WO2020011754A1

WO2020011754A1 - Chimeric vaccinia viruses

Info

Publication number: WO2020011754A1
Application number: PCT/EP2019/068340
Authority: WO
Inventors: Philippe Erbs; Johann Foloppe; Benoît GRELLIER
Original assignee: Transgene
Priority date: 2018-07-09
Filing date: 2019-07-09
Publication date: 2020-01-16

Abstract

The present invention relates to a chimeric vacciniaviruswith improved anticancer activity (higher cancer cell killing capacities and better tumor selectivity), which may further be recombinant and thus comprise one or more nucleic acid(s) of interest, and which may be included in a composition and used for the prophylaxis or the treatment of a disease, in particular proliferative disease (notably cancers) or of a disease associated with an increased osteoclast activity.

Description

CHIMERIC VACCINIA VIRUSES

TECHNICAL FIELD OF THE INVENTION

The present invention is in the field of oncolytic viruses and provides new chimeric vaccinia viruses obtained by a directed evolution methodology. More precisely, the invention provides chimeric vaccinia viruses which are obtained by the pooling of different strains of parental vaccinia viruses and the selection of more potent chimeric vaccinia viruses by cellular passages under stringent conditions. These chimeric vaccinia viruses are more oncolytic and safer in primary cells than the parental viruses largely used for vaccination.

The present invention also provides a chimeric vaccinia virus encoding a suicide polypeptide.

The present invention also deals with methods for obtaining chimeric vaccinia viruses.

The chimeric vaccinia viruses of the invention may be used for prophylaxis or treatment of proliferative diseases, like cancers.

BACKGROUND ART

Oncolytic viruses are a class of therapeutic agents that have the unique property of tumor-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 lyse their host cells. 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. Due to its potential to be more effective and less toxic than current therapies due to the viruses' selective growth and amplification in tumor cells, oncolytic virus therapy has been recognized as a promising new therapeutic approach for cancer treatment. Cancer cells are ideal hosts for many viruses because they have the antiviral interferon pathway inactivated or have mutated tumor 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.

Among the oncolytic viruses in clinical development, recombinant oncolytic Vaccinia viruses (VACV) are promising vectors for tumor therapy. The genome organization, lysis capacity and wide tumor tropism of VACV, the most commonly used poxvirus vector for cancer therapy, make it an ideal oncolytic agent for cancer treatment (Haddad et al., 2017, Front Oncol., 7, 96. doi:10.3389/fonc.2017.00096). It appears to target tumors selectively after systemic administration and thus displays natural tumor tropism (McFadden, 2005, Nat Rev Microbiol., 3, 201-213). Several strains of VACV are currently evaluated in preclinical and clinical trials including Wyeth, Western Reserve, Copenhagen and Lister strains (Heo et al., 2013 Nat Med., 19, 329-336. doi: 10.1038/nm.3089, Zeh et al., 2015, Mol Ther., 23, 202-214. doi: 10.1038/mt.2014.194, Foloppe et al., 2008, Gene Ther., 15, 1361-1371. doi: 10.1038/gt.2008.82, Mell et al., 2017, Clin Cancer Res., 23, 5696-5702. doi: 10.1158/1078-0432. CCR-16-3232).

Modifications of the naturally occurring viruses has already been practiced in order to enhance the ability of vaccinia viruses to infect and lyse 100% of the tumor cells, which is difficult to achieve in in vivo context. For this purpose, the main strategies used currently to modify the viruses include: functional deletions in viral genes, the use of tumor- 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. Therefore, oncolytic vaccinia viruses are often "armed" with enzyme-prodrug system that enhance the oncolytic efficacy of the virus therapy by exerting a strong bystander effect and thus permit elimination of neighboring uninfected tumor cells. For example, armament with the so-called FCU1 suicide gene, encoding a bifunctional chimeric polypeptide that combines the enzymatic activities of FCY1 and FUR1, efficiently catalyzed the direct conversion of 5-fluorocytosine (5-FC), a nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil (5-FU) and 5-fluorouridine- 5'monophosphate (5-FUMP), thus bypassing the natural resistance of certain human tumor cells to 5- fluorouracil (Erbs et al., 2000, Cancer Res., 60(14): 3813-22).

Viral modifications can also be used to increase safety. In this regard, thymidine kinase (TK) deleted virus was shown to have decreased pathogenicity compared with wild type virus, but replication in tumor cells was preserved (Buller et al., 1985, Nature, 317(6040):813-5). Foloppe et al. showed that a TK gene-deleted VACV expressing the FCU1 gene has potent anti-tumor effect both in vitro and in vivo in a murine model of a human colon tumor (Foloppe et al., 2008, Gene Ther., 15:1361-1371). The TK- deleted poxviruses expressing the FCU1 fusion suicide gene, combined with the administration of the 5-fluorocytosine (5-FC) prodrug, displayed a highly potent anti-tumor effect both in vitro and in vivo. The potential of FCU1 delivered by cowpox virus as therapeutic agent was also explored (Ricordel et al., 2017, Molecular Therapy - Oncolytics, 7: 1-11).

Attenuated vaccinia virus strains have thus been developed for therapeutic and diagnostic applications and are being evaluated in clinical studies. However, methods of attenuating vaccinia viruses lead to a decrease of their efficacy. In a therapeutic point of view, this decrease of efficacy can result into a diminution of overall response, a diminution of patient's survival, an increase of mortality, pathology resistances... As a result, the first oncolytic vaccinia viruses tested in clinical trials have been highly safe in patients but have generally fallen short of their expected therapeutic value as monotherapies. Moreover, some tumor cells appeared to be poorly permissive or resistant to infection and replication of oncolytic vaccinia viruses, meaning that some cancers are still refractory or resistant to oncolytic virus-based, or oncolytic vaccinia virus-based therapy (e.g. National Clinical Trial NCT01380600: safety but no efficacy of Pexa-Vec (pexastimogene devacirepvec, JX-594, an oncolytic and immunotherapeutic vaccinia Wyeth (WY) based virus engineered to express GM-CSF) in colorectal carcinoma patients who are refractory to, or intolerant to, oxaliplatin, irinotecan, and Erbitux treatments ; National Clinical Trial NCT01387555: no efficacy of Pexa-Vec in patients suffering from advanced liver cancer who failed sorafenib ; National Clinical Trial NCT01636284 : no efficacy of Pexa- Vec in Sorafenib-nai^'ve advanced liver cancer patients).

Different approaches have been employed to improve the oncolytic potency of vaccinia viruses, including combination therapies with standard and emerging anticancer therapies (Filley et al., 2017, Front Oncol. 7, 106. doi: 10.3389/fonc.2017.00106). For example, Pexa-Vec has now entered a randomized controlled Phase 3 trial in advanced first-line hepatocarcinoma (HCC) comparing the administration of Pexa-Vec and sorafenib to sorafenib alone (National Clinical Trial NCT02562755). TG6002 (Heinrich et al., 2017, Onco Targets Ther., 10, 2389-2401. doi: 10.2147/OTT.S126320), which is a derivative of a Copenhagen (COP) based TK-deleted VACV expressing the FCU1 fusion suicide gene and given in combination with 5-FC, is now entering clinical development in recurrent glioblastoma patients (National Clinical Trial NCT03294486). Combination of vaccinia virus with immune checkpoint inhibitors (ICI) is tested: a phase 1 study is ongoing combining Pexa-Vec with Nivolumab for the treatment of HCC in patients naive to the standard of care, sorafenib (National Clinical Trial NCT03071094).

TECHNICAL PROBLEM AND PROPOSED SOLUTION

However, despite the different current approaches tested, like viral modifications or armaments, the different vaccinia virus platforms are still not oncolytic enough and do not provide satisfying therapeutic results. Moreover, vaccinia viruses are not capable of infecting and replicating in every tumor cells, which means that some cancers are refractory or resistant to oncolytic vaccinia virus- based therapies.

As a result, there is still a need for highly potent oncolytic vaccinia viruses. The use of more oncolytic vaccinia viruses will result in better cancer cell killing capacities, whether they are administrated in monotherapy or combined with other anticancer therapies. The use of these vaccinia viruses will also result in an increased oncolytic effect on cells poorly permissive or resistant to the existing vaccinia viruses. In the context of the present invention, the inventors have generated highly oncolytic chimeric vaccinia viruses via a directed evolution method, which is a method used to mimic the process of natural selection to evolve viruses. Viruses undergo genetic changes by several mechanisms, including point mutation and recombination. Recombination is a widespread phenomenon in viruses and can have a major impact on their evolution. Recombination occurs when at least two viral homologous sequences co-infect the same host cells and exchange genetic fragments. Homologous recombination (HR) occurs in the same site in both parental strands and creates new genetic combinations that may change phenotype of the chimeric viruses. Homologous recombination is the basis for many widely used genetic techniques in virus research, including construction of recombinant vectors (Hruby, 1990, Clin. Microbiol. Rev, 3(2) 153-170). In 1958, experimentations showed that different strains of poxviruses could recombine (Fenner and Comben. 1958, Virology, 5, 530-548). Some characteristics of the obtained chimeras were studied (e.g. virus replication, heat resistance or hemagglutinin production), but no oncolytic power or therapeutic index were explored. Paszkowski et al. have studied the mechanism of poxvirus genetic recombination (Paszkowski et al., 2016, PLOS Pathogens, 12(8) el005824). They have observed multiple genetic exchanges even after one round of selection, showing that homologous intramolecular and intermolecular recombination occurred efficiently; however, no functional characteristics were studied.

The directed evolution methodology is usually employed for the creation of gene libraries (Koerber et al., 2006, Nat. Protocols 1(2) p.701-706). This methodology applied to oncolytic virotherapy has a different purpose. It was recently used for obtaining an oncolytic chimeric orthopoxvirus (O'Leary et al., 2018, Mol. Therap. Vol 9: 13-21) by the pooling of nine strains of orthopoxviruses known to be oncolytic on non-resistant tumor cells. The mix of viruses was grown and shuffled on a non-tumor cell line (African green monkey kidney fibroblasts CV-1), a cell line usually used in the research field for poxvirus production, due to its high permissiveness to orthopoxviruses replication. However, in HCT116 colorectal cancer cell line, the generated chimeric CF33 virus secreted less EEV at early stages than IHD parental strain and did not show improved replication than parental Western Reserve (WR) and Elstree strains at 72h post-infection. The tumor specificity of the chimeric virus has not been compared to parental strains.

Therefore, there is still a need for new oncolytic vaccinia viruses with better anti-cancer efficacy than the existing oncolytic vaccinia viruses. These viruses should have improved tumor cell killing capacities in order to treat cancers, especially cancers refractory or resistant to current therapies with oncolytic viruses or oncolytic vaccinia viruses. The viruses should also have an improved tumor selectivity, in order to be of safe use for the treated subjects.

In the context of the invention, the inventors showed that it was possible to create new chimeric vaccinia viruses and new recombinant chimeric vaccinia viruses under selective pressure, but importantly and surprisingly, that these new viruses achieved better anticancer activity than the parental strains: they have both higher cancer cell killing capacities and better tumor selectivity as they replicate less than the parental strains in the primary cells.

In the invention, the starting pool consisted in a mix of different vaccinia virus strains: Modified Vaccinia Virus Ankara (MVA), Copenhagen (COP), Wyeth (WY) and Western Reserve (WR). Three of the parental vaccinia virus strains are known to be oncolytic (COP, WY and WR), but MVA is known not to replicate efficiently in mammalian cells and thus not to be oncolytic (Guse et al., 2011, Expert Opinion Biol. Ther.ll(5): 595-608). Despite the presence of MVA in parental strains, after exposure of this mix to a directed evolution method, the inventors selected a chimeric vaccinia virus, named deVV5 (wild type chimeric virus), with enhanced oncolytic properties in vitro. Unexpectedly, the enhanced anticancer activity (higher cancer cell killing capacities and better tumor selectivity) was present despite the fact that a significant proportion of the genome of the chimeric vaccinia virus was derived from MVA. The inventors also explored the feasibility of this approach using deVV5 as viral vector, and thus constructed an armed oncolytic chimeric vaccinia virus encoding the FCU1 polypeptide. It appeared that deVV5 encoding FCU1 (deVV5-fcul) had also better anticancer activity than the parental strains, suggesting that insertion of a transgene in the chimeric vaccinia virus does not alter its enhanced anticancer activity.

Based on these results, one may anticipate that the chimeric vaccinia viruses of the invention may be successfully used for replacing the existing oncolytic viruses and may have a better efficiency profile in vivo. The chimeric vaccinia viruses of the invention may also be advantageous for treating cancers refractory or resistant to vaccinia virus-based therapy. The chimeras of the invention can also be exploited in combination with additional anticancer therapy/ies.

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.

SUMMARY OF THE INVENTION

The inventors have generated a chimeric vaccinia virus with improved anticancer activity (higher cancer cell killing capacities and better tumor selectivity), which may further be recombinant and thus comprise one or more nucleic acid(s) of interest, and which may be included in a composition and used for the prophylaxis or the treatment of a disease, in particular proliferative disease (notably cancers) or of a disease associated with an increased osteoclast activity.

The chimeric vaccinia virus with improved anticancer activity (higher cancer cell killing capacities and better tumor selectivity) is defined as comprising at least one protein, nucleic acid, or functional feature as described below, or as being obtained or obtainable by a specific method of directed evolution as described below.

In a first aspect, the invention provides a chimeric vaccinia virus (optionally recombinant), wherein said chimeric vaccinia virus encodes:

(a) at least 304 distinct proteins, each of said 304 distinct proteins comprising an amino acid sequence with at least 70% identity with one of SEQ ID NO:6 to 318;

(b) at least 304 distinct proteins, each of said proteins comprising an amino acid sequence with at least 80% identity with one of SEQ ID NO:6 to 318;

(c) at least 301 distinct proteins, each of said proteins comprising an amino acid sequence with at least 90% identity with one of SEQ ID NO:6 to 318;

(d) at least 301 distinct proteins, each of said proteins comprising an amino acid sequence with at least 91% identity with one of SEQ ID NO:6 to 318;

(e) at least 299 distinct proteins, each of said proteins comprising an amino acid sequence with at least 92% identity with one of SEQ ID NO:6 to 318;

(f) at least 298 distinct proteins, each of said proteins comprising an amino acid sequence with at least 93% identity with one of SEQ ID NO:6 to 318;

(g) at least 296 distinct proteins, each of said proteins comprising an amino acid sequence with at least 94% identity with one of SEQ ID NO:6 to 318;

(h) at least 295 distinct proteins, each of said proteins comprising an amino acid sequence with at least 95% identity with one of SEQ ID NO:6 to 318;

(i) at least 293 distinct proteins, each of said proteins comprising an amino acid sequence with at least 96% identity with one of SEQ ID NO:6 to 318;

(j) at least 290 distinct proteins, each of said proteins comprising an amino acid sequence with at least 97% identity with one of SEQ ID NO:6 to 318;

(k) at least 282 distinct proteins, each of said proteins comprising an amino acid sequence with at least 98% identity with one of SEQ ID NO:6 to 318;

(L) at least 255 distinct proteins, each of said proteins comprising an amino acid sequence with at least 99% identity with one of SEQ ID NO:6 to 318; or (m) at least 205 distinct proteins, each of said proteins comprising an amino acid sequence selected from SEQ ID NO:6 to 318.

In a second aspect, the invention provides a chimeric vaccinia virus (optionally recombinant), wherein said chimeric vaccinia virus encodes:

(a) a protein comprising an amino acid sequence with at least 96.3 % of identity with SEQ ID NO:

54,

(b) a protein comprising an amino acid sequence with at least 99.6 % of identity with SEQ ID NO:

62,

(c) a protein comprising an amino acid sequence with at least 98.7 % of identity with SEQ ID NO:

64,

(d) a protein comprising an amino acid sequence with at least 99.4 % of identity with SEQ ID NO:

143,

(e) a protein comprising an amino acid sequence with at least 99,9 % of identity with SEQ ID NO:

147,

(f) a protein comprising an amino acid sequence with at least 99,9 % of identity with SEQ ID NO:

155, or

(g) any combination of proteins (a) to (f).

In a third aspect, the invention provides a chimeric vaccinia virus (optionally recombinant) having at least 66% of identity with SEQ ID NO:l.

In a fourth aspect, the invention provides a chimeric vaccinia virus (optionally recombinant), wherein at least one organ-specific therapeutic index of said chimeric vaccinia virus is higher than the corresponding organ-specific therapeutic indexes of parental vaccinia virus strains Copenhagen (COP), Modified Vaccinia Ankara (MVA), Wyeth (WY), and Western Reserve (WR), wherein for a given organ and a given virus, an organ-specific therapeutic index TI(organ, virus) is defined as:

TI(organ, virus) = (replication of virus in organ tumor cells / replication of virus in organ healthy cells).

In a fifth aspect, the invention provides a chimeric vaccinia virus (optionally recombinant), wherein at least one tumor-specific oncolytic power of said chimeric vaccinia virus is higher than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR, wherein for a given tumor and a given virus, a tumor-specific oncolytic power OP(tumor, virus) is defined as:

OP(tumor, virus) = (100 - percentage of surviving tumor cells after infection by virus). In a sixth aspect, the invention provides a method of directed evolution for obtaining a chimeric vaccinia virus with high oncolytic power, said method comprising:

(i) amplifying a plurality of parental vaccinia virus strains on a tumor cell line, wherein said tumor cell line is poorly permissive or non-permissive for each parental vaccinia virus strain;

(ii) collecting the supernatant containing one or more variant chimeric vaccinia virus(es) having a non zero oncolytic power in the tumor cell line of step (i) after at least 2 days at a supernatant dilution of at least 2, wherein for a given tumor and a given virus, a tumor-specific oncolytic power OP(tumor, virus) is defined as:

OP(tumor, virus) = (100 - percentage of surviving tumor cells after infection by virus);

(iii) amplifying one or more variant chimeric vaccinia virus(es) of the collected supernatant on a tumor cell line, wherein said tumor cell line is poorly permissive or non-permissive for each parental vaccinia virus strain;

(iv) collecting the supernatant containing one or more chimeric vaccinia virus(es) having a non-zero oncolytic power in the tumor cell line of step (iii) after at least 2 days at a supernatant dilution of at least 2;

(v) repeating steps (iii) and (iv), until one or more variant chimeric vaccinia virus(es) having a desired oncolytic power in tumor cell lines used in performed steps (i) and/or (iii) is obtained.

