxicity was quantified by the use of a MTT colorimetric assay (2- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) (Cell Title 96, catalog # G4000, Promega Corporation, Madison Wl 53711 ), monitored at 570 nm, which detects the mitochondrial activity of the enzyme (Mosman, T., 1983, J. Immunol, Methods 65:55). Viability in the wells treated with virus was expressed as a percentage of the activity in the untreated control wells. The data were graphically plotted as PFU / well versus viability as a percentage of control. IC50 was calculated as the amount of virus in PFU / well, causing a 50% reduction in the number of viable cells.

Table 21. Summary of Cytotoxicity Test Results with PPNJROAKIN Cellular Type IC Cell Line, "(UFP / DOZO) Fibrosarcoma HT1080 13.8 Carcinoma of KB 2.4 Head and Neck Fibroblast Normal CCD922sk 1.2 x 104 These results show that PPNJROAKIN demonstrates selective tumor removal from at least two different human tumor cells (HT1080 and KB) relative to normal skin fibroblasts. The IC50 values for the two tumor cell lines are between 800 and 5000 times lower than that for normal cells.

Example 22 Summary of Cytotoxicity Assay Results with Newcastle Disease Virus PPCONN70726 Tumor cells and human normal cells were grown to a confluence of about 80 percent in 24-well tissue culture boxes. The growth medium was removed and PPCONN70726, a purified clone of Newcastle disease virus plate, Connecticut strain 70726-1946 mesogenic, was added in 10-fold dilutions ranging from 107 plaque forming units (PFU) / well to 1 PFU / well. Control wells without virus added in each plate were included. The virus was absorbed for 90 minutes on an oscillating platform at 37 ° C. At the end of the incubation period, the viral dilutions were removed and replaced with 1 milliliter of growth medium. The plates were then incubated for 5 days at 37 ° C in 5 percent C02. Toxicity was quantified by the use of a MTT colorimetric assay (2- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) (Cell Title 96, catalog # G4000, Promega Corporation, Madison Wl 53711 ), monitored at 570 nm, which detects the mitochondrial activity of the enzyme (Mosman, T., 1983, J. Immunol, Methods 65:55). Viability in the wells treated with virus was expressed as a percentage of the activity in the untreated control wells. The data were graphically plotted as PFU / well versus viability as a percentage of control. IC50 was calculated as the amount of virus in PFU / well, causing a 50% reduction in the number of viable cells. Table 22. Summary of Cytotoxicity Test Results with PPCONN70726 These results (Table 22) show that PPCONN70726 demonstrates selective tumor removal 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 times 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 nu / nu, 5 to 6 weeks old) received a subcutaneous injection of 10 7 HT1080 tumor cells. Four days later, when tumors reached a size range of 6 to 8.5 millimeters, mice were treated intratumorally with saline, PPMK107 (at 1 x 108 PFU), PPNJROAKIN (at 1 x 108 PFU), or PPCONN70726 (through 1 x 108 PFU). As shown in Table 23, regression was noted in the tumor in mice treated with these three viruses (PPMK107, PPNJROAKIN, and PPCONN70726). After treatment with PPMK107 of 12 mice, four underwent a complete regression of the tumor, and six underwent a partial regression. After treatment with PPNJROAKIN of 12 mice, one mouse underwent complete regression of the tumor, and two underwent a partial regression. After treatment with PPCONN70726 of 12 mice, three underwent complete regression of the tumor and two underwent a partial regression. No regression of the tumor was noted in any of the animals treated with saline. These results show that the three mesogenic strains of VEN 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 1 x 108 PFU Sun. Saline 11 0 (0%) Example 24 Effects of PPMK107, PPNJROAKIN, PPCONN70726 After Intracerebral Injection in Atomic Immunodeficient Mice (nu / nu) and Incompetent Heterozygous (nu / +) To fifty-six nude mice (nu / nu) and 56 immunocompetent heterozygous (nu / +) mice were given intracerebral stereotactic injections with either saline, PPMK107, PPNJROAKIN, or PPCONN70726. Eight additional mice of each type were used as untreated controls. The viruses were used at one of two dose levels (2 x 104 or 3.5 x 106 PFU / mouse). As shown in Table 24, all the nu / + heterozygous mice, treated with each of the three viruses at the two dose levels, survived until day 39, with the exception of one mouse at the dose level of PPCONN70726 plus Low, who was euthanized to see the non-neurological symptoms. The nu / nu nude 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 percent (1 of 8) of the athymic animals in each group of viruses survived until day 39. In contrast, there was a 75 percent survival of athymic mice treated with PPNJROAKIN at this same dose level (3.5 x 106 PFU / mouse). These data indicate that PPNJROAKIN is better tolerated in the brains of athymic mice than the other two strains of virus.

