WO2019020543A1 - Oncolytic viruses expressing agents targeting metabolic immune modulators - Google Patents

Abstract

The present invention relates to an oncolytic virus encoding at least one agent that degrades one or more metabolic immune modulator (s), a composition comprising it, and their use for prophylactic or therapeutic purposes, and more particularly for the treatment of cancer. The present invention also relates to a method of treating a disease or condition comprising administering such an oncolytic virus or a composition thereof and a method of preparing such an oncolytic virus.

Description

Oncolytic viruses expressing agents targeting metabolic immune modulators

TECHNICAL FIELD OF THE INVENTION

The present invention is in the field of oncolytic viruses and agents targeting metabolic immune modulators. The invention provides oncolytic viruses comprising, inserted in their genomes, a nucleotide sequence encoding at least one agent targeting one or more metabolic immune modulator(s). More precisely, said agents play a role in the adenosine pathway, by decreasing the adenosine concentration. More particularly, said agents have an activity of degradation of adenosine. Even more particularly, said agents are adenosine deaminases (ADA) or have an adenosine deaminase activity. These oncolytic viruses can be used for the prevention and/or the treatment of proliferative diseases like cancers.

BACKGROUND ART

Oncolytic viruses are a class of therapeutic agents that have the unique property of tumour- dependent self-perpetuation (Hermiston et al., 2006, Curr. Opin. Mol. Ther., 8(4):322-30). The benefit of using these viruses is that as they replicate, they kill their host cells by lysis. Oncolytic viruses are capable of selective replication in dividing cells (e.g. cancer cells) while leaving non- dividing cells (e.g. normal cells) unharmed. As the infected dividing cells are destroyed by lysis, they release new infectious particles to infect the surrounding dividing cells. Therefore, oncolytic viruses offer a new area for treating cancer, optionally in association with conventional treatments for cancer (Fisher et al., 2006, Curr. Opin. Mol. Ther., 8(4):301-13). Cancer cells are ideal hosts for many viruses because they have the antiviral interferon pathway inactivated or have mutated tumour suppressor genes that enable viral replication to proceed unhindered (Chernajovsky et al., 2006, BMJ, 332(7534):170-2). Several viruses including adenovirus, reovirus, measles, herpes simplex, Newcastle disease virus and vaccinia virus have now been clinically tested as oncolytic agents.

Some viruses are naturally oncolytic and have an innate ability to selectively infect and kill tumour cells. However, oncolytic viruses can also be engineered by modifying naturally occurring viruses. For this purpose, the main strategies used currently to modify the viruses include: functional deletions in viral genes, the use of tumour- or tissue-specific promoters to control the expression of these viral genes, tropism modification to redirect virus to the cancer cell surface, among many other possibilities. Among naturally oncolytic viruses, Vaccinia virus (a Poxviridae) possess many of the key attributes necessary for an ideal viral backbone for use in oncolytic virotherapy. These include a short lifecycle, with rapid cell-to-cell spread, strong lytic ability, a large cloning capacity and well- defined molecular biology. In addition, although capable of replicating in human cells, they are not considered a natural health problem and are especially well characterized having been delivered to millions of individuals during the campaign to eradicate smallpox. Early clinical results using either vaccinia strains or genetically modified vaccinia strains have demonstrated antitumour effects (Thorne et al., 2005, Curr. Opin. Mol. Then, 7(4):359-65).

Viruses can also be engineered by introducing modifications in the genome in order to improve their ability to infect and lyse tumor cells notably in the in vivo context while preserving safety. Several gene modifications have already been considered in the art to enhance safety in the host organism. However, increasing safety is often detrimental to efficiency in dividing cells. For example, thymidine kinase (TK) deleted virus showed a decreased lytic activity in non-dividing cells compared to wild type virus (Buller et al., 1985, Nature, 317(6040):813-5). Therefore, armament of oncolytic viruses has been considered for several decades and various enzyme-prodrug systems have been widely used to enhance the oncolytic efficacy of the virus therapy. For example, armament with the so-called FCUl suicide gene, encoding a bifunctional chimeric protein that combines the enzymatic activities of cytidine deaminase (CDase) and uracil phosphoribosyl transferase (UPRTase), and catalyses the conversion of nontoxic 5-fluorocytosine (5-FC) into the toxic metabolites 5-fluorouracil (5-FU) and 5-fluorouridine-5'monophosphate (5-FU P), was shown to improve antitumoral effect in the presence of 5-FC, to exert a strong bystander effect, thus permitting elimination of neighbouring uninfected tumor cells and to bypass the natural resistance of certain human tumor cells to 5-fluorouracil (Erbs et al., 2000, Cancer Res., 60(14):3813).A TK gene-deleted VV expressing the FCUl gene showed potent anti-tumour effect both in vitro and in vivo in a murine model of a human colon tumour (Foloppe et al., 2008, Gene Ther., 15:1361-71). However, the suicide gene armaments have some drawbacks since it requires the providing of the prodrug to the host subject together with viral therapy.

In addition to the well-established oncolytic activity, the ability of oncolytic viruses to deregulate tumor microenvironement (TME) remains to be established and offers new approaches to provide anti-tumor protection. It has been showed that TME contributes to tumour growth and spread (Joyce JA, Pollard JW., 2009, Nat Rev Cancer ;9(4):239-252; Quail DF, Joyce JA., 2013, Nat. Med.; 19(11):1423-1437). In the 90's, Blay and colleagues showed that adenosine levels were 10 to 20 times higher in the tumour microenvironment than those measured in healthy tissues, confirming the immunosuppressive properties of adenosine (Allard et al., 2016, Immunotherapy, DOI: 10.2217/imt.l5.106).

Several metabolic immune modulators can be envisioned for their action on the immune system, including adenosine. This purine nucleotide exerts pleiotropic functions in the cell metabolism (Kumar et al., 2009, Eur J Pharmacol., 616(l-3):7-15). Notably, intracellular adenosine is involved in energy metabolism, nucleic acid metabolism, and the methionine cycle, while extracellular adenosine plays an important role in intercellular signalling, affecting diverse physiological functions including neurological, cardiovascular, and immunological systems (Ohta A., 2016, Front. Immunol., 7:109). Upon binding with adenosine receptors present on the surface of various immune cells, extracellular adenosine suppresses pro-inflammatory activities and upregulates a number of anti-inflammatory molecules and immunoregulatory cells (e.g. T cells, NK cells, neutrophils, etc.,), leading to the establishment of a long-lasting immunosuppressive environment (Ohta A., 2016, Front. Immunol., 7:109).

Through these immune-related activities, and others, aberrant or accumulated levels of adenosine is associated with a number of diseases and conditions (WO2016061286). Adenosine has also been shown to constitute one of the important mechanisms used by solid tumours to evade the immune system. The adenosine pathway also regulates cancer growth and dissemination by interfering with cancer cell proliferation, apoptosis and angiogenesis via adenosine receptors that are expressed on cancer cells and endothelial cells, respectively. Solid tumours express high levels of CD39 and CD73, as well as low levels of nucleoside transporters (NTs), ecto-adenosine deaminase and its cofactor CD26, which lead to an increase in adenosine signalling in the cancer environment (Antonioli et al., 2013, Nature Reviews Cancer 13, 842-857).

As a result, several components of the adenosinergic machinery actually constitute potential therapeutic targets in immune-oncology, in order to promote anti-tumour immunity and to enhance the activity of some anti-cancer treatments. Different ways have been explored to reduce the adenosine effects: blocking of adenosine receptors (e.g. A2A, A2B), modulation of CD39, CD73, or of the activity or concentration of adenosine-degradating enzymes such as the adenosine deaminase enzyme (ADA), etc., the two major targets being the blockade of A2A and CD73. Ohta et al. demonstrated that A2A-deficient mice had increased anti-tumour immunity, and that A2A blockade was an effective approach to re-instate anti-tumour immunity (Ohta et al., 2001, Nature 414(6866), 916-920). This pathway was confirmed by several groups who showed that A2A activation on CD8⁺ anti-tumour T cells and NK cells was the predominant pathway exploited by adenosine-rich tumour microenvironment to dampen anti-tumour immunity. Antagonists against A2A receptor are currently being evaluated: PBF-509, a small molecule inhibitor of A2A entered phase I in 2015 for the treatment of NSCLC.

CD73, is another target of the adenosinergic machinery. This enzyme, which converts AMP to adenosine, has immunosuppressive functions and is highly expressed in various types of solid tumours. High rates of CD73 have been correlated with lower survival in colorectal cancers (Wu et al., 2012, J Surg Oncol, DOI: 10.1002/jso.23056) and in gastric cancers (Lu et al., 2013, World J Gastroenterol, 19(12): 1912-1918). The blockade of CD73 is now considered as clinically relevant approach for cancer immunotherapy (Allard et al., 2016, Immunotherapy, DOI: 10.2217/imt.l5.106). MEDI9447, a blocking anti-CD73 monoclonal antibody, is being evaluated in phase 1 clinical trial in adults with advanced solid tumours, as single agent and in combination with anti-PDLl mAb durvalumab.

Inherited variations in ADA activity levels have been described in different types of cancer and in inflammatory diseases such as rheumatoid arthritis, celiac disease, ulcerative colitis, systemic lupus erythematosus, visceral leishmaniasis and inflammatory obesity or infections such as tuberculosis in human serum, saliva or sputum samples (Ungerer et al, 1992, Clin Chem 38/7, 1322- 1326). For example, ADA deficiency causes severe combined immunodeficiency disease (ADA-SCID), in which both B-cell and T-cell development is impaired. Conversely, there are several diseases in which the level of ADA is above normal (Cristalli et al. 2001, Med Res Rev 21(2):105-28). PEG-ADA has been approved for use for treating inherited ADA deficiency (Chaffee et al., 1992, J Clin Invest, 89:1643-1651). More recently, PEG-ADA2 (human adenosine deaminase 2) has been explored for treating solid tumours (Wang et al., 2006, Abstract 1472, AACR Annual Meeting) and reduction of lung metastasis was observed in PEG-ADA2 treated 4T1 animal model, as well as reduction in TME adenosine levels.

However, the ability to combine oncolytic virus therapy and adenosine-targeted therapy permitting to decrease adenosine concentration and activity has not been tested and remains to be established.

There is clearly an important need to develop effective approaches for the treatment of cancer, and more specifically, a need to provide oncolytic viruses acting on adenosine-associated tumor microenvironment, aiming at overcoming immunosuppressive mechanisms and restoring an efficient anti-tumor immunity.

The inventors have now generated an oncolytic virus engineered to express an adenosine degrading agent. Against all expectations, this adenosine deaminase (ADA)-expressing oncolytic vaccinia virus is able to secrete an active enzyme and, upon administration to animals, to delay tumor growth and increase animal survival in two different tumor models compared to empty oncolytic viruses. The inventors thus provide an effective anti-tumoral therapy allowing a combined effect of tumour cells lysis and modification of the TME, resulting in a significant reduction of tumour progression. Based on these results, one may anticipate that the oncolytic viruses of the invention may be successfully used as an alternative to other existing oncolytic viruses and may have a better efficiency profile. The oncolytic viruses of the invention can also be exploited in combination with additional anticancer therapy/ies.

This technical problem is solved by the provision of the embodiments as defined in the claims.

Other and further aspects, features and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.

BRIEF SUMMARY OF THE INVENTION

One aspect of the invention relates to an oncolytic virus comprising a nucleotide sequence encoding at least one agent degrading one or more metabolic immune modulator(s).

In one embodiment, said metabolic immune modulator is an adenosine, and said nucleotide sequence encodes an adenosine deaminase (ADA) polypeptide, or a polypeptide having an adenosine deaminase activity.

In another embodiment, the oncolytic virus of the invention is a poxvirus belonging to the Orthopoxvirus and especially a vaccinia virus or a cowpox virus.

In yet another embodiment, the oncolytic virus of the invention is defective in the thymidine kinase (TK-) encoding J2R locus. In still another embodiment, the oncolytic virus of the invention is defective in the I4L and/or F4L locus (alternatively or in combination with a defective TK- locus).

In another aspect, the present invention further provides a composition comprising the oncolytic virus of the present invention and a pharmaceutically acceptable vehicle. In one embodiment, the oncolytic virus is preferably formulated for intravenous or intra-tumoral administration. In a further aspect, the present invention also concerns a process for preparing the oncolytic virus, comprising at least the steps of introducing said oncolytic virus into a producer cell, culturing the producer cell under conditions that are appropriate for enabling said oncolytic virus to be produced and recovering the produced virus from the cell culture. Optionally, the recovered oncolytic virus can be purified at least partially.

In still a further aspect, the present invention provides the oncolytic virus or the composition of the invention, for use for the prophylaxis and/or the treatment of a disease. In one embodiment, said disease is a proliferative disease such as cancers and restenosis. Said cancer is preferably selected from the group consisting in renal cancer, bladder cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, hepatic cancer, gastric cancer, pancreatic cancer, melanoma, ovarian cancer and glioblastoma, and especially metastatic ones. In another embodiment, said disease is a disease associated with an increased osteoclast activity like rheumatoid arthritis and osteoporosis.

In yet a further aspect, the present invention provides a method of treating a disease which comprises the administration into a host organism in need thereof of a therapeutically effective amount of the oncolytic virus or the composition of the invention. Said method of treatment may be used in conjunction with one or more additional therapies such as ones selected from the group consisting of surgery, radiotherapy, chemotherapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy or cytokine therapy.

DESCRIPTION OF THE FIGURES

Figure 1. Schematic representation of the genetic constructs for the expression of ADAs by vaccinia virus WR TK- RR- and COP TK- RR-

Abbreviations: WR: western reserve; COP: Copenhagen; huADAl: human ADA1; huADA2: human ADA2; cuADA: culex quinquefasciatus ADA; pllk7.5: promoter; SS: signal sequence; FLAG: FLAG sequence.

Figure 2. Evaluation of adenosine deaminase activity expressed by WRTG19009,

WRTG19021 or WRTG19010 infected MCA205 and CT26 cancerous cells The specific adenosine deaminase activity was measured on culture supematants after 24,

48 or 72 hours infection of murine fibrosarcoma MCA205 (Fig 2A) and murine colon carcinoma CT26 (Fig 2B) cell lines by ADA expressing viruses WRTG19009, WRTG19010 and WRTG19021 or the empty vector WRTG18011. MCA205 were infected at OI of 0.01 and as CT26 cells are more resistant to vaccinia virus infection, they were infected at MOI of 0.1. The adenosine degradation is given as the quantity of adenosine deaminated in nmoles per minute per millilitre of culture supernatant. Values are represented in mean (± standard error of mean) of three individual determinations.

Figure 3. Cytotoxic effect of ADAs expressing recombinant vaccinia viruses

In vitro evaluation of the cytotoxicity of viruses, and therefore the oncolytic effect, in MCA205 (Fig 3A) and CT26 (Fig 3B) cells infected at a MOI of 0.0001, 0.001, 0.01 and 0.1 with the indicated viruses at day 5 post infection. Values are represented in mean (± standard error of mean) of three individual determinations.

Figure 4. Evaluation of the effect of ADAs expression on W in vitro replication In vitro replication efficacy of the different viruses was evaluated in MCA205 (Fig 4A and 4B) and CT26 (Fig 4C and 4D) cells infected at a MOI of 0.001 and 0.01 at day 1, 2, and 3 post infection. Values are represented in mean (± standard error of mean) of three individual determinations.

Figure 5. Effect on tumor growth of murine fibrosarcoma MCA205 tumours implanted subcutaneousiy after two intratumoral injections of WRTG 18011, WRTG19009 and WRTG19021

MCA205 cells were implanted subcutaneousiy (s.c.) in C57BL/6 mice. Tumours were then treated with two intratumoral (i.t.) administrations at day 7 and 10 with vehicle alone (Fig 5A) or with lxlO⁷ PFU of TK and RR-defective virus (empty WRTG18011; Fig 5B), expressing the human ADA1 (WRTG19009; Fig 5C) or expressing the human ADA2 (WRTG19021; Fig 5D). Each plot represents one of five treated animals.

