WO2014194433A1 - Recombinant oncolytic virus expressing an ifn binding protein - Google Patents

Abstract

The present disclosure provides a recombinant interferon (IFN)-sensitive oncolytic rhabdovirus that includes a polynucleotide sequence encoding a soluble protein that binds to IFN-α, IFN-β, or both. The soluble protein is referred to herein as an "interferon binding protein". The interferon binding protein is secretable by a cell infected with the oncolytic rhabdovirus.

Description

RECOMBINANT ONCOLYTIC VIRUS EXPRESSING AN IFN BINDING PROTEIN

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent

Application No. 61/832,740 filed June 7, 2013, which is hereby incorporated by reference.

FIELD

[0002] The present disclosure relates generally to recombinant oncolytic viruses that suppress IFN signaling.

BACKGROUND

[0003] The following discussion is not an admission that anything discussed below is citable as prior art or common general knowledge.

[0004] Oncolytic Viruses (OVs) are promising anti-cancer therapeutics engineered or selected to infect and multiply specifically in tumor cells while having attenuated replication capacity in normal tissues [1 ,2].

[0005] OVs are complex biological agents that interact at multiple levels with both tumor and normal tissues. Anti-viral pathways induced by Interferon (IFN) are known to play a critical role in determining tumor cell sensitivity and normal cell resistance to infection with OVs.

[0006] In non-cancerous cells, the attenuation of OV growth in normal tissues is often due to the inability of OVs to antagonize normal cellular interferon (IFN) mediated anti-viral responses. Many tumor cells have acquired defects in their IFN response during their malignant evolution, and are correspondingly excellent hosts for OV growth [3-7]. However the extent of the IFN response deficit in cancers is highly variable and can impair the efficacy of OV therapies [8].

[0007] IFN antiviral defense mechanisms play a critical role in controlling virus infection. These mechanisms thus represent a formidable hurdle for viral therapy and viral manufacturing.

INTRODUCTION

[0008] The following discussion is intended to introduce the reader to the detailed description to follow, and not to limit or define any claimed invention. [0009] It is an object of the present disclosure to provide an OV that obviates or mitigates at least one disadvantage associated with viral therapy or viral manufacturing using previous oncolytic viruses.

[0010] Whether a patient receives therapeutic virus intravenously or through direct intra-tumoral injection, the quantity of virus that reaches the tumor bed is vastly outnumbered by the number of target cells found in the malignancy. Successful OV therapies thus rely upon the ability of a relatively small number of virus particles to initiate an infection and spread within a population of cancer cells.

[0011] Viruses are strictly dependent upon the biosynthetic machinery of the infected host cell to produce progeny particles. Correspondingly, cancer cells that are rapidly dividing and have established robust biosynthetic machinery inherently produce larger numbers of virus particles when compared to cells in normal tissue, which are quiescent and have a restricted ability to synthesize new nucleic acids and proteins. In cancer cells, this effect is exasperated by defects in immune responses, which further enhance viral replication.

[0012] In a simple model of virus infection, the distinction between viral replication kinetics in healthy and tumor tissue is characterized by differences in available biosynthetic machinery and innate immune responses (Fig. lA).

[0013] When virus is delivered to normal tissue, it infects a limited number of cells creating a subpopulation producing both virus progeny and interferon (I FN). As the infection proceeds, slow virus replication enables the IFN-mediated defense response to outpace virus particle production, and restrict infection. Viruses that can prevent the production of IFN from infected cells, or stimulate host cell metabolism, are

correspondingly expected to have an increased capacity to spread within normal tissues. Indeed pathogenic viruses incorporate both these strategies into their life cycle.

[0014] In tumors non-responsive to the anti-viral effects of IFN, invading virus will co-opt the biosynthetic machinery of the cancer cell producing large numbers of virus particles. In this setting, the virus will rapidly spread and destroy the malignancy. In tumors responsive to IFN, virus spread will be favored by the high metabolic rate of the cancer, but at the same time limited by the ability of neighboring cells to mount an innate immune anti-viral response. In this setting, it is the balance between virus production and the extent of initiation of anti-viral responses that will ultimately determine the therapeutic outcome. [0015] While many tumor cells have defects in their IFN and are correspondingly excellent hosts for OV growth, the extent of the IFN response deficit in cancers is highly variable and can impair the efficacy of OV therapies.

[0016] OVs are a very promising new therapy currently being developed for the treatment of cancer. Manufacturing of the large amounts of these viruses that is required to deliver an effective dose to a patient is a problem for the clinical development of OVs since many of the manufacturing cell lines that are approved for viral manufacturing are normal human cells (such as MRC5 and HEK 293), which have an intact IFN response and therefore repress replication of engineered, attenuated interferon-sensitive OVs.

[0017] To demonstrate the potential of model-based rational design of OVs, the authors of the present disclosure combined mathematical modeling and viral genome engineering to design and test strategies that might allow evasion of the IFN response in the tumor microenvironment while at the same time maintaining safety in normal tissues.

[0018] Based on the mathematical analysis of multiple strategies, the authors of the present disclosure hypothesized that a positive feedback loop, established by virus- mediated expression of an interferon-binding protein, increases tumor cytotoxicity without compromising normal cells.

[0019] As discussed herein, using a recombinant IFN-sensitive oncolytic rhabdovirus that includes a polynucleotide sequence encoding an interferon binding protein may increase virus replication in tumor cells that express IFN, when compared to a recombinant IFN-sensitive oncolytic rhabdovirus lacking the polynucleotide sequence encoding the interferon binding protein.

[0020] As discussed herein, using a recombinant IFN-sensitive oncolytic rhabdovirus that includes a polynucleotide sequence encoding an interferon binding protein may increase virus replication in normal cells, when compared to a recombinant IFN-sensitive oncolytic rhabdovirus lacking the polynucleotide sequence encoding the interferon binding protein.

[0021] Oncolytic rhabodviruses engineered to express an interferon antagonist may have improved oncolytic potential in cellular cancer models, and may display improved therapeutic potential in tumor-bearing mice. Oncolytic rhabodviruses engineered to express an interferon antagonist may also show an increased replication in normal cells, which is beneficial for increasing replication in manufacturing cell lines. BRIEF DESCRIPTION OF DRAWINGS

[0022] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0023] Fig. 1 is an illustration of model development. Comparison of virus replication dynamics in three tissue types: Normal cells, I FN non-responsive tumors and I FN responsive tumors. Processes enhanced relative to normal cells are illustrated in green, while those impaired are illustrated in red.

[0024] Fig. 2 is an illustration of a simulation of interferon evasion strategies on rhabdovirus-induced cytotoxicity. The phenomenological model was amended to describe treatment with A) VSV wild-type (WT), C) VSV Δ51 , E) VSV Δ51 in the presence of a Jak kinase inhibitor (JAKi), G) VSV Δ51 in the presence of a soluble I FN binding protein and I) Recombinant VSV A51-mediated expression of a soluble IFN binding protein as an IFN decoy receptor (VSV A51-IDE). Color coding highlights processes impaired (dashed) or gained (blue) relative to VSV Δ51. Each of the above models used a Monte Carlo sampling method to generate the probability distribution of population viability following treatment with B) VSV WT, D) VSV Δ51 , F) VSV A51+JAKi H), VSV A51 + soluble IFN binding protein (eg. B18R or B19R) J) Recombinant VSV A51-IDE for 72h in normal cells (left), and tumors with non-responsive (Tumor IFN-NR; center) or responsive (Tumor IFN-R; right) IFN signalling pathways. Color coding, quantified in pie charts, describes the probability that each of the three cell types has a viability of <10% (red), 10-90% (grey) or >90% (green) at the end of the simulation.

[0025] Figs. 3A to 3D are graphs that illustrate validation of model predictions in vitro. Fig. 3A) Infection of resistant tumors with functional IFN defenses (786-0) and Normal (MRC5) cells with VSV A51-Green Fluorescent Protein (GFP) at an MOI of 0.1 in the presence or absence of 10μΜ of the JAK kinase inhibitor or 0.1 μg/ml of a soluble IFN binding protein (B18R or B19R). Microscopy images were taken 48h post-infection. Scale bar is 2mm in length. Fig. 3B) Cytopathic effects of the Recombinant VSV Δ51- IDE as observed by brightfield microscopy 24 hours post-infection. Fig. 3C) Cytopathic effects of the Recombinant VSV A51-IDE as observed by crystal violet staining 72 hours post infection. Images are from a 12 wells plate (2.5cm diameter). Fig. 3D) Experimental and simulated relationship between MOI and cellular viability 72 hours post-infection. Error bars represent the standard deviation from triplicate technical replicates. Trends represent the simulation which best describes the experimental results. [0026] Figs. 4A to 4C are graphs that illustrate viral spreading. Fig. 4A)

Comparison of virus spreading by crystal violet staining in IFN-responsive 786-0 tumour cells. Scale bar is 2mm in length. Fig. 4B) Quantification of virus spreading surface. Error bars represent the standard deviation from triplicate technical replicates. Fig. 4C) Cover slip spreading assay. IFN-responsive 786-0 tumour cells were plated in a dish containing a round coverslip and infected at MOI of 3 for 4 hours. The round cover slip was transferred onto a monolayer of naive normal MRC5 cells and an agarose overlay was added. 5 days later, cells were fixed and stained with Coomassie brilliant blue. Images are from 6 wells plate (3.5cm diameter).

