WO2014198002A1 - A bacterium producing an interferon binding protein and uses thereof - Google Patents

Abstract

Described herein is a non- invasive bacterium that includes a polynucleotide sequence encoding a soluble interferon binding protein that binds to IFN-α, IFN-β, or both, wherein the soluble protein is secretable by the bacterium. The bacterium may be used to aid in the replication of an IFN-sensitive oncolytic virus in a tumorous cancer in a patient.

Description

A BACTERIUM PRODUCING AN INTERFERON BINDING PROTEIN AND USES

THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

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

Application No. 61/835,453 filed June 14, 2013, which is hereby incorporated by reference.

FIELD

[0002] The present disclosure relates generally to combinations of oncolytic viruses, and bacteria producing an interferon binding protein.

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 (I FN) 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 is often due to the inability of OVs to antagonize normal cellular, interferon (I FN) 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].

INTRODUCTION

[0007] The following discussion is intended to introduce the reader to the detailed description to follow, and not to limit or define any claimed invention.

[0008] It is an object of the present disclosure to provide bacteria that obviates or mitigates at least one disadvantage associated with viral therapy using oncolytic viruses. [0009] Vesicular stomatitis virus (VSV) is an exemplary oncolytic virus. VSV is a rhabdovirus that has a broad cancer cell tropism and is effective when administrated intravenously in murine tumour models. The wild-type strain of VSV expresses a matrix (M) protein that, upon infection, acts as an intracellular antagonist of I FN production by blocking the transport of I FN mRNAs from the nucleus [3]. This M protein results in wild-type VSV being neurotoxic in certain mouse strains. However, an attenuated version of the virus (VSVA51), having a Δ51 mutation in the M protein, retains oncolytic activity but is harmless when administered intravenously since the virus cannot block the transport of I FN mRNAs from the nucleus and only productively infect tumour cells that have a defective interferon response [4].

[0010] While VSVA51 , and other OVs having oncolytic activity in cells having a defective I FN response, are capable of selectively killing some tumor cells, one of the major problems with oncolytic virotherapy is that some tumours, or regions of tumours, have intact or upregulated IFN-mediated antiviral responses. Intra- and inter- tumour heterogeneity can result in incomplete oncolysis following OV therapy. Some human-derived tumour cell lines, such as HT29 colon carcinoma, retain at least partial responsiveness to I FN and only poorly support the spread of OVs, such as VSVA51.

[0011] Localized expression of a soluble protein that binds to IFN-a, IFN-β, or both may be used to antagonize the type I interferon response in cells that otherwise have an intact I FN response. B18R, a gene from Vaccinia virus which encodes a secreted soluble

I FN binding protein that acts as a decoy receptor for IFN-a and IFN-β, is one example of such a soluble protein. Expression of B18R improved the efficacy of VSVA51 to grow and kill tumours [1 , 5, 6].

[0012] Localized expression of the type 1 IFN antagonist within a tumour shields the OV from immune mediated clearance, enhancing the oncolytic potential of the virus to eliminate malignancies [1]. However, such a strategy requires an efficient, specific and safe means to localize expression of the protein to the desired region of tumours. Non-invasive bacterial vectors represent one solution to this problem since they exhibit high-level growth and transgene expression targeted to various tumours [15, 19]. Administration of non- invasive bacteria expressing a soluble protein that binds to IFN-a, IFN-β, or both results in a microenvironment having reduced amounts of bioactive antiviral cytokines, thus 'preconditioning' the tumour to enhance subsequent tumour destruction by the OV. [0013] The authors of the present disclosure engineered bacteria that encode 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 the bacteria.

[0014] The engineered bacteria are useful because the bacteria selectively replicate in tumour stroma following systemic administration. In the case of non-invasive bacteria, tumour selectivity relates to their ability to grow extracellularly within tumour stroma. This tumour-selective replication restricts the production of the bacterially produced interferon binding protein to tumours and antagonizes the type I interferon response in tumour cells that otherwise have an intact I FN response.

[0015] Without wishing to be bound by theory, the bacterial tumour specificity may be a result of the uniqueness of tumour physiology resulting from a combination of factors, such as: local immune suppression, irregular vasculature, relevant nutrient presence in necrotic tissue, and the anaerobic nature of hypoxic/necrotic regions within tumours promoting growth of anaerobic and facultatively anaerobic bacteria [10, 11]. It is desirable to target these regions of tumours since they may be a major source of cells responsible for tumour re- growth post-treatment.

[0016] In the context of the present disclosure, it is desirable to target tumours using non-invasive bacteria (lacking the ability to mediate disease) since doing so will provide local bacterial expression of genes external to tumour cells, without generating a disease in the patient. Examples of non-invasive bacteria include health-promoting or probiotic bacteria, including certain strains of Escherichia coli [12-14]. Tumour-specific replication of E. coli MG1655 in mice has been shown using a luminescence-based tagging system [15].

[0017] As discussed herein, using engineered bacteria that encode a soluble protein that binds to IFN-a, IFN-β, or both, where the protein is secretable by the bacteria, may locally enhance oncolytic virus mediated tumour oncolysis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0019] Figure 1 is a Northern blot illustrating that E. coli can express an introduced heterologous gene encoding B18R. [0020] Figure 2 is a graph illustrating that the B18R expressed by E. coli is functional and supernatant from E. coli cultures expressing B18R increases VSV IFN sensitive virus replication in 786-0 cells.

[0021] Figure 3 is a graph illustrating that the B18R expressed by E. coli is functional and supernatant from E. coli cultures expressing B18R increases VSV IFN sensitive virus replication in HT29 cells.

[0022] Figure 4 is a graph illustrating that E. coli-B18R reduces IFN-a levels in 786-0 cells.

[0023] Figure 5 is a graph illustrating that E. coli-B18R reduces IFN-a levels in HT29 cells.

[0024] Figure 6 shows fluorescent microscopy images of HT29 cells.

[0025] Figure 7 is a graph illustrating the mean percentage of GFP⁺/ VSV-infected

HT29 cells in the presence or absence of E. coli-B18R, quantified by flow cytometry of immunofluorescent cells from Figure 6.

[0026] Figure 8 is a graph illustrating the effect of E. coli-B18R on luminescence of

HT29 cells infected with VSVA51 FLuc.

[0027] Figure 9 is a graph illustrating the cell viability of the HT29 cells of Figure 8.

[0028] Figure 10 is a graph illustrating that IV administered bioluminescent E. coli specifically colonizes tumours in mice.

[0029] Figure 11 is a graph illustrating that E. coli encoding B18R increases

VSVA51 FLuc luminescence in subcutaneously implanted HT29 cells in vivo.

[0030] Figure 12 is a graph illustrating that E. coli encoding B18R increases

VSVA51 FLuc luminescence in subcutaneously implanted LLC cells in vivo.

[0031] Figure 13 is a graph illustrating LLC tumour volume over time.

[0032] Figure 14 is a graph illustrating survival curves for LLC-bearing mice treated with, among other protocols, E. coli-B18R plus VSVA51 FLuc.

[0033] Figure 15 is a graph illustrating the serum profile of IFN- γ,

[0034] Figure 16 is a graph illustrating the serum profile of mKC.

[0035] Figure 17 is a graph illustrating the serum profile of IL-10.

[0036] Figure 18 is a graph illustrating the serum profile of IL-12p70.

[0037] Figure 19 is a graph illustrating the serum profile of I L- 1 β .

[0038] Figure 20 are fluorescent micrographs showing the effect of bacterial B18R production on HSV-1-GFP and JX594-GFP replication in vitro. [0039] Figure 21 is a graph illustrating the effect of bacterial B18R production on

HSV-1-GFP replication in vitro by flow cytometry.

[0040] Figure 22 is a graph illustrating the effect of bacterial B18R production on

JX594-GFP replication in vitro by flow cytometry.

DETAILED DESCRIPTION

[0041] Definitions

[0042] Throughout the present disclosure, several terms are employed that are defined in the following paragraphs.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] "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.

[0048] "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.