Figure 6. Effect on mice survival

C57BL/6 mice bearing MCA205 s.c. tumours were treated with two i.t. administrations at day 7 and 10 (indicated by arrows) with vehicle alone (■: Vehicle) or with lxlO⁷ PFU of a TK and RR- defective virus (A: WRTG18011), expressing the human ADA1 (T: WRTG19009) or expressing the human ADA2 (♦: WRTG19021). Figure 7. Cytotoxic effect of ADAs expressing recombinant COP vaccinia viruses

In vitro evaluation of the cytotoxicity of viruses, and therefore the oncolytic effect, in HCT 116 (Fig 7A) and LoVo (Fig 7B) cells infected at a MOI of 0.0001, 0.001, 0.01 and 0.1 with the indicated viruses at day 3 post infection. Values are represented in mean (± standard deviation) of three individual determinations.

Figure 8. Evaluation of the effect of ADAs expression on COP W in vitro replication

In vitro replication efficacy of the different viruses was evaluated in HCT 116 (Fig 8A and 8B) and LoVo (Fig 8C and 8D) cells infected at a MOI of 0.001 at day 1, 2, 3, 4 and 5 post infection. Values are represented in mean (± standard error of mean) of three individual determinations.

Figure 9. In vitro evaluation of the level of secretion of ADAs by infected cells

The specific detection of huADAl and huADA2 was performed by western blotting on culture supernatants after infection of HCT 116 cells with VVTG17989, COPTG19183 and COPTG19185. The presence of huADAl (41 kDa) and huADA2 (56 kDa) is indicated (arrows).

Figure 10. In vitro evaluation of the level of adenosine deaminase activity secreted by infected cells

The specific adenosine deaminase activity was measured on culture supernatants after 72 hours infection of human HCT 116 and human LoVo cell lines by huADA expressing viruses COPTG19183 and COPTG19185 or the empty vector VVTG17989. HCT 116 were infected at MOI of 0.001. As LoVo cells are more resistant to vaccinia virus infection, they were infected at MOI of 0.01. The adenosine degradation is given as the quantity of inosine produced from adenosine in pmoles per minute per microlitres of culture supernatant. The enzymatic reaction was initiated with addition of adenosine at 2 mM (Fig 10A and Fig 10B) or 20 μΜ (Fig IOC and Fig 10D). Values are represented in mean (± standard error of mean) of three individual determinations.

Figure 11. Intratumoral replication of WTG17989, COPTG19183 and COPTG19185

The intratumoral replication efficacy was evaluated in HCT 116 tumors collected 7 days after treatment with one single intravenous injection of 10⁷ pfu of COPTG19183 and COPTG19185 or the empty vector VVTG17989. Values are represented in mean (± standard error of mean) of three individual determinations.

Figure 12. In situ expression of vectorized huADAl after treatment of HCT 116 tumor bearing mice with COPTG19183

The specific detection of huADAl was performed by western blotting on interstitial fluids collected from HCT 116 tumors 7 days after treatment with one single intravenous injection of 10⁷ pfu of VVTG17989, COPTG19183. The presence of huADAl is indicated (arrow).

Figure 13. Evaluation of in situ ADA activity after treatment of HCT 116 tumor bearing mice with WTG17989, COPTG19183 and COPTG19185

The specific adenosine deaminase activity was measured in interstitial fluids collected from HCT 116 tumors 7 days after treatment with one single intravenous injection of 10⁷ pfu of VVTG17989, COPTG19183 and COPTG19185. The adenosine degradation is given as the quantity of inosine produced from adenosine in pmoles per minute per microlitres of culture supernatant. The enzymatic reaction was initiated with addition of adenosine at 2 mM (Fig 13A) or 20 μΜ (Fig 13B). Values are represented in mean (± standard error of mean) of three individual determinations.

Figure 14. Effect on tumor growth of human HCT 116 tumours implanted subcutaneously after treatment with WTG17989. COPTG19183 and COPTG19185

HCT 116 cells were implanted subcutaneously (s.c.) in Swiss nude mice. Tumours were then treated with a single intravenous (i.v.) administration at day 14 with vehicle alone (Fig 14A) or with lxlO⁷ PFU of TK and RR-defective virus (empty VVTG11989; Fig 14B), expressing the human ADAl (COPTG19183; Fig 14C) or expressing the human ADA2 (COPTG19185; Fig 14D). Each plot represents one of fifteen treated animals.

Figure 15. Effect on mice survival

Swiss nude mice bearing HCT 116 s.c. tumours were treated with one i.v. administrations at day 14 (indicated by arrows) with vehicle alone (■: Vehicle) or with lxlO⁷ PFU of a TK and RR- defective virus (A: VVTG17989), expressing the huADAl (T: COPTG19183) or expressing the human ADA2 (♦: COPTG19185). Mantel-Cox's multiple tests correction was used. * p < 0.001.

5 DETAILED DESCRIPTION OF THE INVENTION

Definitions

The terms used in this specification generally have their ordinary meanings in the art, unless otherwise indicated. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the products and methods of the

10 invention and how to use them. Moreover, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of the other synonyms. The use of examples anywhere in the specification, including examples of any terms discussed herein, is

15 illustrative only, and in no way limits the scope and meaning of the invention or any exemplified term.

As used throughout the entire application, the terms "a" and "an" are used in the sense that they mean "at least one", "at least a first", "one or more" or "a plurality" of the referenced components or steps. For example, the term "an oncolytic virus" encompasses a single oncolytic 20 virus as well as a plurality of oncolytic viruses, including mixtures thereof.

The term "one or more" refers to either one or a number above one (e.g. 2, 3, 4, etc.).

The term "and/or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

The term "about" or "approximately" as used herein means within 20%, preferably within 25 10%, and more preferably within 5% of a given value or range.

As used herein, when used to define products and compositions, the terms "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are open- 30 ended and do not exclude additional, un-recited elements or method steps. The expression "consisting essentially of" means excluding other components or steps of any essential significance. Thus, a composition consisting essentially of the recited components would not exclude traces, contaminants and pharmaceutically acceptable carriers. "Consisting of" shall mean excluding more than trace elements of other components or steps.

The terms "polypeptide", "peptide" and "protein" refer to polymers of amino acid residues which comprise at least nine or more amino acids bonded via peptide bonds. The polymer can be linear, branched or cyclic and may comprise naturally occurring and/or amino acid analogues and it may be interrupted by non-amino acids. As a general indication, if the amino acid polymer is more than 50 amino acid residues, it is preferably referred to as a polypeptide or a protein whereas if it is 50 amino acids long or less, it is referred to as a "peptide". Within the context of the present invention, the terms "nucleic acid", "nucleic acid molecule", "polynucleotide" and "nucleotide sequence" are used interchangeably and define a polymer of any length of either polydeoxyribonucleotides (DNA) (e.g. cDNA, genomic DNA, plasmids, vectors, viral genomes, isolated DNA, probes, primers and any mixture thereof) or polyribonucleotides (RNA) (e.g. mRNA, antisense RNA, siRNA) or mixed polyribo- polydeoxyribonucleotides. They encompass single or double-stranded, linear or circular, natural or synthetic, modified or unmodified polynucleotides. Moreover, a polynucleotide may comprise non- naturally occurring nucleotides and may be interrupted by non-nucleotide components.

The term "agent" refer to a substance that induces or is capable of inducing an effect, like a biological effect or a chemical reaction. Said agents can be biological agents, chemical agents or pharmaceutical agents. Examples of agents include, but are not limited to, polypeptides (e.g. active fragments of biologically active proteins, antigenic proteins or specific antigenic fragments, antibodies, etc.), nucleic acids (e.g.: DNA, RNA, miRNA, siRNA, etc.), gene regulators, lipids, small molecules, cytotoxins, drugs, acids, bases, oxidizing agents, reducing agents, alkaline agents, nucleophilic agents, electrophilic agents, polymers, synthetic materials, viruses, bacterias, archaea, protozoa, fungi, algae, and combinations thereof. Within the context of the invention, the agents encoded by the oncolytic are polypeptides.

In the context of the invention, the term "degradation" or "degrading" refers to the transformation or reduction of a compound to one less complex, as by splitting off one or more groups. One of a result of the degradation of a compound is the diminution of said compound concentration. Examples degradation reactions include, but are not limited to hydrolysis, deamination, dephosphorilation, Reactive Oxygene Species degradations, etc. In one embodiment of the invention, said degradation consists in a deamination, or the removal of at least one amino group from an amino acid or another compound. As used herein, the term "host cell" should be understood broadly without any limitation concerning particular organization in tissue, organ, or isolated cells. Such cells may be of a unique type of cells or a group of different types of cells such as cultured cell lines, primary cells and dividing cells. In the context of the invention, the term "host cells" include prokaryotic cells, lower eukaryotic cells such as yeast, and other eukaryotic cells such as insect cells, plant and mammalian (e.g. human or non-human) cells as well as cells capable of producing the oncolytic virus of the invention. This term also includes cells which can be or has been the recipient of the virus described herein as well as progeny of such cells.

The terms "virus", "viral particle", "viral vector" and virion" are used interchangeably and are to be understood broadly as meaning a vehicle comprising at least one element of a wild-type virus genome and may be packaged into a viral particle or to a viral particle. Although, viral particles may or may not contain nucleic acid (i.e. the viral genome) it is preferred that a virus comprises a DNA or RNA viral genome packaged into a viral particle (or virion) and is infectious (i.e. capable of infecting and entering a host cell or subject). Desirably, the virus of this invention is associated with a DNA genome, and most preferably a double-stranded DNA genome. In the context of the present disclosure, a "virus" includes wild-type and engineered viruses.

The term "naturally occurring" or "wild-type" or "native" is used to describe a biological molecule or organism that can be found in nature as distinct from being artificially produced by man. For example, a virus which can be isolated from a source in nature is wild-type. The present invention also encompasses wild-type viruses that can be obtained from specific collections (e.g. ECCAC, ATCC, CNCM, etc.). A biological molecule or an organism which has been intentionally modified by man in the laboratory is not naturally occurring. Representative examples of non- naturally occurring viruses include, among many others, recombinant viruses engineered by insertion of one or more gene(s) of interest in the viral genome and mutated viruses engineered by total or partial deletion of a viral gene to make the modified virus defective for the encoded gene product (e.g. TK- virus) as well as chimeric viruses containing genomic fragments obtained from different virus origins.

The term "obtained from", "originating" or "originate" is used to identify the original source of a component (e.g. polypeptide, nucleic acid molecule, virus, etc.) but is not meant to limit the method by which the component is made which can be, for example, by chemical synthesis or recombinant means.

As used herein, the term "oncolytic virus" refers to a virus capable of selectively replicating in dividing cells (e.g. a proliferative cell such as a cancer cell) with the aim of slowing the growth and/or lysing said dividing cell, either in vitro or in vivo, while showing no or minimal replication in non-dividing cells (e.g. primary cells). The term "oncolytic virus" encompasses any virus naturally occurring, engineered or otherwise modified.

The term "treatment" (and any form of treatment such as "treating", "treat") as used herein encompasses prophylaxis (e.g. preventive measure in a subject at risk of having the pathological condition to be treated) and/or therapy (e.g. in a subject diagnosed as having the pathological condition), eventually in association with conventional therapeutic modalities. The result of the treatment is to slow down, cure, ameliorate or control the progression of the targeted pathological condition. For example, a subject is successfully treated for a cancer if after administration of an oncolytic virus as described herein, alone or in combination, the subject shows an observable improvement of its clinical status.

The term "administering" (or any form of administration, such as "administered") as used herein refers to the delivery to a subject of a therapeutic agent such as the oncolytic virus described herein. As used herein, the term "proliferative disease" encompasses any disease or condition resulting from uncontrolled cell growth and spread including cancers and some cardiovascular diseases (restenosis that results from the proliferation of the smooth muscle cells of the blood vessel wall, etc.). The term "cancer" may be used interchangeably with any of the terms "tumour", "malignancy", "neoplasm", etc. These terms are meant to include any type of tissue, organ or cell, any stage of malignancy (e.g. from a pre-lesion to stage IV).

As used herein, the term "disease associated with an increased osteoclast activity" encompasses any disease or condition resulting in bone resorption or destruction (e.g. rheumatoid arthritis, osteoporosis, etc.).

The term "subject" generally refers to an organism for whom any product and method of the invention is needed or may be beneficial. Typically, the organism is a mammal, particularly a mammal selected from the group consisting of domestic animals, farm animals, sport animals, and primates. Preferably, the subject is a human who has been diagnosed as having or at risk of having a proliferative disease such as a cancer. The terms "subject" and "patients" may be used interchangeably when referring to a human organism and encompasses male and female. The subject to be treated may be a new-born, an infant, a young adult, an adult or an elderly.

The terms "combination treatment", "combination therapy", "combined treatment" or "combinatorial treatment", may be used interchangeably and refer to a treatment of a subject with an oncolytic virus as described herein and at least an additional therapeutic modality. The additional therapeutic modality may be selected from the group consisting of surgery, radiotherapy, chemotherapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy, cytokine therapy, targeted cancer therapy, gene therapy, photodynamic therapy, transplantation etc. A combinatorial treatment may include a third or even further therapeutic modality. For combination treatment, it is appreciated that optimal concentration of each component of the combination can be determined by the artisan skilled in the art. In a specific embodiment, the oncolytic virus of the invention (e.g. wild type oncolytic virus, or a modified derivative oncolytic virus) may be delivered in combination with adenosine deaminase, wherein said metabolic immune modulator, or said adenosine deaminase is not encoded by the oncolytic virus. Said modulator or adenosine deaminase may be in the form of a polypeptide (e.g. a recombinantly produced adenosine deaminase, or analogue thereof) or of a nucleic acid molecule (e.g. carried by a vector engineered for expressing such adenosine deaminase(s)), as well as mixture of polypeptide(s) and nucleic acid molecule(s) (e.g. adenosine deaminase(s) and expressing vector(s)).

Oncolytic viruses

The "oncolytic virus" of the present invention can be obtained from any member of virus identified at present time provided that it is oncolytic by a higher propensity to replicate in and kill dividing cells as compared to non-dividing cells. It may be a native virus that is naturally oncolytic or may be engineered by modifying one or more viral genes so-as to increase tumour selectivity and/or preferential replication in dividing cells, such as those involved in DNA replication, nucleic acid metabolism, host tropism, surface attachment, virulence, lysis and spread (see for example Kirn et al., 2001, Nat. Med. 7: 781; Wong et al., 2010, Viruses 2: 78-106). One may also envisage placing one or more viral gene(s) under the control of event or tissue-specific regulatory elements (e.g. promoter).

Exemplary oncolytic viruses include without limitation reovirus, Seneca Valley virus (SVV), vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), herpes simplex virus (HSV), morbillivirus, adenovirus, poxvirus, retrovirus, measles virus, foamy virus, alpha virus, lentivirus, influenza virus, Sinbis virus, myxoma virus, rhabdovirus, picornavirus, coxsackievirus, parvovirus or the like.

In one embodiment, the oncolytic virus of the present invention is obtained from a reovirus. A representative example includes Reolysin (under development by Oncolytics Biotech; NCT01166542). In one embodiment, the oncolytic virus of the present invention is obtained from a Seneca Valley virus. A representative example includes NTX-010 (Rudin et al., 2011, Clin. Cancer. Res. 17(4): 888-95).

In one embodiment, the oncolytic virus of the present invention is obtained from a vesicular 5 stomatitis virus (VSV). Representative examples are described in the literature (e.g. Stojdl et al., 2000, Nat. Med. 6(7): 821-5; Stojdl et al., 2003, Cancer Cell 4(4): 263-75).

