Chimeric poxviruses

Abstract

The present invention relates to chimeric poxviruses with improved anti-cancer activity (higher cancer cell killing ability and better tumor selectivity), as well as mutant forms thereof with one or more altered viral genes, recombinant forms thereof comprising one or more nucleic acids of interest and recombinant mutant forms thereof, all of which can be included in compositions and used for the treatment of diseases, in particular proliferative diseases, especially cancer.

Description

[Technical Field]

[0001] The present invention is in the field of oncolytic viruses and provides novel chimeric poxviruses that are particularly useful for the treatment of proliferative diseases such as, but not limited to, cancer. More precisely, the present invention provides chimeric poxviruses obtained by pooling different strains of parental poxviruses and selecting more potent chimeric viruses by cell passaging. These chimeric poxviruses exhibit many improved characteristics compared to the Copenhagen strain of vaccinia virus (COP), known in the art to be a particularly effective oncolytic viral vector, and more particularly compared to their parental poxvirus.

[0002] The chimeric poxviruses according to the present invention can be modified by altering the thymidine kinase-encoding gene (locus J2R) and / or the ribonucleotide reductase-encoding gene (locus I4L and / or F4L) to form mutant chimeric poxviruses.

[0003] The chimeric poxviruses of the present invention can further encode one or more heterologous transgenes to form recombinant chimeric poxviruses that also express transgenes more efficiently than their parental poxviruses.

[0004] The present invention also features a method for obtaining a chimeric poxvirus.

[0005] The chimeric poxviruses of the present invention can be used for the treatment of proliferative diseases such as cancer. [Background technology]

[0006] Oncolytic viruses are a type of therapeutic agent with the unique property of self-replicating in a tumor-dependent manner (Hermiston et al., 2006, Curr. Opin. Mol. Ther., 8(4):322-30). The advantage of using these viruses is that they lyse host cells as they replicate. Oncolytic viruses can selectively replicate in dividing cells (primarily cancer cells) while leaving non-dividing cells (e.g., healthy or primary cells) unharmed. When infected dividing cells are destroyed by lysis, they release new infectious particles to infect surrounding dividing cells. Oncolytic virotherapy has been recognized as a promising cancer treatment because the selective growth and amplification of the virus within tumor cells may be more effective and less toxic than current therapies. Cancer cells are ideal hosts for many viruses because they have inactivated antiviral interferon pathways or tumor-suppressor gene mutations that allow viral replication to proceed unimpeded (Chernajovsky et al., 2006, BMJ, 332(7534):170-2). Several viruses, including adenovirus, herpes simplex virus, reovirus, poxvirus, Newcastle disease virus, measles virus, vesicular stomatitis virus, Seneca Valley virus, and Japanese enveloped hemagglutinating virus, are undergoing clinical trials as oncolytic agents.

[0007] Among these, oncolytic poxviruses have shown promising results in the treatment of various cancers in multiple preclinical tumor models and several clinical trials. Six poxviruses from four genera have been investigated as potential oncolytic viruses: vaccinia virus, raccoon poxvirus, cowpox virus (orthopoxvirus), myxoma virus (leporipoxvirus), yaba monkey tumor virus (yatapoxvirus), and squirrelpox virus (Torres-Domingez et al., 2019, Review Expert Opin Biol Ther.; 19(6):561-573). Poxviruses accounted for approximately 13% of the total number of clinical trials evaluating oncolytic viruses from 2000 to 2020 (Macedo et al., 2020, Journal for ImmunoTherapy of Cancer; 8). Among these, recombinant oncolytic vaccinia virus (VACV) is a promising vector for tumor therapy. The genome organization, lytic potential, and broad tumor tropism of VACV make it an ideal oncolytic agent for cancer treatment and the most widely used poxvirus vector for cancer therapy (Haddad et al., 2017, Front Oncol., 7, 96. doi:10.3389 / fonc.2017.00096). The virus selectively targets tumors after systemic administration, demonstrating natural tumor tropism (McFadden, 2005, Nat Rev Microbiol., 3, 201-213).Currently, several VACV strains are being 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). However, neither rabbitpox nor cowpox virus has been evaluated in clinical studies for different reasons. Rabbitpox virus is too pathogenic to be used as an oncolytic virus to treat human cancers, which may be explained by the presence of three specific virulence genes encoding zinc ring finger proteins, ankyrin repeat family proteins, and chemokine-binding proteins (Li et al., 2005, Journal of General Virology, 86, 2969-2977). Although cowpox virus has shown interesting results in vitro (Ricordel et al. 2017, Mol. Ther. Oncolytics Vol. 7), its in vivo oncolytic activity is too weak to be a potential oncolytic viral platform.