[0027] Figs. 5A to 5E are graphs illustrating the safety and efficacy of a soluble

I FN binding protein (IDE) expressing virus in vivo. Fig. 5A) Biodistribution in CT26 tumor bearing BALB/c mice injected Intravenously (IV) with 1e8 pfu of either VSV Δ51 compared to a Recombinant VSV A51-IDE or MG1 compared to a Recombinant MG1- IDE. Fig. 5B) Maximum Tolerable Dose in tumor naive BALB/c mice treated with either Maraba wild-type, MG1 or a Recombinant MG1-IDE. Fig. 5C) Survival study of immunodeficient mice bearing an HT29 subcutaneous tumor injected IV with 1 e8 pfu of either PBS, MG1 or a Recombinant MG1-IDE (n=5). Fig. 5D) CT26 tumor cells metastasis to the lung in BALB/c mice after IV injection of 1 E7 pfu of either PBS, MG1 or a Recombinant MG1-IDE (n=7). The Box plot illustrates the sample maximum and minimum (error bars), Q1 and Q3 (box defines the interquartile range which is equal to the difference between the upper and lower quartiles; IQR = Q3 - Q1), and the median of the population (central bar). Fig. 5E) CT26 tumor metastasis to the liver in BALB/c mice after IV injection of 1 E8 pfu of either PBS, MG1 or a Recombinant MG1-IDE (PBS, n=6; MG1 , n=7; and MG1-IDE n=7). * denotes a statistical difference between groups (One way Anova pV<0.0001 ; Two-tailed heteroscedastic T-Test pV<0.05).

[0028] Fig. 6 shows graphs illustrating the simulated difference between VSV

Δ51 and VSV A51-IDE at varying MOI in a population of normal cells. The histogram illustrates the distribution of simulated viability after 72 hours incubation at the described MOI. Results were obtained over 1 e3 Monte Carlo randomizations. Color coding, quantified in pie charts, describes the probability that the normal cell population has a viability <10% (red), 10-90% (grey) or >90% (green) at the end of the simulation.

[0029] Fig. 7 is a graph that depicts the impact of increasing exogenous soluble

I FN binding protein dosage on virus mediated cellular cytotoxicity. IFN-responsive 786-0 tumour cells and normal MRC5 cells were pre-treated with increasing amounts of a recombinant soluble I FN binding protein (eg. B18R or B19R). 2 hours later, cells were infected with VSV Δ51. 72 hours post-infection, Alamar blue was added to determine cell viability.

[0030] Figs. 8A to 8C illustrate in vitro validation of recombinant soluble I FN binding protein expressing rhabdoviruses. Fig. 8A) Diagram depicting position of soluble I FN binding protein gene (eg. B18R or B19R) integration in the rhabdovirus genome. Fig. 8B) Supernatants from IFN-responsive 786-0 tumour cells infected with VSV Δ51 , MG1 , Recombinant VSV A51-IDE or Recombinant MG1-IDE were collected 24 hours after virus infection. Collected supernatants were then added to naive IFN-responsive 786-0 tumour cells with VSV Δ51 or MG1 virus following the addition of neutralizing antibody. Pictures were acquired 48 hours later. Scale bar is 1 mm in length. Fig. 8C) Quantification of cellular viability at 48 hours. Alamar blue was added to assess cell survival in Figs. 8B and 8C. Anova analysis detected a statistically significant difference between groups (pV<0.0001). A Tukey's post-hoc test was utilized to assess the statistical significant difference between groups. Errors bars represent standard deviation between triplicate technical replicates.

[0031] Figs. 9A to 9C show the microarray validation of soluble I FN binding protein (IDE) expressing rhabdoviruses. Fig. 9A) Box plot illustrating the log of the fold change in gene expression induced by MG1 infection for loci sub-classified as induced or repressed by IDE expression. Genes repressed by MG1-IDE relative to MG1 , are typically induced upon MG1 infection (pV<4.39E-027, Wilcoxon signed-rank test) and those induced by MG1-IDE are typically repressed upon MG1 infection (pV<1.89E-6; Wilcoxon signed-rank test). Box plot illustrates the sample maximum and minimum (error bars), Q1 and Q3 (box defines the interquartile range which is equal to the difference between the upper and lower quartiles; IQR = Q3 - Q1), the median of the population (red bar) and samples outliers (+). Fig. 9B) Gene-ontology enrichments for genes sub- classified as induced by the MG1 and repressed by MG1-IDE. Schematic illustrates a summary of the processes identified at a pValue threshold of 0.0001 following correction for multiple hypothesis testing (Bonferroni). Fig. 9C) Heatmap illustrating the fold change in gene expression for genes induced by the MG1 virus relative to control and repressed by subsequent treatment with the soluble IFN binding protein expressing virus (MG1- IDE) which are sub-classified as induced by type-l interferon's.

[0032] Fig. 10 illustrates the experimental and simulated relationship between

VSV A51 versus Recombinant VSV A51-IDE and MG1 versus Recombinant MG1-IDE MOI and cellular viability 72 hours post-infection in CT26 tumour cells. The experimental dose response curve was generated by infecting 2.5E5 cells at increasing MOI of the various viruses and assessing cellular viability 72 hours post-infection through an Alamar blue assay. Viability is defined as the strength in signal intensity relative to control. Error bars represent the standard deviation obtained over triplicate technical replicates.

Trends represent the simulation which best describes the experimental results. In these simulations, all parameters are identical between viral strains in a given cell lineage except for the capacity to produce the soluble I FN binding protein.

[0033] Fig. 1 1 depicts virus spreading assays. A) Quantification of VSV Δ51 versus Recombinant VSV A51-IDE and MG1 versus Recombinant MG1-IDE virus spreading surface area as measured by crystal violet staining in the VSV resistant glioma cell line: U251. B) Microscopy images of VSV A51 versus Recombinant VSV A51-IDE immunostaining in IFN-responsive 786-0 tumour cells and CT26 tumour cells. Infected 786-0 or CT26 cells containing VSV particles are characterized by a brown circle around the center of the well. C) Quantification of spreading in CT26 and 786-0 cell lines as assessed by immunostaining. * indicate a statistically significant between strain (Two-tailed heteroscedastic T-Test, pV<1 E-4). Errors bars in panels A and C represent the standard deviation between triplicate technical replicates.

[0034] Fig. 12 shows HT29 tumor volume. BALB/c mice were injected

subcutaneously in their right flank with 3E6 HT29 tumour cells. The two viruses (MG1 versus Recombinant MG1-IDE) were administered at 1 E8 intra-tumorally 7 days post cancer cell implantation. Once the viruses were administered, tumor volume was monitored every 5 days by measuring tumor volume with calipers. Tumor volume in mm3 was graphed and lines represent tumor growth: blue for MG1 and red for MG1- IDE.

[0035] Fig. 13 illustrates the simulated impact of a recombinant wild-type rhabdovirus (VSV) encoding ΙΡΝβ. A) Development of the model to describe the infection cycle of VSV-IFI\^. According to this model all parameters are identical to the VSV model other than the capacity to produce ΙΡΝβ by VSV (νΙΡΝβ), which was assumed to be equivalent to the rate of soluble I FN binding protein production. B) Histograms illustrate the distribution of viability across a population of 1 E5 normal cells (left) and IFN-responsive tumor cells (right) following 72 hours of infection. Results were obtained over 1 E4 Monte Carlo randomizations. Color coding, quantified in pie charts, describes the probability that the described cell population has a viability of <10% (red), 10-90% (grey) or >90% (green) at the end of the simulation.

[0036] Fig. 14 illustrates a phenomenological model describing the infection cycle of an interferon sensitive oncolytic virus. Depending on the concentration of virus and IFN, the population of cells transitions between the "uninfected cell population" (UP), "infected population" (IP), the "activated population" (AP) and the "protected population" (PP). Φ denotes cell death induced in IP/AP due to the viral infection. Green lines denote processes enhanced across all tumor types relative to normal cells. Dashed Red lines highlight processes impaired exclusively in IFN non-responsive tumors.