In one embodiment, the oncolytic virus of the present invention is obtained from a Newcastle disease virus. Representative examples include without limitation the 73-T PV701 and HDV-HUJ strains as well as those described in the literature (e.g. Phuangsab et al., 2001, Cancer 10 Lett. 172(l):27-36; Lorence et al., 2007, Curr. Cancer Drug Targets 7(2):157-67; Freeman et al., 2006, Mol. Ther. 13(l):221-8).

In one embodiment, the oncolytic virus of the present invention is obtained from a herpes virus. The Herpesviridae are a large family of DNA viruses that all share a common structure and are composed of relatively large double-stranded, linear DNA genomes encoding 100-200 genes

15 encapsidated within an icosahedral capsid which is enveloped in a lipid bilayer membrane. Although the oncolytic herpes virus can be derived from different types of HSV, particularly preferred are HSV1 and HSV2. The herpes virus may be genetically modified so-as to restrict viral replication in tumours or reduce its cytotoxicity in non-dividing cells. For example, any viral gene involved in nucleic acid metabolism may be inactivated, such as thymidine kinase (Martuza et al., 1991, Science

20 252: 854-6), ribonucleotide reductase (RR) (Boviatsis et al., Gene Ther. 1: 323-31; Mineta et al., 1994, Cancer Res. 54: 3363-66), or uracil-N-glycosylase (Pyles et al., 1994, J. Virol. 68: 4963-72). Another aspect involves viral mutants with defects in the function of genes encoding virulence factors such as the ICP34.5 gene (Chambers et al., 1995, Proc. Natl. Acad. Sci. USA 92: 1411-5). Representative examples of oncolytic herpes virus include NV1020 (e.g. Geevarghese et al., 2010,

25 Hum. Gene Ther. 21(9): 1119-28) and T-VEC (Harrington et al., 2015, Expert Rev. Anticancer Ther.

15(12):1389-1403).

In one embodiment, the oncolytic virus of the present invention is obtained from a morbillivirus which can be obtained from the paramyxoviridae family, with a specific preference for measles virus. Representative examples of oncolytic measles viruses include without limitation MV- 30 Edm (McDonald et al., 2006; Breast Cancer Treat. 99(2): 177-84) and HMWMAA (Kaufmann et al., 2013, J. Invest. Dermatol. 133(4): 1034-42)

In one embodiment, the oncolytic virus of the present invention is obtained from an adenovirus. Methods are available in the art to engineer oncolytic adenoviruses. An advantageous strategy includes the replacement of viral promoters with tumour-selective promoters or modifications of the El adenoviral gene product(s) to inactivate its/their binding function with p53 or retinoblastoma (Rb) protein that are altered in tumour cells. In the natural context, the adenovirus ElB55kDa gene cooperates with another adenoviral product to inactivate p53 (p53 is frequently dysregulated in cancer cells), thus preventing apoptosis. Representative examples of oncolytic adenovirus include ONYX-015 (e.g. Khuri et al., 2000, Nat. Med 6(8): 879-85) and H101 also named Oncorine (Xia et al., 2004, Ai Zheng 23(12): 1666-70).

In one and preferred embodiment, the oncolytic virus of the present invention is a poxvirus. As used herein the term "poxvirus" or "poxviral vector" refers to a virus belonging to the Poxviridae family, with a specific preference for a poxvirus belonging to the Chordopoxviridae subfamily and more preferably to the Orthopoxvirus genus. Sequences of the genome of various poxviruses, for example, the vaccinia virus, cowpox virus, canarypox virus, ectromelia virus, myxoma virus genomes are available in the art and specialized databases such as Genbank (accession number NC_006998, NC_003663 or AF482758.2, NC_005309, NC_004105, NC_001132 respectively). Advantageously, the oncolytic poxvirus is an oncolytic cowpox virus, and can derive from any cowpox strain, like for example CPXV_GER1980_EP4 (Genbank HQ420895), CPXV_GER2002_MKY (Genbank HQ420898), CPXV_GER1991_3 (Genbank DQ 437593), CPXV_FRA2001_NANCY (Genbank HQ420894), CPXV_GER1990_2 (Genbank HQ420896), CPXV_UK2000_K2984 (Genbank HQ420900), CPXV_BR (Genbank AF482758.2 or NC 003663) and CPXV_NOR1994-MAN (Genbank HQ420899), CPXV_GER1998_2 (Genbank HQ420897), CPXV_gri (Genbank X94355), CPXV_FIN2000_MAN (Genbank HQ420893) and CPXV_AUS1999_867 (Genbank HQ407377).

Desirably, the oncolytic poxvirus is an oncolytic vaccinia virus. Vaccinia viruses are members of the poxvirus family characterized by a 200 kb double-stranded DNA genome that encodes numerous viral enzymes and factors that enable the virus to replicate independently from the host cell machinery. The majority of vaccinia virus particles is intracellular (IMV for intracellular mature virion) with a single lipid envelop and remains in the cytosol of infected cells until lysis. The other infectious form is a double enveloped particle (EEV for extracellular enveloped virion) that buds out from the infected cell without lysing it. Although it can derive from any vaccinia virus strain, Elstree, Wyeth, Copenhagen and

Western Reserve strains are particularly preferred. The gene nomenclature used herein is that of Copenhagen and Western Reserve vaccinia strains. It is also used herein for the homologous genes of other poxviridae unless otherwise indicated. However, gene nomenclature may be different according to the pox strain but correspondence between Copenhagen or Western Reserve and other vaccinia strains are generally available in the literature.

Preferably, the oncolytic vaccinia virus of the present invention is modified by altering one or more viral gene(s). Said modification(s) preferably lead(s) to the synthesis of a defective protein unable to ensure the activity of the protein produced under normal conditions by the unmodified gene (or lack of synthesis). Exemplary modifications are disclosed in the literature with the goal of altering viral genes involved in DNA metabolism, host virulence, IFN pathway (e.g. Guse et al., 2011, Expert Opinion Biol. Ther.ll(5): 595-608) and the like. Modifications for altering a viral locus encompass deletion, mutation and/or substitution of one or more nucleotide(s) (contiguous or not) within the viral gene or its regulatory elements. Modification(s) can be made by a number of ways known to those skilled in the art using conventional recombinant techniques.

More preferably, the oncolytic virus of the present invention is modified by altering the thymidine kinase-encoding gene (locus J2R). The TK enzyme is involved in the synthesis of deoxyribonucleotides. TK is needed for viral replication in normal cells as these cells have generally low concentration of nucleotides whereas it is dispensable in dividing cells which contain high nucleotide concentration.

Alternatively, or in combination, the oncolytic virus of the present invention is modified by altering at least one gene or both genes encoding ribonucleotide reductase (RR). In the natural context, this enzyme catalyses the reduction of ribonucleotides to deoxyribonucleotides that represents a crucial step in DNA biosynthesis. The viral enzyme is similar in subunit structure to the mammalian enzyme, being composed of two heterologous subunits, designed Rl and R2 encoded respectively by the I4L and F4L locus. Sequences for the I4L and F4L genes and their locations in the genome of various poxvirus are available in public databases, for example via accession number DQ437594, DQ437593, DQ377804, AH015635, AY313847, AY313848, NC_003391, AF482758.2, NC_003389, NC_003310, AY243312, DQ011157, DQ011156, DQ011155, DQ011154, DQ011153, Y16780, AF438165, U60315, AF410153, AF380138, L22579, NC_006998, DQ121394 and NC_008291. In the context of the invention, either the I4L gene (encoding the Rl large subunit) or the F4L gene (encoding the R2 small subunit) or both may be inactivated.

Alternatively, or in combination, other strategies may also be pursued to further increase the virus tumour-specificity. A representative example of suitable modification includes alteration of the VGF-encoding gene from the viral genome. VGF (for VV growth factor) is a secreted protein which is expressed early after cell infection and its function seems important for virus spread in normal cells. Another example is the alteration of the A56R gene coding for hemagglutinin, eventually in combination with TK deletion (Zhang et al., 2007, Cancer Res. 67:10038-46). Alteration of interferon modulating gene(s) may also be advantageous (e.g. the B8R or B18R gene) or the caspase-1 inhibitor B13R gene. Another suitable modification comprises the alteration of the F2L gene which encodes the viral dUTPase involved in both maintaining the fidelity of DNA replication and providing the precursor for the production of TMP by thymidylate synthase (Broyles et al., 1993, Virol. 195: 863-5). Sequence of the vaccinia virus F2L gene is available in Genbank via accession number M25392 and a poxvirus defective in F2L locus is available from WO2009/065547.

In a preferred embodiment, the oncolytic virus of this invention is a poxvirus defective for TK activity, resulting from alteration of the J2R locus. In another preferred embodiment, the oncolytic virus of this invention is a poxvirus defective in both TK and RR activities resulting from alteration of both the J2R locus and at least one of the RR-encoding I4L and/or F4L locus carried by the viral genome (e.g. as described in WO2009/065546 and Foloppe et al., 2008, Gene Ther., 15: 1361-71).

In another embodiment, the oncolytic virus of this invention is a poxvirus defective for dUTPase resulting from alteration of the F2L gene (e.g. as described in WO2009/065547), eventually in combination with alteration of at least one of TK and RR activities or both (resulting in a virus with alterations in the F2L; F2L and J2R gene; F2L and I4L; or F2L, J2R and I4L).

Metabolic immune modulators targeting agents, adenosine degrading agents and adenosine deaminases

In one embodiment, the oncolytic virus described herein comprises a nucleotide sequence encoding at least one agent targeting one or more metabolic immune modulator(s).

As used herein, the term "metabolic immune modulator(s)", also designated as "metabolic immune checkpoint(s)", refers to any metabolite (e.g. amino acids, nucleotides, nucleosides, alcohols, vitamins, polyols, organic acids) that may directly or indirectly modulate the functioning of the immune system. The term "metabolic immune modulator(s)" refers to any metabolite directly or indirectly involved in an immune pathway that under normal physiological conditions is crucial for preventing uncontrolled immune reactions and thus for the maintenance of self-tolerance and/or tissue protection. In the context of the invention, the one or more metabolic immune modulator(s) described herein may independently act by blocking the activation and effectiveness of stimulatory immune cells (e.g. NK cells, macrophages, dendritic cells, Thl cells), or by increasing the number of inhibitory immune cells (e.g. regulatory T-cells (T-regs)) that act to suppress immune cells from responding to the tumor. In normal conditions, each of these reactions are regulated by counterbalancing stimulatory and inhibitory signals that in fine tune the response. A preferred metabolic immune modulator in the context of the invention is a nucleoside, and more precisely, a purine nucleoside with a specific preference for adenosine. For illustrative purposes, adenosine is composed of a molecule of adenine attached to a ribose sugar molecule (ribofuranose) moiety via a β-Ng-glycosidic bond. However, the present invention also encompasses adenosine analogue such as 2'deoxyadenosine.

"Agents targeting metabolic immune modulators" have, in the context of the invention, the ability to modify the activity or the concentration of said one or more metabolic immune modulator(s). Preferably said agents exert a degradation activity on the modulator(s). In preferred embodiments, the agent for use herein is an enzymatic polypeptide exerting a degradative action on adenosine. Such a degradative action can result from any reaction which inhibits adenosine effects, and especially its action on the immune system (e.g. adenosine degradation, inactivation, capture, complexation, sequestration, etc.). Representative examples of suitable agents include, but are not limited to, adenosine deaminase, adenosine monophosphate deaminase, adenosine kinase, adenosine nucleosidase, etc. or any analogue thereof. A particularly appropriate agent, in the context of the invention, is an adenosine deaminase (ADA) polypeptide, or a polypeptide having an adenosine deaminase activity, encompassing naturally-occurring adenosine deaminase or analogues thereof. As used herein, an analogue refers to a polypeptide retaining a substantial adenosine deaminase enzymatic activity (at least 50% of the wild-type counterpart). The term "analogue" encompasses fragment of ADA (e.g. a truncated ADA) as well as mutated ADA comprising one or more amino acid modification(s) preferably outside the catalytic site. Examples of ADA analogues are ADA2-K374D and ADA2-E182T (Wang et al., 2006, Abstract 1472, AACR Annual Meeting). Preferred are analogues that retain a degree of sequence identity of at least 80%, advantageously at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 98% identity with the sequence of the naturally occuring counterpart. In a general manner, the term "identity" refers to an amino acid to amino acid, or nucleotide to nucleotide correspondence between two polypeptides or nucleic acid sequences. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps which need to be introduced for optimal alignment and the length of each gap. Various computer programs and mathematical algorithms are available in the art to determine the percentage of identity between amino acid sequences, such as for example the Blast program available at NCBI or ALIGN in Atlas of Protein Sequence and Structure (Dayhoffed, 1981, Suppl., 3: 482-9). Programs for determining identity between nucleotide sequences are also available in specialized data base (e.g. Genbank, the Wisconsin Sequence Analysis Package, BESTFIT, FASTA and GAP programs).

Adenosine deaminase - also termed "ADA", "adenosine deaminase polypeptide" or "adenosine aminohydrolase" - is a polymorphic enzyme of the purine metabolism which catalyses the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively (EC 3.5.4.4). ADA is involved in the differentiation and maturation of the immune cells including lymphocytes and monocyte-macrophage cell lines.

Adenosine deaminase is present in mammalians and in a wide variety of microorganisms, plants, and invertebrates (Cristalli et al. 2001, Med Res Rev 21(2):105-28), and two different isoenzymes of ADA, designated as "ADAl" and "ADA2", were found in mammals and lower vertebrates (Zavialov et Engstrom, 2005, Biochem J. 391(Pt 1): 51-57). It is the case for example in chickens, which livers contain two kinetically distinct ADA isozymes with different molecular weights.

In humans, almost all adenosine deaminase activity is attributed to a single-chain Zn- binding protein ADAl, while in plasma and liver of lower vertebrates, significant component of overall ADA activity comes from ADA2 (Zavialov et Engstrom, 2005, Biochem J. 391(Pt 1): 51-57). Human ADAl has a molecular mass of 35 kDa, an optimal pH of 7-7.5, a Km of 5.2xl0^"5M, and a similar affinity for both adenosine and 2'-deoxyadenosine. It is present in all tissues, in erythrocytes and, in a lower amount, in plasma. ADAl is also present in the cytosol and nucleus of lymphocytes and macrophages, and on the cell membrane as an ecto-form (ADAl complexed with a dimer of DPPIV or CD26). Two molecules of ADAl can also be connected via a combining protein, forming dimers of 280 kDa (Ungerer et al, 1992, Clin Chem 38/7, 1322-1326). Human ADA2 originates mainly from the monocyte-macrophage system. It is not ubiquitous but is more abundant than ADAl in serum. ADA2 has a molecular mass of 100 kDa, an optimum pH of 6.5, a Km of 200xl0^"5 M, and a weak affinity for 2'deoxyadenosine.

In humans, both ADAl and ADA2 have similar affinity for adenosine (Gakis et al., 1996, Eur Respir J, 9, 632-633) while ADAl also guarantees the downregulation of 2'deoxyadenosine.

Various adenosine deaminases can be used in the context of the present invention, including mammalian (e.g. human, bovine, guinea-pig, dog, cat, rabbit, rat, mouse), insects (e.g. mosquito, drosophila, phlebotomus), bacterial (e.g. E. coli) or lower eukaryotic ADA.

In one embodiment, the oncolytic virus of the present invention encodes or is used in combination with an ADA of human origin. Representative examples of human adenosine deaminases include, without limitation, human ADA1 (Genbank access. NO X02994), human ADA2 (Genbank access NO AF190746) and any human ADA analogues as defined above.

In another embodiment, the oncolytic virus encodes or is used in combination with an ADA of insect origin. Representative examples of insect adenosine deaminases include, but are not limited to, Culex quinquefasciatus ADA (Genbank access. NO. AF298886), Drosophila melanogaster ADA (Genbank access. NO. NM_141609.3) Phlebotomus duboscqi ADA (Genbank access. NO. DQ835383.1, and DQ835357.1) and Lutzomyia longipalpis ADA (Genbank access. NO. AF234182.1).