In a seventh aspect, the invention provides a chimeric vaccinia virus, wherein said chimeric vaccinia virus has been or may be obtained by the above method.

The invention also provides a chimeric vaccinia virus comprising several features of the chimeric vaccinia virus described in aspects one to five and seven above.

The invention also provides derivatives of any chimeric vaccinia virus according to aspects one to five and seven above, including recombinant derivatives (i.e. further comprising one or more nucleic acid(s) of interest) and/or derivatives defective in one or more loci, notably deficient in the TK locus, in particular derivatives in which the TK locus has been deleted.

A recombinant vaccinia virus according to the invention may notably encode a suicide polypeptide. More preferably, said suicide polypeptide has a cytosine deaminase (CDase) activity or a uracyl phosphoribosyl transferase (UPRTase) activity or both CDase and UPRTase activities. In this embodiment, said vaccinia virus can be used with a pharmaceutically acceptable quantity of prodrug. A recombinant vaccinia virus according to the invention may alternatively or also encode one or more polypeptide(s) of therapeutic interest, which a preferably selected from polypeptides capable of reinforcing the oncolytic nature of the chimeric vaccinia virus, polypeptides capable of potentiating anti-tumor efficacy, antigens for inducing or activating an immune humoral and/or cellular response, and permease.

In a further aspect, the present invention also concerns a process for producing a chimeric vaccinia virus, comprising at least the steps of:

(i) infecting a host cell with the chimeric vaccinia virus according to the invention as disclosed herein;

(ii) culturing said host cell under conditions which are appropriate for enabling chimeric vaccinia virus particles to be produced, and;

(iii) recovering said chimeric vaccinia virus particles from the cell culture.

Optionally, the recovered chimeric vaccinia virus can be purified at least partially.

In another aspect of the invention is provided a composition comprising the chimeric vaccinia virus of the invention and a pharmaceutical acceptable vehicle. In one embodiment, the chimeric vaccinia virus is preferably formulated for intra-venous or intra-tumoral administration.

Another aspect of the present invention relates to the chimeric vaccinia 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 a cancer.

Another aspect of the present invention relates to a method for treating a disease in a subject in need thereof comprising the administration to said subject of the chimeric vaccinia virus or the composition according to the invention. In one embodiment, said disease is a proliferative disease such as a cancer.

DESCRIPTION OF THE FIGURES

Figure 1: Oncolytic activities during the different steps of the directed evolution process

Oncolytic activities during the different steps of the directed evolution process (a) Percentage of surviving cells 5 days after infection with LP6, LP9 and COP. Human tumor cells were infected at a MOI of lO ⁵ and 10⁴ with COP and the indicated passages and cell viability was determined by trypan blue exclusion. The parental COP was used as reference. The results are presented as a mean of triplicate experiments ± SD. (b) Percentage of surviving cells after infection of MIA PaCa-2 tumor cell line with the indicated passages at MOI 10⁵ and 10⁴. The parental COP was used as reference. MIA PaCa-2 was infected at MOI of 10⁵ and 10⁴ and the cell viability was determined 5 days after infection. The results are presented as a mean of triplicate experiments ± SD. (c) Percentage of surviving cells 5 days after infection of MIA PaCa-2 tumor cell line with the indicated clones at MOI 10⁴ and 10⁵. The parental COP and MP12 were used as references. The results are presented as a mean of triplicate experiments ± SD.

Figure 2: Homology maps between deVV5 and each parental genome.

(a) De novo sequenced viral genomes are shown around the circos plot (COP, MVA, WY, WR, and deVV5). Only one ITR is displayed (black). Blast results of deVV5 regions greater than lkb with 100% identity are linked by grey-coded ribbons to the corresponding hit. (b) deVV5 genome annotated with results from global pairwise alignments. Each annotation corresponds to the longest region with 100% identity to the corresponding parental genome. deVV5 core and single ITR region are also displayed (c) Percentage of nucleic acid identity between the deVV5 and the parental strains sequences, obtained via global pairwise alignments (MAAFT).

Figure 3: Functionality and efficacy of FCU1 expressed by deVV5.

(a) Conversion of 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) and release of 5-FU in the cell culture supernatant. HCT116 cells were infected with the indicated vector at a MOI of 10 ^s and then incubated with 0.1 mM 5-FC from day 2 to day 5 post infection. The relative concentration of 5-FC and 5-FU in the culture supernatant was measured by HPLC. The results are expressed as the percentage of 5-FU released relative to the total amount of 5-FC + 5-FU. Each data point represents the mean of triplicate determinations ± S.D. (b) In vitro sensitivities of infected human tumor cells to 5-FC. HCT116 human tumor cells were mock-infected or infected with deVV5 and deVV5-fcul at a MOI of 10 ^s. After 48h, cells were treated by increasing concentrations of 5-FC. Cell survival was determined 3 days later as described in Materials and methods section. Cell viability results are expressed as the percentage of viable cells in the presence and absence of the prodrug. Values are represented as mean ± S.D. of triplicate determinations.

Figure 4. Both chimeric vaccinia virus and recombinant chimeric vaccinia virus are able to infect and kill cancer cells leaving primary cells intact.

(a) Oncolytic activities of COP, deVV5 and deVV5-fcul by measuring the cell viability 5 days after infection of different cancer cell lines. Tumor cells were infected with the indicated viruses and cell viability was determined as described in the Materials and Methods section. The MOI used for the infection was MOI 10 ^s for UM-UC-3, SK-OV-3, CAL33, A549 and Hep G2 cells, MOI 10⁴ for OE19, MIA PaCa-2 and HCT 116 cells and MOI 10-₃ for KATO III cells (b) Replication in tumor cell lines and in primary human cells. Tumor cells were infected at MOI 10⁵ and harvested 3 days post infection. Human primary hepatocytes were infected with 100 PFU/well and harvested 3 days post infection. 3D Phenion FT skin models were infected with 1.10^s PFU and harvested 7 days post infection. Viral progeny production was determined by plaque titration. Results are expressed as viral fold amplification (corresponding to output/input ratio) (c) Therapeutic index calculated by ratio between viral production obtained in Hep G2 hepatocarcinoma cells and hepatocytes.

DETAILED DESCRIPTION OF THE INVENTION

GENERAL 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 invention and how to use them. Moreover, alternative language and synonyms may be used for any one of the terms 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 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 "a chimeric vaccinia virus" encompasses a single chimeric vaccinia virus as well as a plurality of chimeric vaccinia 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 "at least" refers to either the number preceded by the expression "at least", considered as the minimum, or a number above said minimum.

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 10%, and more preferably within 5% of a given value or range.

The term "distinct" means "not identical", "distinguished as not being the same". For example, two distinct proteins have different amino-acid sequences, different shapes, etc.

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-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. In the present description, each time the term "comprising" (or any of its derivatives such as "comprise” and "comprises") is used, the invention also relates to the same embodiment in which "comprising" (or any of its derivatives such as "comprise” and "comprises") is replaced by "consisting essentially of" or "consisting of".

The terms "protein", "polypeptide", and "peptide" are used interchangeably and 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". Proteins, polypeptides and peptides are defined by amino acid sequences.

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 "nucleotide" refers to any of various compounds consisting of a sugar, usually ribose or deoxyribose, a purine or pyrimidine base, and one or more phosphates. The expression "nucleotide" designates both ribonucleotides and deoxyribonucleotides.

In a general manner, the term "identity" or "identical" in the context of a virus sample refers to an amino acid to amino acid, or nucleotide to nucleotide correspondence between a polypeptide of the virus and another polypeptide of reference or between a nucleic acid sequence of the virus and another nucleic acid sequence of reference respectively. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences after optimal global alignment, taking into account the number of gaps which need to be introduced for optimal alignment of the two entire sequences 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 after optimal global alignment, 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), and the algorithm of Needleman et Wunsh (J.Mol. Biol. 48,443-453, 1970). Programs for determining identity between nucleotide sequences optimal global alignment are also available in specialized data base (e.g. Genbank, the Wisconsin Sequence Analysis Package, BESTFIT, FASTA and GAP programs).

The term "cluster" or "gene cluster" refer to a group of two or more genes, found within an organism's DNA. The size of gene clusters can vary significantly, from a few genes to several hundred genes.

The term "recombinant" when used with reference, e.g. to a nucleic acid, a protein or a virus, indicates that the nucleic acid, the protein or the virus has been modified by the introduction of a heterologous nucleic acid or protein, or the alteration of a native nucleic acid. Thus, for example, a recombinant chimeric virus expresses at least one gene that is not found in the native form of the chimeric virus.

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 chimeric vaccinia virus of the invention (also designated as producer cells). Producer cells are permissive for infection and replication of the chimeric vaccinia virus of the invention. This term also includes cells which can be or have been the recipient of the virus described herein as well as progeny of such cells.

The term "therapeutic index" represents the ratio between the viral replication on tumor and corresponding healthy cells. For a given organ and a given virus, an organ-specific therapeutic index TI(organ, virus) is defined as follows:

This therapeutic index is improved by the increase of the replicative activity on tumor cells and/or by the decrease of the replication on corresponding healthy cells.

The term "oncolytic" as used herein refers to the ability of a virus to replicate 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. An oncolytic virus may be characterized by its oncolytic power. Said oncolytic power for a given tumor and a given virus is a tumor-specific oncolytic power OP(tumor, virus), defined as:

OP(tumor, virus) = (100 - percentage of surviving tumor cells after infection by virus).

The oncolytic power is expressed as a percentage and represents the percentage of tumor cells lysed by a given virus in specific conditions. For example, a vaccinia virus strain Copenhagen has an oncolytic power of 56,4% on U-87-MG tumor cells, meaning that 56.4% of U-87-MG tumor cells were lysed by COP, more preferably 5 days post infection and at a MOI of 10 ^s, even more preferably wherein U-87- MG are cultured at 37° C, 5% C02, in Dulbecco's Modified Eagle Medium (DMEM) with 10% of Fetal Calf Serum (FCS). The higher the percentage is, the more oncolytic a given virus for a given tumor is.

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 a chimeric vaccinia virus, a recombinant chimeric vaccinia virus or a composition thereof 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 vaccinia 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 "tumor", "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 a chimeric vaccinia 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.

"CHIMERIC VACCINIA VIRUS". OR "VACCINIA VIRUS CHIMERA"

The terms "chimeric vaccinia virus" or "vaccinia virus chimera", "chimeric virus", "virus chimera" or "chimera" are interchangeable and used according to their ordinary meaning in virology: they refer to a hybrid vaccinia virus created by joining nucleic acid fragments from two or more different virus strains, said viruses belonging to the vaccinia virus species.

The terms "vaccinia virus", "vaccinia virus particle", "vaccinia virus vector" and "vaccinia virus virion" (also designated under VACV) are used interchangeably and are to be understood as meaning a vehicle comprising at least one element of a wild-type vaccinia virus genome. It is preferred that the vaccinia virus particle is infectious (i.e. capable of infecting and entering a host cell or subject). This term encompasses both native as well as genetically modified (e.g. engineered) VACV viruses.

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 are intracellular (IMV for intracellular mature virion) with a single lipid envelope and remains in the cytosol of infected cells until lysis. The extracellular forms are enveloped particles with an additional membrane that buds out from the infected cell (e.g. CEV for cell-associated enveloped virus, and EEV for extracellular enveloped virus).

The nucleic acid sequences of vaccinia viruses are composed by a core sequence and two inverted terminal repeats (ITR). The terms "core", "core region" or "core sequence" are used interchangeably and designate a nucleic acid region of a vaccinia virus which is the main viral nucleic acid sequence, flanked by the two ITRs. The length of the core region differs from a vaccinia virus strain to another. The terms "inverted terminal repeats" or "ITR" designate nucleic acid regions which are duplicated and inverted at both 5' and 3' ends of the viral genome. ITRs are composed of non-coding repeated patterns (e.g.: short tandem repeat, microsatellites, minisatellites, etc.) at their extremities that can vary between two viruses. A vaccinia virus comprises two ITRs, one located on the 5' end and the other located on the 3' end of the viral nucleic acid sequence, each one being the reverse complement of the other. The length of the ITRs differs from a vaccinia virus strain to another.

The term "Copenhagen" or "COP" is used according to its common, ordinary meaning and refers to virus strains of the same and similar names and functional fragments and homologs thereof. The term includes naturally or recombinant occurring forms of vaccinia virus strain COP or variants thereof that maintain COP activity. The term includes naturally or recombinant occurring forms of vaccinia virus strain COP or variants thereof whose genome has sequence identity to the vaccinia virus strain COP genome (e.g. about 97%, 98%; 99% or 100%). In the embodiment, the COP genome corresponds to SEQ ID NO: 2, representing the core region (comprised between nucleotide 1 and 167587) and one of the two ITRs (comprised between nucleotide 167588 and 175766).

The term "Modified Vaccinia Virus" or "MVA" is used according to its common, ordinary meaning and refers to virus strains of the same and similar names and functional fragments and homologs thereof. The term includes naturally or recombinant occurring forms of vaccinia virus strain MVA or variants thereof that maintain MVA activity. The term includes naturally or recombinant occurring forms of vaccinia virus strain MVA or variants thereof whose genome has sequence identity to the vaccinia virus strain MVA genome (e.g. about 97%, 98%; 99% or 100%). In the embodiment, the MVA genome corresponds to SEQ ID NO: 3, representing the core region (comprised between nucleotide 1 and 159359) and one of the two ITRs (comprised between nucleotide 159360 and 163668). In the embodiment, the vaccinia virus strain MVA genome expresses the eGFP gene under the control of the pllk7.5 promoter (MVA-GFP) and was constructed and characterized previously (Erbs et al., 2008, Cancer Gene Ther. 2008, 15, 18-28).

The term "Wyeth" or "WY" is used according to its common, ordinary meaning and refers to virus strains of the same and similar names and functional fragments and homologs thereof. The term includes naturally or recombinant occurring forms of vaccinia virus strain Wyeth or variants thereof that maintain Wyeth activity. The term includes naturally or recombinant occurring forms of vaccinia virus strain Wyeth or variants thereof whose genome has sequence identity to the vaccinia virus strain Wyeth genome (e.g. about 97%, 98%; 99% or 100%). In the embodiment, the WY genome corresponds to SEQ ID NO: 4, representing the core region (comprised between nucleotide 1 and 166640) and one of the two ITRs (comprised between nucleotide 166641 and 182933).

The term "Western Reserve" or "WR" is used according to its common, ordinary meaning and refers to virus strains of the same and similar names and functional fragments and homologs thereof. The term includes naturally or recombinant occurring forms of vaccinia virus strain Western Reserve or variants thereof that maintain Western Reserve activity. The term includes naturally or recombinant occurring forms of vaccinia virus strain Western Reserve or variants thereof whose genome has sequence identity to the vaccinia virus strain Western Reserve genome (e.g. about 97%, 98%; 99% or 100%). In the embodiment, the WR genome corresponds to SEQ ID NO: 5, representing the core region (comprised between nucleotide 1 and 174338) and one of the two ITRs (comprised between nucleotide 174339 and 181350).

Chimeric vaccinia virus defined by protein features

In a first aspect, the invention provides a chimeric vaccinia virus (optionally recombinant), wherein said chimeric vaccinia virus encodes one of (a) to (m) below:

(a) at least 304 distinct proteins, each of said 304 distinct proteins comprising an amino acid sequence with at least 70% identity with one of SEQ ID NO:6 to 318.

Preferably, said chimeric vaccinia virus encodes at least 305, at least 306, at least 307, at least 308, at least 309, at least 310, at least 311, at least 312, or even 313 distinct proteins, each of said proteins comprising an amino acid sequence with at least 70% identity with one of SEQ ID NO:6 to 318.

(b) at least 304 distinct proteins, each of said proteins comprising an amino acid sequence with at least 80% identity with one of SEQ ID NO:6 to 318.

Preferably, said chimeric vaccinia virus encodes at least 305, at least 306, at least 307, at least 308, at least 309, at least 310, at least 311, at least 312, or even 313 distinct proteins, each of said proteins comprising an amino acid sequence with at least 80% identity with one of SEQ ID NO:6 to 318.

(c) at least 301 distinct proteins, each of said proteins comprising an amino acid sequence with at least 90% identity with one of SEQ ID NO:6 to 318. Preferably, said chimeric vaccinia virus encodes at least 302, at least 303, at least 304, at least 305, at least 306, at least 307, at least 308, at least 309, at least 310, at least 311, at least 312, or even 313 distinct proteins, each of said proteins comprising an amino acid sequence with at least 90% identity with one of SEQ ID NO:6 to 318.

(d) at least 301 distinct proteins, each of said proteins comprising an amino acid sequence with at least 91% identity with one of SEQ ID NO:6 to 318.

Preferably, said chimeric vaccinia virus encodes at least 302, at least 303, at least 304, at least 305, at least 306, at least 307, at least 308, at least 309, at least 310, at least 311, at least 312, or even 313 distinct proteins, each of said proteins comprising an amino acid sequence with at least 91% identity with one of SEQ ID NO:6 to 318.

(e) at least 299 distinct proteins, each of said proteins comprising an amino acid sequence with at least 92% identity with one of SEQ ID NO:6 to 318.

Preferably, said chimeric vaccinia virus encodes at least 300, at least 301, at least 302, at least 303, at least 304, at least 305, at least 306, at least 307, at least 308, at least 309, at least 310, at least 311, at least 312, or even 313 distinct proteins, each of said proteins comprising an amino acid sequence with at least 92% identity with one of SEQ ID NO:6 to 318.

