RECOMBINANT ONCOLYTIC VIRUSES FOR TREATMENT OF METASTATIC CANCERS

Abstract

Disclosed are recombinant oncolytic viruses that express one or more reovirus fusion-associated small transmembrane FAST) proteins and uses thereof. The oncolytic activity of the recombinant oncolytic viruses expressing FAST proteins can be used to treat primary and metastatic cancers, especially from breast and colon cancers.

Claims

1. A recombinant oncolytic virus that expresses one or more reovirus fusion-associated small transmembrane (FAST) proteins.

2. The recombinant oncolytic virus of claim 1, wherein the FAST protein is p10 derived from avian reoviruses or bat reoviruses; p13 protein derived from bat reoviruses; p14 protein derived from reptilian reoviruses; p15 protein derived from baboon reoviruses; p16 protein derived from an aquareoviruses; p22 protein derived from an aquareoviruses, or combinations thereof.

3. The recombinant oncolytic virus of claim 2, wherein the FAST protein is p14, p15 or a combination of domains from p14 and p15.

4. The recombinant oncolytic virus of claim 1 or 2, wherein the oncolytic virus is derived from herpesvirus, adenovirus, reovirus, measles virus, Newcastle disease virus, vaccina virus and vesicular stomatitis virus.

5. The recombinant oncolytic virus of claim 4, wherein the oncolytic virus is vesicular stomatitis virus.

6.-10. (canceled)

11. A method for treating cancer comprising administering the recombinant oncolytic virus of claim 1 to a subject in need thereof.

12. The method of claim 11, wherein the cancer is a breast or colon cancer.

13. The method of claim 12, wherein the cancer is a metastatic cancer.

14. The method of claim 11, wherein the FAST protein is p10 proteins derived from avian reovirus or Nelson Bay reovirus; p14 protein derived from reptilian reovirus; p15 protein derived from baboon reovirus; or combinations thereof.

15. The method of claim 14, wherein the FAST protein is p14, p15 or a combination of domains from p14 and p15.

16. The method of claim 14 or 15, wherein the oncolytic virus is derived from herpesvirus, adenovirus, reovirus, measles virus, Newcastle disease virus, vaccina virus and vesicular stomatitis virus.

17. The method of claim 16, wherein the oncolytic virus is vesicular stomatitis virus.

18.-23. (canceled)

24. A recombinant oncolytic virus that expresses one or more non-enveloped viral membrane fusion protein, the one or more non-enveloped viral membrane fusion protein comprising: a transmembrane (TM) domain, a cytosolic, membrane-proximal cluster of three or more basic amino acids, an at least one post-translational fatty acid modification, and an N-terminal ectodomain.

25. The recombinant oncolytic virus of claim 24, wherein the one or more non-enveloped viral membrane fusion protein primary sequence is 200 amino acids or fewer.

26. The recombinant oncolytic virus of claim 24, wherein the one or more non-enveloped viral membrane fusion protein, when expressed, induces cell-cell fusion and/or syncytium.

27. The recombinant oncolytic virus of claim 24, in which the one or more non-enveloped viral membrane fusion protein are reovirus fusion-associated small transmembrane (FAST) proteins.

28. The recombinant oncolytic virus of claim 24, wherein the one or more non-enveloped viral membrane fusion protein is p10 derived from avian reoviruses or bat reoviruses; p13 protein derived from bat reoviruses; p14 protein derived from reptilian reoviruses; p15 protein derived from baboon reoviruses; p16 protein derived from an aquareoviruses; p22 protein derived from an aquareoviruses, or combinations thereof.

29. The recombinant oncolytic virus of claim 28, wherein the one or more non-enveloped viral membrane fusion protein is p14, p15 or a combination of domains from p14 and p15.

30. The recombinant oncolytic virus of claim 28, wherein the oncolytic virus is derived from herpesvirus, adenovirus, reovirus, measles virus, Newcastle disease virus, vaccina virus and vesicular stomatitis virus.

31. The recombinant oncolytic virus of claim 30, wherein the oncolytic virus is vesicular stomatitis virus.

32. The recombinant oncolytic virus of claim 31, wherein the vesicular stomatitis virus comprises a deletion at position 51 in a matrix protein.