Adenosine deaminase can also originate from microorganisms, like fungi, yeast, virus or bacteria. Examples of non-human adenosine deaminase originate from S. cerevisiae (GenBank access NO: Z46843), Candida glabrata (GenBank access NO: CR380956), E. coli (GenBank access NO: M59033), etc.

In a preferred embodiment, the oncolytic virus of the invention encodes the human adenosine deaminase (huADAl), wherein said huADAl comprises an amino acid sequence having at least 80%, preferably greater than 90%, and more preferably greater than 95% identity with SEQ ID NO: 1. In this embodiment, said oncolytic virus comprises a nucleic acid sequence having at least 80%, preferably greater than 90%, and more preferably greater than 95% identity with SEQ ID NO: 2. A representative example of such an embodiment is illustrated in the Example section (VVTK-RR- /huADAl)

In another preferred embodiment, the oncolytic virus of the invention encodes the human adenosine deaminase 2 (huADA2), wherein said huADA2 comprises an amino acid sequence having at least 80%, preferably greater than 90%, and more preferably greater than 95% identity with SEQ ID NO: 3. In this embodiment, said oncolytic virus comprises a nucleic acid sequence having at least 80%, preferably greater than 90%, and more preferably greater than 95% identity with SEQ ID NO: 4

The concentration of ADA expressed from the oncolytic virus of the present invention can be measured using high performance liquid chromatography or enzymatic or colorimetric techniques. One example of colorimetric technique is the measurement of the ammonia released from adenosine when broken down to inosine (NewLife BioChemEX LLC). For example, one may proceed by incubating a sample of infected host cells (e.g. cultured host cells or supernatant) with a buffered solution of adenosine, the ammonia is reacted with a Berthelot reagent to form a blue colour which is proportionate to the amount of enzyme activity.

The "adenosine deaminase encoding nucleic acid molecule" may be easily obtained by cloning, by PCR or by chemical synthesis based on the information provided herein and the general knowledge of the skilled person. The ADA-encoding nucleic acid molecule for use herein may be native ADA-encoding sequences (e.g. cDNA) or analogue thereof derived from the latter by mutation, deletion, substitution and/or addition of one or more nucleotides. Moreover, the ADA- encoding nucleic acid molecule can be optimized for providing high level expression in a particular host cell or subject as described hereinafter. The nucleic acid molecules encoding human adenosine deaminase (huADA) can be inserted at any location of the viral genome with a specific preference for a non-essential locus and placed under the control of appropriate regulatory elements as described hereinafter. For example, the J2R locus, the I4L locus, the F4L locus or intergenic zones are particularly appropriate for insertion in oncolytic vaccinia virus. In a preferred embodiment, the present invention relates to a vaccinia virus defective in viral TK and RR-encoded activities and encoding a human ADA, such as WTK-RR-/huADAl or VVTK-RR-/huADA2 illustrated in the Example section. In a more preferred embodiment, the present invention relates to a vaccinia virus defective in viral TK and RR-encoded activities and encoding a human ADA1, such as VVTK-RR-/huADAl.

Nucleic acid(s) of interest

According to a specific embodiment, the oncolytic virus of the invention may further comprise, inserted in its genome, one or more nucleic acid(s) of interest different from ADA- encoding nucleic acid molecule. According to the invention, the nucleic acid(s) of interest can be homologous or heterologous to the host organism into which it is introduced. More specifically, it can originate from Prokaryotes (comprising the kingdoms of Bacteria, Archaea), Acaryotes (comprising the viruses) or Eukaryotes (comprising the kingdoms of Protista, Fungi, Plantae, Animalia). Advantageously, said nucleic acid of interest encodes all or part of a polypeptide. A polypeptide is understood to be any translational product of a polynucleotide regardless of size, and whether glycosylated or not, and includes peptides and proteins. In one embodiment, the nucleic acid of interest encodes a polypeptide of therapeutic or prophylactic interest which is capable of providing a biological activity when administered appropriately to a subject or which is expected to cause a beneficial effect on the course or a symptom of the pathological condition to be treated. A vast number of nucleic acid of interest may be envisaged in the context of the invention such as those encoding polypeptides that can compensate for defective or deficient proteins in the subject, or those that act through toxic effects to limit or remove harmful cells from the body or those that encode immunity conferring polypeptides. They may be native or obtained from the latter by mutation, deletion, substitution and/or addition of one or more nucleotides. Representative examples of suitable polypeptides of therapeutic interest include, without limitation, polypeptides capable of potentiating anti-tumor efficacy such as immunostimulatory polypeptides and antigens (for inducing or activating an immune humoral and/or cellular response), as well as polypeptides encoded by suicide genes which are capable of reinforcing the oncolytic nature of the virus of the present invention.

Immunostimulatory polypeptide

A specific embodiment of the invention is directed to an oncolytic virus further comprising an immunostimulatory polypeptide. As used herein, the term "immunostimulatory polypeptide" refers to a polypeptide, or protein, which has the ability to stimulate the immune system, in a specific or non-specific way. A vast number of proteins are known in the art for their ability to exert an immunostimulatory effect. Examples of suitable immunostimulatory proteins in the context of the invention include, without limitation, immune checkpoint inhibitors, including, but not limited to anti-PDl, anti-PDLl, anti-PDL-2, anti-CTLA4, anti-Tim3, anti-LAG3, anti-BTLA; cytokines, like alpha, beta or gamma interferon, interleukins or tumour necrosis factor; agents that affect the regulation of cell surface receptors such as, e.g. inhibitors of Epidermal Growth Factor Receptor (in particular cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab, gefitinib, erlotinib or lapatinib) or inhibitors of Human Epidermal Growth Factor Receptor-2 (in particular trastuzumab); agents that affect angiogenesis such as, e.g. inhibitor of Vascular Endothelial Growth Factor (in particular bevacizumab or ranibizumab) ; agents that stimulates stem cells to produce granulocytes, macrophages such as, e.g. granulocyte macrophage - colony stimulating factor and B7 proteins.

Antigens

The term "antigen" generally refers to a substance that is recognized and selectively bound by an antibody or by a T cell antigen receptor, in order to trigger an immune response. It is contemplated that the term antigen encompasses native antigen as well as fragment (e.g. epitopes, immunogenic domains, etc.) and analogue thereof, provided that such fragment or analogue is capable of being the target of an immune response. Suitable antigens in the context of the invention are preferably polypeptides (e.g. peptides, polypeptides, post translationally modified polypeptides, etc.) including one or more B cell epitope(s) or one or more T cell epitope(s) or both B and T cell epitope(s) and capable of raising an immune response, preferably, a humoral or cell response that can be specific for that antigen. Typically, the one or more antigen(s) is selected in connection with the disease to treat. Preferred antigens for use herein are cancer antigens and antigens of tumour-inducing pathogens.

In certain embodiments, the antigen(s) contained in or encoded by the oncolytic virus is/are cancer antigen(s) (also called tumour-associated antigens) that is associated with and/or serve as markers for cancers. Cancer antigens encompass various categories of polypeptides, e.g. those which are normally silent (i.e. not expressed) in normal cells, those that are expressed only at low levels or at certain stages of differentiation and those that are temporally expressed such as embryonic and foetal antigens as well as those resulting from mutation of cellular genes, such as oncogenes (e.g. activated ras oncogene), proto-oncogenes (e.g. ErbB family), or proteins resulting from chromosomal translocations. The cancer antigens also encompass antigens encoded by pathogenic organisms (bacteria, viruses, parasites, fungi, viroids or prions) that are capable of inducing a malignant condition in a subject (especially chronically infected subject) such as RNA and DNA tumour viruses (e.g. HPV, HCV, EBV, etc.) and bacteria (e.g. Helicobacter pilori).

Some non-limiting examples of cancer antigens include, without limitation, MART-l/Melan- A, gplOO, Dipeptidyl peptidase IV (DPPIV), cyclophilin b, Colorectal associated antigen, Carcinoembryonic Antigen (CEA) , Prostate Specific Antigen (PSA) , prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family of tumour antigens, GAGE-family of tumour antigens, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family (e.g. MUC1, MUC16, etc.; see e.g. US6,054,438; WO98/04727; or WO98/37095), HER2/neu, p21ras, alpha-fetoprotein, E-cadherin, catenin family, and viral antigens such as the HPV-16 and HPV-18 E6 and E7 antigens.

Other antigens suitable for use in this invention are marker antigens (beta-galactosidase, luciferase, green fluorescent proteins, etc.). The present invention also encompasses oncolytic viruses expressing two or more polypeptides of interest as described herein, e.g. at least two antigens, at least one antigen and one cytokine, at least two antigens and one cytokine, etc.

Suicide genes In one embodiment, the oncolytic virus of the invention may further comprise at least a suicide gene. The term "suicide gene" refers to a gene coding for a polypeptide able to convert a precursor of a drug, also named "prodrug", into a cytotoxic compound. Examples of suicide genes suitable for use herein and corresponding prodrugs are disclosed in the following table: Suicide gene prodrug

Thymidine Kinase Ganciclovir;

Ganciclovir elaidic acid ester;

penciclovir;

Acyclovir;

Valacyclovir;

(E)-5-(2-bromovinyl)-2'-deoxyuridine;

zidovudine;

2'-Exo-methanocarbathymidine

Cytosine deaminase 5-Fluorocytosine

Purine nucleoside phosphorylase 6-Methylpurine deoxyriboside;

Fludarabine

uracil phosphoribosyl transferase 5-Fluorocytosine;

5-Fluorouracil

thymidylate kinase. Azidothymidine

Preferably, the oncolytic virus of the invention carries in its genome a suicide gene encoding a polypeptide having at least cytosine deaminase (CDase) activity. Alternatively, or in combination, the oncolytic virus of the invention carries in its viral genome a suicide gene encoding a polypeptide having uracil phosphoribosyl transferase (UPRTase) activity. CDase converts 5-fluorocytosine (5-FC), thereby forming cytotoxic 5-fluorouracil (5-FU), which is then converted into the even more toxic 5- fluoro-UMP (5-FUMP).

Preferably, the suicide gene inserted in the viral genome of the oncolytic virus of the present invention encodes a polypeptide engineered by fusion of two enzymatic domains, one having the CDase activity and the second having the UPRTase activity. Exemplary polypeptides include without limitation fusion polypeptides codA::upp, FCY1::FUR1 and FCYl::FURl[Delta] 105 (FCUl) and FCUl-8 described in W096/16183, EP998568 and WO2005/07857. Of particular interest is the FCUl suicide gene (or FCYl::FURl[Delta] 105 fusion) encoding a polypeptide comprising the amino acid sequence represented in the sequence identifier SEQ ID NO: 1 of WO2009/065546.

Other nucleic acids of interest: Other nucleic acids of interest include, but not limited to: - Nucleoside pool modulators (e.g.: cytidine deaminase) apoptotic genes, including pro-apoptotic genes inhibitors of pro-apoptotic genes anti- apoptotic genes (and inhibitors of anti-apoptotic genes,

nucleic acid coding for endonuclease, like restriction enzymes, C ISPR/Cas9;

RNA, including but not limited to target-specific miRNA, shRNA, siRNA. As mentioned in connection with the ADA-encoding nucleic acid sequences, the nucleic acid(s) of interest sequences may be easily obtained by cloning, by PCR or by chemical synthesis using conventional techniques. They may be native nucleic acid(s) sequences (e.g. cDNA) or sequences derived from the latter by mutation, deletion, substitution and/or addition of one or more nucleotides. Moreover, their sequences are described in the literature which can be consulted by persons skilled in the art. The nucleic acids sequences can be inserted at any location of the viral genome, with a specific preference for a non-essential locus, at the same location of the ADA- encoding nucleic acid (e.g. within J2R locus) or at another location (within I4L locus).

Expression of the nucleic acid(s) sequences The ADA nucleic acid sequence and, if any, nucleic acid(s) of interest can be independently optimized for providing high level expression in a particular host cell or subject. It has been indeed observed that, the codon usage patterns of organisms are highly non-random, and the use of codons may be markedly different between different hosts. As such nucleic acid(s) might be from bacterial or lower eukaryote origin, they may have an inappropriate codon usage pattern for efficient expression in higher eukaryotic cells (e.g. human). Typically, codon optimization is performed by replacing one or more "native" (e.g. bacterial or yeast) codon, corresponding to a codon infrequently used in the host organism of interest, by one or more codon encoding the same amino acid which is more frequently used. It is not necessary to replace all native codons corresponding to infrequently used codons since increased expression can be achieved even with partial replacement.

Further to optimization of the codon usage, expression in the host cell or subject can further be improved through additional modifications of the nucleic acid sequence. For example, it may be advantageous to prevent clustering of rare, non-optimal codons being present in concentrated areas and/or to suppress or modify "negative" sequence elements which are expected to negatively influence expression levels. Such negative sequence elements include without limitation the regions having very high (>80%) or very low (<30%) GC content; AT -rich or GC-rich sequence stretches; unstable direct or inverted repeat sequences; and/or internal cryptic regulatory elements such as internal TATA-boxes, chi-sites, ribosome entry sites, and/or splicing donor/acceptor sites.

In accordance with the present invention, the oncolytic virus comprises the elements necessary for the expression of the ADA-encoding nucleic acid molecule(s) and/or the nucleic acid(s) of interest in a host cell subject. Specifically, such nucleic acid(s) is/are operably linked to suitable regulatory elements that allow, contribute or modulate expression in a given host cell or subject, including replication, duplication, transcription, splicing, translation, stability and/or transport of the nucleic acid(s) or its derivative (i.e. mRNA). As used herein, "operably linked" means that the elements being linked are arranged so that they function in concert for their intended purposes. For example, a promoter is operably linked to a nucleic acid molecule if the promoter effects transcription from the transcription initiation to the terminator of said nucleic acid molecule.

It will be appreciated by those skilled in the art that the choice of the regulatory sequences can depend on such factors as the nucleic acid molecule itself, the virus into which it is inserted, the host cell or subject, the level of expression desired, etc. The promoter is of special importance. In the context of the invention, it can be constitutive directing expression of the encoded product (e.g. ADA and/or polypeptide(s) of interest) in many types of host cells or specific to certain host cells (e.g. liver-specific regulatory sequences) or regulated in response to specific events or exogenous factors (e.g. by temperature, nutrient additive, hormone, etc.) or according to the phase of a viral cycle (e.g. late or early). One may also use promoters that are repressed during the production step in response to specific events or exogenous factors, in order to optimize virus production and circumvent potential toxicity of the expressed polypeptide(s).

Well known promoters such as cytomegalovirus (CMV) immediate early promoter (US 5,168,062), the RSV promoter, the adenovirus major late promoter, the phosphoglycero kinase (PGK) promoter (Adra et al., 1987, Gene 60: 65-74), the thymidine kinase (TK) promoter of herpes simplex virus (HSV)-l and the T7 polymerase promoter (WO98/10088) may be used in the context of the present invention. Vaccinia virus promoters are particularly adapted for expression in oncolytic poxviruses. Representative examples include without limitation the vaccinia 7.5K, H5R, 11K7.5 (Erbs et al., 2008, Cancer Gene Ther. 15(1): 18-28), TK, p28, pll Prl3.5 (WO2014/063832), pB8R, pFUL, pA44L, pCllR (WO2011/128704) and KIL promoter, as well as synthetic promoters such as those described in Chakrabarti et al. (1997, Biotechniques, 23: 1094-7; Hammond et al., 1997, J. Virol. Methods, 66: 135-8; and Kumar and Boyle, 1990, Virology, 179: 151-8) as well as early/late chimeric promoters (e.g. US 8,394,385; US 8,772,023). Cowpox promoters are also suitable as well (e.g. the ATI promoter). In a preferred embodiment, the ADA nucleic acid molecule is inserted in the TK locus of the oncolytic virus of the invention and placed under the control of the vaccinia pllK7.5 promoter. In another preferred embodiment, the ADA nucleic acid molecule is inserted in the TK locus of the oncolytic virus of the invention and placed under the control of the vaccinia p7.5K or pH5R promoter.