[0008] Modification of naturally occurring viruses has already been performed to enhance the ability of poxviruses, more specifically vaccinia viruses, to infect and lyse 100% of tumor cells, which is difficult to achieve in vivo. Many strategies for modifying viruses are currently being used for this purpose (e.g., tropism modification to redirect the virus to the surface of cancer cells). Oncolytic vaccinia viruses are often "armed" with enzyme-drug systems to enhance the oncolytic effect of virotherapy by exerting a strong bystander effect, allowing the elimination of adjacent uninfected tumor cells. For example, the so-called FCU1 suicide gene, encoding a bifunctional chimeric polypeptide combining the enzymatic activities of FCY1 and FUR1, efficiently catalyzed the direct conversion of the nontoxic antifungal agent 5-fluorocytosine (5-FC) to its toxic metabolites 5-fluorouracil (5-FU) and 5-fluorouridine-5'-monophosphate (5-FUMP), circumventing the natural resistance of certain human tumor cells to 5-fluorouracil (Erbs et al., 2000, Cancer Res., 60(14): 3813-22). Foloppe et al. demonstrated that VACV expressing the FCU1 gene had effective antitumor effects both in vitro and in vivo in a mouse model of human colon tumors (Foloppe et al., 2008, Gene Ther., 15:1361-1371). Vaccinia viruses expressing the FCU1 fusion suicide gene, combined with the administration of a 5-fluorocytosine (5-FC) prodrug, demonstrated highly effective antitumor effects both in vitro and in vivo. The therapeutic potential of FCU1 delivered by oncolytic cowpox virus was also explored (Ricordel et al., 2017, Molecular Therapy - Oncolytics, 7: 1-11). Cowpox viruses expressing the FCU1 fusion suicide gene, combined with the administration of a prodrug, also demonstrated antitumor effects in vitro.

[0009] Viral modifications can also be used to enhance safety. In this regard, poxviruses lacking thymidine kinase (TK) have been shown to be less virulent than wild-type poxviruses, while still replicating in tumor cells (Buller et al., 1985, Nature, 317(6040):813-5). Thus, attenuated poxviruses, particularly vaccinia virus strains, have been developed for therapeutic and diagnostic use and are being evaluated in clinical trials. However, methods for attenuating vaccinia viruses result in reduced efficacy. From a therapeutic perspective, this reduced efficacy can lead to reduced overall response, decreased patient survival, increased mortality, and pathological resistance. As a result, the first oncolytic vaccinia viruses tested in clinical trials demonstrated high safety in patients but generally fell short of the therapeutic value expected as monotherapy. Furthermore, some tumor cells appear to be less tolerant or resistant to infection and replication by oncolytic poxviruses, meaning that some cancers remain refractory or resistant to oncolytic poxvirus-based therapies (e.g., National Clinical Trial NCT01380600: Safety but not efficacy of Pexa-Vec (pexastimogene devacirepvec, JX-594, an oncolytic, immunotherapeutic vaccinia Wyeth (WY)-based virus engineered to express GM-CSF) was confirmed in patients with colorectal cancer who were refractory or intolerant to oxaliplatin, irinotecan, and erbitux treatment; National Clinical Trial NCT01387555: No efficacy of Pexa-Vec in patients with advanced liver cancer who had not responded to sorafenib; National Clinical Trial NCT01636284: No efficacy of Pexa-Vec in patients with advanced liver cancer who had not been treated with sorafenib).

[0010] Another approach to obtaining evolved viruses is to generate novel chimeric oncolytic viruses through directed evolution, a method used to mimic and accelerate the process of natural selection. Viruses undergo genetic change through several mechanisms, including point mutation and recombination. Recombination is a widespread phenomenon among viruses and can significantly impact their evolution. Recombination occurs when homologous sequences from at least two viruses co-infect the same host cell and exchange gene segments. Homologous recombination (HR) occurs at the same site in both parental strands, creating new gene combinations that can alter the phenotype of the chimeric virus. Homologous recombination is the basis for many genetic techniques widely used in virus research, including the construction of recombinant vectors (Hruby, 1990, Clin. Microbiol. Rev, 3(2) 153-170). In 1958, experiments demonstrated that different strains of poxviruses could recombine (Fenner and Comben, 1958, Virology, 5, 530-548). Some characteristics of the resulting chimeras (e.g., viral replication, thermostability, or hemagglutinin production) were examined, but neither oncolytic potential nor therapeutic index was explored. Paszkowski et al. studied the mechanism of genetic recombination in poxviruses (Paszkowski et al., 2016, PLOS Pathogens, 12(8) e1005824). They observed multiple gene exchanges even after a single selection, indicating that homologous intra- and intermolecular recombination occurs efficiently, but functional characteristics were not examined.