Table 24. Survival of Mice After Intracerebral Injection of PPMK107, PPCONN70726, and PPNJROAKIN * - The non-surviving mouse in this treatment group was euthanized for non-neurological symptoms.

Example 25 Summary of Sindbis Cytotoxicity Assay Results PPSINDBIS-Ar339 Tumor cells and human normal cells were grown to a confluence of about 80 percent in 24-well tissue culture boxes. The growth medium was removed and PPSINDBIS-Ar339, a purified clone of Sindbis Ar-339 plate, was added in 10-fold dilutions ranging from 107 plaque forming units (PFU) / well to 1 PFU / well. Control wells without virus added in each plate were included. The virus was absorbed for 90 minutes on an oscillating platform at 37 ° C. At the end of the incubation period, the viral dilutions were removed and replaced with 1 milliliter of growth medium. The plates were then incubated for 5 days at 37 ° C in 5 percent C02. Toxicity was quantified by the use of a MTT colorimetric assay (2- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) (Cell Title 96, catalog # G4000, Promega Corporation, Madison Wl 53711 ), monitored at 570 nm, which detects the mitochondrial activity of the enzyme (Mosman, T., 1983, J. Immunol, Methods 65:55). Viability in the wells treated with virus was expressed as a percentage of the activity in the untreated control wells. The data were graphically plotted as PFU / well versus viability as a percentage of control. IC50 was calculated as the amount of virus in PFU / well, causing a 50% reduction in the number of viable cells.

Table 25. Summary of Cytotoxicity Test Results with PPSINDBIS-Ar339 Cell Type Cell Line ICín (PFU / well) Pancreatic Carcinoma Panc-1 * 69 Colorectal Carcinoma SW620 * 13 Colorectal Carcinoma SW1463 1.8 x 10.5 Non-Small Cell Lung Carcinoma A427 > 1 x 106 Non-Small Cell Lung Carcinoma A549 5.2 x 10.4 Renal Carcinoma A498 2.4 x 104 Renal Carcinoma Caki-1 3.4 x 10 Fibrosarcoma HT1080 7.4 105 Normal Keratinocyte QEHN 2.0 x 105 Normal Fibroblast CCD922sk 1.6 x 10.5 * Cells known to overexpress the .ARNm for the high affinity laminin receptor.

The cellular receptor for Sindbis virus in mammalian cells is the high affinity laminin receptor, which is expressed mainly in cells of the epithelial lineage, but is often overexpressed in many metastatic cancer cells such as the pancreatic carcinoma line. -1, and the colon adenocarcinoma cell line SW620 (Campo et al., (1992) Am. J. Pathol. 141, 1073-1083; Yow et al., (1988) Proc. Nati Acad Sci, 85, 6394-6398). In contrast, it is known that the rectal adenocarcinoma cell line SW1423 expresses very low levels of high affinity laminin receptor mRNA (Yow et al., (1988) Proc. Nati Acad Sci, 85, 6394-6398), and more than 4 orders of magnitude more resistant to removal by PPSINDBIS-Ar399 than SW620 cells. These results (Table 25) demonstrate that cells that are tumorigenic, and express high levels of high affinity laminin receptor, are more sensitive to elimination by Sindbis, Clone PPSINDBIS-Ar399 than other tumors or normal cells.