(f) at least 298 distinct proteins, each of said proteins comprising an amino acid sequence with at least 93% identity with one of SEQ ID NO:6 to 318.

Preferably, said chimeric vaccinia virus encodes at least 299, at least 300, at least 301, at least 302, at least 303, at least 304, at least 305, at least 306, at least 307, at least 308, at least 309, at least 310, at least 311, at least 312, or even 313 distinct proteins, each of said proteins comprising an amino acid sequence with at least 93% identity with one of SEQ ID NO:6 to 318.

(g) at least 296 distinct proteins, each of said proteins comprising an amino acid sequence with at least 94% identity with one of SEQ ID NO:6 to 318.

Preferably, said chimeric vaccinia virus encodes at least 297, at least 298, at least 299, at least 300, at least 301, at least 302, at least 303, at least 304, at least 305, at least 306, at least 307, at least 308, at least 309, at least 310, at least 311, at least 312, or even 313 distinct proteins, each of said proteins comprising an amino acid sequence with at least 94% identity with one of SEQ ID NO:6 to 318.

(h) at least 295 distinct proteins, each of said proteins comprising an amino acid sequence with at least 95% identity with one of SEQ ID NO:6 to 318.

Preferably, said chimeric vaccinia virus encodes at least 296, at least 297, at least 298, at least 299, at least 300, at least 301, at least 302, at least 303, at least 304, at least 305, at least 306, at least 307, at least 308, at least 309, at least 310, at least 311, at least 312, or even 313 distinct proteins, each of said proteins comprising an amino acid sequence with at least 95% identity with one of SEQ ID NO:6 to 318.

(i) at least 293 distinct proteins, each of said proteins comprising an amino acid sequence with at least 96% identity with one of SEQ ID NO:6 to 318.

Preferably, said chimeric vaccinia virus encodes at least 295, at least 300, at least 301, at least 302, at least 303, at least 304, at least 305, at least 306, at least 307, at least 308, at least 309, at least 310, at least 311, at least 312, or even 313 distinct proteins, each of said proteins comprising an amino acid sequence with at least 96% identity with one of SEQ ID NO:6 to 318.

(j) at least 290 distinct proteins, each of said proteins comprising an amino acid sequence with at least 97% identity with one of SEQ ID NO:6 to 318.

Preferably, said chimeric vaccinia virus encodes 295, at least 300, at least 301, at least 302, at least 303, at least 304, at least 305, at least 306, at least 307, at least 308, at least 309, at least 310, at least 311, at least 312, or even 313 distinct proteins, each of said proteins comprising an amino acid sequence with at least 97% identity with one of SEQ ID NO:6 to 318.

(k) at least 282 distinct proteins, each of said proteins comprising an amino acid sequence with at least 98% identity with one of SEQ ID NO:6 to 318.

Preferably, said chimeric vaccinia virus encodes at least 285, at least 290, at least 295, at least 300, at least 301, at least 302, at least 303, at least 304, at least 305, at least 306, at least 307, at least 308, at least 309, at least 310, at least 311, at least 312, or even 313 distinct proteins, each of said proteins comprising an amino acid sequence with at least 98% identity with one of SEQ ID NO:6 to 318.

(L) at least 255 distinct proteins, each of said proteins comprising an amino acid sequence with at least 99% identity with one of SEQ ID NO:6 to 318.

Preferably, said chimeric vaccinia virus encodes at least at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least 301, at least 302, at least 303, at least 304, at least 305, at least 306, at least 307, at least 308, at least 309, at least 310, at least 311, at least 312, or even 313 distinct proteins, each of said proteins comprising an amino acid sequence with at least 99% identity with one of SEQ ID NO:6 to 318.

(m) at least 205 distinct proteins, each of said proteins comprising an amino acid sequence selected from SEQ ID NO:6 to 318.

Preferably, said chimeric vaccinia virus encodes at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least 301, at least 302, at least 303, at least 304, at least 305, at least 306, at least 307, at least 308, at least 309, at least 310, at least 311, at least 312, or even 313 distinct proteins, each of said proteins comprising an amino acid sequence selected from SEQ ID NO:6 to 318.

Amino acid sequences SEQ ID NO:6 to 318 encoded by chimeric vaccinia virus deVV5 are listed in Table 1 below.

Table 1: Amino acid sequences of the proteins encoded by deVV5

The chimeric vaccinia virus of the invention has been obtained from shuffling of nucleic acid sequences of 4 parental vaccinia virus strains: Copenhagen (COP), Modified Vaccinia Ankara (MVA), Wyeth (WY), and Western Reserve (WR). In one embodiment, the chimeric vaccinia virus of the invention described in aspect one above encodes at least two open-reading frames (ORFs) clusters, wherein said clusters are selected from clusters of at least two distinct parental vaccinia virus strains (COP, MVA, WY and WR). In a specific embodiment, the chimeric vaccinia virus of the invention encodes at least three open-reading frames (ORFs) clusters, wherein said clusters are selected from clusters of at least three distinct parental vaccinia virus strains (COP, MVA, WY and WR). In another specific embodiment, the chimeric vaccinia virus of the invention encodes at least four open-reading frames (ORFs) clusters, wherein said clusters are selected from clusters of the four distinct parental vaccinia virus strains (COP, MVA, WY and WR). In these embodiments, the clusters of parental vaccinia virus strains (COP, MVA, WY and WR) are the following:

(a) COP clusters:

(i) ORFs encoding proteins of amino acid sequences SEQ ID NO:173 to 203, and

(ii) ORFs encoding proteins of amino acid sequences SEQ ID NO:208 to 273;

(b) MVA clusters:

(i) ORFs encoding proteins of amino acid sequences SEQ ID NO:65 to 142, and

(ii) ORFs encoding proteins of amino acid sequences SEQ ID NO:148 to 154;

(c) WY clusters:

(i) ORFs encoding proteins of amino acid sequences SEQ ID NO:6 to 53,

(ii) ORFs encoding proteins of amino acid sequences SEQ ID NO:55 to 61, (iii) ORFs encoding proteins of amino acid sequences SEQ ID NO:164 to 172,

(iv) ORFs encoding proteins of amino acid sequences SEQ ID NO:204 to 207, and

(v) ORFs encoding proteins of amino acid sequences SEQ ID NO:280 to 318;

(d) WR clusters:

(i) ORFs encoding proteins of amino acid sequences SEQ ID NO:143 to 145,

(ii) ORFs encoding proteins of amino acid sequences SEQ ID NO:157 to 163, and

(iii) ORFs encoding proteins of amino acid sequences SEQ ID NO:274 to 279.

In the present invention, the chimeric vaccinia virus may encode original proteins (specific to the chimeric vaccinia virus, i.e. not encoded by any of the parental viruses, corresponding to SEQ ID NO:54, 62, 64, 143, 147, and 155), and/or similar proteins (70 % to 99.9 % of identity with proteins encoded by at least one of the parental viruses), and/or identical proteins (100 % of identity with proteins encoded by at least one of the parental viruses). In particular, the chimeric vaccinia virus of the invention may encode at least 1 original protein, more preferably at least 2, at least 3, at least 4, at least 5, at least 6 original proteins. Therefore, in a second aspect, the invention provides a chimeric vaccinia virus (optionally recombinant), wherein said chimeric vaccinia virus encodes:

54,

62,

64,

143,

147,

155, or

(g) any combination of proteins (a) to (f).

In a specific embodiment of aspect two, the chimeric vaccinia virus of the invention encodes:

a) a protein comprising the amino acid sequence SEQ ID NO: 54;

b) a protein comprising the amino acid sequence SEQ ID NO: 62,

c) a protein comprising the amino acid sequence SEQ ID NO: 64,

d) a protein comprising the amino acid sequence SEQ ID NO:143, e) a protein comprising the amino acid sequence SEQ ID NO: 147,

f) a protein comprising the amino acid sequence SEQ ID NO: 155, or

g) any combination of proteins a) to f).

In embodiments of aspect two, the chimeric vaccinia virus of the invention may encode 1, 2, 3, 4, 5 or all 6 of proteins (a) to (f) or a) to f) as defined above.

The genomic sequence of chimeric vaccinia virus deVV5 is as displayed in SEQ ID NO:l, representing the core region (comprised between nucleotide 1 and 162698) and one of the two ITRs (comprised between nucleotide 162699 and 179732).

The ORFs of chimeric vaccinia virus deVV5, their position in deVV5 genomic sequence SEQ ID NO:l, the sequence identifier of the corresponding encoded protein, and the corresponding position in the genomic sequence and percentage of identity with corresponding protein of parental vaccinia virus strains COP, MVA, WY and WR are described in Table 2 below.

Table 2: ORFs of deVV5, their position in genomic sequence SEQ ID NO:l, sequence identifier of the corresponding encoded protein, and corresponding position in the genomic sequence and percentage of identity with corresponding protein of parental vaccinia virus strains COP, MVA, WY and WR. c: core; i: ITR; N.A.: Non Applicable (i.e. the parental strain does not contain a corresponding ORF).

In embodiments, similar (70.0% to 99.9% of identity) or identical (100% of identity) proteins encoded by the chimeric vaccinia virus of the invention and by at least one parental virus can be expressed at the same level or at different levels in the chimera and in the parental virus(es). A similar or identical protein can be less expressed in the chimera than in the parental virus(es) or can be more expressed in the chimera than in the parental virus(es).

In embodiments, similar (70.0% to 99.9% of identity) or identical (100% of identity) proteins encoded by the chimeric vaccinia virus of the invention and by at least one parental virus can be expressed at the same time or at different times in the chimera and in the parental virus(es). A similar or identical protein can be expressed before in the chimera and after in the parental virus(es) or can be expressed after in the chimera and before in the parental virus(es).

These levels of protein expression and timings of protein expression can be evaluated by the skilled person, e.g. using transcriptomics, which is the study of the complete set of RNA transcripts that are expressed from the genome - also called transcriptome - under specific circumstances in a specific cell, using high-throughput methods (Costa et al., 2010, J Biomed Biotechnol, 2010:1-19). Transcriptomics allows to have information linking a genome (e.g. viral genome, cellular genome) to its phenotype. In one embodiment, viral transcriptomes are evaluated at different time points after viral infection of primary or tumor cells. In another embodiment, cellular transcriptomes are evaluated at different time points after viral infection of primary or tumor cells.

Chimeric vaccinia virus defined by nucleic acid features

In a third aspect, the invention provides a chimeric vaccinia virus having at least 66% of identity with SEQ ID NO:l. Preferably, the chimeric vaccinia virus has a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90% of identity with the nucleic acid sequence of SEQ ID NO: 1. More preferably, the chimeric vaccinia virus has a nucleic acid sequence having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% or at least 97% of identity with the nucleic acid sequence of SEQ ID NO: 1. Even more preferably, the chimeric vaccinia virus encodes an amino acid sequence having at least 97,4%, at least 97,5%, at least 97,6%, at least 97,7%, at least 97,8%, at least 97,9%, at least 98,0%, at least 98,1%, at least 98,2%, at least 98,3%, at least 98,4%, at least 98,5%, at least 98,6%, at least 98,7%, at least 98,8%, at least 98,9%, at least 99,0%, at least 99,1%, at least 99,2%, at least 99,3%, at least 99,4%, at least 99,5%, at least 99,6%, at least 99,7%, at least 99,8%, at least 99,9%, or even 100% of identity with the amino acid sequence encoded by SEQ ID NO: 1.

The chimeric vaccinia virus of the invention has been obtained from shuffling of nucleic acid sequences of 4 parental vaccinia virus strains: Copenhagen (COP), Modified Vaccinia Ankara (MVA), Wyeth (WY), and Western Reserve (WR), and thus preferably comprises nucleic acid sequences derived from at least one parental vaccinia virus strain. In one embodiment of aspect three, the chimeric vaccinia virus thus comprises:

(a) at least one Copenhagen (COP)-derived nucleic acid sequence selected from:

o a nucleic acid sequence consisting of nucleotides 96234 to 117168 of SEQ ID NO: 2, and

o a nucleic acid sequence consisting of nucleotides 119868 to 159589 of SEQ ID NO: 2;

(b) at least one Modified Vaccinia Virus Ankara (MVA)-derived nucleic acid sequence selected from:

o a nucleic acid sequence consisting of nucleotides 28840 to 70320 of SEQ ID NO: 3, and o a nucleic acid sequence consisting of nucleotides 73727 to 77829 of SEQ ID NO: 3;

(c) at least one Wyeth (WY)-derived nucleic acid sequence selected from:

o a nucleic acid sequence consisting of nucleotides 321 to 25490 of SEQ ID NO: 4, o a nucleic acid sequence consisting of nucleotides 26305 to 29837 of SEQ ID NO: 4, o a nucleic acid sequence consisting of nucleotides 91820 to 99894 of SEQ ID NO: 4, o a nucleic acid sequence consisting of nucleotides 120781 to 123439 of SEQ ID NO: 4, and

o a nucleic acid sequence consisting of nucleotides 165165 to 182675 of SEQ ID NO: 4;

(d) at least one Western Reserve (WR)-derived nucleic acid sequence selected from:

o a nucleic acid sequence consisting of nucleotides 81430 to 82968 of SEQ ID NO: 5, o a nucleic acid sequence consisting of nucleotides 88514 to 96205 of SEQ ID NO: 5, and o a nucleic acid sequence consisting of nucleotides 167675 to 171367 of SEQ ID NO: 5; or

(e) any combination of (a) to (d).

Preferably, the chimeric vaccinia virus comprises nucleic acid sequences derived from at least two of the parental vaccinia virus strains. The nucleic acid fragments from at least two parental vaccinia virus strains contain the essential genes necessary for replication. The chimeric vaccinia virus can also comprise nucleic acid sequences derived from at least three, or even the four parental vaccinia virus strains. In particular, the chimeric vaccinia virus may comprise:

(a) at least one Copenhagen (COP)-derived nucleic acid sequence selected from:

o a nucleic acid sequence consisting of nucleotides 119868 to 159589 of SEQ ID NO: 2; (b) at least one Modified Vaccinia Virus Ankara (MVA)-derived nucleic acid sequence selected from:

(c) at least one Wyeth (WY)-derived nucleic acid sequence selected from:

o a nucleic acid sequence consisting of nucleotides 165165 to 182675 of SEQ ID NO: 4; and

o a nucleic acid sequence consisting of nucleotides 81430 to 82968 of SEQ ID NO: 5, o a nucleic acid sequence consisting of nucleotides 88514 to 96205 of SEQ ID NO: 5, and o a nucleic acid sequence consisting of nucleotides 167675 to 171367 of SEQ ID NO: 5.

Chimeric vaccinia virus defined by functional features

In a fourth aspect, the invention provides a chimeric vaccinia virus, wherein at least one organ-specific therapeutic index of said chimeric vaccinia virus is higher than the corresponding organ-specific therapeutic indexes of parental vaccinia virus strains Copenhagen (COP), Modified Vaccinia Ankara (MVA), Wyeth (WY), and Western Reserve (WR), wherein for a given organ and a given virus, an organ- specific therapeutic index TI(organ, virus) is defined as:

In a specific embodiment of aspect four, the hepatic therapeutic index of said chimeric vaccinia virus is higher than the corresponding hepatic therapeutic indexes of parental vaccinia virus strains COP, MVA, WY and WR wherein for a given virus, an hepatic therapeutic index Tl(liver, virus) is defined as: Tl(liver, virus) = (replication of virus in Hep G2 tumor cells/ replication of virus in primary hepatocytes). Preferably, the hepatic therapeutic index of said chimeric vaccinia virus is at least 20 times higher than, more preferably at least 30 times higher than the corresponding hepatic therapeutic indexes of parental vaccinia virus strains COP, MVA, WY and WR. More preferably, in this embodiment, the hepatic therapeutic index is measured in vitro in a setting in which the chimeric vaccinia virus is added respectively to the HepG2 tumor cells and to the primary hepatocytes at a MOI of 10 ^s and the replication of the chimeric vaccinia virus in the HepG2 tumor cells and in the primary hepatocytes is measured 7 days post infection. Even more preferably, in this embodiment, the HepG2 tumor cell lines are cultured at 37° C, 5% CO2, in Dulbecco's Modified Eagle Medium (DMEM) with 10% of Fetal Calf Serum (FCS), and the primary hepatocytes are cultured at 37°C, 5% C02, in basal hepatic cell medium (BIOPREDICS catalogue reference MIL600) and additives for hepatocyte culture medium (BIOPREDICS catalogue reference ADD222C).

In a fifth aspect, the invention provides a chimeric vaccinia virus, wherein at least one tumor-specific oncolytic power of said chimeric vaccinia virus is higher than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR, wherein for a given tumor and a given virus, a tumor-specific oncolytic power OP(tumor, virus) is defined as:

In a specific embodiment of aspect five, the tumor-specific oncolytic power of said chimeric vaccinia virus is higher than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR in at least one tumor cell line selected between UM-UC-3, A549, SKOV-3, Flep G2, Cal-33, MIA PaCa-2, OE 19, HCT 116 and KATO III.