[0011] Directed evolution is typically used to create gene libraries (Koerber et al., 2006, Nat. Protocols 1(2) pp.701-706). The purpose of this methodology is different when applied to oncolytic virotherapy. Recently, it was used to obtain the oncolytic chimeric poxviruses CF33 and CF17 by pooling nine poxvirus strains known to exhibit oncolytic activity in non-resistant tumor cells (WO2018 / 031694; O'Leary et al., 2018, Mol. Therap. Vol. 9: 13-21; Chaurasiya et al., 2020, Cancer Gene Therapy 27:125-135; Hammad et al., 2020, Mol. Ther. Oncolytics, vol.19 pp. 278-282). This mixture of viruses was propagated and shuffled on a non-tumor cell line (African green monkey kidney fibroblast CV-1), a cell line commonly used in poxvirus production research due to its high permissiveness for poxvirus replication. However, when infected with the HCT116 colorectal cancer cell line, the resulting chimeric CF33 virus secreted less extracellular enveloped virus (EEV) at an early stage than the IHD parental strain, reflecting a lower ability of CF33 virus to spread within tumor cells compared to at least one of the parental strains. Furthermore, the overall viral titer of CF33 in HCT116 cell lysates was found to be equivalent to that obtained with the parental Western Reserve (WR) and Elstree strains at 72 hours postinfection, indicating that the CF33 virus did not replicate beyond the level of at least two parental viruses. Furthermore, O'Leary et al. did not provide data on the effect of the chimera on healthy cells (preferably primary cells). Therefore, the tumor specificity of the chimera is unknown and cannot be compared to that of the parental strains.

[0012] The oncolytic chimeric vaccinia virus DeVV5 was also generated by directed evolution (WO2020011754, Ricordel et al., 2018 Cancers, Jul 10;10(7):231). Four different vaccinia virus strains (Modified Vaccinia Virus Ankara (MVA), Copenhagen (COP), Wyeth (WY), and Western Reserve (WR)) were pooled and amplified in resistant cancer cell lines, and then serially passaged under stringent conditions for selection. However, the selected chimeric vaccinia virus deVV5 showed enhanced oncolytic properties and tumor selectivity compared to its parent virus in vitro, but in vivo results were not demonstrated.

[0013] Combination therapy with standard and novel anticancer therapies (e.g., chemotherapy, immune checkpoint inhibitors [ICIs]) has also been used to improve the oncolytic efficacy of oncolytic poxviruses (Filley et al., 2017, Front Oncol. 7, 106. doi: 10.3389 / fonc.2017.00106). For example, Pexa-Vec was evaluated in a randomized controlled phase 3 trial comparing Pexa-Vec plus sorafenib with sorafenib alone in advanced, first-line hepatocellular carcinoma (HCC) (Japan Clinical Trial NCT02562755). TG6002 (Heinrich et al., 2017, Onco Targets Ther., 10, 2389–2401. doi: 10.2147 / OTT.S126320), a derivative of the Copenhagen (COP)-based TK-deleted VACV expressing an FCU1-fusion suicide gene, has entered clinical development in patients with recurrent glioblastoma when administered in combination with 5-FC (Domestic Clinical Trial NCT03294486). BT-001 (a vaccinia virus encoding GM-CSF and aCTLA4) is currently being tested in a Phase I / IIa trial in combination with pembrolizumab (a-PD1) in patients with metastatic / advanced solid tumors with cutaneous or subcutaneous lesions or easily injectable lymph nodes (Domestic Clinical Trial NCT04725331). A trial evaluating the combination of Pexa-Vec and nivolumab for the treatment of HCC in sorafenib-naive patients was stopped early because Pexa-Vec and nivolumab failed to respond in their respective pivotal trials (Domestic Clinical Trial NCT03071094). Summary of the Invention

[0014] Technical Problems and Proposed Solutions Despite various current attempts being tested, such as virus engineering, virus arming, chimera creation, or combination with standard or novel therapies, known poxvirus platforms do not possess sufficient lytic potency to provide satisfactory therapeutic results for cancer treatment. Furthermore, poxviruses cannot infect and replicate in all tumor cells, which means that some cancers are refractory or resistant to oncolytic poxvirus-based therapies. Furthermore, physical barriers within the tumor microenvironment can limit intratumoral spread of oncolytic poxviruses, meaning that neutralizing antibodies can also hinder systemic delivery of poxviruses.