33. The recombinant oncolytic virus of claim 24, wherein the one or more non-enveloped viral membrane fusion protein is a chimeric protein.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] These and other features, aspects and advantages of the present invention will become better understood with regard to the following description and accompanying drawings wherein:

[0028] FIG. 1 represents the membrane topology and repertoire and arrangement of structural motifs in the known fusion-associated small transmembrane (FAST) proteins. NOTE: avian and bat reoviruses encode homologous p10 proteins (p10/ARV and p10/NBV, respectively) with the same repertoire and arrangement of structural motifs, but they share only 30% amino acid identity.

[0029] FIG. 2 represents recombinant oncolytic vesicular stomatitis virus (VSV) constructs encoding GFP (VSV-GFP) or the p14 FAST protein (VSV-p14) and their effects on cell-cell fusion and virus replication. (A) Schematic of recombinant VSV containing gene insertions encoding either GFP or the p14 FAST protein. (B) Vero cells were mock-infected or infected with the indicated recombinant viruses at a MOI=0.1 and Giemsa-stained at 24 hpi to detect syncytium formation. (C) As in panel B, using the supernatant from infected cultures to determine the virus yield by plaque assay at the indicated times post-infection. Results are meanSEM from duplicate experiments (*p<0.05 compared to VSV-GFP);

[0030] FIG. 3 represents the effects of VSV-GFP and VSV-p14 on breast cancer spheroid cell death and virus yields. (A) 4T1 and MCF-7 breast cancer cells growing as spheroids were mock-infected or infected with 110.sup.5 PFU/ml of recombinant VSV-GFP or VSV-p14, and phase-contrast images of the infected spheroids were captured at 24 hpi. (B) As in panel A, quantifying virus yields in the supernatants at the indicated times post-infection by TCID.sub.50 in permissive Vero cells (left panels), or quantifying cell viability at 40 hpi using a phosphatase assay (right panels). Phosphatase assay results are reported as meanSEM percent cell viability from duplicate (MCF-7) or triplicate (4T1) experiments relative to mock-infected spheroid cultures (*p<0.05 compared to VSV-GFP);

[0031] FIG. 4 represents the effects of VSV-GFP and VSV-p14 on the growth of primary mammary tumors and animal survival. (A) Syngeneic 4T1 subcutaneous mammary tumors were established in BALB/c mice and ten days later animals were mock-treated or treated by one intravenous injection of VSV-GFP or VSV-p14 (110.sup.8 pfu) followed by four intratumoral injections at the same virus dose (N=5 per treatment group). (B) Tumor size was monitored over time and average tumor volumesSEM were calculated for each treatment group. Statistical analysis used ANOVA to compare VSV-p14 to VSV-GFP (*p<0.05 compared to control; p<0.05 compared to VSV-GFP). (C) Survival advantage was assessed by the log-rank (Mantel-Cox) (*p<0.05 compared to control);

[0032] FIG. 5 represents the biodistribution of VSV-GFP and VSV-p14 in animals bearing primary mammary tumors. Subcutaneous 4T1 mammary tumors were established in BALB/c mice, and then animals were treated with one intravenous injection (110.sup.8 pfu) of VSV-p14 or VSV-GFP. Mice were sacrificed at (A) 24 hpi (N=3 per treatment group) or (B) 48 hpi (N=2 per treatment group), tumors and the indicated organs were harvested, and virus titers were quantified by plaque assay. Results are the average titer per gm of tissueSEM.

[0033] FIG. 6 represents the effects of VSV-GFP and VSV-p14 on animal survival and lung metastases in metastatic models of breast cancer and colon cancer. (A) Subcutaneous 4T1 mammary tumors were established in BALB/c mice and primary tumors were resected on day 12. Mice were treated on days 13, 15 and 17 with PBS, VSV-GFP or VSV-p14 (N=5 per group). Survival advantage was assessed by the log-rank (Mantel-Cox) (*p<0.016 compared to PBS, .sup.p<0.016 compared to VSV-GFP). (B) CT26LacZ colon carcinoma cells (210.sup.5 cells) were injected intravenously into BALB/c mice to establish lung metastases. Animals were injected intravenously with PBS (Control, N=8) or with 110.sup.7 pfu of VSV-p14 (N=8) or VSV-GFP (N=7) on days 3, 5 and 7, and lungs were removed 7 days following the last virus injection and the meanSEM number of surface lung metastases were visually quantified following staining of the excised lungs for (3-galactosidase (*p<0.05 compared to control);