EXAMPLE 26 Elimination by VSV of Tumorigenic and Non-Tumorigenic Cells in the Presence of Interferon In 96-well plates, tumorigenic KB and HT1080 cells (3 x 10 4 cells per well) and non-tumorigenic WISH cells (2.5 x 10 4 cells per well) were seeded, in the presence of serially diluted a-interferon, which varied from 2800 to 22 Units / milliliter, and allowed to incubate for 24 hours at 37 ° C. The cells were then infected with vesicular stomatitis virus (VSV, Indiana strain) at a mdi of 10. Controls were included for cells without interferon, and cells without interferon or virus. The cells were incubated at 37 ° C for 24 hours. Cytotoxicity was quantified by the use of a MTT colorimetric assay (2- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) (Cell Title 96, catalog # G4000, Promega Corporation, Madison Wl 53711 ), monitored at 570 nm, which detects the mitochondrial activity of the enzyme (Mosman, T., 1983, J. Immunol, Methods 65:55). Viability in the wells treated with virus was expressed as a percentage of the activity in the control wells that did not receive the virus.

Table 26. Comparison of the Cell Removal Activity of VSV in Cells Treated with Exogenous Interferon These results (Table 26) demonstrate that VSV is capable of selectively eliminating tumor cells deficient in interferon responsiveness (see Example 27). WISH cells (human amniotic cells) are a well established cell line for use in interferon bioassays, due to their ability to efficiently respond to interferons.

Example 27 Responsiveness to Interferon in Sensitive or Resistant Cells to Elimination by PPMK107 Individual cell lines were grown to close confluence in 96-well microtiter plates, and treated with between 5 and 5000 U / milliliter of alFNA for 24 hours. The cultures were then infected with PPMK107 at a mdi of 1.0, and cultured for an additional 24 hours. After chemical fixation, the amount of viral expression was quantified by immunohistochemistry, using a soluble indicator dye. The amount of virus is represented as the percentage of P antigen expressed relative to the control cells not treated with interferon (Figure 5). In this assay, cells responsive to interferon show a * decrease of at least 50 percent in the viral antigen, in response to interferon. The cells in Figure 5, which are sensitive to PPMK107 are indicated with solid lines; Less sensitive cells are indicated with 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 elimination by PPMK107 (indicated by the IC50 value shown in parentheses next to the name of the cell line in the legend of the graph, see Figure 45). For example, after pretreatment with 5U / milliliter of interferon, 6 of 7 (86 percent) cell lines not responsive to interferon are sensitive to elimination by PPMK107; when treated with 500 U / milliliter of interferon, all (4 of 4) cell lines not responsive to interferon are sensitive to elimination by PPMK107. The above data also presents an example of a screening assay to identify candidate cells that are likely to be susceptible to elimination by PPMK107 or other interferon-sensitive viruses. For example, infected cells expressing significant viral antigen (eg, more than 50 percent of controls), after prior treatment with exogenous interferon, would be considered interferon deficient, and therefore sensitive to viral oncolysis. The foregoing is intended to be illustrative of the present invention, but not limiting. Numerous variations and modifications can be made without departing from the true spirit and scope of the invention.

Claims (106)