[0049] As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

[0050] 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. [0051] Detailed Description

[0052] Generally, the present disclosure provides a non-invasive bacterium engineered to encode a soluble protein that binds to IFN- , IFN-β, or both, that is secretable by the bacterium. The soluble protein is referred to herein as an "interferon binding protein".

[0053] The present disclosure also provides a method for replicating an IFN-sensitive oncolytic virus in a tumorous cancer in a patient using non-invasive bacteria engineered to encode a soluble protein that binds to IFN-a, IFN-β, or both. Enhanced virus replication may result in improved therapeutic outcome in a patient.

[0054] The non-invasive bacterium may be Escherichia coli (for example MG1655,

Nissle 1917, or other commensal strains); species of the genus Bifidobacterium (for example B. breve, B. infantis, B. longum); species of the genus Lactococcus (for example L. lactis); species of the genus Lactobacillus (for example L. reuteri, L. delbrueckii, L. plantarum); noninvasive species of the genus Listeria (for example L. welshimeri). The non-invasive bacterium may also be a safety-attenuated bacterial pathogen, where attenuation renders the strain non-invasive, such as modified Salmonella enterica Typhimurium or Listeria monocytogenes.

[0055] MG1655 was first described in Blattner, FR et al. (1997) The complete genome sequence of Escherichia coli K-12. Science 277 1453-62. Nissle 1917 is discussed in Cress BF, Linhardt RJ, Koffas MAG. 2013. Draft genome sequence of Escherichia coli strain Nissle 1917 (serovar 06:K5:H1). Genome Announc. 1 (2):e00047-13. doi: 10.1128/genomeA.00047-13. The genomic sequence of Nissle 1917 is deposited in DDBJ/EMBL/GenBank under accession no. CAPM00000000.

[0056] Examples of a soluble protein that is secretable by a bacterium according to the present disclosure, 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 bacterial cells and their decoy effects are mainly extracellular.

[0057] 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. 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.

[0058] In some examples, the interferon binding protein expressed by bacterium according to the present disclosure may be a protein comprising three IgG domains that bind IFN- /β, for example a protein that includes an amino acid sequence of SEQ ID NO: 1 , or a variant thereof.

[0059] In other examples, the interferon binding protein expressed by bacterium according to the present disclosure may be a B19R protein, for example a protein that includes an amino acid sequence of SEQ ID NO: 2, or a variant thereof.

[0060] In still other examples, the interferon binding protein expressed by bacterium according to the present disclosure may be a B18R protein lacking a C-terminal IgG domain, for example a protein that includes an amino acid sequence of SEQ ID NO: 3, or a variant thereof.

[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 bacterium expressing the variant protein enhances viral replication to approximately the same degree as a bacterium expressing 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 Aspartic acid (D) Glutamic acid(E)

3 Asparagine (N) Glutamine (Q)

4 Arginine (R) Lysine (K)

5 Isoleucine (I) Leucine (L) Methionine (M) Valine (V)

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

[0065] The method according to the present disclosure may include administering to a patient a plurality of the non-invasive bacterium described above, and a plurality of IFN- sensitive oncolytic viruses.

[0066] IFN-sensitive oncolytic viruses according to the present disclosure may include rhabdovi ruses; adenoviruses; reoviruses; herpes simplex virus 1 ; Newcastle disease viruses; vaccinia viruses; coxsackieviruses; measles viruses; Seneca Valley Viruses;

influenza viruses; and myxoma viruses.

[0067] 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- and IFN-β.

[0068] The IFN-sensitive oncolytic virus may be, for example, a rhabdovirus such as 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.

[0069] 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].

[0070] 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 anti-viral mRNAs and results in the VSV Δ51 virus being IFN sensitive.

[0071] 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.

[0072] 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.

[0073] 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/2011/070440, 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. The mutated M protein may include a mutation at amino acid number 123 from leucine to tryptophan (L123W), and the mutated G protein may include a mutation at amino acid number 242 from glutamine to arginine (Q242R). 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. [0074] Exemplary adenoviruses are disclosed in Xia, Z. J. et al. (2004). Phase III randomized clinical trial of intratumoral injection of E1 B gene-deleted adenovirus (H101) combined with cisplatin-based chemotherapy in treating squamous cell cancer of head and neck or esophagus. Ai Zheng 23, 1666-1670; and in Wakimoto et al., (2004) Altered expression of antiviral cytokine mRNAs associated with cyclophosphamide's enhancement of viral oncolysis. Gene Therapy 11 , 214-223.

[0075] An exemplary herpes simplex virus 1 is disclosed in Shah et al., (2003).

Oncolytic viruses: clinical applications as vectors for the treatment of malignant gliomas. J. Neurooncol. 65, 203-226.

[0076] An exemplary Newcastle disease virus is disclosed in Pecora, A. L. et al.

(2002) Phase I trial of intravenous administration of PV701 , an oncolytic virus, in patients with advanced solid cancers. J. Clin. Oncol. 20, 2251-2266.

[0077] Exemplary vaccinia viruses are disclosed in Mastrangelo, M. J. et al. (1999)

Intratumoral recombinant GM-CSF-encoding virus as gene therapy in patients with cutaneous melanoma. Cancer Gene Ther. 6(5), 409-422; and in US 2006/0099224.

[0078] An exemplary Seneca Valley Viruses is disclosed in Reddy PS, Burroughs

KD, Hales LM, Ganesh S, Jones BH, Idamakanti N, Hay C, Li SS, Skele KL, Vasko AJ, et al. Seneca Valley virus, a systemically deliverable oncolytic picornavirus, and the treatment of neuroendocrine cancers. Journal of the National Cancer Institute. 2007;99: 1623-1633.

[0079] An exemplary myxoma virus is disclosed in Myers, R. ef al. (2005). Oncolytic activities of approved mumps and measles vaccines for therapy of ovarian cancer. Cancer Gene Ther. 12, 593-599.

[0080] Other specific examples of IFN-sensitive oncolytic viruses that may be used according to the present disclosure include JX594, a vaccinia poxvirus, and HSV-1.

[0081] The method may include infecting cancer cells from the tumorous cancer with the IFN-sensitive oncolytic virus ex vivo, and administering the infected cancer cells to the patient.

[0082] The method may include administering the IFN-sensitive oncolytic virus to the patient intravenously, intradermal ly, transdermal^, parenterally, intramuscularly, intranasally, subcutaneously, regionally, percutaneously, intratracheally, intraperitoneally, intraarterially, intravesically, intratumorally, via inhalation, via perfusion, via lavage, via direct injection, or via oral administration or formulation. The IFN-sensitive oncolytic virus may be administered to the patient in a unit dose between about 1 e3 to 1 e13 plaque forming units (pfu). Unit doses higher than 1e13 may also be administered. A unit dose need not be administered as a single injection but may be continuously infusion over a period of time. Unit dose of the present invention may conveniently be described in terms of plaque forming units (pfu) for a viral construct.

[0083] Alternatively, depending on the virus and the titre attainable, between 1 and

1e15 infectious IFN-sensitive oncolytic viral particles may be administered to the patient or to the patient's cells. More than 1e15 infectious viral particles may also be administered. In particular examples, between 1 and 100 viral particles may be administered, for example to the patient's cells ex vivo. In other examples, between 1e10 and 1e15 infectious viral particles may be administered to the patient.

[0084] The non-invasive bacterium may be administering to the patient in an amount between 1e5 and 1e10 colony forming units. It is desirable to administer the non-invasive bacteria before the IFN-sensitive virus is administered to give it sufficient time for the bacteria to locally express the soluble interferon binding protein that binds to IFN-a, IFN-β, or both and to result in a microenvironment having reduced amounts of bioactive antiviral cytokines. In preferred examples, the non-invasive bacteria is administered between 1 and 14 days prior to administration of the IFN-sensitive virus. In other examples, the non-invasive bacteria is administered to the patient at substantially the same time that the IFN-sensitive virus is administered to the patient.