[0015] As a result, there remains a need for highly potent oncolytic poxviruses that have improved ability to infect and lyse a large number of tumor cells without increasing the injection dose to avoid toxic events.The use of these poxviruses with better anticancer effects would provide better cancer cell killing ability compared to currently developed oncolytic viruses, whether as monotherapy or in combination with other anticancer therapies.In addition, the use of these poxviruses would provide enhanced oncolytic efficacy against cells that are poorly tolerant or resistant to current oncolytic virus, oncolytic poxvirus, or oncolytic vaccinia virus therapy.

[0016] Additionally, there is a need for safe oncolytic viruses, and poxviruses should have improved tumor selectivity so that they can be safely used in therapeutic subjects without affecting or significantly affecting the virus's ability to kill cancer cells.

[0017] Furthermore, since oncolytic poxviruses are rapidly cleared from the body, they must be difficult to neutralize by the host's immune system to avoid a decrease in efficacy.

[0018] In the context of the present invention, the inventors have demonstrated the feasibility of generating and selecting novel chimeric poxviruses and their recombinant forms, which importantly and unexpectedly achieve better anticancer therapy by exhibiting higher cancer cell killing capacity, increased oncolytic potential, increased extracellular enveloped virus (EEV) secretion, and improved spreading capacity, resulting in better antitumor efficacy in both injected tumors (tumors into which the virus is directly injected) and non-injected tumors (tumors into which the virus is not injected but is administered via another route, e.g., intravenously or subcutaneously), and improved survival rates in subjects receiving the chimeric poxviruses. In situations where these chimeric poxviruses carry a transgene encoding a protein of interest, the increased EEV production capacity of the chimeras leads to an increased ability to produce the protein of interest, due to their metabolism, more specifically, their rapid replication and ability to infect a large number of cells. Furthermore, by inducing syncytium formation, the chimeric poxviruses of the present invention enhance viral yield, the spreading and cytopathic effect, and antitumor immunity. The chimeric poxviruses of the present invention also replicate less in healthy cells (preferably primary cells), and therefore have better tumor selectivity and safety profiles. Having both better oncolytic activity and a better safety profile increases the therapeutic index of the chimeric poxviruses. The chimeric poxviruses are also less likely to be neutralized by anti-vaccinia virus antibodies, thus enabling their use in subjects who possess such antibodies (e.g., subjects who have been vaccinated with vaccinia virus and thus have induced an adaptive immune response), and are highly resistant to complement-mediated virus neutralization (e.g., induced by the innate immune system).

[0019] The chimeric poxviruses according to the present invention were generated by directed evolution. The starting pool consisted of a mixture of 16 different poxvirus strains: rabbitpox virus Utrecht (RPX), cowpox virus Brighton (CPX), vaccinia virus Copenhagen (COP), vaccinia virus Western Reserve (WR), vaccinia virus Wyeth (WY), modified vaccinia virus Ankara (MVA), raccoon poxvirus Harman (RCN), orf virus NZ2 (ORF), pseudocowpox virus TJS (PCP), bovine papular stomatitis virus Illinois 721 (BPS), myxoma virus Lausanne (MYX), squirrelpox virus Kilham (SQF), fowlpox virus FP9 (FPV), swinepox virus Kasuza (SPV), Yaba-like disease virus Davis (YLD), and chotiavirus SP An 232 (CTV).

[0020] Most of these parental poxvirus strains are known to have oncolytic properties, although to varying degrees, but this is not the case for MVA, FPV, and SPV, which are known not to replicate efficiently in mammalian cells and therefore are not oncolytic (Ricordel et al., 2018, Oncotarget vol 9, 35891-35906; Guse et al., 2011, Expert Opinion Biol. Ther.11(5): 595-608). On the other hand, MVA and FPV are known to have a high safety profile in humans, which is why they are included in the starting mixture of poxvirus strains.