1. NOVELTY OF THE INVENTION Having described the foregoing invention, it is considered as a novelty, and therefore, property is claimed as contained in the following: CLAIMS 1. A method for infecting a neoplasm in a mammal with a virus, comprising administer a clonal RNA virus, sensitive to interferon, competent for replication, to that mammal.
2. 2. A method for infecting a neoplasm in a mammal with a virus, comprising administering a clone RNA virus, competent for replication, to that mammal, wherein the virus is sensitive to interferon.
3. 3. A method, as in claim 1, wherein the RNA virus replicates at least 100 times less in the presence of interferon, as compared to the absence of interferon.
4. 4. A method, as in claim 1, wherein the RNA virus replicates at least 1000 times less in the presence of interferon, as compared to in the absence of interferon.
5. 5. A method, as in claim 1, wherein the step of administration is systemic.
6. 6. A method, as in claim 1, wherein said neoplasm is a cancer.
7. 7. A method, as in claim 1, wherein the mammal is a human.
8. 8. A method, as in claim 1, wherein the clonal virus is purified by plates.
9. 9. A method, as in claim 1, wherein the clonal virus is of recombinant clonal origin.
10. 10. A method, as in claim 1, wherein the .ARN virus is a Paramyxovirus.
11. 11. A method, as in claim 10, wherein the Paramyxovirus is purified to a level of at least 2 x 109 PFU per milligram of protein.
12. 12. A method, as in claim 10, wherein the Paramyxovirus is purified to a level of at least 1 x 1010 PFU per milligram of protein.
13. 13. A method, as in claim 10, wherein the Paramyxovirus is purified to a level of at least 6 x 1010 PFU per milligram of protein.
14. A method, as in claim 10, wherein the Paramyxovirus is purified to a level at which the proportion of particles per UFP is not greater than 5.
15. 15. A method, as in claim 10, wherein the Paramyxovirus is purified to a level at which the proportion of particles per PFU is not greater than 3.
16. 16. A method, as in claim 10, wherein the Paramyxovirus is purified to a level at which the proportion of particles per PFU is not is greater than 1.2.
17. 17. A method, as in claim 10, wherein the Paramyxovirus is a bird paramyxovirus type 2.
18. 18. A method, as in claim 10, wherein the Paramyxovirus is VEN.
19. 19. A method, as in claim 10, wherein the Paramyxovirus is the mumps virus.
20. 20. A method, as in claim 10, wherein the Paramyxovirus is the human parainfluenza virus.
21. 21. A method, as in claim 1, wherein the RNA virus is selected from the group consisting of a Rhabdovirus, Togavirus, Flavivirus, Reovirus, Picornavirus, and Coronary.
22. 22. A method, as in claim 21, wherein the Togavirus is the Sindbis virus.
23. 23. A method, as in claim 21, wherein the Reovirus has a modification in omega 3.
24. 24. A method, as in claim 21, wherein the Reovirus has an attenuating mutation in omega 1.
25. 25. A method, as in claim 21, wherein said Reovirus is an attenuated rotavirus.
26. 26. A method, as in claim 25, wherein the rotavirus is rotavirus WC3.
27. 27. A method for infecting a neoplasm in a mammal with a virus, comprising administering an interferon-responsive clone-responsive clonal vaccinia virus having one or more mutations in one or more genes selected from the group consisting of K3L, E3L and B18R, to the mammal.
28. 28. A method for infecting a neoplasm in a mammal with a virus, comprising administering a replication-competent clonal vaccinia virus, which has one or more mutations in one or more genes selected from the group consisting of K3L, E3L and B18R, to the mammal, wherein said virus is sensitive to interferon.
29. 29. A method for infecting a neoplasm in a mammal with a virus, comprising administering a Replica competent replication-responsive clonal DNA virus selected from the group consisting of Adenovirus, Parvovirus, Papovavirus, and Iridovirus to the mammal .
30. 30. A method for infecting a neoplasm in a mammal with a virus, comprising administering a replication competent clonal DNA virus, selected from the group consisting of Adenovirus, Parvovirus, Papovavirus, and Iridovirus, to the mammal, wherein the virus has sensitivity to interferon.
31. 31. A method, as in claim 29, wherein the mammal is a human.
32. 32. A method, as in claim 29, wherein the Adenovirus virus has a modification in the VA1 transcripts, causing the Adenovirus to become sensitive to interferon.
33. 33. A method, as in claim 32, wherein the Adenovirus virus is selected from the group consisting of vaccinia strains of Ad-4, Ad-7 and Ad-21.
34. 34. A method for infecting a neoplasm in a mammal with a virus, comprising administering a clonal Herpesvirus responsive to interferon, competent for replication, to the mammal.
35. 35. A method for infecting a neoplasm in a mammal with a virus, comprising administering a replication competent clonal Herpesvirus, to the mammal, wherein the virus is sensitive to interferon.