[0085] The present disclosure also provides a kit that includes a plurality of the noninvasive bacterium described above and a plurality of IFN-sensitive oncolytic viruses, such as those described above.

[0086] Various preclinical therapeutic trials have shown the ability of different bacterial strains to traffic to tumour sites, primarily in the context of delivery of DNA for subsequent tumour cell expression [20]. In general, invasive or pathogenic species have been exploited for this purpose to date [21-23]. However even with safety attenuation, the inherent pathogenicity and immunogenicity of these bacteria has outweighed the therapeutic responses in patients [24]. Therefore, the authors of the present disclosure have chosen to exploit a non-invasive E. coli strain and validated that this strain colonises tumours with efficiencies similar to the best-described species in the field, Salmonella Typhimurium [15]. The E. coli strain employed in the examples disclosed herein is non-invasive, and therefore does not act as a cell transfection agent, but rather replicates within the tumour stroma, external to tumour cells. It is desirable that bacteria according to the present disclosure also do not act as cell transfection agents, but instead replicate within the tumor stroma and secrete a soluble interferon binding protein that binds to IFN-a, IFN-β, or both, external to the tumour cells.

[0087] In mouse experiments, both virus and bacterial replication, even in co-infected animals, was restricted to tumours with no evidence of infection or colonisation of normal tissues detected by imaging and immunohistochemical staining. Furthermore, the safety profile of this combination as illustrated by cytokine profiling demonstrates its potential to be safely used even in immunocompromised hosts. The cytokine profiles showed no overall increase in pro-inflammatory cytokines, in fact the bacteria for unknown reasons appeared to decrease the cytokine levels; this may be due to its commensal nature [25].

[0088] At the treatment doses used, therapy with either microorganism on its own had little impact on rapidly growing tumours and did not impact cumulative survival. However, when E. coli-B18R and VSVA51 were sequentially administered, substantially slower tumour growth resulted. In addition, the combination treatment could significantly extend survival as compared to all other treatment arms, a considerable improvement given the aggressiveness of the LLC tumour model. Local bacterial production of B18R was successfully exploited to create a microenvironment depleted of bioactive antiviral cytokines thus permitting robust replication and spread of VSVA51. This finding was extended to other clinical OVs including HSV-1 , a Herpes Simplex virus, and JX594, an engineered vaccinia virus. HSV-1 has its own IFN response genes but these genes inhibitory IFN effects only act on an intracellular level and could be enhanced by a gene product that acts extracellularly. Likewise JX594 contains an endogenous B18R gene but a natural truncation of this gene in this clinical vaccinia candidate reduces its ability to antagonize IFN.

[0089] The nature of these organisms' individual actions are confined to the tumour as the bacteria are unable to colonise healthy tissue and consequently, the B18R

expression, providing the interferon depleted environment necessary for VSVA51 replication and spread, is tumour specific. Combination of these microorganisms, potentially including additional therapeutic genes targeting multiple or sequential pathways, could further enhance efficacy and prevent the development of resistance with no added toxicity. Furthermore, the tumour-specific nature of both of these modalities when administered intravenously permits safe targeting of systemic metastases. Thus, the potential high degree of safety and efficacy predicted for combination therapy of cancer warrants further investigation at both preclinical and clinical levels.

[0090] MATERIALS METHODS

[0091] Bacterial Strains

[0092] E. coli K12 MG1655 containing either the control empty pNZ44 plasmid [30] or pNZ44 B18R (B18R PCR amplified from Vaccinia virus) cloned downstream of p44 promoter as Ncol, Xbal insert) plasmid (£. coli-B18R) were grown aerobically at 37°C in LB medium (Sigma, Steinheim, Germany) supplemented with 20 μg/ml chloramphenicol (Cm). The bioluminescent derivative of MG1655 (E. coli-lux) which was used in a parallel group to monitor bacterial growth through bioluminescence was created using the plasmid p16S/t x which contains the constitutive P_HELPluxABCDE operon [31] on the backbone of pGh9: : ISS7, a thermo-sensitive shuttle vector which integrates randomly into the bacterial chromosome as a consequence of the presence of ISS7 [32] and was grown aerobically at 37°C in LB medium supplemented with 300 pg/ml erythromycin (Em). In the examples discussed below, the B18R protein has a sequence that includes the amino acids according to SEQ ID NO: 1.

[0093] Virus Preparation.

[0094] Recombinant AV3 strain of VSV with Green Fluorescent Protein (VSVA51- GFP), or luciferase (VSVA51-Luc) were propagated in Vero cells. Virions were purified as previously described [33]. Two additional OVs were used in in vitro co-incubation studies. HSV-eGFP (HSV1-GFP), the current most clinically relevant OV and JX594-eGFPdB18R (JX594-GFP) a Vaccinia poxvirus with GM-CSF & TK. [0095] Tumour Cell lines and Culture

[0096] Murine Lewis Lung Carcinoma, HT29 adenocarcinoma and 4T1 breast cancer cells were maintained in culture at 37 °C in a humidified atmosphere of 5 % C0₂, in

Dulbecco's Modified Essential Medium (GIBCO, Invitrogen Corp. , Paisley, Scotland) or RPMI Medium (GI BCO, Invitrogen Corp., Paisley, Scotland) supplemented with 10% iron- supplemented donor calf serum (Sigma Aldrich Ireland, Ireland), 300 pg/ml L-glutamine. [0097] B18R Bioassay

[0098] 786-0 and HT-29 tumour cell lines were pre-treated or not with recombinant

B18R (0.1 μς/μΙ, Bioscience) or with supernatant from bacteria either expressing B18R or not expressing B18R. Bacteria from a 24 h culture (OD600nm = 2.0 +/-0.2) were pelleted by centrifugation and supernatant filtrated with 0.22uM filter. Filtrates were used V/V with media on cells. Pre-treatment was performed 4 h prior addition of VSVA51-GFP at MOI 0.05 for 786-0 and 0.001 for HT29. Fluorescent microscopy (Olympus) was performed for GFP 36 h after addition of virus. In parallel, bacterial supernatant effect on IFN-a levels was investigated by ELISA. Pre-treatment were performed 4 h prior addition or not of VSVA51 at MOI 0.05 in 786-0 and 0.001 in HT29. IFN-a ELISA was performed 24 h after virus addition on cell supernatants and was carried out using IFN-a (Mabtech, OH) according to the manufacturer's instructions.

[0099] In Vitro Co-Incubation Studies

[00100] E. coli containing either pNZ44 (backbone vector) or E. coli-B18R were grown to mid-log OD₆₀onm of 0.6, harvested by centrifugation and the supernatant removed. This supernatant was filter sterilised and added to confluent 6-well plates containing the cell line of interest (HT29 or 4TI) for two hours. The HT29 cells were then washed in PBS and DM EM +/- VSVA51-GFP or separate plates with +/- VSVA51-luc2 at 10⁵ pfu added for between 6 hours. The 4TI cells were then washed in PBS and RPMI +/- HSV1-GFP or separate plates with +/- JX594-GFP at 10^s pfu were added for either 6 or 24 hours. Following incubation the cells were imaged for fluorescence or quantified for luminescence using the MS imaging system. Subsets of wells were counted for total cell numbers and processed for flow cytometry. The total cell death in virus treated versus untreated cell lines was examined as follows: culture medium was removed from wells; cells were fixed in 96% ethanol for 10 min and stained with Prodiff solution C (Braidwood Laboratories, UK). Plates were scanned using the Odyssey IR imaging system (Li-Cor, Cambridge, UK) and viable cells quantified.

[00101] FACS Analyses

[00102] The confluent monolayers of HT29 or 4TI cells treated with either media alone or media containing filter sterilised bacterial supernatants (£. co//^'-pNZ44 or E. coli-B18R) and subsequently VSVA51-GFP or HSV1-GFP or JX594-GFP were washed, counted and re- suspended at 1 x 10⁶ cells/ml for flow cytometric analysis. GFP fluorescence intensity was measured using a LSRII cytometer and BD Diva software (Becton Dickinson, Oxford, UK). For each sample, 30,000 events were recorded. The percentage of GFP⁺ cells was calculated in wells which were exposed to either virus alone, virus plus backbone vector supernatant (£. co//^'-pNZ44) or virus plus B18R vector supernatant (£. coli-B18R). The results represent the percentage of positively stained cells in the total cell population exceeding the background staining signal.