[0034] FIG. 7 represents the effects of VSV-GFP and VSV-p14 on activation of splenic T cells and NK cells in a primary breast cancer model. 4T1 tumor-bearing mice (N=9-10 per treatment group) received one intravenous injection of PBS (Control) or VSV-p14 or VSV-GFP (110.sup.8 pfu) on day 12, followed by similar intratumoral inoculations on days 13, 14 and 15. Spleen cells were isolated 24 h following the final injection. The number of splenic CD4 T cells (CD4.sup.+ TcR.sup.+), CD8 T cells (CD8.sup.+ TcR.sup.+), NKT cells (CD1d-tetramer.sup.+ TcR.sup.+) and NK cells (CD49b.sup.+ TcR.sup.) (top row), and expression of CD69 by CD4 T cells, CD8 T cells, NKT cells and NK cells (bottom row) was assessed by flow cytometry (*p<0.05 compared to control);

[0035] FIG. 8 represents the effects of VSV-GFP and VSV-p14 on the frequency of activated T cells in the tumors and draining lymph node in a primary breast cancer model. 4T1 tumor-bearing mice (N=9-10 per treatment group) received one intravenous injection of PBS (Control) or VSV-p14 or VSV-GFP (110.sup.8 pfu) on day 12, followed by similar intratumoral inoculations on days 13, 14 and 15. The draining lymph node and tumors were isolated 24 h following the final injection. The number of CD4 T cells (CD4.sup.+ TcR.sup.+) and CD8 T cells (CD8.sup.+ TcR.sup.+) in the draining lymph node and tumors (top row), or the frequency of the same cells expressing the early activation marker CD69.sup.+ was assessed using flow cytometry (*p<0.05 compared to control; .sup.p<0.016 compared to VSV-GFP);

[0036] FIG. 9 represents the effects of VSV-GFP and VSV-p14 in combination with adoptive immune cell transfer on survival of animals bearing metastatic mammary tumors. Subcutaneous 4T1 mammary tumors were established in BALB/c mice and primary tumors were resected on day 12. Mice were treated on days 13, 15 and 17 with PBS, VSV-GFP or VSV-p14 (N=5 per group) and on day 18 with adoptive immune cell transfer using dendritic cells loaded with -galactosylceramide to activate NKT cells, and survival advantage was assessed by the Kaplan-Meier estimator;

[0037] FIG. 10 represents the effects of VSV-GFP or recombinant VSV expressing different FAST proteins (VSV-p14, VSV-p10/ARV, VSV-p10/NBV or VSV-p15) on 4T1 breast cancer cells growing in cell culture. 4T1 cells were mock-infected or infected with the indicated recombinant viruses at a MOI=0.1 and Giemsa-stained at 15 hpi to detect syncytium formation. Different FAST proteins show different abilities to induce cell-cell fusion and syncytium formation; VSV-p15 is hyperfusogenic, VSV-p14 and VSV-p10/NBV are strongly fusogenic, VSV-p10/ARV is weakly fusogenic but cytotoxic; and

[0038] FIG. 11 represents the effects of VSV-GFP or recombinant VSV expressing different FAST proteins (VSV-p14, VSV-p10/ARV, VSV-p10/NBV or VSV-p15) on the growth of primary mammary tumors in BALBc mice. Syngeneic 4T1 subcutaneous mammary tumors were established in BALB/c mice and ten days later animals were mock-treated or treated by one intravenous injection of VSV-GFP or the recombinant VSV expressing different FAST proteins (110.sup.8 pfu/50 l injection; a separate treatment group also received VSV-p15 at 110.sup.8 pfu/50 l injection) followed by four intratumoral injections at the same virus dose on days 11, 12, 13 and 14 (N=5 per treatment group). Top panel: tumors excised at day 15 from a single animal in each treatment group. Bottom panel: tumor size was monitored over time and average tumor volumesSEM were calculated for each treatment group.

DESCRIPTION OF THE INVENTION

[0039] The following description is of an illustrative embodiment by way of example only and without limitation to the combination of features necessary for carrying the invention into effect.

[0040] The recombinant oncolytic virus described herein expresses one or more reovirus fusion-associated transmembrane (FAST) proteins. The FAST proteins increase cell-cell fusion which enhances cell-cell virus transmission, and they are cytotoxic and induce a pro-inflammatory response (FIGS. 7 and 8).