[0021] After subjecting this mixture to directed evolution, the inventors selected a chimeric poxvirus designated POXSTG19503 (hereinafter referred to as the "wild-type chimeric poxvirus of the present invention") that exhibits improved in vitro tumor lysis and therapeutic index, improved in vivo syncytium formation and EEV secretion capabilities due to improved spread, and unexpectedly high EEV secretion capacity compared to vaccinia virus IHD-J strain and rabbitpox virus, both of which are known to be high-EV producers of poxviruses. Furthermore, the inventors selected the chimeric poxvirus designated POXSTG19503, which induces superior specific T cell responses against tumors.

[0022] More precisely, POXSTG19503 is a chimeric orthopoxvirus because it contains nucleic acid fragments originating exclusively from the orthopoxviruses: rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR), and modified vaccinia virus Ankara strain (MVA). Unexpectedly, improved oncolytic activity was observed despite a significant proportion of the chimeric poxvirus genome being derived from MVA. Importantly, in contrast to deVV5, POXSTG19503 exhibited improved anticancer efficacy in vivo.

[0023] We also demonstrated that it is possible to delete one or more genes of a chimeric poxvirus without altering its ability to destroy cancer cells, secrete a high percentage of EEV particles, or induce syncytia formation. This / these deletions (genes encoding TK and / or RR) further improved the safety of the virus, and the modified virus still possessed more potent oncolytic properties than the corresponding deleted parent strain.

[0024] The present inventors also investigated the feasibility of recombinant approaches using the chimeric poxvirus as a viral vector and constructed armed, oncolytic chimeric poxviruses encoding FCU1 or interleukin (IL-12). Insertion of the transgenes into the chimeric poxviruses did not appear to alter their anticancer activity. Furthermore, due to their metabolism, specifically their rapid replication and the ability to infect a large number of cells due to the chimera's increased EEV production and syncytium formation induction abilities, the recombinant chimeric poxviruses appeared to express the transgene at a higher rate than the parental Copenhagen vaccinia virus strain expressing the same transgene.

[0025] Based on these results, it is expected that the chimeric poxviruses of the present invention can be successfully used as a therapeutic solution, particularly for the treatment of proliferative diseases, and as an alternative to existing oncolytic viruses. The chimeric poxviruses of the present invention have a better efficacy profile in vivo while remaining safe for use. The chimeric poxviruses of the present invention may also be advantageous in the treatment of cancers that are refractory or resistant to poxvirus-based therapy. The chimeras of the present invention can also be used in combination with additional anticancer therapies.

[0026] Other and further aspects, features and advantages of the present invention will become apparent from the following description of the presently preferred embodiments of the invention, which are given for purposes of disclosure.

[0027] Summary of the Invention In a first aspect, the present invention provides a chimeric poxvirus (optionally mutated and / or recombinant), said chimeric poxvirus having a sequence similar to SEQ ID NO: 1 by at least 96.6%, preferably at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, 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%, 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%, at least 99.91%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99%, or even 100% sequence identity.

[0028] In one embodiment, the chimeric poxvirus of the present invention is the chimeric poxvirus POXSTG19503 clone 7, deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) on October 20, 2022 under accession number CNCM I-5913.

[0029] In the present disclosure, the chimeric poxvirus deposited under accession number CNCM I-5913 is also referred to as POXSTG19503.

[0030] In a preferred embodiment, the chimeric poxvirus (optionally mutant and / or recombinant) of the present invention comprises nucleic acid fragments derived from rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR), and modified vaccinia virus Ankara strain (MVA). In a specific embodiment, said chimeric poxvirus (optionally mutant and / or recombinant) may comprise a glutamic acid at position 151 of the protein encoded by the A34R gene, a valine at position 19 of the protein encoded by the A34R gene, or both. In a further specific embodiment, said chimeric virus (optionally mutant and / or recombinant) may be partially or completely deleted at the A56R locus, and in particular may comprise the A56R gene from rabbitpox virus, preferably the parent rabbitpox virus Utrecht strain (RPX). The present invention also provides derivatives of any chimeric poxvirus, including recombinant derivatives (i.e., further comprising one or more heterologous nucleic acids of interest), mutant derivatives with a deletion in one or more loci, in particular the J2R locus (particularly derivatives with a deletion in the J2R locus) or the J2R locus and the I4L and / or F4L loci (particularly derivatives with a deletion in the J2R locus and the I4L and / or F4L loci), as well as recombinant mutant derivatives with a deletion in one or more loci as indicated above and further comprising one or more heterologous nucleic acids of interest. The recombinant (optionally mutant) chimeric poxvirus of the present invention may preferably encode one or more polypeptides of therapeutic interest selected from a polypeptide capable of enhancing the oncolytic activity of the chimeric poxvirus, a polypeptide capable of enhancing antitumor efficacy, an antigen for inducing or activating a humoral and / or cellular immune response, and a permease. In a preferred embodiment, the recombinant chimeric poxvirus of the present invention may encode an interleukin. More preferably, the interleukin is IL-12.