[00103] Animals and Tumour Induction

[00104] Mice were kept at a constant room temperature (22°C) with a natural day/night light cycle in a conventional animal colony. Standard laboratory food and water were provided ad libitum. Before experiments, the mice were afforded an adaptation period of at least 7 days. Female mice in good condition, without fungal or other infections, weighing 16-22 g and of 6-8 weeks of age, were included in experiments. For routine tumour induction, the minimum tumourigenic dose of cells (5 x 10^s LLC; 3 x 10⁶ HT29) suspended in 200 μΙ of serum-free culture medium was injected subcutaneously (s.c.) into the flank of 6-8 week old female athymic MF1-nu/nu mice (Harlan, Oxfordshire, UK) (5 x 10^s LLC). The viability of cells used for inoculation was greater than 95% as determined by visual count using a haemocytometer and Trypan Blue Dye Exclusion (Gibco), or the Nucleocounter system (ChemoMetec, Bioimages Ltd, Cavan, Ireland). Following tumour establishment, tumours were allowed to grow and develop and were monitored twice weekly. Tumour volume was calculated according to the formula V=(ab²) Π/6, where a is the longest diameter of the tumour and b is the longest diameter perpendicular to diameter a. When tumours reached approximately 100 mm³ in volume, the mice were randomly divided into

experimental groups.

[00105] In Vivo Bacterial Administration

[00106] Inocula were prepared by growing E. coli pNZ44, E. coli-B18R or the integrated p16Slux aerobically in 100 ml LB broth containing either 20 g/ml Cm (pNZ derivatives) or 300 μg/ml Em (p16Slux). Cultures were harvested by centrifugation (4,000 ^χ g for 15 min), washed three times with PBS and resuspended in a one-tenth volume of PBS.

The viable count of each inoculum was determined by retrospective plating on LB agar containing the appropriate selective antibiotic. For tumour-related studies, mice were randomly divided into experimental groups when tumours reached approximately 100 mm³ in volume, and administered E. coli or an equal volume of PBS as control. Each animal received 10⁶ E. coli in 100 μΙ injected directly into the lateral tail vein, as previously described [34] [00107] In Vivo Efficacy Studies

[00108] Mice were treated with either E. coli-B18R (1 ^χ 10⁶ cfu/ml), E. coli pNZ44 (1 ^χ 10⁶ cfu/ml) or PBS and 5 days later with VSVA51 (1 ^χ 10^s pfu). Tumours were measured as previously described. At necropsy tumour and healthy tissue were formalin fixed and paraffin embedded for immunohistochemistry. A cardiac bleed was performed and the serum extracted for cytokine profiling using Meso Scale Discovery 7-plex pro-inflammatory cytokine plate.

[00109] Optical Image Acquisition

[00110] 2D in vivo BLI imaging was performed using the IVIS100 (Caliper, a Perkin Elmer company). At defined time points post bacteria and/or virus administration, animals were anesthetised under 3% Isofluorane and whole-body image analysis was performed in the IVIS 100 system for up to 4 minutes at high sensitivity. Regions of interest were identified and quantified using Living Image software (Caliper). To acquire images of the bacterial luciferase signal emission filter wavelengths ranging from 500-580 nm were used with bin 16 acquisition times of 3-4 min per filter to maximize the signal to noise ratio. To acquire images of the firefly luciferase emanating from the virus, d-luciferin (Molecular Imaging Products, Ann Arbor Ml) was injected approximately 10 min prior to imaging using an intraperitoneal injection. Emission filter wavelengths ranging from 580-620 nm were then used with bin 8 acquisition times of 0.5-0.75 min per filter. For each experiment, images were captured under identical exposure, aperture and pixel binning settings, and bioluminescence is plotted on identical colour scales.

[00111] Immunohistochemistry

[00112] Sectioned tissues were processed as previously described with anti-VSV (1 :5,000; 30 minutes) antibody [35]. Tumour images were obtained with an Epson Perfection 2450 Photo Scanner (Epson, Toronto, Canada) whereas magnifications were captured using a Zeiss Axiocam HRM Inverted fluorescent microscope (Zeiss, Toronto, Canada) and analysed using Axiovision 4.0 software. Consecutive sections were stained with H&E for general morphology.

[00113] Cytokine Profiling

[00114] Concentrations of ΙΙ_-1 β, IL-12p70, IFN-γ, IL-6, mKC, IL-10, and TNF-a in serum at 5 days post virus administration were measured by an ultra-sensitive mouse proinflammatory 7-plex kit from Meso Scale Discovery (MSD, Gaithersburg, MD, USA) according to the manufacturer's instructions. Blood was acquired by cardiac puncture allowed to clot and the serum separated and aliquots frozen at -80 until required. The protocol for the assay was as described by MSD, briefly a spot on the base of each plate was pre-coated with a capture antibody for each cytokine. The standard and serum samples (50 μΙ/well) were added to the prepared plates, and allowed to react at room temperature for 2 h. Afterward, the plates were washed three times with washing buffer (1 ^χ PBS with 0.05% Tween 20). Detection antibody was added and allowed to react at room temperature. After washing the plates three times and adding Read Buffer, the plates were analysed on the MSD Sector Image 2400 (MSD). Calculation of cytokine concentrations was subsequently determined by 4-parameter logistic non-linear regression analysis of the standard curve.

[00115] Statistical Analysis

[00116] Two-tailed Student's f-tests were employed to investigate statistical differences. Microsoft Excel 12 (Microsoft) and Prism were used to manage and analyse data.

[00117] EXAMPLES

[00118] Example 1 : E. coli Expresses Functional B18R

[00119] E. coli was engineered to stably express B18R, in addition to a control bacterial construct, containing the plasmid backbone lacking the B18R DNA sequence (E. co//^'-pNZ44). Figures 1 to 3 demonstrate that E. coli expresses a functional B18R. For Figure 1 , E. coli pNZ44 and E. co//^'-B18R were grown for 24 h in LB-Chloramphenicol, before extraction of total RNA. RT-PCR specific for B18R was performed on resultant cDNA. For

Figures 2 and 3, a B18R Bioassay was performed as previously described where 786-0

(Figure 2) and HT-29 (Figure 3) tumour cell lines were either pre-treated or not with recombinant B18R ("Rec B18r VSV" and "VSV", respectively) or the cells were pretreated with supernatant from bacteria either expressing B18R or not expressing B18R ("pNZ44 B18r VSV" and "pNZ44 VSV", respectively). Pre-treatment was performed for 4 h prior to the addition of VSVA51-GFP at MOI 0.05 for 786-0 cells and 0.001 for HT29 cells. GFP images were taken 36 h after addition of virus.

[00120] The new constructs' abilities to express the introduced heterologous gene were demonstrated by RT-PCR (as shown in Figure 1). The activity of the protein produced was examined in an established bioassay for B18R. Supernatant from E. coli cultures either expressing B18R or not expressing B18R were compared with purified recombinant B18R protein for their ability to increase VSV IFN sensitive virus replication in two different cell lines (786-0 renal cell carcinoma or HT29 colorectal carcinoma) (Figures 2 and 3). In both tumour cell lines, a dramatic increase in VSV titres afforded by E. coli-B18R supernatant was observed, indicating the production of biologically active B18R protein.

[00121] Example 2: E. coli-B18R Reduces IFN Levels in vitro

[00122] To validate that the effect on VSV replication was related to type 1 IFN inhibition by B18R expression, the effects of bacterial supernatants on IFN-a levels in VSV infected cell lines were examined by ELISA. Figures 4 and 5 show that reductions in IFN-a levels in both 786-0 (Figure 4) and HT29 (Figure 5) cell cultures was evident in E. coli-B18R supernatant ("pNZ44 B18r VSV", seventh column) and recombinant B18R protein ("Rec-B18r VSV", fifth column) groups.