In a more specific embodiment of aspect five, the tumor-specific oncolytic power of said chimeric vaccinia virus is at least 2 times more lytic than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR in at least one tumor cell line selected between UM-UC-3, A549, SKOV-3, Flep G2 and Cal-33, preferably at least 3 times more lytic than the parental vaccinia virus strain COP, MVA, WY, and WR in at least one tumor cell line selected between A549, SKOV-3, Flep G2 and Cal-33, more preferably at least 4 times more lytic than the parental vaccinia virus strain COP, MVA, WY, and WR in at least one tumor cell line selected between SKOV-3, Flep G2 and Cal-33, more preferably 6 times more lytic than the parental vaccinia virus strain COP, MVA, WY, and WR in at least one tumor cell line selected between Flep G2 and Cal-33, more preferably at least 12 times more lytic than the parental COP vaccinia virus strain than the parental vaccinia virus strain COP, MVA, WY, and WR in Cal-33 tumor cell lines. In a more preferred embodiment, the tumor-specific oncolytic power of said chimeric vaccinia virus is at least 2 times more lytic than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR in UM-UC-3, A549, SKOV-3, Flep G2 and Cal-33, preferably at least 3 times more lytic than the parental vaccinia virus strain COP, MVA, WY, and WR in A549, SKOV-3, Flep G2 and Cal-33, more preferably at least 4 times more lytic than the parental vaccinia virus strain COP, MVA, WY, and WR in SKOV-3, Flep G2 and Cal- 33, more preferably 6 times more lytic than the parental vaccinia virus strain COP, MVA, WY, and WR in Flep G2 and Cal-33, more preferably at least 12 times more lytic than the parental COP vaccinia virus strain than the parental vaccinia virus strain COP, MVA, WY, and WR in Cal-33 tumor cell lines. Preferably, in this embodiment, the lytic power is evaluated 5 days post infection and at a MOI of 10 ⁵. More preferably, in this embodiment, the tumor cell lines are cultured at 37° C, 5% CO2, in Dulbecco's Modified Eagle Medium (DMEM) with 10% of Fetal Calf Serum (FCS).

In another specific embodiment of aspect five, the tumor-specific oncolytic power of said chimeric vaccinia virus is at least 2 times more lytic than the corresponding tumor-specific oncolytic power of parental vaccinia virus strains COP, MVA, WY and WR in at least one tumor cell line selected between MIA PaCa-2, OE 19 and FICT 116, preferably at least 7 times more lytic than the corresponding tumor- specific oncolytic power of parental vaccinia virus strain COP, MVA, WY, and WR in at least one tumor cell line selected between OE 19 and FICT 116, more preferably at least 20 times more lytic than the corresponding tumor-specific oncolytic power of parental vaccinia virus strain COP, MVA, WY, and WR in FICT 116. In a more preferred embodiment, the tumor-specific oncolytic power of said chimeric vaccinia virus is at least 2 times more lytic than the corresponding tumor-specific oncolytic power of parental vaccinia virus strains COP, MVA, WY and WR in MIA PaCa-2, OE 19 and FICT 116, preferably at least 7 times more lytic than the corresponding tumor-specific oncolytic power of parental vaccinia virus strain COP, MVA, WY, and WR in OE 19 and FICT 116, more preferably at least 20 times more lytic than the corresponding tumor-specific oncolytic power of parental vaccinia virus strain COP, MVA, WY, and WR in FICT 116. Preferably, in this embodiment, the lytic power is evaluated 5 days post infection and at a MOI of 10⁴. More preferably, in this embodiment, the tumor cell lines are cultured at 37° C, 5% C02, in Dulbecco's Modified Eagle Medium (DMEM) with 10% of Fetal Calf Serum (FCS).

In another specific embodiment of aspect 5, the tumor-specific oncolytic power of said chimeric vaccinia virus is at least 2 times more lytic, preferably at least 6 times more lytic than the corresponding tumor-specific oncolytic power of parental vaccinia virus strains COP, MVA, WY and WR in tumor cells KATO III. Preferably, in this embodiment, the lytic power is evaluated 5 days post infection and at a MOI of 10³. More preferably, in this embodiment, the tumor cell lines are cultured at 37° C, 5% CO2, in Dulbecco's Modified Eagle Medium (DMEM) with 10% of Fetal Calf Serum (FCS).

In another specific embodiment of aspect 5, the tumor-specific oncolytic power of said chimeric vaccinia virus in UM-UC-3, A549, SKOV-3, Hep G2, Cal-33, MIA PaCa-2, OE 19, HCT 116 and KATO III is higher than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR in the same tumor cells. Preferably, the chimeric vaccinia virus of the invention is at least 2 times more lytic in UM-UC-3, A549, SKOV-3, Hep G2, Cal-33, MIA PaCa-2, OE 19, HCT 116 and KATO III in specific conditions depending on cell lines, said conditions being a chimeric vaccinia virus MOI of lO ³ in KATO III, a chimeric vaccinia virus MOI of 10⁴ in MIA PaCa-2, OE 19 and HCT 116, and a chimeric vaccinia virus MOI of 10⁵ in UM-UC-3, A549, SKOV-3, Hep G2 and Cal-33. More preferably, in this embodiment, the lytic power is evaluated 5 days post infection, and the tumor cell lines are cultured at 37° C, 5% CO2, in Dulbecco's Modified Eagle Medium (DMEM) with 10% of Fetal Calf Serum (PCS).

Chimeric vaccinia virus obtained or obtainable by the method of directed evolution for selecting a chimeric vaccinia virus with high oncolytic power

In another aspect, the invention provides a chimeric vaccinia virus, wherein said chimeric vaccinia virus has been or may be obtained by any embodiment of the method described below.

Chimeric vaccinia virus combining protein, nucleic, functional, and/or method features

As explained above, the chimeric vaccinia virus of the invention may comprise any combination of protein, nucleic acid, functional and/or method features described herein.

METHOD OF DIRECTED EVOLUTION FOR SELECTING A CHIMERIC VACCINIA VIRUS WITH HIGH

ONCOLYTIC POWER

In another aspect, the invention provides a method of directed evolution for obtaining a chimeric vaccinia virus with high oncolytic power, said method comprising:

(iii) amplifying one or more variant chimeric vaccinia virus(es) of the collected supernatant on a tumor cell line, wherein said tumor cell line is poorly permissive or non-permissive for each parental vaccinia virus strain; (iv) collecting the supernatant containing one or more chimeric vaccinia virus(es) having a non-zero oncolytic power in the tumor cell line of step (iii) after at least 2 days at a supernatant dilution of at least 2;

In said method of directed evolution, different vaccinia virus strains undergo selective pressure. In this general manner, the method facilitates the "forced evolution" of a viral genome to encode a desired phenotype which natural selection and evolution has not generated.

Preferably, in the above method of directed evolution, at least two, more preferably at least three, even more preferably at least four different parental vaccinia virus strains are used for infecting a poorly permissive or non-permissive tumor cell of step (i).

In this case, said parental vaccinia virus strains used in first step (i) are preferably selected in the group consisting of Modified Vaccinia Virus Ankara (MVA), Copenhagen (COP), Wyeth (WY), Western Reserve (WR), Elstree, New York City Board of Health (NYCBH), IHD-J, Dryvax, CCSV, CJ-50300, Lister, RIVM, Israel, Lister/CEP, Elstree-BN, Temple of Heaven (Tian Tan), Guang 9, Ecuador-Moscow 1963 (EM-63), Tashkent , B-15, Bern, Paris, Dairen, Ikeda, Ankara, LC16m8, CV1, EM63, ACAM2000, CL, CAM, CTC, Lederle-Chorioallantoic, L-variant, S-variant, rVV-hgplOO and AS. More preferably, said parental vaccinia virus strains used in first step (i) are selected in the group consisting of MVA, COP, WY and WR. Even more preferably, MVA, COP, WY and WR are used, preferably at a dose comprised between 1.5xl0³ PFU and 1.5x10^s PFU, even more preferably between lxlO⁴ PFU and 1x10^s PFU, and even more preferably between 1.5xl0⁴ PFU and 1.5x10^s PFU.

Preferably, in the above method of directed evolution, said poorly permissive or non-permissive tumor cell lines used for generating the chimeric vaccinia virus of the invention are mammalian cells, said cells being poorly permissive or non-permissive to vaccinia virus infection and replication.

The term "permissive" refers to any tumor cell line allowing a virus to replicate. As used herein, the terms "replication," "viral replication" and "virus replication" in the context of a virus refer to one or more or all of the stages of a viral life cycle which result in infection with or propagation of virus. The steps of a viral life cycle include, but are not limited to, virus attachment to the host cell surface, penetration or entry of the host cell (e.g. through receptor mediated endocytosis or membrane fusion), uncoating (the process whereby the viral capsid is removed and degraded by viral enzymes or host enzymes thus releasing the viral genomic nucleic acid), genome replication, synthesis of viral messenger RNA (mRNA), viral protein synthesis, and assembly of viral ribonucleoprotein complexes for genome replication, assembly of virus particles, post-translational modification of the viral proteins, and release from the host cell by lysis or budding and acquisition of a phospholipid envelope which contains embedded viral glycoproteins. In some embodiments, the terms "replication," "viral replication" and "virus replication" refer to the replication of the viral genome. In other embodiments, the terms "replication," "viral replication" and "virus replication" refer to the synthesis of viral proteins. In a cell, there is a viral replication when the viral titer (measured intra- and extra-cellularly) is multiplied by a number comprised in the interval ]1; +¥ [. In a permissive tumor cell line, the level of virus replication can be low (e.g.: 48 hours post-infection, multiplication of the viral titer by a number comprised in the interval ]1; 20000[), medium (e.g.: 48 hours post-infection, multiplication of the viral titer by a number comprised in the interval ]20000; 40000[) or high (e.g.: 48 hours post-infection, multiplication of the viral titer by a number comprised in the interval ]40000; +¥ [).

The term "non-permissive" refers to any tumor cell line in which the viral titer (measured intra- and extra-cellularly) is comprised between [0; 1], while the expression "poorly permissive" refers to any tumor cell line allowing a low level of replication, as defined above.

In the above method, the tumor cell line(s) used in steps (i) and (iii) are, for each of the parental vaccinia virus strains used in step (i), either poorly permissive or non-permissive.

In a preferred embodiment, the tumor cell lines used for generating a chimeric vaccinia virus are poorly permissive for at least 1, at least 2 or at least 3 parental vaccinia virus strains replication.

Steps (i) and (iii) comprise the infection of poorly permissive or non-permissive tumor cell lines with different vaccinia virus strains, and the amplification of said vaccinia virus strains: genetic exchange, also called "shuffling", may happen between various strains, leading to the generation of new chimeric vaccinia viruses. The use of poorly permissive or non-permissive tumor cells results in a stringent selective pressure towards the genetic selection of a rare recombination event. Selective pressure reduces the reproductive success in a portion of the viral population but increases the frequency of genetic changes between the different viral strains, including point mutation and recombination. As a result, the stringent selective pressure allows the enrichment of the generated chimeric vaccinia viruses with new properties (e.g. increased oncolytic power).

Poorly permissive or non-permissive tumor cell lines of steps (i) and (iii) can be the same or different. Poorly permissive or non-permissive tumor cell lines used for the repetition of step (iii) can be the same or different. Examples of tumor cell lines poorly permissive or non-permissive to vaccinia virus replication are LoVo, MIA PaCa-2, U-118 MG, REM 134, OVCAR-3, SNU-5, KATO III, UACC.257, OE 19, AsPC-1, U-87 MG, OE 21, SW780 and Capan-2. Indeed, these cells are poorly permissive for Copenhagen (COP), Wyeth (WY) and Western Reserve (WR), and most other vaccinia virus strains, and non-permissive for MVA. In a preferred embodiment, the poorly permissive or non-permissive tumor cell lines used for generating a chimeric vaccinia virus are chosen between LoVo and MIA PaCa-2 cell lines.

In a preferred embodiment, the method of directed evolution includes infecting LoVo tumor cell line with Modified Vaccinia Virus Ankara (MVA), Copenhagen (COP), Wyeth (WY) and Western Reserve (WR) in step (i), infecting LoVo tumor cell lines with one or more variant chimeric vaccinia virus(es) in step (iii), and further infecting MIA PaCa-2 tumor cell lines with one or more variant chimeric vaccinia virus(es) in step (iii). More preferably, step (i) of said method includes infecting LoVo tumor cell line with 1.5x10^s PFU of Modified Vaccinia Virus Ankara (MVA), 1.5xl0⁴ PFU of Copenhagen (COP), 1.5xl0⁴ PFU of Wyeth (WY) and 1.5xl0⁴ PFU of Western Reserve (WR).

Steps (ii) and (iv) comprise the collection of the supernatant comprising one or more oncolytic chimeric vaccinia virus. In a specific embodiment, said supernatant is diluted before the following step of amplification. Preferably, the fold dilution is comprised between 2 and 2000, more preferably between 5 and 1500, more preferably between 10 and 1000.

In embodiments, steps (iii) and (iv) are repeated between 1 and 30 times, more preferably between 1 and 25 times, more preferably between 1 and 20 times, more preferably between 1 and 15 times, more preferably between 1 and 10 times, more preferably between 1 and 5 times, and even more preferably between 1 and 3 times.

In one embodiment, the chimeric vaccinia virus having a desired oncolytic power is obtained after 1,

2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 repetition(s) of step (v).

In one embodiment, the method of directed evolution further comprises the use of at least one mutagenic agent in step(s) (i), (ii), (iii) and/or (iv), in order to increase selective pressure. For instance, said mutagenic agents may be selected from physical, chemical and biological agents. More preferably, said physical agents are selected in the group consisting of ultraviolet radiations, ionizing radiations and radioactive decays, said chemical agents are selected in the group consisting of urea, nitrosourea, reactive oxygen species, deaminating agents, polycyclic aromatic hydrocarbon, alkylating agents, aromatic amines, alkaloid, bromine, sodium azide and benzene, and said biological agents are selected in the group consisting of DNA base analogues and transposons. Other physic, chemical and biological mutagenic agents known by the skilled person may be used in the context of the invention. RECOMBINANT CHIMERIC VACCINIA VIRUS ENCODING NUCLEIC ACID(S) OF INTEREST

In another aspect, the chimeric vaccinia virus of the present invention is a recombinant chimeric vaccinia virus, which means that it is intentionally modified by a man in the laboratory.

In an embodiment, the chimera 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.

In the context of the invention, the chimeric vaccinia virus can be rendered defective for a particular locus by a number of ways including substitution(s), deletion(s) and/or insertions of one or more nucleotide(s) present in this locus. For example, insertion of a polynucleotide in the locus may disrupt the open reading frame (ORF) encoded by the nucleic acid sequence of the locus. Partial or total deletion of a particular locus is also appropriate to generate a defective chimeric vaccinia virus.

In the present invention, the chimeric vaccinia virus is partially or totally defective in one or more specific loci.

Preferably, the chimeric vaccinia virus is defective in the thymidine kinase (TK) locus, where is located the TK encoding-gene (similar to J2R gene in COP). In the invention, the TK encoding-gene corresponds to ORF_522. In this embodiment, the chimeric vaccinia virus of the invention does not encode any protein comprising the amino acid sequence SEQ ID NO: 171. 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 (e.g. tumor cells) that contain high nucleotide concentration. The deletion of the TK encoding gene may improve the tumor selectivity of the chimeric vaccinia virus with the reduction of said virus replication in non-tumor cells and. In a preferred embodiment, the deletion of the TK encoding gene has no or low impact on the lytic activity of said virus. Consequently, the deletion of a TK locus in the chimeric vaccinia virus may improve the therapeutic index of said virus. In this embodiment, the chimeric vaccinia virus defective for TK activity has a higher organ-specific therapeutic index than the corresponding organ-specific therapeutic indexes of parental vaccinia virus strains COP, MVA, WY and WR. In a specific embodiment, the hepatic therapeutic index of said chimeric vaccinia virus is higher than the corresponding hepatic therapeutic indexes of parental vaccinia virus strains COP, MVA, WY and WR wherein for a given virus, an hepatic therapeutic index Tl(liver, virus) is defined as: Tl(liver, virus) = (replication of virus in Hep G2 tumor cells/ replication of virus in primary hepatocytes). Preferably, the hepatic therapeutic index of said chimeric vaccinia virus is at least 100 times higher than, more preferably at least 150 times, more preferably at least 200 times higher than the corresponding hepatic therapeutic indexes of parental vaccinia virus strains COP, MVA, WY and WR. More preferably, in this embodiment, the hepatic therapeutic index is measured in vitro in a setting in which the chimeric vaccinia virus is added respectively to the HepG2 tumor cells and to the primary hepatocytes at a MOI of 10 ^s and the replication of the chimeric vaccinia virus in the HepG2 tumor cells and in the primary hepatocytes is measured 7 days post infection. Even more preferably, in this embodiment, the HepG2 tumor cell lines are cultured at 37° C, 5% C02, in Dulbecco's Modified Eagle Medium (DMEM) with 10% of Fetal Calf Serum (FCS), and the primary hepatocytes are cultured at 37°C, 5% C02, in basal hepatic cell medium (BIOPREDICS catalogue reference MIL600) and additives for hepatocyte culture medium (BIOPREDICS catalogue reference ADD222C). Optionally, the chimeric vaccinia virus defective for TK activity has a higher organ-specific therapeutic index than the corresponding organ-specific therapeutic indexes of the chimeric vaccinia virus not defective for TK activity. In another aspect of this embodiment, the tumor-specific oncolytic power of a vaccinia virus defective for TK activity is higher than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR.

In a preferred embodiment, the chimeric vaccinia virus of this invention is defective for TK activity, resulting from alteration of the TK locus.

Alternatively, or in combination, the chimeric vaccinia virus of the invention is defective in at least one of the ribonucleotide reductase (RR) loci, where are located the RR encoding-genes: R1 encoding-gene (similar to I4L gene in COP) and R2 encoding-gene (similar to F4L gene in COP). In the invention, the R1 and R2 encoding-genes correspond respectively to ORF_607 and ORF_773. In the natural context, the ribonucleotide reductase 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 R1 and R2. In the context of the invention, either the locus encoding the R1 large subunit, or the locus encoding the R2 small subunit, or both may be defective. In this embodiment, the chimeric vaccinia virus does not encode any protein comprising the amino acid sequence SEQ ID NO:195 and/or any protein comprising the amino acid sequence SEQ ID NO:243. In a preferred embodiment, the chimeric vaccinia virus of this invention may be defective in both TK and RR activities resulting from alteration of both the TK locus, and R1 and/or R2 locus carried by the viral genome (e.g. as described in W02009/065546 and Foloppe et al., 2008, Gene Ther., 15: 1361- 1371). In this embodiment, the chimeric vaccinia virus does not encode any protein comprising the amino acid sequence SEQ ID NO:171, any protein comprising the amino acid sequence SEQ ID NO:195, and/or any protein comprising the amino acid sequence SEQ ID NO:243.