[0041] By polypeptide or protein is meant any chain of amino acids, regardless of length or post-translational modification (e.g. glycosylation or phosphorylation). Both terms are used interchangeably in the present application.

[0042] FAST proteins are the smallest known membrane fusion proteins, rendering them weakly immunogenic, and their small size facilitates incorporation of FAST protein genes into almost any OV platform, alone or in combination with other immunostimulatory genes. FAST proteins are also not reliant on specific cell receptors and thus fuse numerous cell types, humans have no pre-existing immunity against FAST proteins (they derive from non-human viruses), they promote localized and disseminated virus transmission via syncytium formation at physiological pH, and they are cytocidal and disrupt calcium homeostasis, two mechanisms likely to increase immunogenic cell death and trigger anti-tumor immune responses. Here it is shown that addition of p14 to VSV increases cancer cell death and virus transmission within tumors, reduces tumor growth and metastases, stimulates more robust innate and adaptive immune responses, and improves outcomes in primary and metastatic models of cancer, all while maintaining a favorable safety profile. The FAST proteins therefore provide a novel approach to enhance oncolytic virotherapy by increasing cytocidal and immune-mediated tumor cell killing.

[0043] In one aspect of the invention, FAST proteins are provided which are encoded by the genome of viruses in the Reoviridae. FAST proteins are an evolutionarily related family of viral membrane fusion proteins (Nibert and Duncan, PLOS One 8:e68607, 2013), and the only family of nonenveloped virus membrane fusion proteins. Defining features of all family members are: (1) small size (<200 amino acids); (2) a single transmembrane (TM) domain that functions as a reverse signal-anchor to direct a bitopic, N-out/C-in membrane topology; (3) a cytosolic, membrane-proximal cluster of three or more basic amino acids; (4) a post-translational fatty acid modification involving either a myristoylated N-terminus or one or more palmitoylated cysteine residues; (5) a small (<50 residues), N-terminal ectodomain containing a membrane destabilizing motif sharing features of fusion peptides (FPs). Additional features present in some, but possibly not all, FAST proteins include: (1) a cytosolic, membrane-proximal amphipathic helix motif; (2) an intrinsically disordered C-terminal tail.

[0044] The family Reoviridae includes the genus Orthoreovirus, which includes avian, mammalian and reptilian reoviruses, as well as the genus Aquareovirus. For example, the FAST proteins are one or more of p10 proteins derived from species Avian reovirus (ARV) or bat reoviruses in the species Nelson Bay orthoreovirus (NBV); p13 protein derived from a bat reovirus in the proposed new species Broome orthoreovirus (BroV); p14 protein derived from isolates in the species Reptilian orthoreovirus (RRV); p15 protein derived from isolates in the species Baboon orthoreovirus; p16 protein derived from isolates in the species Aquareovirus-C or Aquareovirus-G; p22 protein derived from isolates in the species Aquareovirus-A, and combinations thereof, such as the p14/p15 fusion described in US Patent Publication No. 2014/0314831 (the contents of which is incorporated in its entirety herein). In some cases, a single type of FAST protein may be expressed, such as, but not limited to, p14 or p15, whereas, in other cases, a hybrid oncolytic virus may be produced that expresses a recombinant of more than one type of FAST protein, such as, but not limited to, a combination of different domains from p14 and p15.

[0045] Oncolytic viruses (OVs) are naturally occurring or genetically engineered viruses that preferentially replicate within and kill cancer cells due to signaling defects in cellular metabolism and innate immunity (Bell and McFadden, Cell Host Microbe 15: 260-265, 2014). A number of viruses can be used for this purpose, including, but not limited to, herpesvirus, adenovirus, reovirus, measles virus, Newcastle disease virus, vaccina virus and vesicular stomatitis virus.

[0046] In one embodiment, the recombinant oncolytic virus is vesicular stomatitis virus (VSV) and the FAST protein is p14, p15 or a recombinant peptide thereof. Preferably, the VSV contains a mutation in the matrix (M) gene that renders the virus highly-susceptible to interferon (IFN) responses (VSV51)

[0047] As used herein, the terms pharmaceutically acceptable, physiologically tolerable and grammatical variations thereof, as they refer to compositions, carriers, diluents, and reagents, are used interchangeably and represent that the materials are capable of administration to a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset, and the like.