Those skilled in the art will appreciate that the regulatory elements controlling the nucleic acid expression may further comprise additional elements for proper initiation, regulation and/or termination of transcription (e.g. a transcription termination sequences), mRNA transport (e.g. nuclear localization signal sequences), processing (e.g. splicing signals), and stability (e.g. introns and non-coding 5' and 3' sequences), translation (e.g. an initiator Met, tripartite leader sequences, IRES ribosome binding sites, signal peptides, etc.), targeting sequences, transport sequences, secretion signal, and sequences involved in replication or integration. Said sequences have been reported in the literature and can be readily obtained by those skilled in the art.

Process for preparing an oncolytic virus

The invention also relates to a process for preparing an oncolytic virus of the invention, in which process:

(i) an oncolytic virus of the invention is introduced into a producer cell,

(ii) said producer cell is cultured under conditions which are appropriate for enabling said oncolytic virus to be produced, and

(iii) said oncolytic virus is recovered from the cell culture.

Typically, the oncolytic virus of the present invention is produced into a suitable host cell line using conventional techniques including culturing the transfected or infected host cell under suitable conditions so-as to allow the production of infectious viral particles and recovering the produced infectious viral particles from the culture of said cell and optionally purifying said recovered infectious viral particles. Suitable host cells for production of the oncolytic virus include without limitation human cell lines such as HeLa (ATCC), 293 cells (Graham et al., 1997, J. Gen. Virol. 36: 59-72), HER96, PER-C6 (Fallaux et al., 1998, Human Gene Ther. 9: 1909-17), Monkey cells such as Vero (ATCC CCL-081), CV1(ATCC CCL-70) and BSC1(ATCC CCL-26) cell lines, avian cells such as those described in WO2005/042728, WO2006/108846, WO2008/129058, WO2010/130756, WO2012/001075, etc.), hamster cell lines such as BHK-21 (ATCC CCL-10) as well as primary chicken embryo fibroblasts (CEF) prepared from chicken embryos obtained from fertilized eggs. Host cells are preferably cultivated in a medium free of animal-or human-derived products, using a chemically defined medium with no product of animal or human origin. Culturing is carried out at a temperature, pH and oxygen content appropriate for the producer cell. Such culturing conditions are within the expertise of one of ordinary skill in the art. If growth factors are present, they are preferably recombinantly produced and not purified from animal material. Suitable animal-free medium media are commercially available, for example VP-SFM medium (Invitrogen) for culturing CEF producer cells. Producer cells are preferably cultivated at a temperature comprised between +30°C and +38°C (more preferably at about +37°C) for between 1 and 8 days (preferably for 1 to 5 days for CEF and 2 to 7 days for immortalized cells) before infection. If needed, several passages of 1 to 8 days may be made in order to increase the total number of cells.

Producer cells are infected by the oncolytic virus with an appropriate multiplicity of infection (MOI), which can be as low as 0.001 (more preferably between 0.05 and 5) to permit productive infection. In step ii), infected producer cells are then cultured under appropriate conditions well known to those skilled in the art until progeny viral vector is produced. Culture of infected producer cells is also preferably performed in a chemically defined medium (which may be the same as or different from the medium used for culture of producer cells and/or for infection step) free of animal- or human-derived products at a temperature between +30°C and +37°C, for 1 to 5 days. In step iii), the viral particles may be collected from the culture supernatant and/or the producer cells. Recovery from producer cells (and optionally also from culture supernatant), may require a step allowing the disruption of the producer cell membrane to allow the liberation of the virus from producer cells. The disruption of the producer cell membrane can be induced by various techniques well known to those skilled in the art, including but not limited to, freeze/thaw, hypotonic lysis, sonication, microfluidization, or high-speed homogenization.

The recovered oncolytic virus can be at least partially purified before being used according to the present invention. Various purification steps can be envisaged, including clarification, enzymatic treatment (e.g. endonuclease such as benzonase, protease), ultracentrifugation (e.g. sucrose gradient or caesium chloride gradient), chromatographic and filtration steps. Appropriate methods are described in the art (e.g. WO2007/147528; WO2008/138533, WO2009/100521, WO2010/130753, WO2013/022764). Oncolytic virus composition

The invention also relates to a composition which comprises a therapeutically effective amount of the oncolytic virus of the present invention or prepared according to the process described herein. Preferably, the composition further comprises a pharmaceutically acceptable vehicle.

A "therapeutically effective amount" corresponds to the amount of each of the active agents comprised in the composition of the invention that is sufficient for producing one or more beneficial results. Such a therapeutically effective amount may vary as a function of various parameters, e.g. the mode of administration; the disease state; the age and weight of the subject; the ability of the subject to respond to the treatment; kind of concurrent treatment; the frequency of treatment; and/or the need for prevention or therapy. When prophylactic use is concerned, the composition of the invention is administered at a dose sufficient to prevent or to delay the onset and/or establishment and/or relapse of a pathologic condition (e.g. a proliferative disease such as cancer), especially in a subject at risk. For "therapeutic" use, the composition of the invention is administered to a subject diagnosed as having a pathological condition (e.g. a proliferative disease such as cancer) with the goal of treating the disease, eventually in association with one or more conventional therapeutic modalities. In particular, a therapeutically effective amount could be that amount necessary to cause an observable improvement of the clinical status over the baseline status or over the expected status if not treated, as described hereinafter. An improvement of the clinical status can be easily assessed by any relevant clinical measurement typically used by physicians and skilled healthcare staff. For example, techniques routinely used in laboratories (e.g. flow cytometry, histology, imaging techniques, etc.) may be used to perform tumour surveillance. A therapeutically effective amount could also be the amount necessary to cause the development of an effective non-specific (innate) and/or specific anti-tumour response. Typically, development of an immune response, in particular a T cell response, can be evaluated in vitro, in suitable animal models or using biological samples collected from the subject. One may also use various available antibodies so-as to identify different immune cell populations involved in anti-tumour response that are present in the treated subjects, such as cytotoxic T cells, activated cytotoxic T cells, natural killer cells and activated natural killer cells. The appropriate dosage of oncolytic virus can be adapted as a function of various parameters and may be routinely determined by a practitioner in the light of the relevant circumstances. Suitably, individual doses for the oncolytic virus may vary within a range extending from approximately 10³ to approximately 10¹²vp (viral particles), iu (infectious unit) or PFU (plaque- forming units) depending on the virus and the quantitative technique used. For illustrative purposes, a suitable dose of oncolytic vaccinia virus for human use is comprised between approximately 10⁴ to approximately 10¹¹ PFU, preferably between approximately 10⁵ PFU to approximately 10¹⁰ PFU; doses of approximately 10⁶ PFU to approximately 5xl0⁹ PFU being particularly preferred (e.g. dose of 10⁶, 2xl0⁶, 3xl0⁶, 4xl0⁶, 5xl0⁶, 6xl0⁶, 7xl0⁶, 8xl0⁶, 9xl0⁶, 10⁷, 2xl0⁷, 3xl0⁷, 4xl0⁷, 5xl0⁷, 6xl0⁷, 7xl0⁷, 8xl0⁷, 9xl0⁷, 10⁸, 2xl0⁸, 3xl0⁸, 4xl0⁸, 5xl0⁸, 6xl0⁸, 7xl0⁸, 8xl0⁸, 9xl0⁸, 10⁹, 2xl0⁹, 3xl0⁹, 4xl0⁹ or 5xl0⁹ PFU). The quantity of virus present in a sample can be determined by routine titration techniques, e.g. by counting the number of plaques following infection of permissive cells (e.g. BHK-21 or CEF), immunostaining (e.g. using anti-virus antibodies; Caroll et al., 1997, Virology 238: 198-211), by measuring the A260 absorbance (vp titers), or still by quantitative immunofluorescence (iu titers).

The term "pharmaceutically acceptable vehicle" is intended to include any and all carriers, solvents, diluents, excipients, adjuvants, dispersion media, coatings, antibacterial and antifungal agents, absorption agents and the like compatible with administration in mammals and in particular human subjects.

The oncolytic virus of the invention can independently be placed in a solvent or diluent appropriate for human or animal use. The solvent or diluent is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength. Representative examples include sterile water, physiological saline (e.g. sodium chloride), Ringer's solution, glucose, trehalose or saccharose solutions, Hank's solution, and other aqueous physiologically balanced salt solutions (see for example the most current edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams&Wilkins).

In other embodiments, oncolytic viruses are suitably buffered for human use. Suitable buffers include without limitation phosphate buffer (e.g. PBS), bicarbonate buffer and/or Tris buffer capable of maintaining a physiological or slightly basic pH (e.g. from approximately pH 7 to approximately pH 9).

The composition of the invention may also contain other pharmaceutically acceptable excipients for providing desirable pharmaceutical or pharmacodynamic properties, including for example osmolarity, viscosity, clarity, colour, sterility, stability, rate of dissolution of the formulation, modifying or maintaining release or absorption into a human or animal subject, promoting transport across the blood barrier or penetration in a particular organ. In a further embodiment, the composition of the invention may be adjuvanted to further enhance immunity (especially a T cell-mediated immunity) or facilitate infection of tumour cells upon administration. Representative examples of suitable adjuvants include, without limitation, alum, mineral oil emulsion such as, Freunds complete and incomplete (IFA), lipopolysaccharide or a derivative thereof (Ribi et al., 1986, Plenum Publ. Corp.,407-419), saponins such as QS21 (Sumino et al., 1998, J.Virol. 72: 4931; W098/56415), imidazo-quinoline compounds such as Imiquimod (Suader, 2000, J. Am Acad Dermatol. 43: S6), S-27609 (Smorlesi, 2005, Gene Ther. 12: 1324) and related compounds such as those described in WO2007/147529, polysaccharides such as Adjuvax and squalenes, oil in water emulsions such as MF59, double-stranded RNA analogs such as poly(l:C), single stranded cytosine phosphate guanosine oligodeoxynucleotides (CpG) (Chu et al., 1997, J. Exp. Med., 186: 1623; Tritel et al., 2003, J. Immunol., 171: 2358) and cationic peptides such as IC-31 (Kritsch et al., 2005, J. Chromatogr. Anal. Technol. Biomed. Life Sci., 822: 263-70).

In one embodiment, the composition of the invention may be formulated with the goal of improving its stability, in particular under the conditions of manufacture and long-term storage (i.e. for at least 6 months, with a preference for at least two years) at freezing (e.g. -70°C, -20°C), refrigerated (e.g. 4°C) or ambient temperatures. Various virus formulations are available in the art either in frozen, liquid form or lyophilized form (e.g. WO98/02522, WO01/66137, WO03/053463, WO2007/056847 and WO2008/114021, etc.). Solid (e.g. dry powdered or lyophilized) compositions can be obtained by a process involving vacuum drying and freeze-drying. For illustrative purposes, buffered formulations including NaCI and/or sugar are particularly adapted to the preservation of viruses (e.g. Tris 10 mM pH 8 with sucrose 5 % (W/V), Sodium glutamate 10 mM, and NaCI 50 mM or phosphate-buffered saline with glycerol (10%) and NaCI).

The oncolytic virus composition is preferably formulated in a way adapted to the mode of administration to ensure proper distribution and release in vivo. For example, gastro-resistant capsules and granules are particularly appropriate for oral administration, suppositories for rectal or vaginal administration, eventually in combination with absorption enhancers useful to increase the pore size of the mucosal membranes. Such absorption enhancers are typically substances having structural similarities to the phospholipid domains of the mucosal membranes (such as sodium deoxycholate, sodium glycocholate, dimethyl-beta-cyclodextrin, lauryl-l-lysophosphatidylcholine). Another and particularly appropriate example is a formulation adapted to the administration through microneedle means (e.g. transcutaneous or intradermal patches). Such a formulation may comprise resuspension of the immunotherapeutic product in endotoxin-free phosphate-buffered saline (PBS). Administration

The oncolytic virus, or the composition of the invention, may be administered in a single dose or multiple doses. If multiples doses are contemplated, administrations may be performed by the same or different routes and may take place at the same site or at alternative sites. Intervals between each administration can be from several hours to 8 weeks (e.g. 24h, 48h, 72h, weekly, every two or three weeks, monthly, etc.). Intervals can also be irregular. It is also possible to proceed via sequential cycles of administrations that are repeated after a rest period (e.g. cycles of 3 to 6 weekly administrations followed by a rest period of 3 to 6 weeks). The dose can vary for each administration within the range described above.

Any of the conventional administration routes are applicable in the context of the invention including parenteral, topical or mucosal routes. Parenteral routes are intended for administration as an injection or infusion and encompass systemic as well as local routes. Common parenteral injection types are intravenous (into a vein), intra-arterial (into an artery), intradermal (into the dermis), subcutaneous (under the skin), intramuscular (into muscle) and intratumoural (into a tumour or at its proximity). Infusions typically are given by intravenous route. Topical administration can be performed using transdermal means (e.g. patch and the like). Mucosal administrations include without limitation oral/alimentary, intranasal, intratracheal, intrapulmonary, intravaginal or intra-rectal route. In the case of intranasal, intrapulmonary and intratracheal routes, it is advantageous for administration to take place by means of an aerosol or by means of instillation. Preferred routes of administration for the oncolytic virus of the invention include intravenous and intratumoral routes.

Administrations may use conventional syringes and needles (e.g. Quadrafuse injection needles) or any compound or device available in the art capable of facilitating or improving delivery of the active agent(s) in the subject. Transdermal systems are also appropriate, e.g. using solid, hollow, coated or dissolvable microneedles (e.g. Van der Maaden et al., 2012, J. Control release 161: 645-55) and preferred are silicon and sucrose microneedle patches (see, e.g., Carrey et al., 2014, Sci Rep 4: 6154 doi 10.1038; and Carrey et al., 2011, PLoS ONE, 6(7) e22442).

A particularly preferred composition comprises 10⁶ PFU to 5xl0⁹ PFU of an oncolytic virus comprising a ADA-encoding nucleic acid molecule and preferably an oncolytic vaccinia virus defective in J2R locus (TK-), in I4L and/or F4L locus (RR-) or both in J2R locus (TK-) and in I4L/ F4L locus (TK- RR-), with a specific preference for a vaccinia having the ADA nucleic acid molecule inserted in place of the TK locus and placed under the pllK7.5 promoter such as VVTK-RR-/ADA1, VVTK-RR-/ADA2 or VVTK-RR-/cuADA described herein; formulated for intravenous or intratumoral administration.

Methods and use In another aspect, the present invention provides an oncolytic virus or a composition thereof (in particular a pharmaceutical composition) for use for treating or preventing a disease or a pathologic condition in a subject in need thereof. The present invention also relates to the use of an oncolytic virus or composition thereof for the manufacture of a medicament for treating or preventing a disease or a pathologic condition in a subject in need thereof. The present invention also relates to a method of treatment comprising administering the oncolytic virus or the composition thereof in an amount sufficient for treating or preventing a disease or a pathologic condition in a subject in need thereof. The present invention also relates to the use of an oncolytic virus or composition thereof for treating or preventing a disease or a pathologic condition in a subject in need thereof. A "disease" (and any form of disease such as "disorder" or "pathological condition") is typically characterized by identifiable symptoms.

Examples of diseases that may be prevented or treated using the oncolytic virus of the invention or the composition thereof include proliferative diseases such as cancers, tumours or restenosis and diseases associated to an increased osteoclast activity such as rheumatoid arthritis and osteoporosis.