[00123] Example 3: E. coli-B18R Enhances VSV Infection in vitro

[00124] Bacterial supernatants were co-incubated with confluent HT29 cells for 2 h and washed with PBS. VSVA51-GFP was added at 10^s pfu to the appropriate wells. 6 h later, the cells were analysed for GFP expression by fluorescence microscopy and flow cytometry. Figure 6 shows representative fluorescent microscopy images from (i) E. coli-B18R (bacteria alone), (ii) VSVA51 GFP (virus alone), (iii) E. coli pNZ44 plus VSVA51GFP (backbone vector plus virus), (iv) E. coli-B18R plus VSVA51GFP (B18R vector plus virus). Figure 7 shows data that represents the mean percentage of GFP⁺ / VSV-infected HT29 cells in the presence or absence of E. coli-B18R, which are expressed as the mean ± SEM of 2-4 samples per group. Statistical significance was determined by unpaired Students T test,*P < 0.05, **P < 0.01 , ***P < 0.001 , ****P < 0.0001. [00125] When E. coli-B18R supernatant incubation was followed by infection with VSVA51-GFP, a rapid spread of VSVA51 was readily detected by fluorescence microscopy. The microscopic examination revealed increased oncolysis due to sensitization of neighbouring cells to VSVA51 infection (Figure 6). Total GFP levels were quantified by flow cytometry 6 h post VSVA51 incubation and a significant increase in the total GFP-positive cells was observed in combination treated samples (Figure 7). The combination treatment resulted in a 1048 +/- 396 % increase in GFP positive cells in comparison with virus alone treated wells and a 1497 +/- 528 % increase over the backbone bacterial vector (E. coli pNZ44) plus virus treated wells (Figure 7).

[00126] Example 4: Effect of E. coli-B18R on VSV Oncolytic Activity in vitro.

[00127] To further quantitatively the effects of bacterial-produced B18R on

VSVA51 replication levels, a firefly luciferase expressing VSVA51 FLuc was employed and examined by luminescence imaging. HT29 cells were incubated with supernatant from either E. coli pNZ44 ("pNZ44") or E. coli-B18R ("pNZ44B18R"), alone or in combination with VSVA51 FLuc. Infection of HT29 cells by VSVA51 was significantly enhanced by E. coli-B18R (p=0.032) but not E. coli pNZ44 (p=0.545), as evidenced by VSVA51 FLuc luminescence with a 2.33 +/- 0.01 fold increase in luminescence (Figure 8). LLC cells were also examined in a similar assay, but due to the rapid oncolysis induced by VSVA51 alone, no improvements due to E. coli -B18R could be measured within the limits of the in vitro assay (data not shown). HT29 cell viability of VSV-treated cell lines was examined immediately post luminescence imaging. Cells were stained with Prodiff solution C and cell viability quantified using the Odyssey IR imaging system. Results are presented as integrated intensity ± SEM. Bacterial supernatant in the absence of VSV ("pNZ44B18R") had no significant effect on cell viability versus PBS (p > 0.388), in contrast to bacterial supernatant in the presence of VSV ("pNZ44B18R & VSV") (Figure 9).

[00128] Example 5: Tumour-specific Growth of E. coli Following IV Administration in vivo

[00129] Bacterial homing to, and replication within, subcutaneous tumours following IV administration was visualised over time using 2D whole body BioLuminescence Imaging

(BLI) of mice. Subcutaneous LLC tumours were induced in MF1 nu/nu mice (n=6) and bacteria administered upon tumour development (12-14 days; average tumour volume 100 mm³). Each animal (bearing subcutaneous LLC tumours) received 1 x 10⁶ E. coli-lux injected directly into the lateral tail vein. In vivo BLI was performed at various times post bacterial administration. Lux signal was detected specifically in tumours of mice 3-14 days post IV- administration of E. coli (Figure 10). Bacterial replication in tumours was confirmed by ex vivo bacterial culture (Figure 10) and viable bacterial numbers correlated with bioluminescence. Figure 10 shows recovery from subcutaneous tumour tissue (bars, y-axis) and bacterial lux expression in vivo in live mice (black circles, z-axis and images). Time scale (x axis) is time in days post bacterial administration. Increase in bacterial numbers and lux gene expression specifically in tumours was observed over time. There was no detectable luminescence in organs of treated animals, data not shown. Viable bacterial culture indicated E. coli numbers of < 100 cfu/g tissue in liver and spleen at the various timepoints, consistent with our previous findings using this strain [16], confirming tumour specific replication of the bacteria and no off target growth, validating its safety profile. [00130] Example 6: E. coli-B18R Enhances VSVA51 Replication Specifically in Tumours and Effects Tumour Growth

[00131] The temporal growth pattern of the E. coli in tumours, based on the above E. coli-lux experiments, was used to design the timing strategy for combination treatments. Nude mice bearing either HT29 or LLC subcutaneous tumours were monitored for tumour development and once tumours reached an average volume of 100 mm³, animals were randomly assigned to the various treatment groups. E. coli containing either an empty vector (pNZ44) or the therapeutic B18R plasmid were administered IV at10⁶ cfu in 100 μΙ. One week post bacterial administration, 10⁷ pfu VSVA51 FLuc were injected IV, and VSVA51 FLuc luminescence measured by BLI intermittently, Figures 11 and 12 show VSV-related luminescence measurements and representative images from (i) HT29 (Figure 11) or (ii) LLC (Figure 12) xenograft bearing mice in the absence or presence of bacterial B18R expression. Representative data shown relate to 40 h post VSV administration. Figure 13 shows LLC tumour growth over time. ^* indicates significant difference in tumour volumes at day 32 (p < 0.05). Figure 14 illustrates the Kaplan-Meier survival curves for LLC-bearing athymic mice treated with (i) E. coli-B18R plus VSVA51 FLuc (combination treatment, solid line - square),

(ii) E. coli pNZ44 plus VSVA51 FLuc (backbone E. coli vector plus VSV, solid line - triangle),

(iii) VSVA51 FLuc alone (dotted line - circle), (iv) E. coli-B18R alone (solid line - diamond), (v)

E. coli-lux (reporter strain, dotted line), or (vi) PBS (untreated, solid line - circle). Survival was significantly prolonged by E. coli-B18R, with mean survival of the E. coli-B 18R plus

VSVA51 FLuc group significantly higher than any of the other groups (p < 0.0211).

[00132] A non-lux tagged E. coli was used in these experiments, with VSVA51 - luciferase the sole source of luminescence, used for quantitation of VSVA51 replication and bio-distribution. A parallel group was run concurrently and injected with the bacterial lux reporter strain (£. coli-lux). Following bioluminescence imaging of the reporter group we observed bacterial tumour colonisation as expected at 5 days post-injection of the bacteria. At this time, VSVA51-luciferase was administered intravenously and expression of the virally encoded luciferase reporter monitored over time.

[00133] Significant VSVA51 -associated luciferase expression was observed in tumours only in doubly treated animals (Figures 1 1 and 12), and was significantly higher than all other groups (p<0.04). The VSVA51 -luciferase signal peaked at approximately 5 days after infection and remained detectable in the tumour for up to 1 1 days after infection. It should be noted that inevitable cell death of infected cells ultimately reduces VSVA51 levels over time, affecting the luminescence read out. Importantly, during daily monitoring of infected animals, no "off-target" VSV infection of healthy tissues was observed.

[00134] The effects of E. coli-B18R on VSVA51 -mediated tumour destruction was monitored by tumour volume measurements over time. While the virus was capable of replication and LLC cell killing in the absence of B18R, its potency mirrored the in vitro studies described above, with dramatic improvement in anti-tumour activity in the presence of B18R. Reduced tumour growth and prolonged survival of the combination treatment group in comparison to the backbone vector treatment group (p=0.002) or the virus alone treatment group (p=0.006) confirmed the ability of the bacterial B18R to enhance the oncolytic potential of VSVA51 in this aggressive tumour model (Figures 13 and 14). Survival was significantly prolonged by E. coli -B18R, with mean survival of the E. coli-B 18R plus VSVA51 FLuc group significantly higher than any of the other groups (p <0.021 1).