Alternatively, or in combination with alteration of at least one of TK or RR activities or both, the chimeric vaccinia virus of this invention may be defective for dUTPase resulting from alteration of the dUTPase encoding-gene (similar to F2L gene in COP). In the invention, the dUTPase encoding gene corresponds to ORF_788. In this embodiment, the chimeric vaccinia virus does not encode any protein comprising the amino acid sequence SEQ ID NO: 248.

Alternatively, or in combination, other strategies may also be pursued to further increase the virus tumor-specificity. A representative example of suitable modification includes disruption of the hemagglutinin encoding-gene (similar to A56R gene in COP and corresponding to ORF_109 in the present invention), optionally in combination with TK deletion (Zhang et al., 2007, Cancer Res. 67:10038-46). Disruption of interferon modulating gene(s) may also be advantageous (similar to B8R or B18R genes in COP and corresponding to ORF_52 and ORF_4 respectively in the present invention) or the caspase-1 inhibitor (similar to B13R gene in COP and corresponding to ORF_35 in the present invention). Another suitable modification comprises the disruption of the gene encoding 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).

Alternatively, or in combination with altering one or more viral gene(s) as described above, any chimeric vaccinia virus of the invention may further comprise one or more nucleic acid(s) of interest inserted in its genome. 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 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, suicide polypeptides which are capable of reinforcing the oncolytic nature of the chimeric vaccinia virus of the present invention, as well as polypeptides capable of potentiating anti-tumor efficacy (such as immunostimulatory polypeptides), antigens for inducing or activating an immune humoral and/or cellular response, or permease to increase the cellular nucleoside or nucleotide pool among many others.

Suicide polypeptide

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

In this embodiment, the chimeric vaccinia virus encoding a suicide polypeptide preferably has a higher tumor-specific oncolytic power than the corresponding tumor-specific oncolytic power of parental vaccinia virus strains COP, MVA, WY and WR. Alternatively, or in combination, the chimeric vaccinia virus encoding a suicide polypeptide preferably has a higher organ-specific therapeutic index than the corresponding organ-specific therapeutic indexes of parental vaccinia virus strains COP, MVA, WY and WR.

Preferably, the chimeric vaccinia virus of the invention carries in its genome a gene encoding a suicide polypeptide having at least cytosine deaminase (CDase) activity. Alternatively, or in combination, the chimeric vaccinia virus of the invention carries in its viral genome a gene encoding a suicide 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 chimeric vaccinia virus of the invention encodes a suicide 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 (FCU1) and FCU1-8 described in W096/16183, EP998568 and W02005/07857. Of particular interest is the FCU1 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 W02009/065546.

In a preferred embodiment of the invention, the chimeric vaccinia virus is defective in the TK locus and further encodes a suicide polypeptide. In a more preferred embodiment, the chimeric vaccinia virus is defective in the TK locus and further encodes a FCU1 suicide gene.

Immunostimulatory polypeptide

A specific embodiment of the invention is directed to a chimeric vaccinia 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- PD1, anti-PDLl, anti-PDL-2, anti-CTLA4, anti-Tim3, anti-LAG3, anti-BTLA; cytokines, like alpha, beta or gamma interferon, interleukins or tumor 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 Fluman 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

Another embodiment of the invention is directed to a chimeric vaccinia virus encoding an antigen. 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 tumor-inducing pathogens.

In certain embodiments, the antigen(s) encoded by the chimeric vaccinia virus is/are cancer antigen(s) (also called tumor-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 fetal 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 tumor viruses (e.g. HPV, HCV, EBV, etc.) and bacteria (e.g. Helicobacter pilori). Cancer antigens can also be neoantigens, which are specific for a patient's tumor, and used for personalized medicine (EP2018/066668).

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 tumor antigens, GAGE-family of tumor 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 chimeric vaccinia 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.

Permease

According to another embodiment, the chimeric vaccinia virus of the invention may comprise a nucleic acid of interest encoding a permease.

As used herein, the term "permease" refers to trans-membranous protein involved in the translocation of nucleoside and nucleobases. Examples of permeases which are involved in the translocation of nucleosides, nucleoside analogues and nucleobases are hCNTl, hCNT2, hCNT3, hENTl and hENT2. hCNTl, hCNT2 and hCNT3 proteins translocate nucleosides in a Na+ coupled manner with high affinity and some substrate selectivity, being hCNTl and hCNT2 pyrimidine - and purine - preferring, respectively, and hCNT3 abroad selectivity transporter. hENTl and hENT2 are unequivocally implicated in the translocation of nucleosides and nucleobases (Pastor-Anglada et al, 2015, Front. Pharmacol., 6(13):1-14).

Other nucleic acids of interest:

Other nucleic acids of interest include, but are not limited to:

Nucleoside pool modulators (e.g.: cytidine deaminase, like yeast cytidine deaminase (CDD1) or human cytidine deaminase (hCD) (see EP16306831.5))

Agents targeting metabolic immune modulators (e.g.: adenosine deaminase like human adenosine deaminase (huADAl or huADA2) (see EP17306012.0))

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, CRISPR/Cas9

RNA, including but not limited to target-specific miRNA, shRNA, siRNA.

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 (e.g. within TK, R1 or R2 loci).

Expression of the nucleic acid(s) sequences

The 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 a specific embodiment of the present invention, the recombinant chimeric vaccinia virus comprises the elements necessary for the expression of nucleic acid 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. polypeptide(s) encoded by a suicide gene) 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 the chimeric vaccinia virus production and circumvent potential toxicity of the expressed polypeptide(s).

Vaccinia virus promoters are particularly adapted in the context of the invention. Representative examples include without limitation the vaccinia 7.5K, H5R, llk7.5 (Erbs et al., 2008, Cancer Gene Ther. 15(1): 18-28), TK, p28, pll Prl3.5 (WO2014/063832), pB8R, pFUL, pA44L, pCUR (W02011/128704) and K1L 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 suicide gene is inserted in the TK locus of the chimeric vaccinia virus of the invention and placed under the control of the vaccinia pllk7.5 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 PRODUCING A CHIMERIC VACCINIA VIRUS

The invention also relates to a process for producing a chimeric vaccinia virus of the invention, said process comprising:

(i) infecting a producer cell with the chimeric vaccinia virus of the invention (ii) culturing said producer cell is cultured under conditions which are appropriate for enabling said chimeric vaccinia virus particles to be produced, and

(iii) recovering said chimeric vaccinia virus particles from the producer cell culture.

The chimeric vaccinia 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 chimeric vaccinia 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), CV-1 (ATCC CCL-70) and BSC1(ATCC CCL-26) cell lines, avian cells such as those described in W02005/042728, W02006/108846, W02008/129058, W02010/130756,

W02012/001075, 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 chimeric vaccinia 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 chimeric vaccinia 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 cesium chloride gradient), chromatographic and filtration steps. Appropriate methods are described in the art (e.g. WO2007/147528; WO2008/138533, W02009/100521, W02010/130753,

WO2013/022764).

CHIMERIC VACCINIA VIRUS COMPOSITION

The invention also relates to a composition (preferentially a pharmaceutical composition) that comprises a therapeutically effective amount of the chimeric vaccinia 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 tumor 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-tumor 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 tumor 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 the chimeric vaccinia 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 chimeric vaccinia virus may vary within a range extending from approximately 10³ to approximately 10¹² vp (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 chimeric vaccinia virus for human use is comprised between approximately 10⁴ to approximately 10¹¹ PFU, preferably between approximately 10^s PFU to approximately 10¹⁰ PFU; doses of approximately 10^s PFU to approximately 5xl0⁹ PFU being particularly preferred (e.g. dose of 10^s, 2x10^s, 3x10^s, 4x10^s, 5x10^s, 6x10^s, 7x10^s, 8x10^s, 9x10^s, 10⁷, 2xl0⁷, 3xl0⁷, 4xl0⁷, 5xl0⁷, 6xl0⁷, 7xl0⁷, 8xl0⁷, 9xl0⁷, 10^s, 2x10^s, 3x10^s, 4x10^s, 5x10^s, 6x10^s, 7x10^s, 8x10^s, 9x10^s, 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. BFIK-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 chimeric vaccinia 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, Flank'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, chimeric vaccinia 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 tumor 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, WOOl/66137, WO03/053463, W02007/056847 and W02008/114021, 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 chimeric vaccinia 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 chimeric vaccinia 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 intratumoral (into a tumor 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 chimeric vaccinia 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 10^s PFU to 5xl0⁹ PFU of a chimeric vaccinia virus according to the invention comprising a suicide gene (preferably defective in TK locus (TK-)), with a specific preference for a chimeric vaccinia virus according to the invention having the suicide gene inserted in place of the TK locus and placed under the pllK7.5 promoter such as VV TK-/fcul described herein; formulated for intravenous or intratumoral administration.

METHODS AND USES

In another aspect, the present invention provides a chimeric vaccinia 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 a chimeric vaccinia 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 chimeric vaccinia 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 a chimeric vaccinia 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 chimera of the invention, or the composition thereof include proliferative diseases such as cancers, tumors 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 present invention is particularly suited for treating or preventing lung cancer, renal cancer, bladder cancer, prostate cancer, breast cancer, colorectal cancer, hepatic cancer, gastric cancer, pancreatic cancer, melanoma, ovarian cancer and glioblastoma. In another preferred embodiment, the present invention is particularly suited for treating or preventing cancers refractory or resistant to at least one oncolytic virus-based therapy, or to at least one oncolytic vaccinia virus-based therapy.

A particularly preferred method comprises 1 to 6 intravenous or intratumoral administrations of the chimeric vaccinia virus of the invention or the 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 10^s to 5xl0⁹ PFU of a chimeric vaccinia virus, the latter being preferably defective in TK locus (TK-) and encoding a suicide polypeptide, more preferably encoding FCU1.

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 tumor growth, proliferation and metastasis, preventing or delaying tumor invasion (spread of tumor cells in neighboring tissues), reducing the tumor number; reducing the tumor 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-tumor response (e.g. non-specific (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 chimeric vaccinia 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, tumors 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 tumor cell growth in vivo comprising administering a chimeric vaccinia virus or a composition thereof in a subject in need thereof so-as to inhibit the growth of a tumor. For general guidance, inhibition of tumor cell growth can be evaluated routinely, for example by radiography means. The administration(s) of the chimeric vaccinia virus or the composition thereof desirably result(s) in at least a 10% decrease of the tumor mass.

EXAMPLES

EXAMPLE 1: GENERATION AND CHARACTERIZATION OF DEVV5, A CHIMERIC VACCINIA VIRUS

Materials and Methods

Cell lines and viruses

Human colon cancer cell lines LoVo (CCL-229™) and HCT 116 (CCL-247™), human lung cancer cell line A549 (CCL-185™), human hepatocarcinoma cell line Hep G2 (HB 8065™), human glioblastoma cancer cell line U-87 MG (HTB-14™), human gastric carcinoma cell line KATO III (HTB-103™), human pancreatic cancer cell line MIA PaCa-2 (CRL-1420™), human ovarian cancer cell line SK-OV-3 (HTB-77™) and human bladder cancer cell line UM-UC-3 (CRL-1749™) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Human esophagus cancer cell line OE19 (n°96071721) was obtained from European Collection of Cell Culture (ECACC). Human head and neck cancer cell line CAL33 was kindly provided by Dr. G. Milano (Centre Antoine-Lacassagne, Nice, France). All cell lines were grown in recommended media supplemented with 10% fetal calf serum (FCS).

Primary chicken embryo fibroblasts (CEF) were used for recombination, production and titration of viral vectors. CEF cells were prepared from chicken embryos obtained from fertilized eggs (Charles River SPAFAS) previously incubated 11 or 12 days at 37°C in a humid atmosphere. Chicken embryos were dissected and treated with a 2.5% (w/v) solution of trypsin. CEF cells were maintained in Eagle- based Medium (MBE) supplemented with 5 % fetal calf serum. Wild type vaccinia viruses Wyeth strain (WY, VR-1536™) and Western Reserve strain (WR, VR-119™) were obtained from ATCC. Wild type vaccinia virus Copenhagen strain (COP) used in the work described here comes from the Institut Merieux (Marcy I'Etoile, France). MVA expressing the eGFP gene under the control of the pllk7.5 promoter (MVA-GFP) was constructed and characterized previously (Erbs et al., 2008, Cancer Gene Ther., 15, 18-28).

Directed Evolution: in vitro selection of chimeric Vaccinia virus deVV5

Viral strains of vaccinia virus: COP, WY and WR were pooled at MOI 10 ² and MVA-GFP was added at MOI 10 ¹. The blend was used to infect human LoVo cells (1,5 x 10^s cells/well in a 6 well-plate) and constituted the passage 1 (LP1). LP1 was used in its entirety to infect T-75 tissue culture flask (T75). Supernatant from the second passage was then used in a 10-fold dilution series to infect confluent T75 of LoVo cells. Amplification and selection of viral progeny was done by 9 successive passages on LoVo cells by supernatant dilution. The infected T75 were observed for the first signs of cytopathic effect (CPE). To harvest the most potent viruses, cell culture supernatant was harvested from the flask infected with the most concentrated inocula in the 10-fold dilution series that did not show any sign of potent CPE. LP2 and LP3 were harvested after 24h of infection. Next passages (LP4 to LP9) were harvested at 72h post-infection. LP9 served as crude lysate for a new round of selection on MIA PaCa- 2 cells. Twelve passages were performed on MIA PaCa-2 cells as described above. Viral selection and amplification of the strongest chimera was undertaken by decimal dilutions.

Generation of deVV5-fcul deVV5 -fcul was generated by insertion of the FCU1 gene into the deVV5 TK locus. Briefly, CEF were infected with deVV5 at a MOI of 10 ² and incubated at 37°C for 2h, then transfected with a shuttle plasmid containing the FCU1 gene under the control of the synthetic pllk7.5 promoter and surrounded by the flanking sequence of the vaccinia virus TK gene. The cells were then incubated for 48h at 37°C. Double recombination occurred between TK homologous regions in the shuttle plasmid and the wild-type virus, resulting in the insertion of the FCU1 gene into the TK locus of the deVV5. Recombinant virus deVV5 -fcul was isolated and submitted to additional plaque purification cycles on CEF. Insertion of the FCU1 sequence into the TK locus was confirmed by multiple PCRs and DNA sequencing.

In vitro cytotoxicity assay

The viral lytic capacity was measured using the trypan blue exclusion method. Fluman tumor cells were transduced in suspension by respective chimeric viruses at the indicated MOI. A total of 3 x 10^s cells/well were plated in 6-well culture dishes in 2 ml of medium supplemented with 10% FCS. Cells were then cultured at 37°C for 5 days and the viable cells were counted by trypan blue exclusion using a Vi-Cell Cell Counter (Beckmann Coulter, CA). All samples were analyzed in triplicate. Mock- infected cells served as negative control and established the 100% survival point for the given assay.

In vitro virus yield

Growing Flep G2, A549 and OE19 tumor cells were seeded onto 6-well plates at 3 x 10^s cells/well. After 24h in DMEM with 10 % FCS, the cells were infected with individual virus at MOI of 10 ⁵ and cells were incubated in fresh growth medium supplemented with 10 % FCS until harvesting. Supernatants and cells collected 72 hours post-infection were submitted to a quick freeze-thaw cycle and sonication to release intracellular viral particles and viral progeny were quantified on CEF by plaque assay. All samples were analyzed in triplicate. To evaluate viral replication human primary cells, human primary hepatocytes were infected in 6-well plates (1 x 10^s cells/well) by COP, deVV5 and deW5-/o/l at a MOI of 10⁴ (100 PFU/well). Cells were incubated in fresh growth medium supplemented with 10 % FCS until harvesting. At 48h post-infection, supernatant and cells were collected, freeze-thawed and sonicated and viral progeny were quantified on CEF by plaque assay. To evaluate viral replication on human skin tissue, human reconstituted Phenion skin were infected. The Phenion full-thickness (FT) skin model, a three-dimensional tissue construct that simulates histological and physiological properties of human skin, was purchased from Flenkel AG&Co. KGaA (DOsseldorf, Germany). This organotypic epithelial raft culture model was maintained in tissue culture medium according to the manufacturer's instructions. Each Phenion FT skin model was infected with 1 x 10^s PFU of COP, deVV5 -fcul and deVV5 by infusion (viruses were added directly into the medium). Cultures were incubated for 7 days at 37°C and medium was changed twice a week. Viral replication both in medium and reconstituted skin was quantified on CEF by plaque assay after 2 cycles of sonication in PBS.

In vitro cell sensitivity to 5-FC

Fluman FICT 116 tumor cells in suspension were transduced by deVV5 and deVV5 -fcul viruses at a MOI of 10⁵. A total of 3 x 10^s cells/well were plated in 6-well culture dishes in 2 mL of medium supplemented with 10 % FCS. After 48h of infection, cells were exposed to various concentrations of 5-FC ranging from 10⁷ to 10³ M. Three days later, cell viability was determined by trypan blue exclusion using a Vi-Cell cell counter. Results are expressed as percentage of viable cells, 100% corresponding to infected cells without 5-FC.