DESCRIPTION

[0037] Definitions

[0038] Throughout the present disclosure, several terms are employed that are defined in the following paragraphs.

[0039] As used herein, the words "desire" or "desirable" refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be desirable, under the same or other circumstances.

Furthermore, the recitation of one or more desired embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.

[0040] As used herein, the word "include," and its variants, is intended to be non- limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this technology. Similarly, the terms "can" and "may" and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.

[0041] Although the open-ended term "comprising," as a synonym of non- restrictive terms such as including, containing, or having, is used herein to describe and claim embodiments of the present technology, embodiments may alternatively be described using more limiting terms such as "consisting of" or "consisting essentially of." Thus, for any given embodiment reciting materials, components or process steps, the present technology also specifically includes embodiments consisting of, or consisting essentially of, such materials, components or processes excluding additional materials, components or processes (for consisting of) and excluding additional materials, components or processes affecting the significant properties of the embodiment (for consisting essentially of), even though such additional materials, components or processes are not explicitly recited in this application. For example, recitation of a composition or process reciting elements A, B and C specifically envisions embodiments consisting of, and consisting essentially of, A, B and C, excluding an element D that may be recited in the art, even though element D is not explicitly described as being excluded herein.

[0042] As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. Disclosures of ranges are, unless specified otherwise, inclusive of endpoints and include all distinct values and further divided ranges within the entire range. Thus, for example, a range of "from A to B" or "from about A to about B" is inclusive of A and of B. Disclosure of values and ranges of values for specific parameters (such as temperatures, molecular weights, weight percentages, etc.) are not exclusive of other values and ranges of values useful herein. It is envisioned that two or more specific exemplified values for a given parameter may define endpoints for a range of values that may be claimed for the parameter. For example, if Parameter X is exemplified herein to have value A and also exemplified to have value Z, it is envisioned that Parameter X may have a range of values from about A to about Z. Similarly, it is envisioned that disclosure of two or more ranges of values for a parameter (whether such ranges are nested, overlapping or distinct) subsume all possible combination of ranges for the value that might be claimed using endpoints of the disclosed ranges. For example, if Parameter X is exemplified herein to have values in the range of 1-10, or 2-9, or 3-8, it is also envisioned that Parameter X may have other ranges of values including 1-9, 1-8, 1-3, 1-2, 2-10, 2-8, 2-3, 3-10, and 3-9.

[0043] "A" and "an" as used herein indicate "at least one" of the item is present; a plurality of such items may be present, when possible.

[0044] "About" when applied to values indicates that the calculation or the measurement allows some slight imprecision in the value (with some approach to exactness in the value; approximately or reasonably close to the value; nearly). If, for some reason, the imprecision provided by "about" is not otherwise understood in the art with this ordinary meaning, then "about" as used herein indicates at least variations that may arise from ordinary methods of measuring or using such parameters. [0045] As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

[0046] As used herein, a virus that is "interferon sensitive" would be understood to refer to a virus that exhibits slower viral replication in cells with intact interferon defense mechanism when compared to the viral replication in cells without an intact IFN defense mechanism.

[0047] As used herein, the expression "IDE" refers to "Interferon DEcoy receptor". Accordingly, a "maraba-IDE" virus refers to a recombinant maraba virus that includes a polynucleotide that encodes an interferon decoy receptor. In the context of the present disclosure, an "interferon decoy receptor" is equivalent to an "interferon binding protein".

[0048] Detailed Description

[0049] Generally, the present disclosure provides a recombinant interferon (IFN)- sensitive oncolytic rhabdovirus that includes a polynucleotide sequence encoding a soluble protein that binds to IFN-a, IFN-β, or both. The soluble protein is referred to herein as an "interferon binding protein". The interferon binding protein is secretable by a cell infected with the oncolytic rhabdovirus.

[0050] Like many animal viruses, the Rhabdovirus family are able to antagonize the type I interferon response and cause disease in mammalian hosts. Though these negative- stranded RNA viruses are very simple and code for as few as five proteins, they have been seen to completely abrogate the type I interferon response early in infection. Type I interferon response includes IFN-a and IFN-β.

[0051] The IFN-sensitive rhabdovirus may be an IFN-sensitive ephemerovirus, an IFN-sensitive vesiculovirus, an IFN-sensitive cytorhabdovirus, an IFN-sensitive nucleorhabdovirus, an IFN-sensitive lyssavirus, an IFN-sensitive paramyxovirus, or an IFN-sensitive novirhabdovirus. The IFN-sensitive vesiculovirus may be, for example, an IFN-sensitive Vesicular stomatitis virus (VSV) or an IFN-sensitive maraba virus.

[0052] Vesicular stomatitis virus (VSV) is a member of the Rhabdovirus family and is classified in the Vesiculovirus Genus. VSV has been shown to be a potent OV capable of inducing cytotoxicity in many types of human tumour cells in vitro and in vivo (WO

2001/19380). However, wild type- VSV is not interferon sensitive and VSV's functional matrix (M) protein blocks IFN production to enable viral evasion of the immune response [25]. [0053] A recombinant IFN-sensitive VSV may include a polynucleotide sequence encoding a mutated matrix (M) protein. The polynucleotide sequence may encode an M protein with a Δ51 mutation. An exemplary recombinant IFN-sensitive VSV that encodes an M protein with a Δ51 mutation is described in WO 2004/085658, which is

incorporated herein by reference. VSV Δ51 is an engineered attenuated mutant of the natural wild-type isolate of VSV. The Δ51 mutation renders the virus sensitive to IFN signaling via a mutation of the Matrix or M protein. In VSV Δ51 , the M protein mutation renders it incapable of interfering with host gene transcription and nuclear export of antiviral mRNAs and results in the VSV Δ51 virus being IFN sensitive.

[0054] In another example, a recombinant IFN-sensitive VSV may include a polynucleotide sequence encoding interferon-β ("VSV INF-β"). An exemplary IFN- sensitive VSV that encodes interferon-β is described in Jenks N, et al.. "Safety studies on intrahepatic or intratumoral injection of oncolytic vesicular stomatitis virus expressing interferon-beta in rodents and nonhuman primates." Hum Gene Ther. 2010 Apr;

21 (4):451-62, which is incorporated herein by reference.

[0055] Maraba is another member of the Rhabdovirus family and is also classified in the Vesiculovirus Genus. Wild type-Maraba virus has also been shown to have a potent oncolytic effect on tumour cells in vitro and in vivo (WO 2009/016433) and, like wild type- VSV, wild type-Maraba is capable of blocking innate IFN-mediated immune responses.

[0056] A recombinant IFN-sensitive maraba virus may include a polynucleotide sequence encoding a mutated matrix (M) protein, a polynucleotide sequence encoding a mutated G protein, or both. An exemplary IFN-sensitive maraba virus that encodes a mutated M protein and a mutated G protein is described in WO/201 1/070440, which is incorporated herein by reference. This attenuated variant of Maraba virus (MG1) has a mutation in the M protein and a mutation in the G protein. MG1 is attenuated in normal cells but hypervirulent in cancer cells. Some of the attenuation in normal cells can be attributed to defects in the mutated viruses ability to block IFN production.

[0057] As discussed above, a recombinant IFN-sensitive rhabodivirus according to the present disclosure also includes a polynucleotide sequence encoding a soluble protein, secretable by a cell infected by the oncolytic rhabdovirus, that binds to IFN-a, IFN-p, or both.

[0058] Examples of a soluble protein that is secretable by a cell infected by the oncolytic rhabdovirus, and that binds to IFN-a, IFN-β, or both, include B18R and B19R proteins. B18R and B19R proteins are proteins that antagonize the antiviral effect of IFN- α/β. B18R and B19R are soluble IFN-α/β binding proteins that act as decoy receptors to block the activity of IFN-α/β, inhibiting them from binding to their proper receptor. The B18R and B19R proteins are released outside of the cells and their decoy effects are mainly extracellular. The Vaccinia virus Western Reserve strain B18R encodes a secreted protein with 3 IgG domains that functions as a soluble binding protein/receptor for IFN-α/β. The Copenhagen strain of Vaccinia Virus has a B19R gene that is a homolog of the B18R gene. The sequences of B18R and B19R, as used herein, are shown in SEQ ID NOs: 1 and 2, respectively.

[0059] The Wyeth strain of Vaccinia virus expresses a truncated B18R protein lacking the C-terminal IgG domain. The sequence of this truncated B18R protein is shown in SEQ ID NO: 3.