[00135] Example 7: Tumour Specificity of VSVA51 Replication in Combination Treated Mice

[00136] Tumours and organs (liver, spleen, kidney and brain) from the various groups were harvested at necropsy. Immunohistochemistry (IHC) specific for VSV was used to examine the presence of VSV in tumour as well as various organs following treatments. VSV staining was detected by IHC 5 days post virus administration only in tumours of combination treated animals. Consecutive sections of all tissues were also stained by H&E, with tumour tissue in combination treatment groups displaying denucleation and evidence of cell death.

[00137] Example 8: Immune Responses to Combination Treatment with E. coli- B18R and VSVA51

[00138] Mindful that this strategy involves the systemic administration of two replication-competent biological agents to the body, the authors of the present disclosure sought to examine potential immune responses to the treatments. Five days post-virus administration (= 11 days post administration of E. coli), a subset of animals which received either PBS, bacteria alone, virus alone, or the combination treatment (E. coli-B18R plus VSVA51 FLuc) were euthanised and serum collected following cardiac puncture. Serum was analysed using the 7-plex pro-inflammatory cytokine plate from Meso Scale Discovery to measure concentrations of (i) IFN-γ, (ii) mKC, (iii) IL-10, (iv) IL-12p70 and (v) IL-1 β in serum. Neither TNFa nor IL-6 were at detectable levels in any samples. Concentrations of (i) IFN-γ, (ii) mKC/IL8, (iii) IL10, (iv) IL12p70 and (v) IL1 β were quantified (Figures 15 to 19). While increased levels of the neutrophil chemoattractant mKC (the murine homologue of IL8) was detected in all groups, no treatment group displayed levels statistically significantly higher than the PBS group (P<0.9024). For the other cytokines profiled IFN-a, IL10, IL12, and ΙΙ_1β, there was no up-regulation. Overall, these data suggest a lack of sustained pro-inflammatory response to either the bacterial or viral vectors.

[00139] Example 9: E. coli-B18R enhances Other OVs Replication in vitro

[00140] The effect of E. coli +/- B18R supernatant on HSV1-GFP or JX594-GFP replication within 4T1 cells was examined. Bacterial supernatants were co-incubated with confluent 4T1 cells for 2 h and washed with PBS. The relevant OV was added at 10⁵ pfu to the appropriate wells. Either 6 or 24 h later, the cells were analysed for GFP expression by fluorescence microscopy and flow cytometry. Representative fluorescent microscopy images are shown in Figure 20 at 6 hours from (i) HSV1-GFP (virus alone), (ii) JX594-GFP (virus alone), (iii) E. coli-B18R plus HSV1-GFP (B18R vector plus HSV-1 virus) (iv) E. coli-B18R plus JX594-GFP (B18R vector plus JX594 virus). Figures 21 and 22 are graphs showing the analysis of cells for GFP expression by flow cytometry. The bars represent the percentage of GFP⁺/ virus-infected cells in the presence or absence of E. coli-B18R. Data represent the mean percentage of GFP⁺ cells and are expressed as the mean ± SEM of 2 samples per group.

[00141] 4T1 cell monolayers were unaffected by incubation with the supernatant of E. coli +/- B18R in the absence of an OV and no evidence of cell killing was detected using Prodiff (Braidwood Lab, UK) cell stain (data not shown). When E. coli-B18R supernatant incubation was followed by infection with HSV1-GFP or JX594-GFP, a rapid spread of virus was readily detected by fluorescent microscopy. However it should be noted the virus alone was also capable of significant oncolytic potential as was expected given their superb efficacy as oncolytic agents alone. The microscopic examination revealed increased oncolysis due to sensitization of neighbouring cells to virus infection (Figure 20).

[00142] Total GFP levels were quantified by flow cytometry at both 6 and 24 hours post virus incubation. There was a slight enhanced benefit to the combination treatment at the 6 hour time point which was less significant at 24 hour time point due to the efficacy of the oncolytics alone in this particular tumour model. The combination treatment with JX594- GFP at 6 hours resulted in 6 +/- 0.01 % increase (data not shown) while at 24 hours it resulted in an increase of 9 +/- 2.5 % (Figure 22) of GFP positive cells in comparison with virus alone treated wells was observed. For HSV1-GFP the combination treatment at 6 hours resulted in a 3.2 +/- 0.9 % increase (Figure 21) and at 24 hours resulted in a 3.5 +/- 0.5 % increase (data not shown) in GFP positive cells in comparison with virus alone treated wells.

[00143] 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.

[00144] 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.

Le Boeuf, F, Diallo, JS, McCart, JA, Thome, S, Falls, T, Stanford, M, et al. (2010) Synergistic interaction between oncolytic viruses augments tumor killing. Mol Ther 18: 888-895.

Russell, SJ, Peng, KW, and Bell, JC (2012). Oncolytic virotherapy. Nat Biotechnol 30: 658-670.

Stojdl, DF, Lichty, BD, tenOever, BR, Paterson, JM, Power, AT, Knowles, S, et al. (2003). VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents. Cancer Cell 4: 263-275.

Liu, TC, Hwang, T, Park, BH, Bell, J, and Kirn, DH (2008). The targeted oncolytic poxvirus JX-594 demonstrates antitumoral, antivascular, and anti-FIBV activities in patients with hepatocellular carcinoma. Mol Ther 16: 1637-1642.

Vaha-Koskela, MJ, Le Boeuf, F, Lemay, C, De Silva, N, Diallo, JS, Cox, J, et al.

(2013). Resistance to two heterologous neurotropic oncolytic viruses, Semliki Forest virus and vaccinia virus, in experimental glioma. J Virol 87: 2363-2366.

Le Boeuf, F, Batenchuk, C, Vaha-Koskela, M, Breton, S, Roy, D, Lemay, C, et al.

(2013). Model -based rational design of an oncolytic virus with improved therapeutic potential. Nat Commun 4: 1974.

Le Boeuf, F, and Bell, JC (2010). United virus: the oncolytic tag-team against cancer! Cytokine Growth Factor Rev 21 : 205-211.

Janelle, V, Poliquin, L, and Lamarre, A (2013). [Vesicular stomatitis virus in the fight against cancer]. Med Sci (Paris) 29: 175-182.

Potts, KG, Hitt, MM, and Moore, RB (2012). Oncolytic viruses in the treatment of bladder cancer. Adv Urol 2012: 404581.

Morrissey, D, O'Sullivan, GC, and Tangney, M (2010). Tumour targeting with systemically administered bacteria. Curr Gene Ther 10: 3-14.

Baban, CK, Cronin, M, O'Hanlon, D, O'Sullivan, GC, and Tangney, M (2010). Bacteria as vectors for gene therapy of cancer. Bioeng Bugs 1: 385-394.

Martins, FS, Silva, AA, Vieira, AT, Barbosa, FH, Arantes, RM, Teixeira, MM, et al. (2009). Comparative study of Bifidobacterium animalis, Escherichia coli, Lactobacillus casei and Saccharomyces boulardii probiotic properties. Arch Microbiol 191: 623-630.

13. Schultz, M (2008). Clinical use of E. coli Nissle 1917 in inflammatory bowel disease.

Inflamm Bowel Dis . 1012-1018.

14. Stritzker, J, Weibel, S, Hill, PJ, Oelschlaeger, TA, Goebel, W, and Szalay, AA (2007).

Tumor-specific colonization, tissue distribution, and gene induction by probiotic Escherichia coli Nissle 1917 in live mice. Int J Med Microbiol 297 ^': 151-162.

15. Cronin, M, Akin, AR, Collins, SA, Meganck, J, Kim, JB, Baban, CK, et al. (2012).

High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting. PLoS One 7: e30940.