Cytosine deaminase enzymatic assay

Cytosine deaminase (CDase) activity was quantified by measuring the amount of 5-FU released in the culture media. FICT 116 cells were infected with the different vectors at a MOI of 10 ^s and plated in 6-well culture dish (1 x 10^s cells/well). After 48h, 0.1 mM 5-FC was added to the culture medium. Every day for 4 days, 5-FC and 5-FU concentrations in the media were measured by H PLC. Fifty pL of media were quenched with 50 pL of acetonitrile. The samples were vortexed and centrifuged. The organic supernatant was evaporated to dryness and reconstituted in 50 pL of water and analyzed by H PLC using a mobile phase of 50 mM phosphoric acid adjusted to pH 2.1. Results are expressed as the percentage of 5-FU relative to the total amount of 5FC + 5FU after various incubation times with 5-FC. DNA sequencing

Samples from viral DNA were purified with AMPure XP (Beckman Coulter, Inc.) beads kit to remove residual cellular DNA and sent for sequencing to the GenomEast platform (IGBMC Microarray and Sequencing platform, lllkirch-Graffenstaden, France). Data from lllumina HiSeq4000 in 2xl00bp paired-end runs were quality trimmed using a Phred score threshold of Q30. Contigs were de novo assembled with SPAdes v3.11.1 (Nurk et al., 2013, J Comput Biol., 20, 714-737. doi: 10.1089/cmb.2013.0084) following authors instructions and scaffolding was performed with custom script to generate the longest consensus sequence for each viral genome. BLAST 2.7.1 pairwise alignments were used to locate homologous regions between deVV5 and each of the parental strains (Altschul et al., 1990, J Mol Biol., 215, 403-410). Global pairwise alignments were performed using MAFFT v7.017 to measure the genomic homology between deVV5 and each parental genome, and to highlight the longest regions with strict identity between deVV5 and the corresponding parental genome (Katoh et al., 2002, Nucleic Acids Res. 30, 3059-3066). Results were reported using Circos vO.69-3 (Krzywinski, et al., 2009, Genome Res., 19, 1639-1645. doi: 10.1101/gr.092759.109) and Geneious v8.1.9, www.geneious.com (Kearse et al., 2012, Bioinformatics., 28, 1647-1649. doi: 10.1093/bioinformatics/btsl99) software.

Percentage of identity of the proteins

The percentages of identity between the proteins encoded by the chimeric vaccinia virus of the invention and the corresponding proteins encoded by the parental viruses (VACV Copenhagen, MVA, VACV Wyeth and VACV Western Reserve) were calculated via the alignment of the amino acid sequences by the algorithm of Needleman et Wunsch with affine gap penalty (Needleman et Wunsh. J.Mol., 1970, Biol. 48,443-453) using the Blosum62 similarity matrix. The percentage of identity is deducted from the alignment of the longest sequence. The proteins for which the study was non- applicable were truncated proteins (a protein shortened by a mutation which specifically induces premature termination of messenger RNA translation), or proteins having less than 30% of identity. It is well known in the art that models based on <30% sequence identity have significant alignment errors, resulting in large errors in main chain positions (Vitkup et al., 2001, Nature Struct Biol, volume 8 number 6, p 559-566). Below 30%, sequence identity, the alignment errors increase rapidly and become the most substantial origin of errors in comparative models.

Analysis of viral and cellular transcriptomes of de\/V5-FCUl or COP-FCU1 infecting normal

(hepatocytes) or tumor (HCT116) cells

Human colorectal carcinoma HCT116 cells (ATCC-CCL-185), are cultured using Me Coy's 5A ATCC (30-2007) + 10% FBS (Lifetch #10101-145) + 1% Gentamicin (4g/ml). Human hepatocytes are prepared by Biopredic (2 x 10^s cells per well). Cells are sed 24 hours before being infected by VVTG17111 (COP-eGFP-FCUl) or by VVSTG18974 (deVV5-eGFP-FCUl) with a MOI of 2 for 2 or 6 hours. Infections are performed in quadruplicates.

RNA extractions are performed using RNeasy Plus Mini Kit (Qiagen, cat#74134) and RNAse free DNAse I Set (Qiagen, cat#79254). RNA concentrations are measured using DO260 and DO280 absorbances with Nanodrop 2000/2010. Samples are stored at -80°C prior to sequencing.

For library preparation, SMART-Seq v4 UltraLow Input RNA kit + Nextera XT DNA sample preparation kit are used at the Genomeast platform (IGBMC, lllkirch-Graffenstaden, France).

RNASeq is performed using lllumina HiSeq4000 technology in 2x100 bp paired-end mode. All samples are sequenced in an average of 35 million reads, with a quality Phred score Q30 above 83%.

Results

Oncolytic activity of the four VACV parental strains

To determine the oncolytic activity of the four parental VACV strains as reference, the percentage of surviving cells in a panel of tumor cells was calculated after infection at MOI 10⁵ with the strains used for shuffling: MVA, WY, WR and COP. 100% of cell survival corresponds to the mock-infected cells. At the MOI of 10 ^s, COP demonstrated the lowest number of surviving cells across all tumor cell lines indicating that COP is the strongest oncolytic strain tested. Thus, throughout the remaining study, COP served as the reference for oncolytic potency (Table 3). As expected and as previously demonstrated (Erbs et al., 2008, Cancer Gene Ther., 15, 18-28), MVA had no cytotoxic effect on tumor cell lines. Table 3 also shows that the tumor cell lines LoVo and MIA PaCa-2 are among the most resistant to all three oncolytic viruses: WY, WR and COP.

MVA WY WR COP

A549 94.5 ± 3.6 90.2 ± 3.2 58.6 ± 3.7 47.8 ± 4.8

LoVo 98.6 ± 2.7 90.4 ± 5.9 70.8 ± 6.2 73.1 ± 5.2

MIA PaCa-2 103.2 ± 4.2 92.8 ± 2.9 89.4 ± 2.4 78.1 ± 1.5 U-87 MG 102.8 ± 4.8 64.6 ± 1.6 41.0 ± 0.8 43.6 ± 0.3 Hep G2 97.3 ± 3.9 66.8 ± 4.9 70.9 ± 9.4 13.6 ± 2.7 HCT 116 99.2 ± 5.4 91.5 ± 3.4 94.6 ± 3.4 55.1 ± 7.5 OE19 105.2 ± 6.2 78.9 ± 2.2 77.6 ± 3.3 70.3 ± 0.7 SK-OV-3 95.4 ± 7.3 59.8 ± 1.8 99.7 ± 1.5 42.9 ± 1.3

Table 3. Percentage of surviving cells infected by four strains of VACV used in this study. Human tumor cell lines were infected by the indicated viruses at MOI 10⁵. Survival was determined 5 days later, as described in the Materials and Methods section. Cell viability results are expressed as the percentage of viable cells relative to non-infected cells. Values are expressed as mean ± SD of three individual infections.

VACV shuffling: deVV5 is a chimeric virus with enhanced oncolytic potency in vitro

A directed evolution strategy was employed to generate chimeric VACV with increased oncolytic potency and tumor selectivity. The method comprised 2 steps. First, a library of viruses was generated by infecting LoVo cells by the 4 strains of virus. Second, amplification and selection of viral progeny was done by 9 successive passages on LoVo cells followed by 12 successive passages on MIA PaCa-2 cells. Virus pools with improved properties were passaged on LoVo and MIA PaCa-2 cells under stringent conditions towards clonal isolation of a more potent virus, as described in the Materials and Methods section. Sequential passages on LoVo and MIA PaCa-2 cells was chosen as these tumor cell lines are known to be rather resistant to oncolytic viruses compared to other tumor cell lines (see also Table 1). In the first step, LoVo cells were infected with a pool of VACV strains including COP, WY, WR and MVA. After the primary infection, 9 passages (LP1 - LP9) were performed on LoVo cells.

During the whole process of chimeric virus selection and amplification, several passages were tested in comparison to the parental COP to evaluate the selection and improvement of oncolytic potency (Figure la). At passage 6 (LP6), no improvement was observed. At passage 9 (LP9), an important gain of oncolytic potency was recorded on LoVo cells and a slight gain was observed in OE19 and U-87 MG cells. In contrast, no improvement of oncolytic activity was observed in SK-OV-3, HCT 116, Hep G2 and MIA PaCa-2 cells. Considering these results, LP9 was chosen for another cycle of genetic selection on MIA PaCa-2 which was the least permissive cell line to the LP9 pool, thereby imposing stringent selective pressure towards the genetic selection of a rare recombination event. Selection by dilution was performed under the same conditions as described above and in the Materials and Methods section. During the selection process in the MIA PaCa-2 cell line, three passages were tested for their lytic potential. MP6, MP10 and MP12 were compared at low MOI, with the parental COP (Figure lb). MP12 were found to be more effective than all others. Based on these results MP12 was selected to proceed to clone isolation. Eighteen individual plaque-purified viruses were isolated and screened for their oncolytic potential on the MIA PaCa-2 tumor cell line. The oncolytic potency of individual plaque clones was compared to that of the parental COP and the pool MP12 (Figure lc). The clones were found heterogenic in potency compared to MP12 pool, suggesting that numerous viruses were present in the pool. The most potent and the only clone surpassing MP12 at both MOI 10⁵ and 10⁴ was clone C5. With this clone and using a MOI of 10⁵, we have obtained a superior oncolytic activity compared to COP used at MOI 10⁴. The clone purity was assessed by evaluation of 20 plaque-purified clones originated from C5, all of which displayed the same phenotype in different tumor cells (data not shown). This clone, termed deVV5, was chosen for further characterization.

Genome analysis

DNA from deVV5 and four parental virus strains were purified and sequenced by Next Generation Sequencing. The use of paired-end short reads and whole genome de novo assembly led to the generation of viral genomes with the core region and one copy of the Inverted Terminal Repeats (ITR) domain. These single-ITR versions of the genomes are due to the use of unique k-mers in the contigs assembly process, but the presence of both ITR regions in every viral genome was confirmed by the mean depth coverage which was higher than the one from the core region, with a 2-fold factor (data not shown). Single-ITR versions of the genomes were retained for the sake of clarity in the subsequent paragraph. The resulting genome sequences, MVA (163,668 bp), COP (175,766 bp), WR (181,350 bp), WY (182,933 bp) and deVV5 (172,732 bp) are shown in Figure 2a around the circos plot. The links between the genomes reflect identical DNA sequences longer than 1,000 bp between the deVV5 sequence and each of the 4 parental genomes. Some regions of deVV5 seem to be conserved in several parental strains but long regions (including ITR) appeared to be specific to only one parental genome. Nevertheless, almost all the deVV5 genome was found to be 100% identical in at least one parental genome. To further investigate the composition of the deVV5 genome, global pairwise alignments were performed to identify the longest identical DNA regions between deVV5 and any parental genome (Figure 2b). Starting at the 5' end of the core region, the first 30 kilobases of deVV5 are almost identical to WY sequence, followed by MVA (42 kb). The middle of the core region appears to be a patchwork of regions around 5 kb derived from the 4 strains. The 3' end of the core domain is mainly composed of sequences identical to COP (56 kb). Finally, as described above, the 17-kb ITR domain is identical to WY. These observations confirmed that the shuffling resulted in an assembly of three main regions, derived from WY, MVA and COP for the core domain, surrounded by ITR regions derived from WY. The percentage of nucleic acid identity was also explored between deVV5 and each of the parental vaccinia virus strains (Figure 2c), using the method of global pairwise alignment (MAAFT). The deVV5 nucleic acid sequence was 89.15% identical to MVA nucleic acid sequence, 91.26% identical to WR nucleic acid sequence, 96.82% identical to COP nucleic acid sequence, and 97.39% identical to WY nucleic acid sequence.

Arming ofdeVV5 leads to increased potency

To evaluate the potential of the chimeric virus to carry and express functional transgene, deVV5 was armed with a therapeutic gene. The FCU1 suicide gene (Erbs et al., 2000, Cancer Res., 60, 3813-3822) under the control of the strong pllk7.5 VACV promoter, was inserted in the TK locus of deVV5 to generate the deVV5 -fcul. Expression of functional FCU1 by deVV5 -fcul was confirmed by quantification of the 5-FU released in supernatant of infected cells. The analysis of FICT 116 cells supernatants by H PLC showed a progressive release of 5-FU in extracellular medium of FICT 116 cells transduced with the indicated viruses at MOI of 10⁵ and incubated with 0.1 mM 5-FC (Figure 3a). No 5-FU was detected in supernatants of COP or deVV5-infected cells. Five days after infection, approximately 60% of 5-FC was converted to 5-FU in supernatants of deVV5 -fcul transduced cells, confirming functionality and efficacy of the FCU1 protein expressed by the recombinant deVV5 -fcul vector. To compare the antitumoral activity of deVV5 -fcul vector alone or combined with 5-FC treatment, FICT 116 cells were infected at MOI 10 ^s. Two days post-infection, 5-FC was added to the culture supernatants at a range of concentrations (10 ³ to 10⁷M). Cell viability was determined three days later by trypan blue exclusion (Figure 3b). The addition of 5-FC had no impact on the viability of mock or deVV5 infected tumor cells. In contrast, the 5-FC conferred increased cytotoxicity in a prodrug dose-dependent manner to deVV5 -fcul infected human tumor cells. The combination of a low amount of deW5-/o/l with 1 mM of 5-FC induced 70% mortality of FICT 116 cells. These results indicate that recombinant deVV5 -fcul acquired an enhanced in vitro anti-tumor activity as compared to deVV5 in the presence of 5-FC prodrug. Together, these in vitro enzymatic activity results demonstrate that deVV5 -fcul can express a functional therapeutic FCU1 gene and is an efficient vector for viral directed enzyme prodrug therapy (VDEPT) in vitro.

Increased oncolytic and replicative efficiency of the novel evolved VACV

To determine whether the armed and wild type evolved viruses can replicate and kill cancer cells, a screen of tumor cells from various origins was undertaken (Figure 4a). At low MOI, all tested cancer cells were more susceptible to the chimeric virus deVV5. Numerous cancer cell lines (UM-UC-3, FICT 116, A549 and SK-OV-3) were destroyed by deVV5 while staying resistant to COP oncolysis. The deletion of the TK gene in deVV5 had a slight impact on the virus capacity to destroy cancer cells. In most of the tested cell lines, deW5-/o/l demonstrated similar lytic activity than the parental deVV5. These results were correlated with those observed during the replication potency assay in tumor cells (Figure 4b). All human cancer cell lines tested showed significant deVV5 replication. Compared to parental COP, deVV5 produced 5 times more viral particles in Flep G2 cells, 30 times more in A549 cells and 90 times more in OE19 cells. These results confirmed the oncolytic benefit obtained through the process of directed evolution resulting in genomic blending and the possibility to delete genes without major impact on lytic and replication features of the virus in tumor cells. Enhanced safety of the chimeric deVV5 and the recombinant armed deVV5-fcul on human primary cells

The therapeutic index of COP, deVV5 -fcul and deVV5 was determined using the yield of replication in human tumor cells and human primary cells. Human hepatocytes and human reconstituted skin (Phenion full-thickness skin model) were used as primary healthy cells. As compared to COP, the chimeric wild type deVV5 demonstrated an enhanced replicative-capacity on human tumor cells while reducing replication on primary cells (Figure 4b). A 2-fold and 10-fold reduction of replication were observed respectively in Phenion full-thickness skin model and hepatocytes. Moreover, TK deletion had a clear benefit on deVV5 -fcul allowing a supplementary 10-fold reduction of replication on human skin and hepatocytes. These results confirmed, and are consistent with previously reports demonstrating, the benefit of deleting TK gene for safety improvement (Ricordel et al., 2017, Mol Ther Oncolytics., 7, 1-11, doi: 10.1016/j.omto.2017.08.003).

Regarding the therapeutic index calculated with the ratio of viral progeny produced on Hep G2 tumor cells and human primary hepatocytes, a significant improvement in favor of the chimeric viruses, notably the TK-deleted virus, was demonstrated (Figure 4c).

Analysis of viral and cellular transcriptomes of deVV5-FCUl or COP-FCU1 infecting normal (hepatocytes) or tumor (HCT116) cells

Transcriptome experiment is designed to characterize the transcriptome of deVV5 and to get information linking its genome to its phenotype. The viruses used in this experiment are modified versions of deVV5 and vaccinia virus Copenhagen strain, both bearing the fusion protein eGFP-FCUl at the Thymidine Kinase locus. In this study, deep RNA is used to analyze the transcriptome at two timepoints (2 hours and 6 hours post-infection) following the infection of a human colorectal carcinoma cell line (HCT116) and human primary hepatocytes. Control conditions are non-infected cells (HCT116 and hepatocytes) at 6 hours.

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Claims

1. A chimeric vaccinia virus, wherein said chimeric vaccinia virus encodes:

(L) at least 255 distinct proteins, each of said proteins comprising an amino acid sequence with at least 99% identity with one of SEQ ID NO:6 to 318; or

2. The chimeric vaccinia virus of any one of claim 1, wherein said chimeric vaccinia virus encodes:

54, (b) a protein comprising an amino acid sequence with at least 99.6 % of identity with SEQ ID NO: 62,

64,

143,

147,

155; or

(g) any combination of proteins (a) to (f); preferably said chimeric vaccinia virus encodes: a) a protein comprising the amino acid sequence SEQ ID NO: 54;

b) a protein comprising the amino acid sequence SEQ ID NO: 62;

c) a protein comprising the amino acid sequence SEQ ID NO: 64;

d) a protein comprising the amino acid sequence SEQ ID NO:143;

e) a protein comprising the amino acid sequence SEQ ID NO: 147;

f) a protein comprising the amino acid sequence SEQ ID NO: 155; or

g) any combination of proteins a) to f).

3. The chimeric vaccinia virus of claim 1 or claim 2, wherein said chimeric vaccinia virus comprises a nucleic acid sequence having at least 66 % of identity with SEQ ID NO: 1, preferably having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, more preferably at least 97,4% of identity, at least 97,5%, at least 97,6%, at least 97,7%, at least 97,8%, at least 97,9%, at least 98%, at least 98,1%, at least 98,2%, at least 98,3%, at least 98,4%, at least 98,5%, at least 98,6%, at least 98,7%, at least 98,8%, at least 98,9%, at least 99%, at least 99,1%, at least 99,2%, at least 99,3%, at least 99,4%, at least 99,5%, at least 99,6%, at least 99,7%, at least 99,8%, at least 99,9%, or even 100% of identity with SEQ ID NO: 1.