[0048] Methods for the preparation of a pharmacological composition that contains active ingredients, such as the recombinant oncolytic virus described herein, dissolved or dispersed therein is well known in the art. Typically such compositions are prepared as injectables either as liquid solutions or suspensions; however, solid forms suitable for solution, or suspension, in liquid prior to use can also be prepared. The preparation can also be emulsified.

[0049] The active ingredient can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, as well as combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance the effectiveness of the active ingredient.

[0050] The therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable nontoxic salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid, and the like.

[0051] Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials other than the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline, or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol, and other solutes.

[0052] Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.

[0053] A therapeutically effective amount is a predetermined amount calculated to achieve the desired effect. The required dosage will vary with the particular treatment and with the duration of desired treatment; however, it is anticipated that dosages between about 10 micrograms and about 1 milligram per kilogram of body weight per day will be used for therapeutic treatment. In some instances, it may be particularly advantageous to administer such compounds in depot or long-lasting form. A therapeutically effective amount is typically an amount of a fusion protein according to the invention, or polypeptide fragment thereof that, when administered in a physiologically acceptable composition, is sufficient to achieve a plasma concentration of from about 0.1 g/ml to about 100 g/ml, preferably from about 1.0 g/ml to about 50 g/ml, more preferably at least about 2 g/ml and usually 5 to 10 g/ml.

[0054] Unless otherwise specified, all references cited are incorporated herein.

[0055] It will be understood that numerous modifications thereto will appear to those skilled in the art. Accordingly, the above description and accompanying drawings should be taken as illustrative of the invention and not in a limiting sense. It will further be understood that it is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein set forth, and as follows in the scope of the appended claims.

EXAMPLES

[0056] Cells: African green monkey (Vero) cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% Fetal Bovine Serum (FBS). QM5 (Quail muscle fibrosarcoma) cells were cultured in Medium 199 supplemented with 10% FBS. Mouse mammary epithelial (4T1) tumor cells were maintained in complete Roswell Park Memorial Institutes Media (RPMI-1640) supplemented with 10% FBS. MCF-7 cells were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% FBS. Mouse colon carcinoma (CT26LacZ) cells were cultured in DMEM with 10% FBS. All culture reagents were obtained from Gibco, and all cells were cultured as monolayers at 37 C. with 5% CO2.

[0057] Mice: Female BALB/c mice were purchased form Charles River Laboratories (Senneville, Canada) and used at 8-12 weeks of age. All animal protocols followed the guidelines of the Canadian Council on Animal Care, and were approved by the University Committee on Laboratory Animals.

[0058] Generation of recombinant VSV: The p14 FAST protein gene in pcDNA3 and the EGFP gene in pEGFP-N1 were amplified by PCR and subcloned into the Xhol and Nhel sites located between the G and L genes in pVSV51-XN to generate pVSV51-XN-p14 and pVSV51-XN-GFP (hereinafter referred to as VSV-p14 and VSV-GFP). QM5 cells were infected with the modified vaccinia virus Ankara strain expressing T7 RNA polymerase (MVA-T7), and 4 hours later co-transfected with four plasmids at a ratio of 2:2:1.25:0.25 g: pVSV51-XN-p14 or pVSV51-XN-GFP, and pBS-N, pBS-P and pBS-L, encoding the VSV N, P and L proteins, respectively, under the control of a CMV promoter. Two days later, cell culture supernatants were harvested, filtered through a 0.2 m filter to remove vaccinia virus, and then used to infect Vero cells. Vero cell supernatants were harvested 3 days post-infection, and the recombinant VSV particles were isolated by plaque purification on Vero cells. The identities of the recombinant viruses were confirmed by sequencing cDNA amplicons obtained by PCR using primers complementary to VSV sequences flanking the insertion site. Virus stocks were amplified and titered by plaque assay using Vero cells. Similar approaches were used to clone the genes encoding the p10/ARV, p10/NBV and p15 FAST proteins into the same pVSV51-XN plasmid and to generate and isolate recombinant VSV51 encoding these FAST proteins.