The present invention is particularly suited for treating or preventing cancers, and particularly Adrenocortical Carcinoma, Adrenal Cortex Cancer, Anal Cancer, Gastrointestinal Carcinoid Tumors (for example Appendix Cancer and Carcinoid Tumor), Bile Duct Cancer (for example Cholangiocarcinoma), Bladder Cancer, Bone Cancer (for example Ewing Sarcoma, Malignant Fibrous Histiocytoma of Bone and Osteosarcoma), Brain Tumors (for example Astrocytomas, Embryonal Tumors, Germ Cell Tumors, Central Nervous System Atypical Teratoid/Rhabdoid Tumor, Craniopharyngioma, Ependymoma, Gliomas and Glioblastoma), Breast Cancer (for example Ductal Carcinoma In Situ), Bronchial Tumors, Carcinoma of Unknown Primary, Cardiac (Heart) Tumors, Cervical Cancer, Chordoma, Chronic Myeloproliferative Neoplasms, Colorectal Cancer (for example Rectal Cancer), Esthesioneuroblastoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Retinoblastoma, Gallbladder Cancer, Gastrointestinal Carcinoid Tumor, Testicular Cancer, Gestational Trophoblastic Disease, Head and Neck Cancer (for example Hypopharyngeal Cancer, pharyngeal Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Metastatic Squamous Neck Cancer with Occult Primary, Mouth Cancer, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Salivary Gland Cancer, Throat Cancer, Esophageal Cancer), Hepatocellular (Liver) Cancer, Histiocytosis, Langerhans Cell, Kidney cancer (for example Wilms Tumor, Renal Cell Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter), Langerhans Cell Histiocytosis, Laryngeal Cancer and Papillomatosis, Leukemia (for example Hairy Cell Leukemia, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL)), Liver Cancer, Lung Cancer (Small Cell Lung Cancer and Non-Small Cell Lung Cancer), Lymphoma (for example AIDS-Related Lymphoma, Primary CNS Lymphoma, Cutaneous T-Cell Lymphoma, Hodgkin Lymphoma, Burkitt Lymphoma, Primary Lymphoma, Mycosis Fungoides, Non-Hodgkin Lymphoma, Macroglobulinemia, Waldenstrom, Primary Central Nervous System (CNS) Lymphoma, Sezary Syndrome, T-Cell Lymphoma), Intraocular Melanoma, Mesothelioma, Midline Tract Carcinoma Involving NUT Gene, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/Plasma Cell Neoplasms Myelodysplastic Syndromes, Chronic Myeloproliferative Neoplasms, Neuroblastoma, Ovarian Cancer (for example Primary Peritoneal Cancer and Fallopian Tube Cancer), Pancreatic Cancer and Pancreatic Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis, Paraganglioma, Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Prostate Cancer, Retinoblastoma, Vascular Tumors, Skin Cancer (for example Basal Cell Carcinoma, Melanoma, Squamous Cell Carcinoma and Merkel Cell Carcinoma), Small Intestine Cancer, Soft Tissue Sarcoma (for example Gastrointestinal Stromal Tumors (GIST), AIDS-Related Cancers Kaposi Sarcoma, Kaposi Sarcoma and Rhabdomyosarcoma), Stomach (Gastric) Cancer, Testicular Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Urethral Cancer, Uterine Cancer, Endometrial and Uterine Sarcoma, Vaginal Cancer and Vulvar Cancer. The present invention is also useful for treatment of metastatic cancers.

In a preferred embodiment, the oncolytic virus or a composition of the invention is used for treating gastro-intestinal cancers, comprising cancers of oesophagus, gallbladder, liver, pancreas, stomach, small intestine, bowel (large intestine or colon and rectum), and anus. A particularly preferred method comprises 1 to 6 intravenous or intratumoral administrations of the oncolytic virus of the invention or composition thereof given at weekly to monthly intervals with a specific preference for 3 bi-weekly administrations (e.g. at approximately Dl, D14 and D29) of a composition comprising 10⁶ to 5xl0⁹ PFU of an oncolytic vaccinia virus comprising inserted in its genome a ADA-encoding nucleic acid molecule (e.g. placed under the pllK7.5 promoter), preferably defective in J2R locus (TK-), in I4L and/or F4L locus (RR-) or both in J2R locus (TK-) and in I4L/ F4L locus (TK- RR-), such as VVTK-RR-/huADAl or VVTK-RR-/huADA2.

The beneficial effects provided by the methods of the present invention can be evidenced by an observable improvement of the clinical status over the baseline status or over the expected status if not treated according to the modalities described herein. An improvement of the clinical status can be easily assessed by any relevant clinical measurement typically used by physicians and skilled healthcare staff. In the context of the invention, the therapeutic benefit can be transient (for one or a couple of months after cessation of administration) or sustained (for several months or years). As the natural course of clinical status which may vary considerably from a subject to another, it is not required that the therapeutic benefit be observed in each subject treated but in a significant number of subjects (e.g. statistically significant differences between two groups can be determined by any statistical test known in the art, such as a Tukey parametric test, the Kruskal- Wallis test the U test according to Mann and Whitney, the Student's t-test, the Wilcoxon test, etc.).

In a particular embodiment, as the methods according to the present invention are particularly appropriate for treating cancer, such methods can be correlated with one or more of the followings: inhibiting or slowing tumour growth, proliferation and metastasis, preventing or delaying tumour invasion (spread of tumour cells in neighbouring tissues), reducing the tumour number; reducing the tumour size, reducing the number or extent of metastases, providing a prolonged overall survival rate (OS), increasing progression free survival (PFS), increasing the length of remission, stabilizing (i.e. not worsening) the state of disease, providing a better response to the standard treatment, improving quality of life and/or inducing an anti-tumour response (e.g. nonspecific (innate) and/or specific such as a cytotoxic T cell response) in the subject treated in accordance with the present invention.

The appropriate measurements that can be used to assess a clinical benefit such as blood tests, analysis of biological fluids and biopsies as well as medical imaging techniques are evaluated routinely in medical laboratories and hospitals and a large number of kits is available commercially. They can be performed before the administration (baseline) and at various time points during treatment and after cessation of the treatment.

The present invention also relates to a method for treating a disease or a pathologic condition in a subject in need thereof comprising administering the oncolytic virus or the composition of the present invention or prepared according to the process described herein. In one embodiment, said disease is a proliferative disease such as cancers, tumours and restenosis. In another embodiment, said disease is a disease associated with an increased osteoclast activity like rheumatoid arthritis and osteoporosis. More precisely, the present invention relates to a method for inhibiting tumour cell growth in vivo comprising administering an oncolytic virus or a composition thereof in a subject in need thereof so-as to inhibit the growth of a tumour. For general guidance, inhibition of tumour cell growth can be evaluated routinely, for example by radiography means. The administration(s) of the oncolytic virus or a composition thereof desirably result(s) in at least a 10% decrease of the tumour mass.

Combination therapy

In any of the methods according to the invention, the oncolytic virus or composition can be administered in association with any conventional therapeutic modalities which are available for treating or preventing the targeted disease or pathological condition. Representative examples of conventional therapies include, without limitation, surgery, radiotherapy, chemotherapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy, cytokine therapy, transplantation (e.g. stem cell), hyperthermia and photodynamic therapy. Such conventional therapy/ies is/are administered to the subject in accordance with standard practice before, after, essentially simultaneously or in an interspersed manner with the oncolytic virus or composition thereof.

In one embodiment, the oncolytic virus, composition or method according to the invention can be used in association with radiotherapy. Those skilled in the art can readily formulate appropriate radiation therapy protocols and parameters (see for example Perez and Brady, 1992, Principles and Practice of Radiation Oncology, 2nd Ed. JB Lippincott Co; using appropriate adaptations and modifications as will be readily apparent to those skilled in the field). The types of radiation that may be used are well known in the art and include electron beams, high-energy photons from a linear accelerator or from radioactive sources such as cobalt or caesium, protons, and neutrons. Dosage ranges for radio-isotopes can be defined by the practitioners depending on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. Regular X-rays doses for prolonged periods of time (3 to 6 weeks), or high single doses are contemplated by the present invention.

In other embodiments, the methods may be used in conjunction with surgery. For example, the oncolytic virus or composition thereof may be administered upon excision of the tumour (e.g. by local application within the excised zone for example).

In further embodiments of any of the methods of the invention, the oncolytic virus or composition thereof may be used in combination with one or more substances effective in anticancer therapy, like chemotherapeutic drugs or immunotherapeutic products. In a specific embodiment, the oncolytic virus or composition of the invention may be used in conjunction with chemotherapeutic drugs currently used for treating cancer. Although any anticancer chemotherapy drug may be used in combination with the oncolytic virus of the present invention or the composition thereof, there may be mentioned more specifically alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, platinum derivatives, inhibitors of tyrosine kinase receptors, antimetabolites and antimitotic agents.

In still further embodiments, the oncolytic virus or composition of the invention may be used in conjunction with immunotherapy, and especially with anti-neoplastic antibodies as well as siRNA and antisense polynucleotides. Representative examples include, among others, monoclonal antibodies blocking immune checkpoint (e.g. Ipilimumab, tremelimumab pembrolizumab, nivolumab, pidilizumab, AMP-224MEDI4736, MPDL3280A, BMS-936559, etc.), alpha, beta or gamma interferon, interleukin (in particular IL-2, IL-6, IL-10 or IL-12) or tumour necrosis factor; agents that affect the regulation of cell surface receptors such as, e.g. monoclonal antibodies blocking Epidermal Growth Factor Receptor (in particular cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab, trastuzumab (Herceptin™), gefitinib, eriotinib, lapatinib, etc.) and monoclonal antibodies blocking Vascular Endothelial Growth Factor (in particular bevacizumab and ranibizumab). Other representative examples of such immunotherapeutic products suitable for use in the present invention are plasmid DNA vector, vaccinia virus (e.g. Copenhagen, WR, Wyeth, MVA, etc.), adenovirus, lentivirus, herpes virus, recombinant polypeptides, among many others. Another aspect of the invention concerns the combination of an oncolytic virus (e.g. wild type oncolytic virus, or modified derivative oncolytic virus) with at least one agent degrading a metabolic immune modulator (e.g. polypeptide, nucleic acid, gene regulator, lipid, small molecule, cytotoxin, drug, acid, base, oxidizing agent, reducing agent, alkaline agent, nucleophilic agent, electrophilic agent, polymer, synthetic material, virus, bacteria, archaea, protozoa, fungi, algae, and combinations thereof), wherein said degrading agent is not encoded by the oncolytic virus. In a specific embodiment of the invention, said degrading agent is a polypeptide, more specifically an ADA polypeptide, or a polypeptide having an adenosine deaminase activity. ADA may be encoded by another vector, like for example DNA molecules (plasmids, non-oncolytic viral vectors, cosmids and artificial chromosomes), RNA molecules, nanoparticles, etc. Adenosine deaminase may also be in its polypeptide form. The polypeptide can be of any origin, e.g. human, humanized, animal, insects, microorganisms or chimeric. In addition, the polypeptide may be glycosylated or non- glycosylated. In a further embodiment, the oncolytic virus or composition thereof may also be used in combination with substances which enhance adenosine deaminase activity.

One may provide the subject with the oncolytic virus and the additional anti-cancer therapy sequentially or in an interspersed way but concomitant administrations of both therapies within the same period of time are also contemplated. The course of treatment may be routinely determined by a practitioner and various protocols are encompassed by the present invention. For example, 1 to 10 administrations of the oncolytic virus (e.g. 3 to 6 biweekly injections) may be interspersed within one or multiple administrations of the additional anti-cancer therapy (for example chemotherapy may be given in one or more cycles of one or several weeks). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no anti-cancer treatment is administered before repetition of treatment cycle(s).

All of the above cited disclosures of patents, publications and database entries are specifically incorporated herein by reference in their entirety. Other features, objects, and advantages of the invention will be apparent from the description, drawings and from the claims. The following examples are given solely for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned above, as well as those objects, ends and advantages inherent herein. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

EXAMPLES

These examples illustrate oncolytic vaccinia virus engineered for expressing at least one agent degrading one or more metabolic immune modulator(s). Preclinical evidence for the beneficial effects of expressing agents degrading immune modulators from viral vectors was to be demonstrated in mouse tumour models. This implies the use of i) metabolic immune modulators degrading agents and ii) an oncolytic poxvirus capable of infecting murine cells with a higher efficacy. The following examples are given solely for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned above, as well as those objects, ends and advantages inherent herein. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Vectorization of adenosine degrading agents in oncolytic TK^' RR vaccinia virus

All vaccinia viruses used in this study are of the Western Reserve strain or Copenhagen strain with disrupted TK and RR genes for enhanced cancer cell specificity.

The human ADA1 (huADAl), ADA2 (huADA2) and the ADA from culex quinquefasciatus (cuADA) were cloned under the control of the viral promoter pllk7.5. While huADA2 and cuADA possess a signal sequence allowing for their secretion, huADAl is not naturally actively transported outside the cell. Therefore, the signal sequence from Gaussia luciferase (Stern, B. and L. Olsen, 2007, Trends Cell Mol. Biol, 2:1-17) was added to the corresponding construct. Additionally, a FLAG tag was added to the cuADA coding gene to allow for detection. The constructs are summarized below and illustrated in Figure 1:

huADAl encoding WRTG19009 corresponding to pllK7.5-ss-huADAl

huADA2 encoding WRTG19021 corresponding to pllK7.5-huADA2

cuADA encoding WRTG19010 corresponding to pllK7.5-cuADA-FLAG

huADAl encoding COPTG19183 corresponding to pllK7.5-ss-hADAl

huADA2 encoding COPTG19185 corresponding to pllK7.5-huDA2

Construction of WRTG19009, WRTG19010 and WRTG19021

Fragments containing huADAl, huADA2 and cuADA were generated synthetically (Geneart, Germany) and cloned in in the vaccinia transfer plasmid pTG18495 restricted by Pst\ and fcoRI to give pTG19009, pTG19021 and pTG19010 respectively.

The vaccinia transfer plasmid, pTG18495, is designed to permit insertion of the nucleotide sequence to be transferred by homologous recombination in TK gene of the VV genome. It contains the flanking sequences (BRGTK and BRDTK) surrounding the J2R gene and the pllK7.5 promoter followed by multiple cloning sites.

The amino acid sequences of the huADAl, huADA2 and cuADA are given in SEQ ID NO: 1,

SEQ ID NO: 3 and SEQ ID NO: 5 respectively. The nucleic acid sequence of the huADAl, huADA2 and cuADA are given in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. Generation of the WR vaccinia viruses was performed by homologous recombination in primary chicken embryos fibroblasts (CEF) infected with a RR-deleted WR and transfected by nucleofection with pTG19009, pTG19010 or pTG19021 (according to Amaxa Nucleofector technology). Viral selection was performed by plaque purification after growth in Thymidine kinase- 5 deficient (TK~) 143B cells cultured in the presence of bromodeoxyuridine. This selection allows only TK^~ recombinant WR to remain viable. Absence of contamination by parental WR was verified by PCR.

In vitro expression of ADAs by vaccinia virus WR TK- RR- infected cultured cells

10 The recombinant oncolytic vectors described above were tested to assess the production of the encoded adenosine deaminase. For this, a total of 2xl0⁵ mouse sarcoma MCA205 cells (Rosenberg et al., 1985, J Immunol 135(4): 2895-2903) or mouse colon carcinoma CT26 cells (ATCC^® CRL-2638™) per well were plated in six-well culture dishes in 2 mL of culture medium and infected with either WRTG18011 (empty VV), WRTG19009, WRTG19010 or WRTG19021 at a MOI of either

15 0.1, 0.01 or 0.001. Culture supernatants were collected after 24, 48 or 72 hours of infection at 37°C.