4. The chimeric vaccinia virus of any one of claims 1 to 3, wherein said chimeric vaccinia virus comprises:

(a) at least one Copenhagen (COP)-derived nucleic acid sequence selected from:

o a nucleic acid sequence consisting of nucleotides 96234 to 117168 of SEQ ID NO: 2, and o a nucleic acid sequence consisting of nucleotides 119868 to 159589 of SEQ ID NO: 2;

(c) at least one Wyeth (WY)-derived nucleic acid sequence selected from:

(e) any combination of (a) to (d).

5. The chimeric vaccinia virus of any one of claims 1 to 4, wherein at least one organ-specific therapeutic index of said chimeric vaccinia virus is higher than the corresponding organ-specific therapeutic indexes of parental vaccinia virus strains Copenhagen (COP), Modified Vaccinia Ankara (MVA), Wyeth (WY), and Western Reserve (WR), wherein for a given organ and a given virus, an organ- specific therapeutic index TI(organ, virus) is defined as:

6. The chimeric vaccinia virus of claim 5, wherein the hepatic therapeutic index of said chimeric vaccinia virus is higher than, preferably at least 20 times higher than, at least 30 times higher than the corresponding hepatic therapeutic indexes of parental vaccinia virus strains COP, MVA, WY and WR wherein for a given virus, an hepatic therapeutic index Tl(liver, virus) is defined as:

Tl(liver, virus) = (replication of virus in Hep G2 tumor cells/ replication of virus in primary hepatocytes).

7. The chimeric vaccinia virus of any one of claims 1 to 6, wherein at least one tumor-specific oncolytic power of said chimeric vaccinia virus is higher than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR, wherein for a given tumor and a given virus, a tumor-specific oncolytic power OP(tumor, virus) is defined as: OP(tumor, virus) = (100 - percentage of surviving tumor cells after infection by virus).

8. The chimeric vaccinia virus of claim 7, wherein tumor-specific oncolytic power of said chimeric vaccinia virus is higher than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR in at least one tumor cells line selected between UM-UC-3, A549, SKOV-3, Hep G2, Cal-33, MIA PaCa-2, OE 19, HCT 116 and KATO III.

9. The chimeric vaccinia virus of any one of claims 1 to 8, which has been or may be obtained by a method of directed evolution, said method comprising:

(i) amplifying a plurality of parental vaccinia virus strains on a poorly or non-permissive tumor cell line;

(ii) collecting the supernatant containing one or more variant chimeric vaccinia virus(es) having an oncolytic power in the poorly or non-permissive tumor cell line of step (i) wherein for a given tumor and a given virus, a tumor-specific oncolytic power OP(tumor, virus) is defined as:

(iii) amplifying one or more variant chimeric vaccinia virus(es) of the supernatant on a poorly or non- permissive tumor cell line;

(iv) collecting the supernatant containing one or more chimeric vaccinia virus(es) having an oncolytic power in the poorly or non-permissive tumor cell line of step (iii);

(v) repeating steps (iii) and (iv), until one or more variant chimeric vaccinia virus(es) having a desired oncolytic power in poorly or non-permissive tumor cell lines used in performed steps (i) and/or (iii) is obtained.

10. The chimeric vaccinia virus of claim 9, wherein said parental vaccinia virus strains used in first step (i) are selected in the group consisting of Modified Vaccinia Virus Ankara (MVA), Copenhagen (COP), Wyeth (WY), Western Reserve (WR), Elstree, New York City Board of Health (NYCBH), IHD-J, Dryvax, CCSV, CJ-50300, Lister, RIVM, Israel, Lister/CEP, Elstree-BN, Temple of Heaven (Tian Tan), Guang 9, Ecuador-Moscow 1963 (EM-63), Tashkent , B-15, Bern, Paris, Dairen, Ikeda, Ankara, LC16m8, CV1, EM63, ACAM2000, CL, CAM, CTC, Lederle-Chorioallantoic, L-variant, S-variant, rVV-hgplOO and AS, preferably said parental vaccinia virus strains used in first step (i) are selected in the group consisting of MVA, COP, WY and WR.

11. The chimeric vaccinia virus of claim 9 or claim 10, wherein said poorly permissive or non-permissive tumor cell lines used in steps (i) and/or (iii) are from higher mammal's origins, preferably said poorly permissive or non-permissive tumor cell lines used in steps (i) and/or (iii) are selected in the group consisting of LoVo, MIA PaCa-2, U-118 MG, REM 134, OVCAR-3, SNU-5, KATO III, UACC.257, OE 19, AsPC-1, U-87 MG, OE 21, SW780 and Capan-2.

12. The chimeric vaccinia virus of any one of claims 9 to 11, wherein said method further comprises the use of at least one mutagenic agent in step(s) (i), (ii), (iii) and/or (iv), wherein said mutagenic agents are preferably selected from physical agents, chemical agents and biological agents.

13. A chimeric vaccinia virus, wherein said chimeric vaccinia virus encodes:

(a) a protein comprising an amino acid sequence with at least 96.3 % of identity with SEQ ID NO: 54,

(b) a protein comprising an amino acid sequence with at least 99.6 % of identity with SEQ ID NO: 62,

(c) a protein comprising an amino acid sequence with at least 98.7 % of identity with SEQ ID NO: 64,

(d) a protein comprising an amino acid sequence with at least 99.4 % of identity with SEQ ID NO: 143,

(e) a protein comprising an amino acid sequence with at least 99,9 % of identity with SEQ ID NO: 147,

(f) a protein comprising an amino acid sequence with at least 99,9 % of identity with SEQ ID NO: 155, or

(g) any combination of proteins (a) to (f); preferably said chimeric vaccinia virus encodes: a) a protein comprising the amino acid sequence of SEQ ID NO: 54,

b) a protein comprising the amino acid sequence of SEQ ID NO: 62,

c) a protein comprising the amino acid sequence of SEQ ID NO: 64,

d) a protein comprising the amino acid sequence of SEQ ID NO: 143,

e) a protein comprising the amino acid sequence of SEQ ID NO: 147,

f) a protein comprising the amino acid sequence of SEQ ID NO: 155, or

g) any combination of proteins a) to f).

14. The chimeric vaccinia virus of claim 13, wherein said chimeric vaccinia virus encodes:

(b) at least 304 distinct proteins, each of said proteins comprising an amino acid sequence with at least 80% identity with one of SEQ ID NO:6 to 318; (c) at least 301 distinct proteins, each of said proteins comprising an amino acid sequence with at least 90% identity with one of SEQ ID NO:6 to 318;

15. The chimeric vaccinia virus of any one of claims 13 to 14, wherein said chimeric vaccinia virus comprises a nucleic acid sequence having at least 66 % of identity with SEQ ID NO: 1, preferably having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, more preferably at least 97,4% of identity, at least 97,5%, at least 97,6%, at least 97,7%, at least 97,8%, at least 97,9%, at least 98%, at least 98,1%, at least 98,2%, at least 98,3%, at least 98,4%, at least 98,5%, at least 98,6%, at least 98,7%, at least 98,8%, at least 98,9%, at least 99%, at least 99,1%, at least 99,2%, at least 99,3%, at least 99,4%, at least 99,5%, at least 99,6%, at least 99,7%, at least 99,8%, at least 99,9%, or even 100% of identity with SEQ ID NO: 1.

16. The chimeric vaccinia virus of any one of claims 13 to 15, wherein said chimeric vaccinia virus comprises: (a) at least one Copenhagen (COP)-derived nucleic acid sequence selected from: o a nucleic acid sequence consisting of nucleotides 96234 to 117168 of SEQ ID NO: 2, and

(c) at least one Wyeth (WY)-derived nucleic acid sequence selected from:

(e) any combination of (a) to (d).

17. The chimeric vaccinia virus of any one of claims 13 to 16, wherein at least one organ-specific therapeutic index of said chimeric vaccinia virus is higher than the corresponding organ-specific therapeutic indexes of parental vaccinia virus strains Copenhagen (COP), Modified Vaccinia Ankara (MVA), Wyeth (WY), and Western Reserve (WR), wherein for a given organ and a given virus, an organ- specific therapeutic index TI(organ, virus) is defined as:

TI(organ, virus) = (replication of virus in organ tumor cells / replication of virus in organ healthy cells ).

18. The chimeric vaccinia virus of claim 17, wherein the hepatic therapeutic index of said chimeric vaccinia virus is higher than, preferably at least 20 times higher than, at least 30 times higher than the corresponding hepatic therapeutic indexes of parental vaccinia virus strains COP, MVA, WY and WR, wherein for a given virus, a hepatic therapeutic index Tl(liver, virus) is defined as:

19. The chimeric vaccinia virus of any one of claims 13 to 18, wherein at least one tumor-specific oncolytic power of said chimeric vaccinia virus is higher than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR, wherein for a given tumor and a given virus, a tumor-specific oncolytic power OP(tumor, virus) is defined as:

20. The chimeric vaccinia virus of claim 19, wherein tumor-specific oncolytic powers of said chimeric vaccinia virus in tumor cells UM-UC-3, A549, SKOV-3, Hep G2, Cal-33, MIA PaCa-2, OE 19, HCT 116 and KATO III are higher than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR in the same tumor cells.

21. The chimeric vaccinia virus of any one of claims 13 to 20, wherein said chimeric vaccinia virus has been or may be obtained by a method of directed evolution, said method comprising:

22. The chimeric vaccinia virus of claim 21, wherein said parental vaccinia virus strains used in first step (i) are selected in the group consisting of Modified Vaccinia Virus Ankara (MVA), Copenhagen (COP), Wyeth (WY), Western Reserve (WR), Elstree, New York City Board of Health (NYCBH), IHD-J, Dryvax, CCSV, CJ-50300, Lister, RIVM, Israel, Lister/CEP, Elstree-BN, Temple of Heaven (Tian Tan), Guang 9, Ecuador-Moscow 1963 (EM-63), Tashkent , B-15, Bern, Paris, Dairen, Ikeda, Ankara, LC16m8, CV1, EM63, ACAM2000, CL, CAM, CTC, Lederle-Chorioallantoic, L-variant, S-variant, rVV-hgplOO and AS, preferably said parental vaccinia virus strains used in first step (i) are selected in the group consisting of MVA, COP, WY and WR.

23. The chimeric vaccinia virus of claim 21 or claim 22, wherein said poorly permissive or non- permissive tumor cell lines used in steps (i) and/or (iii) are from higher mammal's origins, preferably said poorly permissive or non-permissive tumor cell lines used in steps (i) and/or (iii) are selected in the group consisting of LoVo, MIA PaCa-2, U-118 MG, REM 134, OVCAR-3, SNU-5, KATO III, UACC.257, OE 19, AsPC-1, U-87 MG, OE 21, SW780 and Capan-2.

24. The chimeric vaccinia virus of any one of claims 21 to 23, wherein said method further comprises the use of at least one mutagenic agent in step(s) (i), (ii), (iii) and/or (iv), wherein said mutagenic agents are preferably selected from physical agents, chemical agents and biological agents.

25. A chimeric vaccinia virus comprising a nucleic acid sequence having at least 66 % of identity with SEQ ID NO: 1, preferably having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, or at least 97% of identity with SEQ ID NO: 1.

26. The chimeric vaccinia virus of claim 25, comprising a nucleic acid sequence having at least 97,4% of identity, at least 97,5%, at least 97,6%, at least 97,7%, at least 97,8%, at least 97,9%, at least 98%, at least 98,1%, at least 98,2%, at least 98,3%, at least 98,4%, at least 98,5%, at least 98,6%, at least 98,7%, at least 98,8%, at least 98,9%, at least 99%, at least 99,1%, at least 99,2%, at least 99,3%, at least 99,4%, at least 99,5%, at least 99,6%, at least 99,7%, at least 99,8%, at least 99,9%, or even 100% of identity with SEQ ID NO: 1.

27. The chimeric vaccinia virus of any one of claims 25 to 26, wherein said chimeric vaccinia virus comprises:

(a) at least one Copenhagen (COP)-derived nucleic acid sequence selected from:

(c) at least one Wyeth (WY)-derived nucleic acid sequence selected from:

(e) any combination of (a) to (d).

28. The chimeric vaccinia virus of any one of claims 25 to 27, wherein said chimeric vaccinia virus encodes:

(k) at least 282 distinct proteins, each of said proteins comprising an amino acid sequence with at least 98% identity with one of SEQ ID NO:6 to 318; (L) at least 255 distinct proteins, each of said proteins comprising an amino acid sequence with at least 99% identity with one of SEQ ID NO:6 to 318; or

29. The chimeric vaccinia virus of any one of claims 25 to 28, wherein said chimeric vaccinia virus encodes:

54,

62,

64,

143,

147,

155, or

b) a protein comprising the amino acid sequence of SEQ ID NO: 62,

c) a protein comprising the amino acid sequence of SEQ ID NO: 64,

d) a protein comprising the amino acid sequence of SEQ ID NO: 143,

e) a protein comprising the amino acid sequence of SEQ ID NO: 147,

f) a protein comprising the amino acid sequence of SEQ ID NO: 155, or

g) any combination of proteins a) to f).

30. The chimeric vaccinia virus of any one of claims 25 to 29, wherein at least one organ-specific therapeutic index of said chimeric vaccinia virus is higher than the corresponding organ-specific therapeutic indexes of parental vaccinia virus strains Copenhagen (COP), Modified Vaccinia Ankara (MVA), Wyeth (WY), and Western Reserve (WR), wherein for a given organ and a given virus, an organ- specific therapeutic index TI(organ, virus) is defined as: TI(organ, virus) = (replication of virus in organ tumor cells / replication of virus in organ healthy cells).

31. The chimeric vaccinia virus of claim 30, wherein the hepatic therapeutic index of said chimeric vaccinia virus is higher than, preferably at least 20 times higher than, at least 30 times higher than the corresponding hepatic therapeutic indexes of parental vaccinia virus strains COP, MVA, WY and WR, wherein for a given virus, a hepatic therapeutic index Tl(liver, virus) is defined as:

32. The chimeric vaccinia virus of any one of claims 25 to 31, wherein at least one tumor-specific oncolytic power of said chimeric vaccinia virus is higher than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR, wherein for a given tumor and a given virus, a tumor-specific oncolytic power OP(tumor, virus) is defined as:

33. The chimeric vaccinia virus of claim 32, wherein tumor-specific oncolytic powers of said chimeric vaccinia virus in tumor cells UM-UC-3, A549, SKOV-3, Hep G2, Cal-33, MIA PaCa-2, OE 19, HCT 116 and KATO III are higher than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR in the same tumor cells.

34. The chimeric vaccinia virus of any one of claims 25 to 33, wherein said chimeric vaccinia virus has been or may be obtained by a method of directed evolution, said method comprising:

35. The chimeric vaccinia virus of claim 34, wherein said parental vaccinia virus strains used in first step (i) are selected in the group consisting of Modified Vaccinia Virus Ankara (MVA), Copenhagen (COP), Wyeth (WY), Western Reserve (WR), Elstree, New York City Board of Health (NYCBH), IHD-J, Dryvax, CCSV, CJ-50300, Lister, RIVM, Israel, Lister/CEP, Elstree-BN, Temple of Heaven (Tian Tan), Guang 9, Ecuador-Moscow 1963 (EM-63), Tashkent , B-15, Bern, Paris, Dairen, Ikeda, Ankara, LC16m8, CV1, EM63, ACAM2000, CL, CAM, CTC, Lederle-Chorioallantoic, L-variant, S-variant, rVV-hgplOO and AS, preferably said parental vaccinia virus strains used in first step (i) are selected in the group consisting of MVA, COP, WY and WR.

36. The chimeric vaccinia virus of claim 34 or claim 35, wherein said poorly permissive or non- permissive tumor cell lines used in steps (i) and/or (iii) are from higher mammal's origins, preferably said poorly permissive or non-permissive tumor cell lines used in steps (i) and/or (iii) are selected in the group consisting of LoVo, MIA PaCa-2, U-118 MG, REM 134, OVCAR-3, SNU-5, KATO III, UACC.257, OE 19, AsPC-1, U-87 MG, OE 21, SW780 and Capan-2.

37. The chimeric vaccinia virus of any one of claims 34 to 36, wherein said method further comprises the use of at least one mutagenic agent in step(s) (i), (ii), (iii) and/or (iv), wherein said mutagenic agents are preferably selected from physical agents, chemical agents and biological agents.

38. A chimeric vaccinia virus, wherein at least one organ-specific therapeutic index of said chimeric vaccinia virus is higher than the corresponding organ-specific therapeutic indexes of parental vaccinia virus strains Copenhagen (COP), Modified Vaccinia Ankara (MVA), Wyeth (WY), and Western Reserve (WR), wherein for a given organ and a given virus, an organ-specific therapeutic index TI(organ, virus) is defined as:

39. The chimeric vaccinia virus of claim 38, wherein the hepatic therapeutic index of said chimeric vaccinia virus is higher than, preferably at least 20 times higher than, at least 30 times higher than the corresponding hepatic therapeutic indexes of parental vaccinia virus strains COP, MVA, WY and WR, wherein for a given virus, a hepatic therapeutic index Tl(liver, virus) is defined as:

40. The chimeric vaccinia virus of any one of claims 38 to 39, wherein said chimeric vaccinia virus encodes:

41. The chimeric vaccinia virus of any one of claims 38 to 40, wherein said chimeric vaccinia virus encodes:

54,

62,

64,

143,

147, (f) a protein comprising an amino acid sequence with at least 99,9 % of identity with SEQ ID NO: 155, or

b) a protein comprising the amino acid sequence of SEQ ID NO: 62,

c) a protein comprising the amino acid sequence of SEQ ID NO: 64,

d) a protein comprising the amino acid sequence of SEQ ID NO: 143,

e) a protein comprising the amino acid sequence of SEQ ID NO: 147,

f) a protein comprising the amino acid sequence of SEQ ID NO: 155, or

g) any combination of proteins a) to f).