36. 36. A method, as in claim 1, wherein the neoplasm is a cancer selected from the group consisting of lung, colon, prostate, breast and brain cancer.
37. 37. A method, as in claim 1, wherein the neoplasm is a solid tumor.
38. 38. A method, as in claim 36, wherein the brain cancer is a glioblastoma.
39. 39. A method, as in claim 1, wherein the virus contains a gene encoding interferon, to allow viral expression of interferon.
40. 40. A method, as in claim 1, wherein the virus contains a gene encoding a prodrug activating enzyme.
41. 41. A method, as in claim 1, characterized in that it also comprises administering IFN, before, during or after the administration of said virus.
42. 42. A method, as in claim 41, wherein the interferon is selected from the group consisting of C.-IFN, β-IFN, β-IFN, β-IFN, and synthetic consensus forms of IFN.
43. 43. A method, as in claim 1, characterized in that it also comprises administering a tyrosine kinase inhibitor before, during or after the administration of the virus.
44. 44. A method, as in claim 1, characterized in that it also comprises administering a compound selected from the group of compound comprising a purine nucleoside analogue, tyrosine kinase inhibitor, cimetidine, and mitochondrial inhibitor.
45. 45. A method, as in claim 1, characterized in that it also comprises administering a chemotherapeutic agent before, during or after administration of the virus.
46. 46. A method, as in claim 1, characterized in that it also comprises administering a cytokine before, during or after the administration of the virus.
47. 47. A method, as in claim 1, characterized in that it also comprises administering an immunosuppressant before, during or after the administration of said virus.
48. 48. A method, as in claim 1, characterized in that it also comprises administering a viral replica that controls the amount of compound, selected from the group consisting of IFN, ribavirin, acyclovir, and ganciclovir.
49. 49. A method, as in claim 1, wherein the administration is intravenous or intratumoral.
50. 50. A method for infecting a neoplasm, which is at least 1 centimeter in size in a mammal with a virus, which comprises administering a clonal virus, selected from the group consisting of (1) RNA virus; (2) Hepadanovirus; (3) Parvovirus; (4) Papovavirus; (5) Herpesvirus; (6) Poxvirus; and (7) Iridovirus, to the mammal.
51. 51. A method, as in claim 50, wherein the neoplasm is at least 300 cubic millimeters in volume.
52. 52. A method, as in claim 50, wherein the 7RNA virus is a Paramyxovirus.
53. 53. A method, as in claim 52, wherein the Paramyxovirus is VEN.
54. 54. A method, as in claim 50, wherein the mammal is a human.
55. 55. A method, as in claim 50, wherein the administration is intravenous or intratumoral.
56. 56. A method, as in claim 53, wherein the paramyxovirus is purified to a level of at least 2 x 109 PFU per milligram of protein.
57. 57. A method, as in claim 53, wherein the VEN is mesogenic.
58. 58. A method for classifying tumor cells or tissue freshly removed from the patient, to determine the sensitivity of those cells or tissue to elimination by a virus, which comprises subjecting a tissue sample to a differential cytotoxicity assay, using a virus sensitive to interferon.
59. 59. A method, as in claim 58, characterized in that it also comprises the step of classifying the cells or tissue to view the protein, or the protein encoding the mRNA, selected from the group consisting of p68 protein kinase, c -Myc, c-Myb, FGEI-3, FRI-1, IFN receptor, and p58.
60. 60. A method for identifying a virus with antineoplastic activity in a mammal, comprising: (a) using the test virus to infect i) cells deficient in an interferon-mediated antiviral activity, and ii) competent cells in a mediated antiviral activity by interferon, and b) determining whether the test virus kills the cells deficient in an interferon-mediated antiviral activity, preferentially to the competent cells in interferon-mediated antiviral activity.
61. 61. A method, as in claim 60, wherein said cells deficient in an interferon-mediated antiviral activity are human head and neck carcinoma cells KB.
62. 62. A method, as in claim 60, wherein the cells competent in an interferon-mediated antiviral activity are human skin fibroblasts.
63. 63. A method for making viruses for use in antineoplastic therapy, comprising: a) modifying an existing virus by decreasing or removing a viral mechanism for inactivation of the antiviral effects of IFN, and optionally b) creating an attenuating mutation.
64. 64. A method for controlling viral replication in a mammal treated with a virus selected from the group consisting of .ARN, Adenovirus, Poxvirus, Iridovirus, Parvovirus, Hepadnavirus, Varicellavirus, Betaherpesvirus, and Gamaherpesvirus, which comprises administering a virus. antiviral compound.
65. 65. A method, as in claim 64, wherein the antiviral compound is interferon.
66. 66. A method, as in claim 64, wherein the antiviral is selected from the group consisting of ribavirin, acyclovir, and ganciclovir.