[0059] Oncolytic activity in cell culture: Recombinant viruses were tested for their cytolytic activity in cell culture using Vero cells, 4T1 breast cancer cells, and breast cancer spheroids. Vero cells cultured in 12-well plates were infected with recombinant viruses at a multiplicity of infection (MOI) of 0.1 for 1 hr at 37 C., then cells were washed with PBS to remove unbound virus. Cells were then cultured in fresh medium for 24 hrs. Culture supernatants were harvested to quantify virus yield by plaque assay, and monolayers were stained with Wright-Giemsa to view cell death and syncytium formation under bright-field microscopy. To obtain spheroid cultures, MCF-7 and 4T1 breast cancer cells were seeded into ultra-low attachment Costar 6-well plates using 310.sup.4 cells/well and cultured in a mammosphere medium (DMEM/F-12 supplemented with 20 ng/mL bFGF, 20 ng/mL EGF, 100 U/mL penicillin, 100 g/mL streptomycin and 1B27 serum-free supplement), and cultured for 7-9 days, replacing the medium with fresh medium every 72 hrs. Spheroid cultures were infected using 110.sup.5 PFU/well of VSV-GFP or VSV-p14, culture supernatants were harvested at 16-24 hrs post-infection (hpi), and virus yields were determined by TCID.sub.50 in permissive Vero cells. Cell viability was assessed by incubating resuspended spheroids in PBS at a 1:1 ratio (final volume 1 ml) with phosphatase solution (0.1M sodium acetate, pH 5.5, 0.1% Triton X-100, 4 mg/ml phosphatase substrate) for 90 min at 37 C. in the dark. After incubation, 50 M of 1 N NaOH was added to each sample to stop the reaction, samples were cleared by centrifugation at 1000g for 5 min, and supernatants were transferred to 96-well plates to measure absorbance at 405 nm using an Asys Expert 96 Microplate Reader

[0060] Immunocompetent animal tumor models: Primary breast cancer model: 4T1 breast cancer cells were harvested in the logarithmic growth phase, resuspended in saline, and injected subcutaneously (210.sup.5 cells in 50 l) into the mammary fat pad of female BALB/c mice (n=5/treatment group). Palpable tumors formed within 10 d after seeding. Mice were injected intravenously with VSV-GFP or VSV-p14 (110.sup.8 PFU/mouse in 50 l), followed by four similar intratumoral injections one day apart. For the efficacy studies, 4T1 tumors were measured every 2-4 d using an electronic caliper, and tumor volume was calculated as (WWL)/2. For biodistribution studies, BALB/c mice with established 4T1 subcutaneous breast tumors were injected intravenously with VSV-GFP or VSV-p14 (110.sup.8 pfu/mouse in 50 l), mice were sacrificed 24-48 hpi, and normal organs (lungs, liver, spleen, heart, brain) and tumor tissues were harvested for virus titration by plaque assay, as previously described.

[0061] Post-Surgical Breast Cancer Metastasis Model:

[0062] 4T1 tumors were established in mice, as described above, and primary tumors were resected 12 days following tumor inoculation, as previously described. On days 13, 15 and 17 mice received 100 l intravenous injections of PBS or 110.sup.8 plaque forming units (PFU) of VSV-GFP or VSV-p14. Survival was monitored over time (FIG. 9).

[0063] Lung Metastasis Model:

[0064] CT26-LacZ colon carcinoma cells (210.sup.5 in 50 l) were injected intravenously into BALB/c mice, and at days 3, 5 and 7 mice were injected intravenously with VSV-GFP or VSV-p14 (110.sup.8 PFU/mouse) or with PBS. Mice were sacrificed 7 days following the last virus injection, lungs were harvested, and lung metastases were quantified visually following staining of the tumors using X-gal (Sigma-Aldrich).

[0065] Immune phenotyping: BALB/c mice were inoculated with 210.sup.5 4T1 cells in the fourth mammary fat pad. Twelve days after inoculation, mice received intravenous injections of PBS, VSV-GFP or VSV-p14. On days 13, 14, 15 mice received intratumoral injections of PBS, VSV-GFP or VSV-p14. Spleens, draining lymph nodes and primary tumors were isolated on day 16. Following mechanical dispersion, tumor infiltrating lymphocytes were enriched by centrifugation through a 33% Percoll gradient (GE Healthcare; Baie dUrfe, Canada). Red blood cells were lysed with ammonium chloride buffer and cells were washed by centrifugation. The immune profile of lymphoid and myeloid populations was examined by flow cytometry (FIGS. 7 and 8).