[0060] Inclusion of a gene encoding a soluble B18R, truncated B18R, B19R protein, or variants thereof, into an I FN sensitive OV (for example VSV Δ51 or MG1) may be used to potentiate the anti-tumour effects of the OV in IFN-responsive tumour cells. Inclusion of a gene encoding a soluble B18R, truncated B18R, B19R protein, or variants thereof into an IFN sensitive OV (for example VSV Δ51 or MG1) may be used to enable higher production of the OV in non-cancerous manufacturing cell lines, which have an intact IFN viral defense mechanism.

[0061] Variant polypeptide sequences that are substantially identical to those provided in the sequence listing can be used in the compositions and methods disclosed herein. Substantially identical or substantially similar polypeptide sequences are defined as polypeptide sequences that are identical, on an amino acid by amino acid basis, with at least a subsequence of a reference peptide. Such polypeptides can include, e.g., insertions, deletions, and substitutions relative to any of those listed in the sequence listing.

[0062] A variant of a reference protein may be a protein having a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the sequence of the reference protein, and the variant protein maintains the same biological function as the reference protein. For example, a variant protein would be considered to maintain the same biological function as the reference protein if a virus which has been modified with the variant protein had the same cytotoxicity and neurotoxicity as a virus with the reference protein. An example of variant that is at least 70% identical to a reference protein has at least 7 out of 10 amino acids within a window of comparison are identical to the reference sequence selected. [0063] The variant peptide sequences may include conservative or non- conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having functionally similar side chains. Conservative substitution tables providing functionally similar amino acids are well known in the art. Table 1 sets forth examples of six groups containing amino acids that are "conservative substitutions" for one another. Other conservative substitution charts are available in the art, and can be used in a similar manner.

[0064] Table 1. Conservative Substitution Chart

Conservative Substitution Group

1 Alanine (A) Serine (S) Threonine (T)

2 As artic acid (D) Glutamic acid(E)

Asparagine (N) Glutamine (Q)

4 Arginine (R) Lysine (K)

5 Iso leucine (I) Leucine (L) Methionine (M) Valine (V)

6 Phenylalanine (F) Tyrosine (Y) Tryptophan (W)

[0065] In specific examples, the present disclosure provides a recombinant IFN- sensitive oncolytic virus that is VSV Δ51 and that includes a polynucleotide sequence encoding proteins according to SEQ ID NOs: 1 , 2, or 3, or variants thereof.

[0066] In other specific examples, the present disclosure provides a recombinant

IFN-sensitive oncolytic virus that is VSV IFN-β and that includes a polynucleotide sequence encoding proteins according to SEQ ID NOs: 1 , 2, or 3, or variants thereof.

[0067] In still other specific examples, the present disclosure provides a recombinant IFN-sensitive oncolytic virus that is MG1 and that includes a polynucleotide sequence encoding proteins according to SEQ ID NOs: 1 , 2, or 3, or variants thereof.

[0068] In another aspect, the present disclosure provides a method of replicating an oncolytic virus in cells. The method includes: infecting the cells with a recombinant IFN-sensitive rhabdovirus as described above.

[0069] The cells may express IFN-a, IFN-β, or both.

[0070] The cells may be cancer cells. The cancer cells may be in a patient and the method may include administering the recombinant IFN-sensitive rhabdovirus to the patient. Alternatively, the cancer cells may be from a patient and the method may include infecting the cancer cells with the recombinant IFN-sensitive rhabdovirus, and administering the infected cancer cells to the patient.

[0071] The cells may be non-cancerous mammalian cells and the method may include expressing the recombinant IFN-sensitive rhabdovirus in the non-cancerous mammalian cells, and separating at least a portion of the expressed recombinant IFN- sensitive rhabdovirus from the non-cancerous mammalian cells.

[0072] The non-cancerous mammalian cells may be MRC5 or HEK 293 cells.

[0073] Materials and Methods

[0074] Viruses, cell lines, reagents

[0075] Vesicular Stomatitis virus: VSV Δ51 [6], VSV A51-GFP, VSV WT and VSV

A51-IDE. Attenuated Maraba double mutant virus MG1 and MG1-IDE were both generated by Dr. Stojdl [35]. The following cell lines used were obtained from the American Type Culture Collection (ATCC) (Manassas, VA): African Green Monkey kidney (Vero), human renal cancer cells (786-0), murine colon cancer cells (CT26), human colon cancer cells (HT29), mouse melanoma cancer cells (B16), human glioma cancer cells (U251), primary human fibroblast cells (GM38) and human (MRC5). All cells were grown in DMEM (Hyclone Laboratories Inc.) with 10% FBS (Invitrogen).

Recombinant B19R (VACWR200) utilized in this study was obtained from eBioscience, Inc., San Diego (CA), and the JAK inhibitor I was purchased from EMD millipore.

[0076] Construct Validation

[0077] Cloning

[0078] mRNA expression of soluble I FN binding protein (decoy receptor) expression from new rhabdovirus constructs was verified by PCR. For primers used, see Table 2.

[0079] Table 2. Primers Used in B19R Cloning

Primer Sequence

VSV B19R Forward 5' AAACTCGAGCTCATGCGATGTGTGTAAAA 3'

VSV B19R Reverse 5' AAAGCTAGCCGCGTTATAGCACAAATACG 3'

MG1 B19R Forward 5' TTAATTAATTACTCCAATACTACTGTA 3'

MG1 B19R Reverse 5' GCTAGCATGACGATGAAAATGATGG 3'. [0080] Microarray analysis.

[0081] RNA extraction was performed 24h post infection in 786-0 cells. Duplicate samples were pooled, and hybridized on Affymetrix human gene 1.0 ST arrays according to manufacturer instructions. Data analysis was performed using AltAnalyze [42]. Briefly, probe set filtering implemented a DABG threshold of 70 with pV<0.05 and utilized constitutively expressed exons. Genes differentially expressed were identified using a combination of a >1.5-fold change in expression and a significance of p<0.05 (T- Test) (Supplementary Data 1 , Processed microarray data. This dataset contains gene names, expression values, as well as the fold change and pValue between conditions.).

[0082] Gene ontology enrichments were performed using GOrilla [43]

(Supplementary Data 2, Enriched gene ontology processes associated with genes sub- categorized as induced by MG1 and repressed by B18R. This dataset contains go-term descriptions, pValues, Bonferroni corrected pValues, fold enrichments, contingency table counts, and associated gene names for categories with a pV<0.01 following correction for multiple hypothesis testing). Identification of genes induced by type-1 interferons was performed using the interferome database [44]. For both cloning and microarray analysis, RNA was collected using an RNeasy kit (Quiagen, Toronto, Ontario, CANADA)

[0083] In vitro analysis

[0084] In vitro cell death and cytopathic effects assay.

[0085] Cell lines were plated in 12 well plates at 3E6 cells per well. 24 hours later, cells were treated with each of the 5 different regimens. 72h post infection, crystal violet (0.1 %) staining was performed to visualize live cells. Brightfield images were taken with a Zeiss Axiovert S100 Inverted Microscope.

[0086] Alamar Blue Assay.

[0087] 5E4 cells were plated in 96 wells plate. After 24 hours, cells were infected with each virus. 48 hours post-infection, 10μΙ_ of the Alamar blue reagent (Invitrogen Burlington, On, CANADA) was added to each well. Reading was done using the

Labsystems Fluoroskan II Fluorescent Microplate Reader using the Em/Ex 590/530nm filters. [0088] Spreading Assay.

[0089] Cells were plated in 6 wells plate at 8E5 cells per well. After 24 hours, the cells were covered with a 0.7% agarose overlay containing antibiotics (Penicillin and Streptomycin) in which a small hole was made in the middle of the well where 5E3 pfu of virus was injected. After 5-6 days incubation, crystal violet staining or VSV

immunostaining assessed virus spread. VSV antibodies (polyclonal antibodies from immunized mouse) targeting whole virus were utilized at a dilution of 1/800 and left for a duration of 30 minutes before being washed off with water.

[0090] Cover slip spreading assay.

[0091] MRC5 cells were plated into a 10cm well dish until 100% confluence was obtained after 24 hours. 786-0 cells were plated in a 6 wells plate containing a sterile cover slip at density to reach 100% confluence after 24 hours. 786-0 cells were then infected at an MOI of 5 for 4 hours. Cover slips were removed and placed on the dish containing MRC5 cells and covered by a 0.5% agarose/2xDMEM overlay. 5 days later virus spreading was assayed by crystal violet staining.

[0092] Safety

[0093] The biodistribution assay was performed by injecting 3E5 CT26-LacZ cells subcutaneously on the right side of a BALB-c mouse. 7 days later, tumors were administered 1 e8 pfu of each virus intravenously (IV). Organs tittered and harvested 0.2, 1 ,4,7, and 9 days post infection include: brain, lungs, ovaries, spleen, liver, kidneys, heart and tumor.