42. The chimeric vaccinia virus of any one of claims 38 to 41, wherein said chimeric vaccinia virus comprises a nucleic acid sequence having at least 66 % of identity with SEQ ID NO: 1, preferably having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, more preferably at least 97,4% of identity, at least 97,5%, at least 97,6%, at least 97,7%, at least 97,8%, at least 97,9%, at least 98%, at least 98,1%, at least 98,2%, at least 98,3%, at least 98,4%, at least 98,5%, at least 98,6%, at least 98,7%, at least

98,8%, at least 98,9%, at least 99%, at least 99,1%, at least 99,2%, at least 99,3%, at least 99,4%, at least 99,5%, at least 99,6%, at least 99,7%, at least 99,8%, at least 99,9%, or even 100% of identity with SEQ ID NO: 1.

43. The chimeric vaccinia virus of any one of claims 38 to 42, wherein said chimeric vaccinia virus comprises:

(a) at least one Copenhagen (COP)-derived nucleic acid sequence selected from:

(c) at least one Wyeth (WY)-derived nucleic acid sequence selected from:

(e) any combination of (a) to (d).

44. The chimeric vaccinia virus of any one of claims 38 to 43, wherein at least one tumor-specific oncolytic power of said chimeric vaccinia virus is higher than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR, wherein for a given tumor and a given virus, a tumor-specific oncolytic power OP(tumor, virus) is defined as:

45. The chimeric vaccinia virus of claim 44, wherein tumor-specific oncolytic powers of said chimeric vaccinia virus in tumor cells UM-UC-3, A549, SKOV-3, Hep G2, Cal-33, MIA PaCa-2, OE 19, HCT 116 and KATO III are higher than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR in the same tumor cells.

46. The chimeric vaccinia virus of any one of claims 38 to 45, wherein said chimeric vaccinia virus has been or may be obtained by a method of directed evolution, said method comprising:

47. The chimeric vaccinia virus of claim 46, wherein said parental vaccinia virus strains used in first step (i) are selected in the group consisting of Modified Vaccinia Virus Ankara (MVA), Copenhagen (COP), Wyeth (WY), Western Reserve (WR), Elstree, New York City Board of Health (NYCBH), IHD-J, Dryvax, CCSV, CJ-50300, Lister, RIVM, Israel, Lister/CEP, Elstree-BN, Temple of Heaven (Tian Tan), Guang 9, Ecuador-Moscow 1963 (EM-63), Tashkent , B-15, Bern, Paris, Dairen, Ikeda, Ankara, LC16m8, CV1, EM63, ACAM2000, CL, CAM, CTC, Lederle-Chorioallantoic, L-variant, S-variant, rVV-hgplOO and AS, preferably said parental vaccinia virus strains used in first step (i) are selected in the group consisting of MVA, COP, WY and WR.

48. The chimeric vaccinia virus of claim 46 or claim 47, wherein said poorly permissive or non- permissive tumor cell lines used in steps (i) and/or (iii) are from higher mammal's origins, preferably said poorly permissive or non-permissive tumor cell lines used in steps (i) and/or (iii) are selected in the group consisting of LoVo, MIA PaCa-2, U-118 MG, REM 134, OVCAR-3, SNU-5, KATO III, UACC.257, OE 19, AsPC-1, U-87 MG, OE 21, SW780 and Capan-2.

49. The chimeric vaccinia virus of any one of claims 46 to 48, wherein said method further comprises the use of at least one mutagenic agent in step(s) (i), (ii), (iii) and/or (iv), wherein said mutagenic agents are preferably selected from physical agents, chemical agents and biological agents.

50. A chimeric vaccinia virus, wherein at least one tumor-specific oncolytic power of said chimeric vaccinia virus is higher than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR, wherein for a given tumor and a given virus, a tumor-specific oncolytic power OP(tumor, virus) is defined as:

51. The chimeric vaccinia virus of claim 50, wherein tumor-specific oncolytic powers of said chimeric vaccinia virus in tumor cells UM-UC-3, A549, SKOV-3, Hep G2, Cal-33, MIA PaCa-2, OE 19, HCT 116 and KATO III are higher than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR in the same tumor cells.

52. The chimeric vaccinia virus of any one of claims 50 to 51, wherein said chimeric vaccinia virus encodes:

53. The chimeric vaccinia virus of any one of claims 50 to 52, wherein said chimeric vaccinia virus encodes:

64,

143,

147,

155, or

b) a protein comprising the amino acid sequence of SEQ ID NO: 62,

c) a protein comprising the amino acid sequence of SEQ ID NO: 64,

d) a protein comprising the amino acid sequence of SEQ ID NO: 143,

e) a protein comprising the amino acid sequence of SEQ ID NO: 147,

f) a protein comprising the amino acid sequence of SEQ ID NO: 155, or

g) any combination of proteins a) to f).

54. The chimeric vaccinia virus of any one of claims 50 to 53, wherein said chimeric vaccinia virus comprises a nucleic acid sequence having at least 66 % of identity with SEQ ID NO: 1, preferably having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, more preferably at least 97,4% of identity, at least 97,5%, at least 97,6%, at least 97,7%, at least 97,8%, at least 97,9%, at least 98%, at least 98,1%, at least 98,2%, at least 98,3%, at least 98,4%, at least 98,5%, at least 98,6%, at least 98,7%, at least

55. The chimeric vaccinia virus of any one of claims 50 to 54, wherein said chimeric vaccinia virus comprises:

(a) at least one Copenhagen (COP)-derived nucleic acid sequence selected from:

(c) at least one Wyeth (WY)-derived nucleic acid sequence selected from:

(e) any combination of (a) to (d).

56. The chimeric vaccinia virus of any one of claims 50 to 55, wherein at least one organ-specific therapeutic index of said chimeric vaccinia virus is higher than the corresponding organ-specific therapeutic indexes of parental vaccinia virus strains Copenhagen (COP), Modified Vaccinia Ankara (MVA), Wyeth (WY), and Western Reserve (WR), wherein for a given organ and a given virus, an organ- specific therapeutic index TI(organ, virus) is defined as:

57. The chimeric vaccinia virus of claim 56, wherein the hepatic therapeutic index of said chimeric vaccinia virus is higher than, preferably at least 20 times higher than, at least 30 times higher than the corresponding hepatic therapeutic indexes of parental vaccinia virus strains COP, MVA, WY and WR, wherein for a given virus, a hepatic therapeutic index Tl(liver, virus) is defined as:

58. The chimeric vaccinia virus of any one of claims 50 to 57, wherein said chimeric vaccinia virus has been or may be obtained by a method of directed evolution, said method comprising:

(i) amplifying a plurality of parental vaccinia virus strains on a poorly or non-permissive tumor cell line; (ii) collecting the supernatant containing one or more variant chimeric vaccinia virus(es) having an oncolytic power in the poorly or non-permissive tumor cell line of step (i) wherein for a given tumor and a given virus, a tumor-specific oncolytic power OP(tumor, virus) is defined as:

(iv) collecting the supernatant containing one or more chimeric vaccinia virus(es) having an oncolytic power in the poorly permissive tumor cell line of step (iii);

59. The chimeric vaccinia virus of claim 58, wherein said parental vaccinia virus strains used in first step (i) are selected in the group consisting of Modified Vaccinia Virus Ankara (MVA), Copenhagen (COP), Wyeth (WY), Western Reserve (WR), Elstree, New York City Board of Health (NYCBH), IHD-J, Dryvax, CCSV, CJ-50300, Lister, RIVM, Israel, Lister/CEP, Elstree-BN, Temple of Heaven (Tian Tan), Guang 9, Ecuador-Moscow 1963 (EM-63), Tashkent , B-15, Bern, Paris, Dairen, Ikeda, Ankara, LC16m8, CV1, EM63, ACAM2000, CL, CAM, CTC, Lederle-Chorioallantoic, L-variant, S-variant, rVV-hgplOO and AS, preferably said parental vaccinia virus strains used in first step (i) are selected in the group consisting of MVA, COP, WY and WR.

60. The chimeric vaccinia virus of claim 58 or claim 59, wherein said poorly permissive or non- permissive tumor cell lines used in steps (i) and/or (iii) are from higher mammal's origins, preferably said poorly permissive or non-permissive tumor cell lines used in steps (i) and/or (iii) are selected in the group consisting of LoVo, MIA PaCa-2, U-118 MG, REM 134, OVCAR-3, SNU-5, KATO III, UACC.257, OE 19, AsPC-1, U-87 MG, OE 21, SW780 and Capan-2.

61. The chimeric vaccinia virus of any one of claims 58 to 60, wherein said method further comprises the use of at least one mutagenic agent in step(s) (i), (ii), (iii) and/or (iv), wherein said mutagenic agents are preferably selected from physical agents, chemical agents and biological agents.

62. A method of directed evolution for selecting a chimeric vaccinia virus with high oncolytic power, said method comprising:

(i) amplifying a plurality of parental vaccinia virus strains on a tumor cell line, wherein said tumor cell line is poorly permissive or non-permissive for each parental vaccinia virus strain; (ii) collecting the supernatant containing one or more variant chimeric vaccinia virus(es) having a non zero oncolytic power in the tumor cell line of step (i) after at least 2 days at a supernatant dilution of at least 2, wherein for a given tumor and a given virus, a tumor-specific oncolytic power OP(tumor, virus) is defined as:

63. The method of claim 62, wherein said parental vaccinia virus strains used in first step (i) are selected in the group consisting of Modified Vaccinia Virus Ankara (MVA), Copenhagen (COP), Wyeth (WY), Western Reserve (WR), Elstree, New York City Board of Health (NYCBH), IHD-J, Dryvax, CCSV, CJ-50300, Lister, RIVM, Israel, Lister/CEP, Elstree-BN, Temple of Heaven (Tian Tan), Guang 9, Ecuador-Moscow 1963 (EM-63), Tashkent , B-15, Bern, Paris, Dairen, Ikeda, Ankara, LC16m8, CV1, EM63, ACAM2000, CL, CAM, CTC, Lederle-Chorioallantoic, L-variant, S-variant, rVV-hgplOO and AS, preferably said parental vaccinia virus strains used in first step (i) are selected in the group consisting of MVA, COP, WY and WR.

64. The method of claim 62 or claim 63, wherein said poorly permissive or non-permissive tumor cell lines used in steps (i) and/or (iii) are from higher mammal's origins, preferably said poorly permissive or non-permissive tumor cell lines used in steps (i) and/or (iii) are selected in the group consisting of LoVo, MIA PaCa-2, U-118 MG, REM 134, OVCAR-3, SNU-5, KATO III, UACC.257, OE 19, AsPC-1, U-87 MG, OE 21, SW780 and Capan-2.

65. The method of any one of claims 62 to 64, wherein said method further comprises the use of at least one mutagenic agent in step(s) (i), (ii), (iii) and/or (iv), wherein said mutagenic agents are preferably selected from physical agents, chemical agents and biological agents.

66. A chimeric vaccinia virus, wherein said chimeric vaccinia virus has been or may be obtained by the method of any one of claims 62 to 65.

67. The chimeric vaccinia virus of claim 66, wherein said chimeric vaccinia virus encodes:

68. The chimeric vaccinia virus of any one of claims 66 to 67, wherein said chimeric vaccinia virus encodes:

54,

62, (c) a protein comprising an amino acid sequence with at least 98.7 % of identity with SEQ ID NO: 64,

143,

147,

155, or

b) a protein comprising the amino acid sequence of SEQ ID NO: 62,

c) a protein comprising the amino acid sequence of SEQ ID NO: 64,

d) a protein comprising the amino acid sequence of SEQ ID NO: 143,

e) a protein comprising the amino acid sequence of SEQ ID NO: 147,

f) a protein comprising the amino acid sequence of SEQ ID NO: 155, or

g) any combination of proteins a) to f).

69. The chimeric vaccinia virus of any one of claims 66 to 68, wherein said chimeric vaccinia virus comprises a nucleic acid sequence having at least 66 % of identity with SEQ ID NO: 1, preferably having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, more preferably at least 97,4% of identity, at least 97,5%, at least 97,6%, at least 97,7%, at least 97,8%, at least 97,9%, at least 98%, at least 98,1%, at least 98,2%, at least 98,3%, at least 98,4%, at least 98,5%, at least 98,6%, at least 98,7%, at least

70. The chimeric vaccinia virus of any one of claims 66 to 69, wherein said chimeric vaccinia virus comprises:

(a) at least one Copenhagen (COP)-derived nucleic acid sequence selected from:

(c) at least one Wyeth (WY)-derived nucleic acid sequence selected from:

(e) any combination of (a) to (d).

71. The chimeric vaccinia virus of any one of claims 66 to 70, wherein at least one organ-specific therapeutic index of said chimeric vaccinia virus is higher than the corresponding organ-specific therapeutic indexes of parental vaccinia virus strains Copenhagen (COP), Modified Vaccinia Ankara (MVA), Wyeth (WY), and Western Reserve (WR), wherein for a given organ and a given virus, an organ- specific therapeutic index TI(organ, virus) is defined as:

72. The chimeric vaccinia virus of claim 71, wherein the hepatic therapeutic index of said chimeric vaccinia virus is higher than, preferably at least 20 times higher than, at least 30 times higher than the corresponding hepatic therapeutic indexes of parental vaccinia virus strains COP, MVA, WY and WR, wherein for a given virus, a hepatic therapeutic index Tl(liver, virus) is defined as:

73. The chimeric vaccinia virus of any one of claims 66 to 72, wherein at least one tumor-specific oncolytic power of said chimeric vaccinia virus is higher than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR, wherein for a given tumor and a given virus, a tumor-specific oncolytic power OP(tumor, virus) is defined as: OP(tumor, virus) = (100 - percentage of surviving tumor cells after infection by virus).

74. The chimeric vaccinia virus of claim 73, wherein tumor-specific oncolytic powers of said chimeric vaccinia virus in tumor cells UM-UC-3, A549, SKOV-3, Hep G2, Cal-33, MIA PaCa-2, OE 19, HCT 116 and KATO III are higher than the corresponding tumor-specific oncolytic powers of parental vaccinia virus strains COP, MVA, WY and WR in the same tumor cells.

75. The chimeric vaccinia virus of any one of claims 1 to 61 and 66 to 74, wherein said virus is further defective in the TK locus.

76. The chimeric vaccinia virus of any one of claims 1 to 61 and 66 to 75, wherein said virus further comprises one or more nucleic acid(s) of interest.

77. The chimeric vaccinia virus of claim 76, wherein said nucleic acid of interest encodes one or more polypeptide(s) of therapeutic interest, said polypeptide(s) of therapeutic interest being preferably selected from polypeptides capable of reinforcing the oncolytic nature of the chimeric vaccinia virus, polypeptides capable of potentiating anti-tumor efficacy such as immunostimulatory polypeptide, antigens for inducing or activating an immune humoral and/or cellular response, and permease.

78. The chimeric vaccinia virus of claim 77, wherein said nucleic acid of interest encodes a suicide polypeptide, more preferably an FCU1 suicide polypeptide.

79. A process for producing a chimeric vaccinia virus, said process comprising:

(i) infecting a producer cell with the chimeric vaccinia virus according to any one of claims 1 to 61 and 66 to 78;

(ii) culturing said producer cell under conditions which are appropriate for enabling chimeric vaccinia virus particles to be produced, and;

80. A composition comprising the chimeric vaccinia virus of any one of claims 1 to 61 and 66 to 78 and a pharmaceutically acceptable vehicle.

81. The composition of claim 80, wherein said composition comprises a dose of chimeric vaccinia virus comprised between 10^s and 5xl0⁹ PFU.

82. The composition of claim 80 or claim 81, wherein said chimeric vaccinia virus is formulated for parenteral route administration, with a preference for intravenous or intratumoral route.

83. The chimeric vaccinia virus of any one of claims 1 to 61 and 66 to 78, or the composition of any one of claims 80 to 82, for use for the prophylaxis or the treatment of a proliferative disease or of a disease associated with an increased osteoclast activity.

84. The chimeric vaccinia virus or the composition for use according to claim 83, wherein said proliferative disease is selected from cancers, tumors and restenosis, preferably said proliferative disease is selected from cancers.

85. The chimeric vaccinia virus or the composition for use according to claim 84, wherein said cancer is selected from lung cancer, renal cancer, bladder cancer, prostate cancer, breast cancer, colorectal cancer, hepatic cancer, gastric cancer, pancreatic cancer, melanoma, ovarian cancer and glioblastoma, and especially metastatic ones.

86. The chimeric vaccinia virus or the composition for use according to claim 83 or claim 84, wherein said cancer is refractory or resistant to at least one oncolytic virus-based therapy, more preferably to at least one oncolytic vaccinia virus-based therapy.

87. The chimeric vaccinia virus or the composition for use according to any one of claims 84 to 86, wherein said chimeric vaccinia virus or composition is administered in combination with one or more substances effective in anticancer therapy.

88. The chimeric vaccinia virus or the composition for use according to claim 83, wherein said disease associated to an increased osteoclast activity is selected from rheumatoid arthritis and osteoporosis.

89. A method for treating a disease in a subject in need thereof comprising the administration to said subject of the chimeric vaccinia virus according to anyone of claims 1 to 61 and 66 to 78 or of the composition of any one of claims 80 to 82.

90. The method of claim 89, wherein said disease is a proliferative disease or a disease associated with an increased osteoclast activity.

91. The method of claim 90, wherein said proliferative disease is selected from cancers, tumors and restenosis, preferably said proliferative disease is selected from cancers.

92. The method of claim 91, wherein said cancer is selected from lung cancer, renal cancer, bladder cancer, prostate cancer, breast cancer, colorectal cancer, hepatic cancer, gastric cancer, pancreatic cancer, melanoma, ovarian cancer and glioblastoma, and especially metastatic ones.

93. The method of claim 91 or claim 92, wherein said cancer is refractory or resistant to at least one oncolytic virus-based therapy, more preferably to at least one oncolytic vaccinia virus-based therapy.

94. The method of any one of claims 91 to 93, further comprising the administration of one or more substances effective in anticancer therapy.

95. The method of claim 90, wherein said disease associated to an increased osteoclast activity is selected from rheumatoid arthritis and osteoporosis.