[0066] The oncolytic activity of recombinant VSV51 encoding the p14 FAST protein (VSV-p14) was compared to a similar construct encoding GFP (VSV-GFP) in cell culture, and in primary and metastatic syngeneic Balb/c tumour models. Compared to VSV-GFP, VSV-p14 increased VSV oncolytic activity in MCF-7 breast cancer spheroids, delayed primary breast cancer tumour growth and prolonged survival in both primary and metastatic breast cancer models, and prolonged survival in a CT26 metastatic colon cancer model (FIGS. 2-6). Survival data and biodistribution results indicate the VSV51 backbone effectively restricted virus replication to the tumor, which was unaffected by p14 (FIG. 4), implying p14 does not compromise the biosafety profile of VSV51.

[0067] The effects of VSV-GFP or recombinant VSV expressing different FAST proteins (VSV-p14, VSV-p10/ARV, VSV-p10/NBV or VSV-p15) on 4T1 breast cancer cells growing in cell culture was also tested (FIG. 10). 4T1 cells were mock-infected or infected with the indicated recombinant viruses at a MOI=0.1 and Giemsa-stained at 15 hpi to detect syncytium formation. Different FAST proteins show different abilities to induce cell-cell fusion and syncytium formation; VSV-p15 is hyperfusogenic, VSV-p14 and VSV-p10/NBV are strongly fusogenic, VSV-p10/ARV is weakly fusogenic but cytotoxic.

[0068] The effects of VSV-GFP or recombinant VSV expressing different FAST proteins (VSV-p14, VSV-p10/ARV, VSV-p10/NBV or VSV-p15) were also evaluated for the growth of primary mammary tumors in BALBc mice (FIG. 11). Syngeneic 4T1 subcutaneous mammary tumors were established in BALB/c mice and ten days later animals were mock-treated or treated by one intravenous injection of VSV-GFP or the recombinant VSV expressing different FAST proteins (110.sup.8 pfu/50 l injection; a separate treatment group also received VSV-p15 at 110.sup.7 pfu/50 l injection) followed by four intratumoral injections at the same virus dose on days 11, 12, 13 and 14 (N=5 per treatment group). Top panel: tumors excised at day 15 from a single animal in each treatment group. Bottom panel: tumor size was monitored over time and average tumor volumesSEM were calculated for each treatment group.

[0069] Flow cytometry: All antibodies were purchased from eBioscience or Biolegend (San Diego, Calif.): purified CD16/32 (clone 97); fluorescein isothiocyanate (FITC)-conjugated CD3 (145-2C11), CD49b (DX5), CD11b (M1/70); phycoerythrin (PE)-labeled CD69 (H1.2F3), CD86 (GL1), Gr-1 (RB6-8C5); peridinin chlorophyll (PERCP)-labeled CD4 (RM4-5), CD11c (H13), TCR- (H57-597), F4/80 (BM8); allophycocyanin (APC)-labeled CD8 (53-6.7), CD80 (16-10A1). To examine NKT cells by flow cytometry, cells were stained with allophycocyanin-labeled CD1d tetramers loaded with the glycolipid PBS57 (NIH Tetramer Core Facility, Emory Vaccine Center at Yerkes, Atlanta, Ga.). All cell samples were pre-incubated with anti-CD16/32 antibody to block non-specific binding. Following Fc-receptor blocking, cells were incubated at 4 C. for 20 min with surface-staining antibody panels, washed, and fixed in 2% paraformaldehyde. Data acquisition was performed using a two laser FACSCalibur flow cytometer (BD Biosciences; San Jose, Calif.) and data analysis was performed using FlowJo (V10.2; FlowJo, LLC; Ashland, Oreg.).

[0070] Statistical analyses: Data are expressed as meanSEM. A non-parametric two-tailed Mann-Whitney U test was used to compare between two groups. Comparisons between more than two groups were made using a Kruskal-Wallis non-parametric ANOVA with Dunn's post-test. Statistical significance was set at p<0.05. Survival data was analyzed by log-rank (Mantel-Cox) significance test and the statistical significance level was set using the Bonferroni corrected threshold (p<(0.05/K), where K is the number of comparisons performed. Statistical computations were carried out using GraphPad Instat 3.02 and GraphPad Prism 7.02.