[0094] The MTD assay was performed using a dose escalation of Maraba, MG1 and MG1-IDE between 5E8 pfu and 5E9 pfu administered IV, and monitoring the dose keeping 100% of mice alive.

[0095] Efficacy

[0096] In the HT29 model, 3E6 cells were injected subcutaneously in nude mice.

7 days post tumor embedding, mice were treated with PBS, MG1 or MG1-IDE at 1 E8 pfu administered IV.

[0097] In the CT26-LacZ model, 1 E5 CT26-LacZ cells were injected via the tail vein into BALB/c mice. Mice were treated with MG1 and MG1-IDE (1e7pfu) IV on day 3. 13 days post tumor implantation, mice were sacrificed and lungs were excised and stained with X-gal solution prior to counting.

[0098] In the liver metastases model, 1 E6 CT26-LacZ cells were administered intrasplenic into BALB/c mice using intrasplenic/portal injection. 4 days after, 1 E8 pfu of virus was injected IV as treatment. 14 days after tumor cell injection, mice were sacrificed and spleen/liver were harvested stained with X-Gal prior to counting.

[0099] Modeling

[00100] Our model describing OV replication dynamics (Fig. 14) is represented by a subset of 8 ordinary differential equations. The first four equations describe the transition between the UP, IP, AP and PP depending on the concentration of virus and IFN in the environment. These equations are:

= -K_VI x [v] x [up -

₀ x [UP] + K_{IFN OFF} x [PP] ,

^ = K_VI X [V] X [UP] - ( ^g^ + K_{IFN ON} ) X [IP] - _Yc X [IP] ,

K, [UP] + Kvc X [AP] - K_{IFN OFF} X [PP] .

[00101] The parameters used in the above equations represent the infection rate ( vi.) , the rate of IFN signaling activation (K_{IFN ON}), the rate of IFN signalling inactivation (K_{IFN 0} ), the EC50 of IFN (EC50), the rate of cell death (y_c), and the rate viral clearance (K_VC) .

[00102] The next subset of equation describe the concentration of virus (V), interferon (IFN), INF binding protein or decoy receptor (DR) and decoy receptor-IFN complex (DR-IFN) in the media. These equations are:

T = K_{BUD IP} x [IP] + K_{BUD AP} x [AP] - K_VI x [V] x [UP] - _Yv x [V] , = K_IFN1 X [IP] + K_{IPN2 L} X [AP] + K_1FN22 X [PP] - _YLFN X IFN - K_F X [DR] X [IFN] + K_R X

[DR - IFN] , ^ = K_IFN1 X [IP] + K_{1PN2 I} X [AP] + K_IFN22 X [PP] - _YLFN X IFN - K_F X [DR] X

[IFN] + K_r X [DR - IFN] = K_IFN1 X [IP] + ¾_FW2.i X ] + K_IFN2,2 X [PP] - _YlFN X IFN - K_F X [DR] X [IFN] + K_r X [DR - IFN] = K_DRJP X [IP] + K_{DR AP} X [AP] - _Ydr X [DR] - K_f X [DR] X [I FN] + K_R X [DR - IFN] ,

d^DR~t "^N = K_f X [DR] X [IFN] - K_r X [DR - IFN] .

[00103] The parameters described in the above equations represent the rate of virus budding from IP and AP (K_{Bud IP} and K_{Bud AP}, respectfully), the infection rate (K_vi), the rate of virus degradation (γ_ν), the rate of IFN production from I P, AP and PP

{K_IFN1, K_{IFN2 1} and K_IFN2.2 respectfully), the rate of I FN degradation (γ_ΙΡΝ), the rate of I FN binding protein or decoy receptor production from IP and PP (K_{DR IP} and K_{DR AP} respectfully), the rate of IFN binding protein or decoy degradation (y_DR) , the forward rate of I FN-Decoy complex formation (K_f), and the reverse rate of complex formation (K_r). I FN responsive tumors were simulated by decreasing K_{Bud IP} and K_{Bud AP} 10 fold relative to normal cells. I FN non-responsive tumors were simulated by equally randomly decreasing all I FN-regulated processes (K_IFN1, K_IFN2.i , K_{IFN2 2 l}K_vc and K_{IFN on}) 2 to 20 fold.

[00104] The Monte Carlo simulation was generated by varying the parameters in the above model within a 1 log window. Model parameter identification was performed using a simulated annealing method were all parameters are identical in a given cell line other than the capacity to produce INF binding protein or IDE. All simulations were generated in Matlab using the ODE solver ode15s under default parameters imposing a none-negativity constraint. A full list of model amendments to describe each I FN evasion strategy, as well as a list of parameter values utilized in our simulations, are available in Tables 3 and 4 respectfully.

[00105] Table 3. Amendments Performed to Recapture Differences Between Viral Genotypes.

Genotype Modification Explanation

VSV A51 K_{DR IP} = o Removed I FN binding protein

KDR_AP = ⁰ kinetics. VSV K_IFN1 = 0.1 - 1% x K_IFN1 Wildtype M-protein impairs I FN

¾FW2.1 ⁼ 0-1^— 1% ^X ¾FW2.1 signaling and defense

K_VC = 0.1 - 1% X K_VC mechanisms by blocking mRNA KDRJP ⁼ 0 export in infected cells. Equally KDR_AP ⁼ 0 removed I FN binding protein kinetics.

VSV A51 +Jakl KlFN on ⁼ 0-1^— 1% ^X ¾FW on The Jak inhibitor prevents

KDRJP ⁼ 0 activation of IFN signaling by KDR_AP ⁼ 0 inhibiting IFN signalling.

Equally removed I FN binding protein kinetics.

VSV A51 +I DE ¾_fl_/P = 0 Removed I FN binding protein

KDR_AP ⁼ 0 production to substitute for a

Initial Decoy=l-10pM pre-determined concentration of the I FN binding protein.

VSV A51 -I DE None

Table 4. List of Parameters Estimates.

Parameter Estimate Range Utilized

K^_! See below * 7.5E-5 to7.5E-4 (VV)

EC50 1 .5pM(45j 0.25e-12 to 2.5e-12 (M)

K_{IFN ON} <ln(2)/1 h (46) ln(2)/(0.2 to 2.0) (h^"1)

K_IFNOFF ln(2)/20h (47) ln(2)/(5 to 50) (IT¹) y_c ln(2)/8-10h (48) ln(2)/(2.5 to 25) (h^"1)

K_VC ln(2)/(2-5h) (49) ln(2)/(1 to 10) (IT¹)

K_{BUD IP} See below ** 0.5 to 5 (V/h)

K_{BUD AP} See below ** K_BUD_IP ^χ 0.1 to 1 % (V/h)

K_IFN1 See below ***** K_IFN2 * 10 to 100% (M/h)

KIFN2.I & KIFN2.2 2.5E-17 M/cell/h*** 8.3e-18 to 8.3e-17 (M/cell/h)

(i.e. 15000 molecules/cell/h) (ie 5000-50000 molecules/cell/h)

_YlFN ln(2)/(5.3 h) in vivo(50) ln(2)/(5 to 50) (h^"1) K_f 1 E7 M^"1s^"V45) 1 e10 to 10e10 (M^"1h^"1)

(ie 4E10 (IWV)

where¾=2.5E-12 to 25E-12 (M)

K_DR ,p See below ****** Keu_djp* 4.15E-16 to 4.15E-15 (M)

K_{DR AP} See below****** KDR_AP_ ⁼ KDRJP ^X

IP

_Ydr ln(2)/(14.8 h) in vivo(52) ln(2)/(5 to 50) (h^"1) y_v ln(2)/20 h in pes******* ln(2)/(2.5 to 25) (h^"1)

*Adjusted such that upon infection of 2.5E5 cells at an MOI=1 , 99% of the viral particles have infected their target cell within 0.5-5 hours. A result recapturing experimental observations [53].

**Adjusted such that infection at an MOI of 0.05 over a 72 hour period leads to death of

-95% (±5% standard deviation) of the tumor cells within the population in the absence of

IFN signaling. A result similar to our experimental evidence (data not shown).

*** Derived from experimental evidence measuring IFN production over time (data not shown).

**** Except for VSV WT.

***** Unknown value, based on notion that IFN signaling can induce a positive feedback [54] and inherently must be slower than or equivalent to the maximal rate of IFN production once positive feedback is induced.

****** This value is based on the assumption that decoy production is proportional to G- Protein production by the virus. Each functional viron contains -1205 molecules of viral G-protein [55] andx500 due to defective interfering particles. This value was in turn converted to mol/l.