16. Cronin, M, Stanton, RM, Francis, KP, and Tangney, M (2012). Bacterial vectors for imaging and cancer gene therapy: a review. Cancer Gene Ther 19: 731-740.

17. Bell, J, and Kirn, D (2010). Recent advances in the development of oncolytic viruses as cancer therapeutics. Foreword. Cytokine Growth Factor Rev 21 : 83-84.

18. Tedcastle, A, Cawood, R, Di, Y, Fisher, KD, and Seymour, LW (2012). Virotherapy- cancer targeted pharmacology. Drug Discov Today 17: 215-220.

19. Cronin, M, Morrissey, D, Rajendran, S, El Mashad, SM, van Sinderen, D, O'Sullivan, GC, et al. (2010). Orally administered bifidobacteria as vehicles for delivery of agents to systemic tumors. Mol Ther 18: 1397-1407.

20. Cummins, J, and Tangney, M (2013). Bacteria and tumours: causative agents or opportunistic inhabitants? Infect Agent Cancer 8: 1 1.

21. Toso, JF, Gill, VJ, Hwu, P, Marincola, FM, Restifo, NP, Schwartzentruber, DJ, et al.

(2002). Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J Clin Oncol 20: 142-152.

22. Nemunaitis, J, Cunningham, C, Senzer, N, Kuhn, J, Cramm, J, Litz, C, et al. (2003).

Pilot trial of genetically modified, attenuated Salmonella expressing the E. coli cytosine deaminase gene in refractory cancer patients. Cancer Gene Ther 10: 737-744. 23. Heppner, F, and Mose, JR (1978). The liquefaction (oncolysis) of malignant gliomas by a non pathogenic Clostridium. Acta Neurochir (Wien) 42: 123-125. Bermudes, D, Low, B, and Pawelek, J (2000). Tumor-targeted Salmonella. Highly selective delivery vectors. Adv Exp Med Biol 465: 57-63.

Karlsson, H, Larsson, P, Wold, AE, and Rudin, A (2004). Pattern of cytokine responses to gram-positive and gram-negative commensal bacteria is profoundly changed when monocytes differentiate into dendritic cells. Infect Immun 72: 2671- 2678.

Lehouritis, P, Springer, C, and Tangney, M (2013). Bacterial-directed enzyme prodrug therapy. J Control Release.

Ahmad, S, Casey, G, Cronin, M, Rajendran, S, Sweeney, P, Tangney, M, et al. (201 1). Induction of effective antitumor response after mucosal bacterial vector mediated DNA vaccination with endogenous prostate cancer specific antigen. J Urol 186: 687- 693.

van Pijkeren, JP, Morrissey, D, Monk, IR, Cronin, M, Rajendran, S, O'Sullivan, GC, et al. (2010). A novel Listeria monocytogenes-based DNA delivery system for cancer gene therapy. Hum Gene Ther 21 : 405-416.

Tangney, M (2010). Gene therapy for cancer: dairy bacteria as delivery vectors. Discov Med 10: 195-200.

McGrath, S, Fitzgerald, GF, and van Sinderen, D (2001). Improvement and optimization of two engineered phage resistance mechanisms in Lactococcus lactis. Appl Environ Microbiol 61: 608-616.

Riedel, CU, Monk, IR, Casey, PG, Morrissey, D, O'Sullivan, GC, Tangney, M, et al. (2007). Improved luciferase tagging system for Listeria monocytogenes allows realtime monitoring in vivo and in vitro. Appl Environ Microbiol 73: 3091-3094.

Burns-Guy dish, SM, Olomu, IN, Zhao, H, Wong, RJ, Stevenson, DK, and Contag, CH (2005). Monitoring age-related susceptibility of young mice to oral Salmonella enterica serovar Typhimurium infection using an in vivo murine model. Pediatr Res 58: 153-158.

Brown, CW, Stephenson, KB, Hanson, S, Kucharczyk, M, Duncan, R, Bell, JC, et al. (2009). The pl4 FAST protein of reptilian reovirus increases vesicular stomatitis virus neuropathogenesis. J Virol 83: 552-561. Baban, CK, Cronin, M, Akin, AR, O'Brien, A, Gao, X, Tabirca, S, et al. (2012). Bioluminescent bacterial imaging in vivo. J Vis Exp.

Clancy, EK, and Duncan, R (2009). Reovirus FAST protein transmembrane domains function in a modular, primary sequence-independent manner to mediate cell-cell membrane fusion. J Virol 83: 2941-2950.

Appendix A - Sequences

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

MTMKM VHIYFVSLLLLLFHSYAIDIENEITEFFNKMRDTLPAKDSKWLNPACMFGGTMNDIAALGEP FSAKCPPIEDSLLSHRYKDYWKWERLEKNRRRQVSNKRVKHGDLWIANYTSKFSNRRYLCTVTTKNG DCVQGIVRSHIRKPPSCIPKTYELGTHDKYGIDLYCGILYAKHYNNITWYKDNKEINIDDIKYSQTGK ELIIHNPELEDSGRYDCYVHYDDVRIKNDIWSRCKILTVIPSQDHRFKLILDPKINVTIGEPA ITC TAVSTSLLIDDVLIEWENPSGWLIGFDFDVYSVLTSRGGITEATLYFENVTEEYIGNTYKCRGHNYYF EKTLTTTWLE (SEQ ID NO: 1 )

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

MTMKM VHIYFVSLSLLLLLFHSYAIDIENEITEFFNKMRDTLPAKDSKWLNPACMFGGTMNDMATLG EPFSAKCPPIEDSLLSHRYKDYWKWERLEKNRRRQVSNKRVKHGDLWIANYTSKFSNRRYLCTVTTK NGDCVQGIVRSHIKKPPSCIPKTYELGTHDKYGIDLYCGILYAKHYNNITWYKDNKEINIDDIKYSQT GKELI IHNPELEDSGRYDCYVHYDDVRIKNDIWSRCKILTVI SQDHRFKLILDPKINVTIGEPANI TCTAVSTSLLIDDVLIEWENPSGWLIGFDFDVYSVLTSRGGITEATLYFENVTEEYIGNTYKCRGHNY YFEKTLTTTWLE (SEQ ID NO: 2 ) The sequence for the vaccinia virus Wyeth strain truncated B18R protein (GenBank Accession No: AAR18044.1) is:

MTMKMMVHIYFVSLSLLLLLFHSYAIDIENEITEFFNKMRDTLPAKDSKWLNPACMFGGTMNDMATLG EPFSAKCPPIEDSLLSHRYKDYWKWERLEKNRRRQVSNKRVKHGDLWIANYTSKFSNRRYLCTVTTK NGDCVQGIVRSHIRKPPSCIPKTYELGTHDKYGIDLYCGILYAKHYNNITWYKDNKEINIDDIKYSQT GKKLI IHNPELEDSGRYDCYVHYDDVRIKNDIVVSRCKILTVIPSQDHRFKLKRNCGYASN (SEQ ID NO: 3)

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

Atgacgatgaaaatgatggtacatatatatttcgtatcattattgttattgctattccacagttacgccatagacatcgaaaa tgaaatcacagaattcttcaataaaatgagagatactctaccagctaaagactctaaatggttgaatc cagcatgtatgttcggaggcacaatgaatgatatagccgctctaggagagccattcagcgcaaagtgt cctcctat gaagacagtctttta cgcacagatataaagactatgtggttaaatgggaaaggctaga aaaaaatagacggcgacaggtttctaataaacgtgttaaacatggtgatttatggatagccaactata catctaaattcagtaaccgtaggtatttgtgcaccgtaactacaaagaatggtgactgtgttcagggt atagttagatctcata tagaaaacctccttcatgcattccaaaaacatatgaactaggtactcatga taagtatggcatagacttatactgtggaattctttacgcaaaacattataataatataacttggtata aagataataaggaaattaatatcgacgacattaagtattcacaaacgggaaaggaattaattatteat aatccagagttagaagatagcggaagatacgactgttacgttcattacgacgacgttagaatcaagaa tgatatcgtagtatcaagatgtaaaatacttacggttataccgtcacaagaccacaggtttaaactaa tactagatccaaaaatcaacgtaacgataggagaacctgccaatataacatgcactgctgtgtcaacg cattattgattgacgatgtactgattgaatgggaaaatccatccggatggcttataggattcgattt tgatgtatactctgttttaactagtagaggcggtattaccgaggcgaccttgtactttgaaaatgtta ctgaagaatatataggtaatacatataaatgtcgtggacacaactattattttgaaaaaacccttaca actacagtagtattggagtaa (SEQ ID NO: 4)