Those supernatants were centrifugated 10 minutes at 500 g at 4°C in order to remove suspended cells and store at -20°C. Adenosine deaminase activity was measured on the culture supernatants in UV transparent 96 wells microplates (Greiner). The supernatants were diluted in 50 mmol ¹ NaH₂P0₄ buffer, pH 7.2, in the microplate wells, and the reaction was initiated by the addition of

20 adenosine at a final concentration of 200 μ . The absorbance (Abs) at 265 nm was read at 60 s intervals using an Infinite MIOOOPro microplate reader thermostated to 30 °C (Tecan, Switzerland), after orbital mixing. One unit of enzyme activity is defined as the amount of enzyme that catalyses the deamination of 1 nmol of adenosine per minute per millilitre of culture supernatant (using AAbs = 8.6 min ¹ ml ¹ at 265 nm (Ribeiro et al., 2001, J. Exp. Biol., 204(Pt 11): 2001-

25 2010).

WRTG19009 and WRTG19021 encoded huADAl and huADA2 were well secreted by MCA205 (Figure 2A) and CT26 (Figure 2B) and active in the culture supernatant. The enzymatic activity was accumulating in the culture medium during the extended culture which showed a certain level of stability of the virus encoded human ADAs. As expected, the lower permissivity of 30 the CT26 cell line resulted in overall lower level of active enzymes produced by the infected cells.

On the other hand, no adenosine deaminase activity could be detected after infection of those two cell lines by the WRTG19010 encoding the ADA from culex quinquefasciatus. These results showed that non-mammalian ADA such as the insect one could not be expressed in an active form by W infected cells. Evaluation of the in vitro cytotoxicity of ADA expressing vaccinia viruses Western Reserve (oncolytic activity)

The oncolytic activity of the different viruses WR expressing huADAl, huADA2 and cuADA was evaluated by cell viability measurements in both MCA205 or CT26 infected cells. A total of 5xl0⁴ cells per well were plated in six-well culture dishes in 2 mL of culture medium and infected with either WRTG18011, WRTG19009, WRTG19010 or WRTG19021 at a MOI of either 0.1, 0.01, 0.001 or 0.0001. Five days after infection, cell viability was determined by trypan blue exclusion with a cell counter (Vi-Cell, Beckman coulter) and the condition of untreated cells was used to set the 100 % of viability.

As shown in Figure 3A, in MCA205, the oncolytic activity of the empty virus and the constructs expressing human ADA resulted in more than 90 % and close to 100 % reduction in viable cell number at a MOI of 0.01 and 0.1, respectively. Similar results were obtained with the CT26 cells (panel B), even though this cell line is less permissive to vaccinia virus WR strain. However, the virus encoding the cuADA showed no cytotoxicity at MOI of 0.01 contrary to the human counterparts.

TK-RR-deleted WR vaccinia virus without transgene and TK-RR deleted WR virus expressing the two human enzymes showed similar cytotoxicity in tumor cells, whereas in contrary, the expression of cuADA did not affect the oncolytic activity of the vaccinia virus towards the cultured tumor cells.

Evaluation of the in vitro replication of the ADA expressing vaccinia viruses WR in cultured cells

The effect of ADAs expression on viral replication was assessed in cultured cells. For that purpose, 2xl0⁵ cells per well of MCA205 and CT26 cells were plated in six-well culture dishes in 2 mL of culture medium and infected with WRTG18011, WRTG19009, WRTG19010 or WRTG19021 at a MOI of 0.01 and 0.001. Cells and medium were harvested after 24, 48 or 72 hours after infection and frozen at -80°C until use. Cell suspensions were thawed and sonicated and treated with benzonase (Novagen) to eliminate non-encapsidated DNA. After inactivation of the nuclease, the virus capsids were disrupted by addition of an equal volume of Tris 20 mM, EDTA 10 mM, SDS 1 % pH7.4 followed by 160 μg of Proteinase K (Qiagen) and an incubation of 30 min at 65°C. After inactivation of Proteinase K, the MVA genome DNA was quantified using a q-PCR. The absolute quantification was allowed using a series of dilutions from 10⁶ to 10² copies of purified MVA genome. A set of oligonucleotides primers and probe that hybridize into the gene encoding for the secreted chemokine-binding protein (SCBP) of MVA were used (forward primer 5'- CGATGATGGAGTAATAAGTGGTAGGA-3' (SEQ ID NO: 7); reverse primer 5'- CACCGACCGATGATAAGATTTG-3' (SEQ ID NO: 8); probe 5' FAM - ACTG ATTCC ACCTCG G G - 3' (SEQ ID NO:9; MGB / NFQ). The Quantitect Multiplex PCR kit from Qiagen was used to perform the q-PCR reactions on a 7300 real-time system from Applied Biosystem.

As shown in Figure 4, no substantial difference could be detected for the replication of all the different viruses. Therefore, the expression of the adenosine deaminases has little or no effect on the infection and multiplication of the recombinant viruses.

In vivo assessment of the ADA expressing vaccinia viruses WR The antitumour activity of the various ADA expressing vaccinia viruses described above were tested in a mouse preclinical model after implantation of tumours followed by injection of the constructs. For the MCA205 tumour model, 8xl0⁵ MCA205 cells in 100 μΐ PBS were injected subcutaneously into the right flanks of C57BL/6 mice. Seven days after inoculation, when tumours volume reached between 20 and 130 mm³, mice were injected intratumorally with vehicle or 10⁷ pfu of WRTG18011, WRTG19009 or WRTG19021. Injections of virus were repeated once 3 days after the first injection and tumours volume were monitored twice per week by calliper measurements for over 40 days. Mice were sacrificed when tumour size reached 2000 mm³. As expected, tumours increased in size very rapidly in the vehicle control group (Figure 5A) whereas the tumour growth was slightly delayed when treated with the empty vaccinia virus (Figure 5B); In contrast, a noticeable improvement was seen in the groups treated with human ADA1 and ADA2 expressing oncolytic viruses (Figures 5C and 5D) where tumor growth is delayed similarly whatever the human ADA expressed.

As shown in Figure 6, the survival of mice is also increased by ADA-expression. More specifically, all mice treated with the vehicle were dead at 32 days (0% survival rate), whereas at this time, 40, 60 and 80 % survival rate were obtained respectively in empty virus control group, huADAl and huADA2 expressing viruses treated group.

Construction of COPTG19183 and COPTG19185

Generation of the vaccinia viruses Copenhagen strain (COP) expressing adenosine deaminase was performed by homologous recombination in primary chicken embryos fibroblasts (CEF) infected with a RR-deleted COP virus and transfected by nucleofection with pTG19183 or pTG19185 (according to Amaxa Nucleofector technology). Viral selection was performed by plaque purification after growth in Thymidine kinase-deficient (TK^") 143B cells cultured in the presence of bromodeoxyuridine. This selection allows only TIC recombinant WR to remain viable. Absence of contamination by parental COP was verified by PCR.

Evaluation of the in vitro cytotoxicity of ADA expressing vaccinia viruses COP (oncolytic activity)

The oncolytic activity of the COP viruses expressing huADAl and huADA2 was evaluated by cell viability measurements in both human colorectal carcinoma HCT116 (ATCC^® CCL-247™) or human colorectal adenocarcinoma LoVo (ATCC^® CCL-229™) infected cells. A total of 5xl0⁴ cells per well were plated in six-well culture dishes in 2 mL of culture medium and infected with either VVTG17989 (empty virus), COPTG19183 and COPTG19185 at a MOI of either 0.1, 0.01, 0.001 or 0.0001. Five days after infection, cell viability was determined by trypan blue exclusion with a cell counter (Vi-Cell, Beckman coulter) and the condition of untreated cells was used to set the 100 % of viability.

As shown in Figure 7A, in HCT116, the oncolytic activity of the empty COP virus and the constructs expressing human ADA resulted in nearly 100 % reduction in viable cell number at a MOI of 0.1. In this very permissive model, the viruses expressing huADAl and huADA2 show a very similar oncolytic activity compared to the empty virus.

Similarly, as shown in Figure 7B, in LoVo, the oncolytic activity of the empty COP virus and the constructs expressing human ADA resulted in similar levels of viable cell number. The overall oncolytic activity was lower in this less permissive cell line.

TK-RR-deleted COP vaccinia virus without transgene and TK-RR- deleted COP virus expressing the two human enzymes showed similar cytotoxicity in tumor cells. There is no impact of huADAl and huADA2 vectorization on in vitro oncolytic activity of VVCOP.

Evaluation of the in vitro replication of the ADA expressing vaccinia viruses COP in cultured cells The effect of ADAs expression on viral replication was assessed in cultured cells. For that purpose, 2xl0⁵ cells per well of LoVo (Figures 8A and 8B) and HCT116 (Figures 8C and 8D) cells were plated in six-well culture dishes in 2 mL of culture medium and infected with VVTG17989, COPTG19183 and COPTG19185 at a MOI of 0.01 and 0.001. Cells and medium were harvested after 24, 48, 72 and 120 hours after infection and frozen at - 80°C until use. Cell suspensions were thawed and sonicated and treated with benzonase (Novagen) to eliminate non-encapsidated DNA. After inactivation of the nuclease, the virus capsids were disrupted by addition of an equal volume of Tris 20 mM, EDTA 10 mM, SDS 1 % pH 7.4 followed by 160 μg of Proteinase K (Qiagen) and an incubation of 30 min at 65°C. After inactivation of Proteinase K, the MVA genome DNA was quantified using a q-PC . The absolute quantification was allowed using a series of dilutions from 10⁶ to 10² copies of purified MVA genome. A set of oligonucleotides primers and probe that hybridize into the gene encoding for the secreted chemokine-binding protein (SCBP) of MVA were used (forward primer 5'-CGATGATGGAGTAATAAGTGGTAGGA-3' (SEQ ID NO: 7); reverse primer 5'- CACCGACCGATGATAAGATTTG-3' (SEQ ID NO: 8); probe 5' FAM - ACTGATTCCACCTCGGG - 3' (SEQ ID NO:9; MGB / NFQ). The Quantitect Multiplex PCR kit from Qiagen was used to perform the q-PCR reactions on a 7300 real-time system from Applied Biosystem. As shown in Figure 8, no substantial difference could be detected for the replication of all the different viruses. Therefore, the expression of the adenosine deaminases has little or no effect on the infection and multiplication of the recombinant viruses.

In vitro expression of ADAs by vaccinia virus COP TK- RR- infected cultured cells The recombinant oncolytic vectors described above were tested in vitro to assess the production of the encoded adenosine deaminases. For this, a total of 2xl0⁵ LoVo or HCT116 cells per well were plated in six-well culture dishes in 2 mL of culture medium and infected with either VVTG17989 (empty virus), COPTG19183 and COPTG19185 at a MOI of 0.01 or 0.001. Culture supernatants were collected after 72 hours of infection at 37°C. Those supernatants were centrifugated 10 minutes at 500 g at 4°C in order to remove suspended cells and store at -20°C.

The expression of both transgenes was first analysed on the HCT116 culture supernatants by western blotting using specific rabbit antibodies for each of the two enzymes (Figure 9). Compared to the infection by the VVTG17989, the infection of HCT116 by COPTG19183 and COPTG19185 leads to the secretion of proteins with an apparent size close to the theoretical mass of 41 kDa (Figure 9A) and 56 kDa (Figure 9B) for huADAl and huADA2 respectively. These results demonstrate that huADAl and huADA2 are expressed and secreted in the extracellular space as full length homogenous products.

Concomitantly, the secreted adenosine deaminase activity was measured on the culture supernatants by quantifying the production of inosine from an excess of adenosine using HPLC and a diode array UV-visible detector. Briefly, the supernatants were diluted into 50 mmol.l^"1 NaH₂P0₄ buffer, pH 7.2, and the reaction was initiated by the addition of adenosine at a final concentration of 20 μΜ, equivalent to physiological conditions, or 2 mM. After incubation at 30°C for 20 min. the reaction was stopped, and the proteins precipitated on ice by adding trifluoroacetic acid at a final concentration of 17%. Samples were centrifugated at 19000g for 15 min. before analysis by HPLC. Inosine and adenosine were separated using a Sinergy Fusion RP C18 column (Phenomenex) using a mobile phase composed of 50 mmol.l ¹ sodium acetate buffer at pH 4.5 and acetonitrile up to 50%. Inosine produced was quantified from the area under peak using a standard curve.

The Figure 10 shows that infection of HCT116 or LoVo cells by either of the two viruses encoding an ADA leads to the accumulation of adenosine deaminase activity in the culture supernatant. In conditions for which adenosine is added in large excess (Figures 10A and 10B), cellules infected by COPTG19185, encoding the huADA2, show higher levels of specific activity. When the reaction is performed with lower concentration of adenosine (20 μΜ), closer to physiological diseased conditions (Figures IOC and 10D), infection by COPTG19183, encoding the huADAl enzyme, results in higher specific ADA activity than after infection by COPTG19185. The former ADA isoform presents a higher affinity for adenosine than the latter (Km equals to 20 μΜ and 2.5 mM respectively) that largely compensate the lower level of secretion of this enzyme. This higher affinity for the targeted metabolite results in higher degrading efficacy for the virus COPTG19183.

In vivo assessment of the ADA expressing vaccinia viruses COP

The antitumour activity of the various ADA expressing vaccinia viruses described above were tested in a xenograft mouse model after implantation of tumours followed by injection of the constructs. For the HCT116 tumour model, 5xl0⁶ HCT116 cells in 100 μί PBS were injected subcutaneously into the right flanks of 15 Swiss nude mice. Seventeen days after inoculation, when tumours volume reached between 100 and 200 mm³, mice were injected once intravenously with vehicle or 10⁷ pfu of COPTG19183 or COPTG19185. Tumours volume were monitored twice per week by calliper measurements for over 120 days and mice were sacrificed when tumour size reached 2000 mm³. After 3, 7 and 14 days, tumors were surgically removed from three mice per treated group and promptly snap-frozen for further analysis.

Immunohistochemical analysis of tumor sections after 3 and 14 days treatment with COPTG19183 and COPTG19185 showed that the expression of both human enzymes was spatially overlapping the replication of the virus (data not shown). The expression of huADAl in tumors treated with COPTG19183 was confirmed by western blotting (Figure 11). Additionally, the Q-PCR viral titration was performed on HCT116 tumors treated with the empty virus or the two viruses encoding an ADA. As observed in vitro, the Figure 12 shows that there is no significant difference between the amount of viral genome measured after treatment with the empty virus or the two ADA expression constructs. The in situ multiplication of the virus is therefore not impaired by the expression of the adenosine degrading enzymes.

The adenosine deaminase activity was measured in the tumors after 7 days after the single virus injection. The frozen tumors were thawed on ice, washed with PBS, and the interstitial fluids were collected by centrifugation for 10 min. at 500g after placing the tissues on a cell strainer with nylon mesh presenting 70 μηι pores. The ADA specific activity was quantified by HPLC, as described above, with an adenosine concentration of 2 mM and 20 μΜ. Figure 13A illustrates the amount of inosine produced per minutes per microliter of tumor interstitial fluid from a large excess of adenosine. This figure shows that the intratumoral expression of huADAl induced by treatment with COPTG19183 leads to a 7-fold increase of the basal adenosine degradation activity observed after treatment with the empty virus; similarly, the expression of huADA2 results in a 9.6-fold increase of specific enzymatic activity.

At a lower concentration of substrate (Figure 13B), simulating a diseased physiological state, the higher affinity of huADAl for adenosine leads to a higher accumulation of degrading activity than huADA2 encoded by COPTG19183. These data demonstrate that the replication of the two viruses COPTG19183 and COPTG19185 is followed by in situ accumulation of adenosine degrading activity, indispensable for lowering local extracellular adenosine concentration and lift the immunosuppressor role of this metabolite. Additionally, those results confirm the observations made in vitro showing that the expression of huADAl is advantageous compared to the 2^nd isoform for a higher activity of the oncolytic virus against intratumoral adenosine.