[0031] In a second aspect, the present invention provides a chimeric poxvirus (optionally mutant and / or recombinant), which for at least one tumour has an oncolytic potency higher than the oncolytic potency of at least one of the oncolytic parent poxvirus strains (optionally mutant and / or recombinant), namely Rabbitpox virus Utrecht strain (RPX), Cowpox virus Brighton strain (CPX), Vaccinia virus Copenhagen strain (COP), Vaccinia virus Wyeth strain (WY) and Vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post infection, such that for a given tumour, a given virus, given conditions and a given time post infection, the oncolytic potency OP(tumour, virus, condition, time post infection) is: OP(tumor, virus, condition, time post-infection) = (100 - percentage of surviving tumor cells after infection with virus) It is defined as follows.

[0032] In a preferred embodiment, the oncolytic potential of said chimeric poxvirus (optionally mutant and / or recombinant) for at least one tumor is higher than the oncolytic potential of the parental vaccinia virus Copenhagen strain (COP) (optionally mutant and / or recombinant) or the parental rabbitpox virus Utrecht strain (RPX) (optionally mutant and / or recombinant), in particular two oncolytic poxvirus strains, measured under the same conditions and at the same time post-infection. More preferably, for at least one tumor, the oncolytic activity of the chimeric poxvirus (optionally mutant and / or recombinant) is higher than that of at least two of the oncolytic parental poxvirus strains (optionally mutant and / or recombinant), i.e., rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post-infection. For example, for at least one tumor, the oncolytic activity of the chimeric poxvirus (optionally mutant and / or recombinant) is higher than that of the parental vaccinia virus Copenhagen strain (COP) (optionally mutant and / or recombinant) and the parental rabbitpox virus Utrecht strain (RPX) (optionally mutant and / or recombinant), measured under the same conditions and at the same time post-infection. In a more preferred embodiment, the oncolytic potency of said chimeric poxvirus (optionally mutant and / or recombinant) for at least one tumor is higher than the oncolytic potency of at least three, more preferably higher than the oncolytic potency of at least four, and even more preferably higher than the oncolytic potency of each of five oncolytic parental poxvirus strains (optionally mutant and / or recombinant), namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post-infection.

[0033] In a third aspect, the present invention provides a chimeric poxvirus (optionally mutant and / or recombinant), wherein in at least one type of healthy cell (preferably a primary cell), the viral replication of said chimeric poxvirus (optionally mutant and / or recombinant) in the healthy cell is lower than the viral replication of at least one of five oncolytic parental poxvirus strains (optionally mutant and / or recombinant), namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR).

[0034] Preferably, for at least one type of healthy cell (preferably primary cell), the viral replication of said chimeric poxvirus (optionally mutant and / or recombinant) in the healthy cell is lower than the viral replication of the parental vaccinia virus Copenhagen strain (COP) (optionally mutant and / or recombinant). More preferably, for at least one type of healthy cell (preferably primary cell), the viral replication of said chimeric poxvirus (optionally mutant and / or recombinant) in the healthy cell (preferably primary cell) is lower than the viral replication of at least two, more preferably at least three, more preferably at least four, and even more preferably each of five oncolytic parental poxvirus strains (optionally mutant and / or recombinant), namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR).

[0035] In a fourth aspect, the present invention provides a chimeric poxvirus (optionally mutant and / or recombinant), wherein for at least one organ the therapeutic index of said chimeric poxvirus (optionally mutant and / or recombinant) is higher than the therapeutic index of at least one of five oncolytic parental poxvirus strains (optionally mutant and / or recombinant), namely Rabbitpox virus Utrecht strain (RPX), Cowpox virus Brighton strain (CPX), Vaccinia virus Copenhagen strain (COP), Vaccinia virus Wyeth strain (WY) and Vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post infection, and wherein for a given organ, a given tumor, a given virus, a given condition and a given time post infection, the therapeutic index TI(organ, tumor, virus, condition, time post infection) is: TI (organ, tumor, virus, condition, time post infection) = (viral replication in tumor cells of organ / viral replication in healthy cells of organ) It is defined as follows.