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

Atgacgatgaaaatgatggtacatatatatttcgtatcat atcatta tgttattgctattccacag ttacgccatagacatcgaaaatgaaatcacagaattcttcaataaaatgagagatactctaccagcta aagactctaaatggttgaatccagcatgtatgttcggaggcacaatgaatgatatggccactctagga gagccattcagtgcaaagtgtcctcctattgaagacagtcttttatcgcacagatataaagactatgt ggttaaatgggagaggctagaaaagaatagacggcgacaggtttctaataaacgtgttaaacatggtg atttatggatagccaactatacatctaaattcagtaaccgtaggtatttgtgcaccgtaactacaaag aatggtgactgtgttcagggtatagttagatctcatattaaaaaacctccttcatgcattccaaaaac atatgaactaggtactcatgataagtatggcatagacttatactgtggaattctttacgcaaaacatt ataataatataacttggtataaagataataaggaaattaatatcgacgacattaagtattcacaaacg ggaaaggaattaatta tcataatccagagttagaagatagcggaagatacgactgttacgttcatta cgacgacgttagaatcaagaatgatatcgtagtatcaagatgtaaaatacttacggttataccgtcac aagaccacaggtttaaactaatactagatccgaaaatcaacgtaacgataggagaacctgccaatata acatgcactgctgtgtcaacgtca tattgattgacgatgtactgattgaatgggaaaatccatccgg atggcttataggattcgattttgatgtatactctgttttaactagtagaggcggtatcaccgaggcga ccttgtactttgaaaatgttactgaagaatatataggtaatacatataaatgtcgtggacacaactat tattttgaaaaaacccttacaactacagtagtattggagtaa (SEQ ID NO: 5)

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

Atgacgatgaaaatgatggtacatatatatttcgtatcat atcatta tgttattgctattccacag ttacgccatagacatcgaaaatgaaatcacagaattcttcaataaaatgagagatactctaccagcta aagactctaaatggttgaatccagcatgtatgttcggaggcacaatgaatgatatagccgctctagga gagccattcagcgcaaagtgtcctcctattgaagacagtcttttatcgcacagatataaagactatgt ggttaaatgggaaaggctagaaaagaatagacggcgacaggtttctaataaacgtgttaaacatggtg atttatggatagccaactatacatctaaattcagtaaccgtaggtatttgtgcaccgtaactacaaag aatggtgactgtgttcagggtatagttagatctcatattagaaaacctccttcatgcattccaaaaac atatgaactaggtactcatgataagtatggcatagacttatactgtggaattctttacgcaaaacatt ataataatataacttggtataaagataataaggaaattaatatcgacgatattaagtattcacaaacg ggaaagaaattaattattcataatccagagttagaagatagcggaagatacgactgttacgttcatta cgacgacgttagaatcaagaatgatatcgtagtatcaagatgtaaaatacttacggttttaccgtcac aagaccacaggtttaaactaaaaagaaattgcggatatgcgtcaaattaa (SEQ ID NO: 6)

Claims

WHAT IS CLAIMED IS:

1. A non-invasive bacterium comprising a polynucleotide sequence encoding a soluble interferon binding protein that binds to IFN- , IFN-β, or both, wherein the soluble protein is secretable by the bacterium.

2. The non-invasive bacterium according to claim 1 or 2 wherein the non-pathogenic bacterium is Escherichia coli; a bacterial species of the genus Bifidobacterium; a bacterial species of the genus Lactococcus; a bacterial species of the genus Lactobacillus; a non- invasive bacterial species of the genus Listeria; a non-invasive safety-attenuated Salmonella enterica Typhimurium pathogen; or a non-invasive safety-attenuated Listeria monocytogenes pathogen.

3. The non-invasive bacterium according to claim 2, wherein the Escherichia coli is strain MG 1655 or strain Nissle 1917.

4. The non-invasive bacterium according to claim 2, wherein the species of the genus Bifidobacterium is B. breve, B. infantis, or B. long urn).

5. The non-invasive bacterium according to claim 2, wherein the species of the genus Lactococcus is L lactis.

6. The non-invasive bacterium according to claim 2, wherein the species of the genus Lactobacillus is L. reuteri, L. delbrueckii, or L. plantarum.

7. The non-invasive bacterium according to claim 2, wherein the species of the genus Listeria is L. welshimeri.

8. The non-invasive bacterium according to any one of claims 1-7, wherein the interferon binding protein is a protein comprising three IgG domains that bind IFN-α/β.

9. The non-invasive bacterium according to claim 8, wherein the interferon binding protein comprises an amino acid sequence of SEQ ID NO: 1 , or a variant thereof.

10. The non-invasive bacterium according to any one of claims 1-7, wherein the interferon binding protein is a B19R protein.

11. The non-invasive bacterium according to claim 10, wherein the interferon binding protein comprises an amino acid sequence of SEQ ID NO: 2, or a variant thereof.

12. The non-invasive bacterium according to any one of claims 1-7, wherein the interferon binding protein is a B18R protein lacking a C-terminal IgG domain.

13. The non-invasive bacterium according to claim 12, wherein the interferon binding protein comprises an amino acid sequence of SEQ ID NO: 3, or a variant thereof.

14. A method of replicating an IFN-sensitive oncolytic virus in a tumorous cancer in a patient, the method comprising:

administering to the patient a plurality of the non-invasive bacterium according to any one of claims 1-13; and

administering to the patient a plurality of the IFN-sensitive oncolytic virus.

15. The method according to claim 14, wherein the IFN-sensitive oncolytic virus is an IFN-sensitive rhabdovirus, preferably 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.

16. The method according to claim 15, wherein the IFN-sensitive rhabdovirus is an IFN- sensitive vesiculovirus.

17. The method according to claim 16 wherein the IFN-sensitive vesiculovirus is an IFN- sensitive vesicular stomatitis virus (VSV), or an IFN-sensitive maraba virus.

18. The method according to claim 17, wherein the IFN-sensitive VSV comprises a polynucleotide sequence encoding a mutated matrix protein.

19. The method according to claim 18, wherein the polynucleotide sequence encodes a Δ51 mutation.

20. The method according to claim 17, wherein the IFN-sensitive VSV comprises a polynucleotide sequence encoding interferon-β.

21. The method according to claim 17, 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.

22. The method according to claim 21 , 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).

23. The method according to claim 14, wherein the IFN-sensitive oncolytic virus is a pox virus, preferably a vaccinia virus such as JX594; or a herpesvirus, preferably herpes simplex virus 1.

24. The method according to any one of claims 14-23, wherein the method comprises: infecting cancer cells from the tumorous cancer with the IFN-sensitive onclytic virus ex vivo, and

administering the infected cancer cells to the patient.

25. The method according to any one of claims 14-24, wherein the non-invasive bacteria is administered to the patient before the IFN-sensitive oncolytic virus is administered to the patient.

26. The method according to claim 25 wherein the non-invasive bacteria is administered to the patient between 1 and 14 days before the IFN-sensitive oncolytic virus is administered to the patient.

27. The method according to any one of claims 14-26, wherein the non-invasive bacteria is administered to the patient in an amount between 1e5 and 1e10 colony forming units.

28. The method according to any one of claims 14-27, wherein the IFN-sensitive oncolytic virus is administered in an amount between 1 and 1e15 infectious IFN-sensitive oncolytic viral particles.

29. A kit comprising a plurality of the non- invasive bacterium according to any one of claims 1-13 and a plurality of IFN-sensitive oncolytic viruses.