The tumor growth and survival were monitored after treatment of mice bearing HCT116 tumors by VVTG17989, COTG19183 and COPTG19185 (Figure 14 and Figure 15). As expected, tumours increased in size very rapidly in the vehicle control group (Figure 14A) whereas the tumour growth was largely controlled when treated with the empty vaccinia virus (Figure 14B) with 3 tumor-free mice at the end of the study. Similarly, a noticeable improvement, compared to the untreated group, was seen in the groups treated with human ADA1 and ADA2 expressing oncolytic viruses (Figures 14C and 14D) where tumor growth is controlled similarly whatever the human ADA expressed, with 2 and 3 tumor-free mice respectively at the closure of the study.

As shown in Figure 15, the survival of mice is also significantly increased by the treatment with the oncolytic viruses. More specifically, all mice treated with the vehicle were dead at 57 days (0% survival rate), whereas at this time, 78%, 89% and 100 % survival rates were obtained respectively in empty virus control group, huADAl and huADA2 expressing viruses treated group respectively. The use of xenograft models based on human cancer cells implanted in nude immunodeficient animals allows for the evaluation of the oncolytic activity of the VV constructs, as those cells are relatively permissive to those viruses, compared to mice cell lines. Nevertheless, the impaired adaptive immune response associated to those models prevent to appreciate the overall activity of the viruses armed with immunomodulators.

Antitumour activity in an immunocompetent subcutaneous tumour model

Antitumour activity of the ADA-expressing vaccinia viruses described above may be tested in immunocompetent preclinical models after implantation of tumour followed by injection of the constructs. For example, said anti-tumour activity can be tested in a mouse model composed of female NOD/Shi-scid/IL-2Rpull immunodeficient mouse strain (NOG) humanized using hematopoietic stem cells (CD34⁺) isolated from human cord blood and engrafted subcutaneously with human derived cancer cell line (e.g. HCT116). Based on tumor volume, body weight and CD34+ cell donor, 45 mice are subsequently randomized into 3 groups of treatment (n=15 per group). Treatments start when tumors reach an average volume of 100 mm³. 50 μί of vehicle, reference empty VVTG18058 or COPTG19183 are injected (10⁷ ρίυ/50μΙ-Λ ου5θ). Body weight and tumor volume are monitored three times a week. Seven days after the VV injection, tumors are harvested to evaluate viral replication, adenosine deaminase activity, the immune landscape by measuring immune cells populations (e.g.: CD4+, CD8+ T cells, T reg, NK cells, MDSCs, M1/M2 macrophages, etc.); soluble markers of the intratumoral immune response are also evaluated (e.g. IFNg, Granzyme B, IL12, VEGF, etc.). One expects that the replication of the oncolytic virus accompanied by the release of localized ADA activity will result in lowering the local concentration of extracellular adenosine. This effect on the immunosuppressor metabolite will trigger a remodeling of the immune intratumoral landscape: increased infiltration of CD8+ T cells (Beavis et al., 2013, Oncoimmunology 2(12): e26705) and activated NK cells (Hausler et al., 2011, Cancer immunology, immunotherapy: Cll 60(10): 1405-1418), reduced presence of M2 macrophages. One expects that the increased infiltration and activation of anti-tumoral immune cells will be followed by increased IFNg and Granzyme B levels (lannone et al., 2014, American journal of cancer research 4(2): 172- 181), for instance, and the potential impact of lower adenosine levels on angiogenesis will be measurable through lower levels of VEGF (Allard et al., 2014, International Journal of Cancer 134(6): 1466-1473). The overall reduction of the immunosuppressor activity of adenosine will lead to a better control of the tumor growth by the ADA expressing oncolytic virus as well as a benefit in terms of survival. Evaluation of the activity on the immune microenvironment

The effect and benefit of ADA-expressing vaccinia virus on the immune microenvironment can be assessed in an ex vivo environment reconstituted from human tumors by quantifying leucocytes (e.g. granulocytes, monocytes, T lymphocytes, T-helper cells, T regulatory cells, cytotoxic T cells, B lymphocytes, thrombocytes, NK cells) by using markers (ex: CD45⁺, CD3⁺, CD4⁺, CD8⁺, etc.), quantifying intracellular Thl cytokines (e.g. IFNy, TNFa), assessing the activation status of myeloid cells (e.g. DC, MDSC), etc. The level of relevant metabolites can also be evaluated (e.g. ATP, ADP, AMP, ADO, INO and HX) and compared with the expression level of the key enzymes involved in the ADO levels modulation (e.g. CD39, CD73, ADA). One expects that the bioactivity of secreted huADAs will result in a measurable decrease of adenosine levels. Additionally, the treatment with the armed oncolytic vaccinia virus will lead to a higher activation of effector T cells and NK cells and an increase of the ratio CD8+ T cells / Treg (Fend et al., 2017, Cancer Res., 77(15):4146-4157).

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific method and reagents described herein, including alternatives, variants, additions, deletions, modifications and substitutions. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

BIBLIOGRAPHIC REFERENCES

Adra et al., 1987, Gene 60: 65-74

Allard et al., 2016, Immunotherapy, DOI: 10.2217/imt.l5.106

Andersen et al., 1992, European J. Biochem. 204: 51-56

Antonioli et al., 2013, Nature Reviews Cancer 13, 842-857

Beavis et al., 2013, Oncoimmunology 2(12): e26705

Boviatsis et al., Gene Ther. 1: 323-31

Broyles et al., 1993, Virol. 195: 863-5

Buller et al., 1985, Nature, 317(6040): 813-5

Caroll et al., 1997, Virology 238: 198-211

Carrey et al., 2011, PLoS ONE, 6(7) e22442)

Carrey et al., 2014, Sci Rep 4: 6154 doi 10.1038 Chaffee et al., 1992, J Clin Invest, 89: 1643-1651

Chambers et al., 1995, Proc. Natl. Acad. Sci. USA 92: 1411-5

Chakrabarti et al., 1997, Biotechniques, 23: 1094-7

Chernajovsky et al., 2006, BMJ, 332(7534): 170-2

Chu et al., 1997, J. Exp. Med., 186: 1623

Cristalli et al. 2001, Med Res Rev 21(2): 105-28

Dayhoffed, 1981, Suppl., 3: 482-9

Erbs et al., 2000, Cancer Res., 60(14):3813

Erbs et al., 2008, Cancer Gene Ther. 15(1): 18-28

Fallaux et al., 1998, Human Gene Ther. 9: 1909-17

Fend et al., 2017, Cancer Res., 77(15):4146-4157

Fisher et al., 2006, Curr. Opin. Mol. Ther., 8(4):301-13

Foloppe et al., 2008, Gene Ther., 15:1361-71

Freeman et al., 2006, Mol. Ther. 13(l):221-8)

Gakis et al., 1996, Eur Respir J, 9, 632-633

Geevarghese et al., 2010, Hum. Gene Ther. 21(9): 1119-28

Goubran et al., Cancer Growth Metastasis. 2014; 7: 9-18

Graham et al., 1997, J. Gen. Virol. 36: 59-72

Guse et al., 2011, Expert Opinion Biol. Ther.ll(5): 595-608

Hammond et al., 1997, J. Virol. Methods, 66: 135-8

Harrington et al., 2015, Expert Rev. Anticancer Ther. 15(12):1389-1403

Hausler et al., 2011, Cancer immunology, immunotherapy: Cll 60(10): 1405-1418

Hermiston et al., 2006, Curr. Opin. Mol. Ther., 8(4):322-30

Joyce JA, Pollard JW., 2009, Nat Rev Cancer ;9(4):239-252

Kaufmann et al., 2013, J. Invest. Dermatol. 133(4): 1034-42

Kern et al. (1990, Gene, 88: 149-57

Khuri et al., 2000, Nat. Med 6(8): 879-85

Kirn et al., 2001, Nat. Med. 7: 781

Kritsch et al., 2005, J. Chromatogr. Anai. Technol. Biomed. Life Sci., 822: 263-70 Kumar and Boyle, 1990, Virology, 179: 151-8

Kumar et al., 2009, Eur J Pharmacol., 616(1-3): 7-15

Lorence et al., 2007, Curr. Cancer Drug Targets 7(2):157-67

Lu et al., 2013, World J Gastroenterol, 19(12): 1912-1918 Martinussen et al., 1994, J. Bacteriol. 176: 6457-63

Martinussen et al., 1995, J. Bacteriol. 177: 271-4

Martuza et al., 1991, Science 252: 854-6

McDonald et al., 2006; Breast Cancer Treat. 99(2): 177-84

Mineta et al., 1994, Cancer Res. 54: 3363-66

Ohta et al., 2001, Nature 414(6866), 916-920

Ohta A., 2016, Front. Immunol., 7:109

Oldham and Dillman, 2009, Principles of cancer biotherapy, Fifth Edition, Springer

Phuangsab et al., 2001, Cancer Lett. 172(1): 27-36

Pyles et al., 1994, J. Virol. 68: 4963-72

Quail DF, Joyce JA., 2013, Nat. Med. 19(11):1423-1437

Ribeiro et al., 2001, J. Exp. Biol., 204(Pt 11): 2001-2010

Ribi et al., 1986, Plenum Publ. Corp.,407-419

Rosenberg et al., 1985, J Immunol 135(4): 2895-2903

Rudin et al., 2011, Clin. Cancer. Res. 17(4): 888-95

Smorlesi, 2005, Gene Ther. 12: 1324

Stern, B. and L. Olsen, 2007, Trends Cell Mol. Biol, 2:1-17

Stojdl et al., 2000, Nat. Med. 6(7): 821-5

Stojdl et al., 2003, Cancer Cell 4(4): 263-75

Suader, 2000, J. Am Acad Dermatol. 43: S6

Sumino et al., 1998, J.Virol. 72: 4931

Thorne et al., 2005, Curr. Opin. Mol. Ther., 7(4):359-65

Tritel et al., 2003, J. Immunol., 171: 2358

Ungerer et al, 1992, Clin Chem 38/7, 1322-1326

Van der Maaden et al., 2012, J. Control release 161: 645-55

Wang et al., 2006, Abstract 1472, AACR Annual Meeting

Wong et al., 2010, Viruses 2: 78-106

Wu et al., 2012, J Surg Oncol, DOI: 10.1002/jso.23056

Xia et al., 2004, Ai Zheng 23(12): 1666-70

Zavialov et Engstrom, 2005, Biochem J. 391(Pt 1): 51-57

Zhang et al., 2007, Cancer Res. 67:10038-46

EP998568

US 5,168,062 US6,054,438 US 8,394,385 US 8,772,023 W096/16183 WO98/02522 WO98/04727 WO98/10088 WO98/37095 W098/56415 WO01/66137 WO03/053463 WO2005/042728 WO2005/07857 WO2006/108846 WO2007/056847 WO2007/147528 WO2007/147529 WO2008/114021 WO2008/129058 WO2008/138533 WO2009/065546 WO2009/065547 WO2009/100521 WO2010/130753 WO2010/130756 WO2011/128704 WO2012/001075 WO2013/022764 WO2014/063832 WO2016061286

Claims

An oncolytic virus comprising a nucleotide sequence encoding at least one agent degrading one or more metabolic immune modulator(s).

The oncolytic virus of claim 1, wherein said metabolic immune modulator is an adenosine.

The oncolytic virus of claim 1, wherein said agent is an adenosine deaminase (ADA) polypeptide, or a polypeptide having an adenosine deaminase activity.

The oncolytic virus of claim 3, wherein said polypeptide is selected from the group consisting of ADA1, ADA2 and adenosine deaminase-like proteins.

The oncolytic virus of claim 3 or 4, wherein said polypeptide is a human adenosine deaminase

The oncolytic virus of claim 5, wherein said human adenosine deaminase is selected in the group composed by huADAl and huADA2.

The oncolytic virus of claim 6, wherein said huADAl comprises an amino acid sequence having at least 80%, preferably greater than 90%, and more preferably greater than 95% identity with SEQ ID NO:l.

The oncolytic virus of claim 6, wherein said huADA2 comprises an amino acid sequence having at least 80%, preferably greater than 90%, and more preferably greater than 95% identity with SEQ ID NO:3.

The oncolytic virus of any of claims 3 to 8, wherein said polypeptide catalyses the deamination of adenosine-based components to inosine-based components, like adenosine to inosine and deoxyadenosine to deoxyinosine.

10. The oncolytic virus of any of claims 1 to 9, wherein said virus is selected from the group consisting of poxvirus, herpes simplex virus (HSV), reovirus, Seneca Valley virus (SVV), vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), morbillivirus, retrovirus, adenovirus, measles virus, foamy virus, alpha virus, lentivirus, influenza virus, Sinbis virus, myxoma virus, rhabdovirus, picornavirus, coxsackievirus, parvovirus or the like.

11. The oncolytic virus of claim 10, wherein said virus is a poxvirus belonging to the Orthopoxvirus genus.

12. The oncolytic virus of claim 11, wherein said poxvirus belongs to the vaccinia virus (VV) species or to the cowpox virus species.

13. The oncolytic virus of claim 12, wherein said Vaccinia virus is selected from the group consisting of Copenhagen, Wyeth and Western Reserve (WR) strains.

14. The oncolytic virus of any of claims 10 to 13, wherein said virus is defective in the J2R locus.

15. The oncolytic virus of any of claims 10 to 14, wherein said virus is defective in the I4L and/or F4L locus.

16. The oncolytic virus of claim 14 or 15, wherein said virus is a vaccinia virus defective in the J2R locus and the I4L and/or F4L locus, and which encodes a human ADA, such as VVTK-RR- /huADAl or VVTK-RR-/huADA2.

17. The oncolytic virus of any of claims 10-16, wherein said virus further comprises one or more nucleic acid(s) of interest.

18. The oncolytic virus of claim 17, wherein said nucleic acid of interest encodes an immunostimulatory polypeptide, an antigen, a suicide gene product or any other nucleic acid of interest.

19. The oncolytic virus of claims 17 or 18, wherein said virus further comprises the elements necessary for the expression of said adenosine deaminase and said one or more nucleic acid(s) of interest.

20. A process for preparing an oncolytic virus, in which process:

(i) An oncolytic virus according to anyone of claims 1 to 19 is introduced into a producer cell; said producer cell is cultured under conditions which are appropriate for enabling said oncolytic virus to be produced, and;

said oncolytic virus is recovered from the cell culture.

21. A composition comprising the oncolytic virus of any one of claims 1 to 19, or pre according to claim 20, which further comprises a pharmaceutically acceptable vehicle.

22. The composition of claim 21, wherein said composition comprises a dose of oncolytic virus comprised between 10⁶ and 5xl0⁹ PFU.

23. The composition of claim 21 or 22, wherein said oncolytic virus is formulated for parenteral route administration with a preference for intravenous or intratumoural route.

24. An oncolytic virus of any one of claims 1 to 19, or prepared according to claim 20, or a composition of any one of claims 21 to 22, for use for the prophylaxis or the treatment of a proliferative disease or a disease associated with an increased osteoclast activity.

25. The oncolytic virus or the composition for use according to claim 24, wherein said proliferative diseases are cancers, tumours and restenosis.

26. The oncolytic virus for use according to claim 25, wherein said cancers are renal cancer, bladder cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, hepatic cancer, gastric cancer, pancreatic cancer, melanoma, ovarian cancer and glioblastoma, and especially metastatic ones.

27. The oncolytic virus for use according to claim 24, wherein said diseases associated to an increased osteoclast activity are rheumatoid arthritis and osteoporosis.

28. The oncolytic virus for use according to any one of claims 25 to 27, further comprising the administration of one or more substances effective in anticancer therapy.

29. The oncolytic virus for use according to claim 28, further comprising the administration of one or more substances which enhance adenosine deaminase activity.

30. A method for treating a disease or a pathologic condition in a subject in need thereof comprising the administration of an oncolytic virus according to anyone of claims 1 to 19, or prepared according to claim 20, or comprised in a composition as defined in claims 21 to 23, to a host organism or cell which is in need of such treatment.

31. The method of claim 30, wherein said disease or pathologic condition is a proliferative disease or a disease associated with an increased osteoclast activity.