[0036] In a preferred embodiment, for at least one organ, the therapeutic index of said chimeric poxvirus (optionally mutant and / or recombinant) is higher than the therapeutic index of the parental vaccinia virus Copenhagen strain (optionally mutant and / or recombinant) measured under the same conditions and at the same time post-infection. More preferably, for at least one organ, the therapeutic index of said chimeric poxvirus (optionally mutant and / or recombinant) is higher than the therapeutic index of at least two, more preferably at least three, more preferably at least four, and even more preferably each of five oncolytic parental poxvirus strains (optionally mutant and / or recombinant), namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post-infection.

[0037] In a fifth aspect, the present invention provides a chimeric poxvirus (optionally mutant and / or recombinant), wherein the extracellular enveloped virus (EEV) secretion capacity (SC) (abbreviated as EEV-SC) of said chimeric poxvirus (optionally mutant and / or recombinant) for at least one producer cell (preferably tumor cell) is higher than the EEV-SC of at least one of six parental poxvirus strains (optionally mutant and / or recombinant), namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara strain (MVA), measured under the same conditions and at the same time post-infection, and wherein for a given virus, a given producer cell, a given condition and a given time post-infection with the virus, said EEV-SC is EEV-SC (virus, producer cells, conditions, time post-infection) = number of EEV particles / number of (EEV + IMV) particles is the ratio of extracellular enveloped virus (EEV) to all forms of virus (the extracellular enveloped virus (EEV) and intracellular mature virus (IMV) forms of the virus), defined as

[0038] In a preferred embodiment, the present invention provides a chimeric poxvirus (optionally mutant and / or recombinant), wherein, for at least one producer cell (preferably a tumor cell), the EEV-SC of said chimeric poxvirus (optionally mutant and / or recombinant) is higher than the EEV-SC of the parental vaccinia virus Copenhagen strain (COP) (optionally mutant and / or recombinant) or parental rabbitpox virus Utrecht strain (RPX) (optionally mutant and / or recombinant), measured under the same conditions and at the same time post-infection. More preferably, for at least one producer cell (preferably a tumor cell), the EEV-SC of said chimeric poxvirus (optionally mutant and / or recombinant) is higher than the EEV-SC of at least two of six parental poxvirus strains (optionally mutant and / or recombinant), namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara strain (MVA), measured under the same conditions and at the same time post-infection. For example, for at least one type of producer cell (preferably a tumor cell), the EEV-SC of said chimeric poxvirus (optionally mutant and / or recombinant) is higher than the EEV-SC of the parental vaccinia virus Copenhagen strain (COP) (optionally mutant and / or recombinant) and the parental rabbitpox virus Utrecht strain (RPX) (optionally mutant and / or recombinant), measured under the same conditions and at the same time post-infection.In a more preferred embodiment, for at least one producer cell (preferably tumor cell), the EEV-SC of the chimeric poxvirus (optionally mutant and / or recombinant) is higher than the EEV-SC of at least three, more preferably at least four, or at least five, more preferably of six parental poxvirus strains (optionally mutant and / or recombinant), namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR), and modified vaccinia virus Ankara strain (MVA), measured under the same conditions and at the same time post-infection. Alternatively, or in combination, for at least one producer cell (preferably tumor cell), the EEV-SC of the chimeric poxvirus (optionally mutant and / or recombinant) is higher than the EEV-SC of vaccinia virus IHD-J strain, measured under the same conditions and at the same time post-infection.

[0039] Preferably, in the fifth aspect, for at least one tumor, the spreading capacity of the chimeric poxvirus (optionally mutant and / or recombinant) is higher than the spreading capacity of at least one of six parental poxvirus strains (optionally mutant and / or recombinant), namely, Rabbitpox virus Utrecht strain (RPX), Cowpox virus Brighton strain (CPX), Vaccinia virus Copenhagen strain (COP), Vaccinia virus Wyeth strain (WY), Vaccinia virus Western Reserve strain (WR), and Modified Vaccinia virus Ankara strain (MVA), measured under the same conditions and at the same time post-infection. For a given virus, producer cell, condition, and time post-infection, spreading capacity is defined as the ability of a virus to spread between cells (e.g., tumor cells) or between tumors (e.g., from an injected tumor to a distant tumor). It is known that spreading capacity is related to the formation of EEV particles; the more EEV a virus can produce, the higher its spreading capacity. The spreading ability can be evaluated by various techniques well known to those skilled in the art, including the viral comet assay, which can evaluate comet tail formation (e.g., comet number, comet tail size). In some cases, an increase in the comet number or comet tail size may indicate an increase in the amount of EEV relative to the IMV form of the virus strain.