******* Derived from experimental measurements measuring VSV titer in PBS at 37°C (data not shown).

[00106] EXAMPLES

[00107] Example 1 : Model Simulations of Different IFN-Evading Strategies

[00108] To simulate virus infection and spread within tumor and normal tissue, the authors of the present disclosure developed a phenomenological model describing the variable ability of different cell types to support virus proliferation. In this model, cells within an "uninfected population" (UP) transition into an "infected population" (IP) upon viral infection. The number of infected cells in the IP increases over time as virus is produced and spreads to neighboring cells. Production of I FN from IP cells allows these cells to transition into an "activated population" (AP) where viral defenses slow virus release and further enhance I FN production. Over time, this population will gradually become a "protected population" (PP) of cells that have cleared the infection and maintain active anti-viral defense programs [22-24].

[00109] The authors of the present disclosure used the phenomenological model in Fig.1 B as the basis for simulating the outcome of different IFN-evasion strategies on three types of cells: normal cells, IFN non-responsive tumor cells, and IFN responsive tumor cells. The authors of the present disclosure assumed that these cell types differ mainly in their ability to facilitate virus replication and to activate IFN. By quantifying cytotoxicity induced 72h post infection in each of these three cell types, the simulations seek to explore how the relationship between virus replication, activation of IFN- mediated defense responses and cytotoxicity induced across the population might be exploited to design improved therapeutic strategies and better OVs for manufacturing purposes. Because of the heterogeneity of tumor and healthy cells, and the

corresponding uncertainty associated with kinetic parameter estimates, the authors of the present disclosure further employed a Monte Carlo sampling method to simulate 1 E4 different combinations of kinetic parameters randomly sampled within one order of magnitude from values reported in the literature. The outcome of this unbiased method are probability distributions describing the susceptibility of each of the three simulated types of cells towards viral infection.

[00110] The authors of the present disclosure first asked if the model could recapitulate the effects associated with an attenuating mutation in the natural wild-type isolate of the vesicular stomatitis virus (VSV WT), which renders the virus sensitive to IFN signaling, here referred to as VSV A51. In VSV WT, functional M-protein blocks IFN production to enable viral evasion of the immune response [25]. As expected, VSV WT is highly efficient in infecting and killing both normal and tumor populations (Fig.2A-B). In agreement with experimental observations in a variety of tumor models [6,26,27], our simulations predict that the VSV Δ51 attenuated virus will eradicate IFN non-responsive tumors while normal populations or IFN responsive tumors will be largely resistant (Fig.2C-D).

[00111] The authors of the present disclosure next tested two scenarios to determine if chemical manipulation of the interferon responses of virus-infected cells could enhance the activity of VSV Δ51 in IFN responsive tumors while maintaining a low impact in normal populations. Simulations of VSV Δ51 infection in the presence of a chemical inhibitor that blocks interferon signaling (e.g. a JAK-inhibitor) predicts a toxicity profile reminiscent of infection with VSV WT (Fig.2E-F). While the inhibition of I FN signaling increases virus effectiveness against tumors, it also causes the normal population to become highly susceptible to infection. This outcome is also predicted to occur in simulations where the population is exposed to a biological agent that prevents I FN interaction with its cognate receptor (e.g. a soluble I FN receptor antagonist) (Fig.2G- H).

[00112] The authors of the present disclosure then tested the idea of coupling the production and secretion of a soluble IFN binding protein (e.g. a soluble IFN receptor antagonist) to virus replication. In this model, VSV Δ51 is engineered to synthesize a soluble IFN binding protein only when viral gene expression is initiated, thereby creating a positive feedback loop. Positive feedback sharpens dose-responses, enables all-or- none switching in cellular signaling pathways [28], and might thus drive specificity towards the tumor environment. The results obtained by simulating the cytotoxicity induced upon infection with the VSV A51 IFN binding protein-expressing virus (VSV A51-IDE) were highly encouraging (Fig.2l-J). Specifically, the simulated efficacy towards tumors was significantly increased compared to the unmodified VSV Δ51 virus without posing additional risk of damage to the normal population even at high-doses of the virus (Fig. 6).

[00113] Example 2: Experimental Testing of Simulation Predictions in vitro

[00114] To test the model predictions, the authors of the present disclosure performed experiments to compare the efficacy and specificity of VSV Δ51 under each of the simulated IFN-evasion strategies. The authors of the present disclosure previously identified the renal carcinoma cell line 786-0 as having a partially intact interferon response and being refractory to killing by VSV Δ51 [9,29]. Correspondingly,

experiments were performed by the co-treatment of interferon responsive tumor cells (786-0) and normal fibroblast (MRC5) cells with the VSV A51-GFP virus in the presence of the JAK kinase inhibitor I or exogenously added recombinant B19R soluble IFN binding protein [30-34]. In agreement with the outcome predicted by our simulations, the presence of the chemical inhibitor increased VSV A51-mediated killing of 786-0 tumor cells, but equally resulted in a loss of specificity as MRC5 normal cells became susceptible to infection (Fig.3A). Similar results were obtained when the two cell types were cultured in the presence of the soluble I FN binding protein prior to VSV Δ51 treatment (Fig.3A and Fig. 7).

[00115] To test the prediction that virus performance can be improved by incorporating an IFN-suppressing positive feedback loop, the authors of the present disclosure incorporated the B19R I FN binding protein into the VSV Δ51 backbone, and a second IFN-sensitive attenuated oncolytic virus derived from the Maraba virus termed MG1 (Fig. 8). This second rhabdovirus was used to evaluate the generality of the strategy, and because MG1 is a more aggressive rhabdovirus with more potent oncolytic activity as compared to VSV Δ51 [35]. The authors of the present disclosure refer to these soluble IFN binding protein expressing (IDE) viruses as VSV Δδΐ-IDE and MG1- IDE, respectively. After confirming the expression and activity of the IFN binding protein produced from these viruses (Fig. 8), the authors of the present disclosure performed a microarray analysis of differential genome-wide transcription upon infection. This analysis confirmed that interferon binding protein expression following viral infection leads to a significant repression of the type I IFN response in IFN producing cells (Fig. 9 and Table 5). Table 5 summarizes the overlap between genes induced by MG1 and repressed by the IFN binding protein and vice versa; the pValues describe the likelihood of significant overlap (Wilcoxon signed-rank test).

[00116] Table 5. Summary describing the overlap between induced genes

Induced by Maraba Repressed by Maraba

and and

Repressed by IDE Induced by IDE

Total Genes n=25084

Maraba Induced Repressed

vs Untreated (n=1463) (n=2862)

IDE vs Repressed Induced

Maraba (n=358) (n=175)

Overlap n=149 n=49

Enrichment 7.14 2.45

pValue 1 .66E-88 1 .46E-09

[00117] As predicted by our simulations, both VSV A51-IDE and MG1-IDE viruses were associated with tumor selective cytotoxicity. In the 786-0 tumor cell line, both IFN binding protein expressing strains were associated with a greater cytopathic effect than their attenuated parental virus strains. However, neither of the IDE viruses caused significant damage to MRC5 normal cell (Fig.3B-C). The authors of the present disclosure next examined the effect of varying initial virus concentrations on cell viability using both our computer model and cell culture experimentation. Remarkably, the authors of the present disclosure were able to achieve excellent quantitative agreement between the model simulations and experimental data in the above cell lines (Fig.3D), or alternative models (Fig. 10).

[00118] To establish if spreading between the cancerous and normal cell compartments compromises safety, the authors of the present disclosure performed a series of co-culture experiments. To first assess if IFN binding protein expressing viruses have increased spreading ability, the authors of the present disclosure deposited virions in the center of a monolayer of 786-0 cells and examined cell death caused by virus spreading 48 hours post-infection (Fig.4A). Both the VSV A51-IDE and MG1-IDE viruses were superior to their parental counterparts in terms of their ability to rapidly spread through a 786-0 monolayer. Quantification of the surface area affected revealed that virus penetrance was increased 7 fold for VSV A51-IDE and 4 fold for MG1-IDE (Fig.4B). Similar results were obtained using alternative tumor cell lines and methods (Fig. 1 1). In contrast, IFN binding protein expressing viruses had no detectable spread in normal GM38 cells. The authors of the present disclosure next performed our co-culture spreading assay by adding pre-infected 786-0 cells onto a monolayer of normal fibroblasts, and monitored viability 72h post infection by crystal violet staining. Parental wild type viruses (VSV WT or Maraba) spread from the infected tumor cells into surrounding normal cell culture causing widespread off-target killing (Fig.4C). On the other hand, VSV Δ51 and MG1 were restricted only to the local tumor microenvironment and had their spreading rapidly blunted by the normal cell monolayer. Finally, the VSV A51-IDE and MG1-IDE viruses were indistinguishable from their attenuated parental counterparts, and lacked the ability to spread into a normal cell monolayer suggesting that the engineered IFN-suppressing positive feedback loop does not compromise normal tissue (Fig.4C) from a toxicity perspective.