67. 67. A method, as in claim 64, wherein the antiviral is a neutralizing antibody to said virus.
68. 68. A Paramyxovirus purified by ultracentrifugation without granule formation.
69. 69 A Paramyxovirus purified to a level of at least 2 x 109 PFU / milligram of protein.
70. 70. A Paramyxovirus, as in claim 69, wherein the paramyxovirus develops in eggs, and is substantially free of contaminating egg proteins.
71. 71. A Paramyxovirus, as in claim 69, wherein the paramyxovirus has a ratio of particles per UFP not greater than 5.
72. 72. A Paramyxovirus, as in claim 69, wherein the paramyxovirus has a ratio of particles per UFP not greater than 3.
73. 73. A Paramyxovirus, as in claim 69, wherein the paramyxovirus has a proportion of particles per UFP not greater than 1.2.
74. 74. A purified Paramyxovirus to a level of at least 1 x 1010 PFU / milligram of protein.
75. 75. A purified Paramyxovirus to a level of at least 6 x 1010 PFU / milligram of protein.
76. 76. A Paramyxovirus, as in claim 69, wherein the virus is cytocidal.
77. 77. A Paramyxovirus, as in claim 69, wherein said Paramyxovirus is the Newcastle disease virus.
78. 78. A VEN as in claim 77, wherein the VEN is cytocidal.
79. 79. A VEN as in claim 77, wherein the VEN is mesogenic.
80. 80. An RNA virus purified to a level of at least 2 x 109 PFU / milligram of protein.
81. 81. An RNA virus, as in claim 80, wherein the virus is competent for replication.
82. 82. A cytocidal virus competent for replication that is sensitive to interferon, and purified to a level of at least 2 x 109 PFU / milligram of protein.
83. 83. A cytocidal virus, as in claim 82, wherein said virus is clonal.
84. 84. A cytocidal DNA virus that is sensitive to interferon, and purified to a level of at least 2 x 109 PFU / milligram of protein.
85. 85. A cytocidal DNA virus, as in claim 84, wherein said virus is a Poxvirus.
86. 86. A Poxvirus, as in claim 85, wherein the Poxvirus is a vaccinia virus having one or more mutations in one or more genes, selected from the group consisting of K3L, E3L and B18R.
87. 87. A replication-competent vaccinia virus having a) one or more mutations in one or more of the K3L, E3L and B18R genes, and b) an attenuating mutation in one or more of the genes encoding the thymidine kinase, reductase of ribonucleotide, vaccinia growth factor, thymidylate kinase, DNA ligase, dUTPase.
88. 88. A vaccinia virus competent for replication having one or more mutations in two or more genes selected from the group consisting of K3L, E3L and B18R.
89. 89. A Herpesvirus that has a modification in the expression of the analogue (2'-5 ') A.
90. 90. A Reovirus that has a mutation in omega 3, and purified to a level of at least 2 x 109 PFU / milligram of protein.
91. 91. A Reovirus that has mutations in omega 1 and omega 3.
92. 92. A method for purifying an RNA virus, comprising the steps of: a) generating a clonal virus, and b) purifying the clonal virus by ultracentrifugation without formation of granules .
93. 93. A method, as in claim 92, wherein the RNA virus is competent for replication.
94. 94. A method for purifying a Paramyxovirus, which comprises purifying that virus by ultracentrifugation without formation of granules.
95. 95. A method, as in claim 94, wherein the purification step further comprises, prior to ultracentrifugation: a) purifying the plate to generate a clonal virus, b) inoculating the eggs with the clonal virus, c) coverting the eggs, d) cool the eggs, e) harvest the allantoic fluid from the eggs, and f) remove the waste from the cell from the allantoic fluid.
96. A method, as in claim 94, wherein said Paramyxovirus virus is VEN.
97. 97. A method for infecting a neoplasm in a mammal with a virus, comprising administering an RNA virus responsive to interferon, competent for replication, to the mammal.
98. 98. A method, as in claim 1, wherein the 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.
99. 99. A method for 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, virus Sindbis, and vesicular stomatitis virus.
100. 100. A method, as in claim 1, or claim 27, or claim 29, wherein the virus is administered as more than one dose.
101. 101. A method, as in claim 100, wherein the first dose is a desensitizing dose.
102. 102. A method, as in claim 101, wherein the first dose is administered intravenously, and a subsequent dose is administered intravenously.
103. 103. A method, as in claim 101, wherein said first dose is administered intravenously, and a subsequent dose is administered intraperitoneally.
104. 104. A method, as in claim 101, wherein the first dose is administered intravenously, and a subsequent dose is administered intra-arterially.
105. 105. A method, as in claim 1, or claim 27, or claim 29, wherein the virus is administered during the course of at least 4 minutes.
106. 106. A method for reducing pain in a mammal, comprising administering a clonal virus sensitive to interferon, competent for replication.