[00119] Example 3: Efficacy and Specificity of IFN Binding Protein

Expressing Viruses in vivo

[00120] To test the in vivo activity of the IFN binding protein expressing viruses, the authors of the present disclosure established subcutaneous tumors in BALB/c mice using an interferon responsive variant of the murine CT26 colon tumor cell line. In our initial studies, animals were treated with OVs intravenously and sacrificed at various times to quantitate the level of virus replication in normal and tumor tissues (Fig.5A). Consistent with earlier studies, virus was cleared from normal organs whether or not it expressed an I FN binding protein by day four. However, the VSV A51-IDE and MG1-IDE viruses persisted longer and grew to higher titers in the tumor tissue than their parental counterparts.

[00121] The authors of the present disclosure also tested the efficacy of MG1-IDE in a number of tumor settings after confirming that the Maximum Tolerated Dose (MTD) was minimally affected by addition of the IFN binding protein (Fig 5B). In a human xenograft model using the HT-29 cell line, MG1-IDE clearly outperformed its parental attenuated IFN-sensitive MG1 strain, effecting long-term cures in approximately 50% of the animals (Fig.5C and Fig. 12). The authors of the present disclosure next established CT-26-LacZ colon tumors in the lungs of BALB/C mice by intravenous infusion to assess the impact of an IFN binding protein on metastatic tumor clearance (Fig.5D). Mice were injected with CT26-LacZ intravenously and 3 days later with 1e7 pfu of virus. Mice were sacrificed 13 days after cells injection. CT26 metastases were counted and results clearly show an increase in efficacy associated with the IFN binding protein expressing virus. This result could be further improved in a liver metastasis model in immunocompetent mice where 100% of the mice treated with MG1-IDE were liver tumor free (Fig.5E). Taken together, these results indicate that IFN binding protein expressing rhabdoviruses improve the therapeutic potential of OVs without compromising toxicity in normal tissue.

[00122] Example 4: IFN Binding Protein from Various Vaccinia Virus Strains and Effects on in vitro Replication of IFN-Sensitive Rhabdovirus

[00123] Supernatants from cells infected with various Vaccinia viruses (Western reserve, Western reserve B18R knock-out (KO), Wyeth, Wyeth B18R knock-out (KO), and Copenhagen) were filtered with a 0.22 μηι filter to remove virus but to retain soluble expressed proteins like B18R. The supernatants were tested on naive cells in combination with VSV Δ51. Supernatants having functional B18R were expected to increase titer (replication) of VSV Δ51. As illustrated in Table 6, the Vaccinia strains having functional B18R were able to increase rhabdovirus IFN sensitive virus replication (titre). This increase was lost when using supernatant from cells infected with B18R KO Vaccinia virus variants. [00124] Table 6. Comparison of replication of VSV Δ51 with supernatant from various Vaccinia virus infected cells

[00125] B18R/B19R IFN-α/β receptor-like secreted glycoprotein sequences were compared strain to strain and Table 7 shows various homology between 73.2% (Wyeth Vaccinia virus Acambis 2000) to 98.7% (Copenhagen Vaccinia virus compared to Western Reserve strain.

[00126] Table 7. Comparison of sequences of various Vaccinia virus strains

[00127] Discussion

[00128] OVs are advancing through late phase randomized clinical trials and it seems likely that one or more products will be approved in the near future [36-40].

Despite encouraging clinical data, it is clear that the genetic heterogeneity of tumors will make it necessary to create novel OVs to maximize their potential as anti-cancer agents.

[00129] Here, the authors of the present disclosure evaluated if model-based rational design could be used to design OVs. After implementing the model to characterize various pre-validated attenuating strategies, such as the Δ51 mutation (6) in VSV or engineering VSV WT to express I FN (Fig. 13) [41], the authors of the present disclosure sought to predict novel methods that would increase tumor killing in IFN responsive tumors without compromising normal tissues. Our model simulations demonstrated that an indirect positive feedback-loop, generated through virus-mediated expression of a soluble IFN binding protein, should selectively enhance virus-mediated cytotoxicity within the tumor microenvironment in excellent agreement with subsequent experimental observations.

[00130] These simulations provide the theoretical framework describing the advantages of incorporating a soluble IFN binding protein (to act as an IFN decoy receptor) into the backbone of oncolytic viruses. By allowing a soluble IFN binding protein to be encoded by the virus, the functional impact of the repressor on virus kinetics is delayed as its expression requires the establishment of a productive viral infection. Repressor-mediated enhancement of viral replication can only occur in the environment surrounding cells pre-disposed towards viral infection and replication. When virus particles enter adjacent normal tissue where viral replication is slow or aborted, levels of the soluble IFN binding protein should be gradually lost thereby preventing the systematic spread of the virus in normal tissue which could lead to toxicity.

[00131] By using a synthetic biology approach to rationally design OVs, the authors of the present disclosure have established a methodology that allows the potential caveats of various immune evasion strategies to be evaluated. This method has allowed us to identify those with the greatest probability of success, and has resulted in the development of two novel OV candidates.

[00132] In the preceding description, for purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the examples.

However, it will be apparent to one skilled in the art that these specific details are not required.

[00133] The above-described examples are intended to be exemplary only.

Alterations, modifications and variations can be effected to the particular examples by those of skill in the art without departing from the scope, which is defined solely by the claims appended hereto. References

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Appendix A - Sequences

The sequence for the vaccinia virus Western Reserve strain B18R protein (NCBI Reference Sequence: YP_233082.1) is:

MTMKJYIMVHIYFVSLLLLLFHSYAIDIENEITEFFNKjyiRDTLPAKDSKWLNPACMFGGTMNDIAAL GEPFSAKCPPIEDSLLSHRYKDYWKWERLEK RRRQVSNKRVKHGDLWIA YTSKFSNRRYLCT VTTK GDCVQGIVRSHIRKPPSCIPKTYELGTHDKYGIDLYCGILYAKHYNNITWYKDNKEINID DIKYSQTGKELIIHNPELEDSGRYDCYVHYDDVRIK DIWSRCKILTVIPSQDHRFKLILDPKI NVTIGEPA ITCTAVSTSLLIDDVLIEWENPSGWLIGFDFDVYSVLTSRGGITEATLYFENVTEE YIGNTYKCRGHNYYFEKTLTTTWLE (SEQ ID NO: 1)

The sequence for the vaccinia virus Copenhagen strain B19R protein (GenBank Accession No: AAA48218.1) is:

MTMKJYIMVHIYFVSLSLLLLLFHSYAIDIENEITEFFNKjyiRDTLPAKDSKWLNPACMFGGTMNDMA TLGEPFSAKCPPIEDSLLSHRYKDYWKWERLEKNRRRQVSNKRVKHGDLWIANYTSKFSNRRYL CTVTTKNGDCVQGIVRSHIKKPPSCIPKTYELGTHDKYGIDLYCGILYAKHYNNITWYKDNKEIN IDDIKYSQTGKELIIHNPELEDSGRYDCYVHYDDVRIKNDIWSRCKILTVIPSQDHRFKLILDP KINVTIGEPANITCTAVSTSLLIDDVLIEWENPSGWLIGFDFDVYSVLTSRGGITEATLYFENVT EEYIGNTYKCRGHNYYFEKTLTTTWLE (SEQ ID NO: 2)

The sequence for the vaccinia virus Wyeth strain truncated B18R protein (GenBank Accession No: AAR18044.1) is:

MTMKJYIMVHIYFVSLSLLLLLFHSYAIDIENEITEFFNKjyiRDTLPAKDSKWLNPACMFGGTMNDMA TLGEPFSAKCPPIEDSLLSHRYKDYWKWERLEKNRRRQVSNKRVKHGDLWIANYTSKFSNRRYL CTVTTKNGDCVQGIVRSHIRKPPSCIPKTYELGTHDKYGIDLYCGILYAKHYNNITWYKDNKEIN IDDIKYSQTGKKLIIHNPELEDSGRYDCYVHYDDVRIKNDIWSRCKILTVIPSQDHRFKLKRNC GYASN (SEQ ID NO: 3)

SEQ ID NO: 1 may be encoded by the polynucleotide sequence:

Atgacgatgaaaatgatggtacatatatatttcgtatcattattgttattgctattccacagttacgccatagacatcga aaatgaaatcacagaattcttcaataaaatgagagatactctaccagctaaagactctaaatggt tgaatccagcatgtatgttcggaggcacaatgaatgatatagccgctctaggagagccattcagc gcaaagtgtcctcctattgaagacagtcttttatcgcacagatataaagactatgtggttaaatg ggaaaggctagaaaaaaatagacggcgacaggtttctaataaacgtgttaaacatggtgatttat ggatagccaactatacatctaaattcagtaaccgtaggtatttgtgcaccgtaactacaaagaat ggtgactgtgttcagggtatagttagatctcatattagaaaacctccttcatgcattccaaaaac atatgaactaggtactcatgataagtatggcatagacttatactgtggaattctttacgcaaaac attataataatataacttggtataaagataataaggaaattaatatcgacgacattaagtattca caaacgggaaaggaattaattattcataatccagagttagaagatagcggaagatacgactgtta cgttcattacgacgacgttagaatcaagaatgatatcgtagtatcaagatgtaaaatacttacgg ttataccgtcacaagaccacaggtttaaactaatactagatccaaaaatcaacgtaacgatagga gaacctgccaatataacatgcactgctgtgtcaacgtcattattgattgacgatgtactgattga atgggaaaatccatccggatggcttataggattcgattttgatgtatactctgttttaactagta gaggcggtattaccgaggcgaccttgtactttgaaaatgttactgaagaatatataggtaataca tataaatgtcgtggacacaactattattttgaaaaaacccttacaactacagtagtattggagta a (SEQ ID NO: 4)

SEQ ID NO: 2 may be encoded by the polynucleotide sequence:

Atgacgatgaaaatgatggtacatatatatttcgtatcattatcattattgttattgctattcca cagttacgccatagacatcgaaaatgaaatcacagaattcttcaataaaatgagagatactctac cagctaaagactctaaatggttgaatccagcatgtatgttcggaggcacaatgaatgatatggcc actctaggagagccattcagtgcaaagtgtcctcctattgaagacagtcttttatcgcacagata taaagactatgtggttaaatgggagaggctagaaaagaatagacggcgacaggtttctaataaac gtgttaaacatggtgatttatggatagccaactatacatctaaattcagtaaccgtaggtatttg tgcaccgtaactacaaagaatggtgactgtgttcagggtatagttagatctcatattaaaaaacc tccttcatgcattccaaaaacatatgaactaggtactcatgataagtatggcatagacttatact gtggaattctttacgcaaaacattataataatataacttggtataaagataataaggaaattaat atcgacgacattaagtattcacaaacgggaaaggaattaattattcataatccagagttagaaga tagcggaagatacgactgttacgttcattacgacgacgttagaatcaagaatgatatcgtagtat caagatgtaaaatacttacggttataccgtcacaagaccacaggtttaaactaatactagatccg aaaatcaacgtaacgataggagaacctgccaatataacatgcactgctgtgtcaacgtcattatt gattgacgatgtactgattgaatgggaaaatccatccggatggcttataggattcgattttgatg tatactctgttttaactagtagaggcggtatcaccgaggcgaccttgtactttgaaaatgttact gaagaatatataggtaatacatataaatgtcgtggacacaactattattttgaaaaaacccttac aactacagtagtattggagtaa (SEQ ID NO: 5)

SEQ ID NO: 3 may be encoded by the polynucleotide sequence:

Atgacgatgaaaatgatggtacatatatatttcgtatcattatcattattgttattgctattcca cagttacgccatagacatcgaaaatgaaatcacagaattcttcaataaaatgagagatactctac cagctaaagactctaaatggttgaatccagcatgtatgttcggaggcacaatgaatgatatagcc gctctaggagagccattcagcgcaaagtgtcctcctattgaagacagtcttttatcgcacagata taaagactatgtggttaaatgggaaaggctagaaaagaatagacggcgacaggtttctaataaac gtgttaaacatggtgatttatggatagccaactatacatctaaattcagtaaccgtaggtatttg tgcaccgtaactacaaagaatggtgactgtgttcagggtatagttagatctcatattagaaaacc tccttcatgcattccaaaaacatatgaactaggtactcatgataagtatggcatagacttatact gtggaattctttacgcaaaacattataataatataacttggtataaagataataaggaaattaat atcgacgatattaagtattcacaaacgggaaagaaattaattattcataatccagagttagaaga tagcggaagatacgactgttacgttcattacgacgacgttagaatcaagaatgatatcgtagtat caagatgtaaaatacttacggttttaccgtcacaagaccacaggtttaaactaaaaagaaattgc ggatatgcgtcaaattaa (SEQ ID NO: 6)

Claims

What is claimed is:

1. A recombinant interferon (IFN)-sensitive oncolytic rhabdovirus comprising a polynucleotide sequence encoding a soluble protein that binds to IFN-a, IFN-β, or both, wherein the soluble protein is secretable by a cell infected with the oncolytic rhabdovirus.

2. The recombinant IFN-sensitive oncolytic rhabdovirus according to claim 1 wherein the IFN-sensitive rhabdovirus is an IFN-sensitive ephemerovirus, an IFN- sensitive vesiculovirus, an IFN-sensitive cytorhabdovirus, an IFN-sensitive nucleorhabdovirus, an IFN-sensitive lyssavirus, an IFN-sensitive paramyxovirus, or an IFN-sensitive novirhabdovirus.

3. The recombinant IFN-sensitive oncolytic rhabdovirus according to claim 2, wherein the rhabdovirus is an IFN-sensitive vesiculovirus.

4. The recombinant IFN-sensitive oncolytic rhabdovirus according to claim 3 wherein the IFN-sensitive vesiculovirus is an IFN-sensitive vesicular stomatitis virus (VSV), or an IFN-sensitive maraba virus.

5. The recombinant IFN-sensitive oncolytic rhabdovirus according to claim 4, wherein the IFN-sensitive VSV comprises a polynucleotide sequence encoding a mutated matrix protein.

6. The recombinant IFN-sensitive oncolytic rhabdovirus according to claim 5, wherein the polynucleotide sequence encodes a Δ51 mutation.

7. The recombinant IFN-sensitive oncolytic rhabdovirus according to claim 4, wherein the IFN-sensitive VSV comprises a polynucleotide sequence encoding interferon-p.

8. The recombinant IFN-sensitive oncolytic rhabdovirus according to claim 4, wherein the IFN-sensitive maraba virus comprises a polynucleotide sequence encoding a mutated matrix (M) protein, and a polynucleotide sequence encoding a mutated G protein.

9. The recombinant IFN-sensitive oncolytic rhabdovirus according to claim 8, wherein the mutated M protein comprises a mutation at amino acid number 123 from leucine to tryptophan (L123W), and wherein the mutated G protein comprises a mutation at amino acid number 242 from glutamine to arginine (Q242R).

10. The recombinant IFN-sensitive oncolytic rhabdovirus according to any one of claims 1-9, wherein the interferon binding protein is a protein comprising three IgG domains that bind IFN-α/β.

11. The recombinant IFN-sensitive oncolytic rhabdovirus according to claim 10, wherein the interferon binding protein comprises an amino acid sequence of SEQ ID NO: 1 , or a variant thereof.

12. The recombinant IFN-sensitive oncolytic rhabdovirus according to any one of claims 1-9, wherein the interferon binding protein is a B19R protein.

13. The recombinant IFN-sensitive oncolytic rhabdovirus according to claim 12, wherein the interferon binding protein comprises an amino acid sequence of SEQ ID NO: 2, or a variant thereof.

14. The recombinant IFN-sensitive oncolytic rhabdovirus according to any one of claims 1-9, wherein the interferon binding protein is a B18R protein lacking a C-terminal IgG domain.

15. The recombinant IFN-sensitive oncolytic rhabdovirus according to claim 14, wherein the interferon binding protein comprises an amino acid sequence of SEQ ID NO: 3, or a variant thereof.

16. A method of replicating an oncolytic virus in cells, the method comprising:

infecting the cells with a recombinant IFN-sensitive rhabdovirus according to any one of claims 1-15.

17. The method according to claim 16, wherein the cells expresses IFN-a, IFN-β, or both.

18. The method according to claim 16 or 17 wherein the cells are cancer cells.

19. The method according to claim 18, wherein the cancer cells are in a patient and the method further comprises administering the recombinant IFN-sensitive rhabdovirus to the patient.

20. The method according to claim 18, wherein the cancer cells are from a patient and the method further comprises:

infecting the cancer cells with the recombinant IFN-sensitive rhabdovirus ex vivo, and

administering the infected cancer cells to the patient.

21. The method according to claim 16 or 17, wherein the cells are non-cancerous mammalian cells and the method further comprises:

expressing the recombinant IFN-sensitive rhabdovirus in the non-cancerous mammalian cells, and

isolating at least a portion of the expressed recombinant IFN-sensitive rhabdovirus from the non-cancerous mammalian cells.

22. The method according to claim 21 wherein the non-cancerous mammalian cells are MRC5 or HEK 293 cells.