[0040] In a preferred embodiment, the spreading capacity of said chimeric poxvirus (optionally mutant and / or recombinant) is higher for at least one tumor than the spreading capacity of the parental vaccinia virus Copenhagen strain (COP) (optionally mutant and / or recombinant) or the parental rabbitpox virus Utrecht strain (RPX) (optionally mutant and / or recombinant), measured under the same conditions and at the same time post-infection. More preferably, for at least one tumor, the spreading capacity of the chimeric poxvirus (optionally mutant and / or recombinant) is higher than that of at least two of six parental poxvirus strains (optionally mutant and / or recombinant), namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR), and modified vaccinia virus Ankara strain (MVA), measured under the same conditions and at the same time post-infection. For example, for at least one tumor, the spreading capacity of the chimeric poxvirus (optionally mutant and / or recombinant) is higher than that of the parental vaccinia virus Copenhagen strain (COP) (optionally mutant and / or recombinant) and the parental rabbitpox virus Utrecht strain (RPX) (optionally mutant and / or recombinant), measured under the same conditions and at the same time post-infection. In a more preferred embodiment, for at least one tumor, the spreading capacity of the chimeric poxvirus (optionally mutant and / or recombinant) is higher than the spreading capacity of at least three, preferably at least four, more preferably at least five, or even more preferably, of six parental poxvirus strains (optionally mutant and / or recombinant), namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara strain (MVA).

[0041] In a sixth aspect, the present invention provides a chimeric poxvirus (optionally mutant and / or recombinant), wherein the neutralization rate of said chimeric poxvirus (optionally mutant and / or recombinant) for at least one poxvirus-specific antibody and tumor is measured under the same conditions and at the same time post-infection, and is compared with six parental poxvirus strains (optionally mutant and / or recombinant), namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (Vaccinia virus Wyeth strain), and / or rabbitpox virus Utrecht strain (RPX). The neutralization rate (NT(virus, tumor, condition, time post infection)) is a measure of the antibody-induced inhibition of the virus's oncolytic activity, and is lower than the neutralization rate of at least one of the following strains: WY, Western Reserve vaccinia virus strain (WR), and modified Ankara vaccinia virus strain (MVA). For a given virus, a given tumor, a given poxvirus-specific antibody, a given condition, and a given time post infection, the neutralization rate (NT(virus, tumor, condition, time post infection)) is calculated as follows: NT(virus, tumor, poxvirus-specific antibody, condition, time post infection) = EC50(with poxvirus-specific antibody) / EC50(without poxvirus-specific antibody). It is defined as follows.

[0042] In a preferred embodiment, for at least one poxvirus-specific antibody and tumor, the neutralization rate of the chimeric poxvirus (optionally mutant and / or recombinant) is lower than the neutralization rate of the parental vaccinia virus Copenhagen strain (COP) (optionally mutant and / or recombinant) measured under the same conditions and at the same time post-infection. More preferably, for at least one poxvirus-specific antibody and tumor, the neutralization rate of the chimeric poxvirus (optionally mutant and / or recombinant) is lower than the neutralization rates of at least two, preferably at least three, more preferably at least four, more preferably at least five, and even more preferably of six parental poxvirus strains (optionally mutant and / or recombinant), namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), Wyeth vaccinia virus (WY), Western Reserve vaccinia virus (WR), and modified vaccinia virus Ankara strain (MVA).

[0043] In a seventh aspect, the present invention provides a chimeric poxvirus (optionally mutant and / or recombinant), wherein the complement-mediated virus neutralization rate of said chimeric poxvirus (optionally mutant and / or recombinant) is lower than the complement-mediated virus neutralization rate of at least one of six parental poxvirus strains (optionally mutant and / or recombinant), namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara strain (MVA), measured under the same conditions; and wherein the complement-mediated virus neutralization rate (CMV-NT(virus, condition)) for a given virus and a given condition measures the complement-induced inhibition of the oncolytic activity of the virus, CMV-NT(virus, condition) = virus titer(human serum) / virus titer(heat-inactivated serum) It is defined as follows.

[0044] In a preferred embodiment, the complement-mediated virus neutralization rate of the chimeric poxvirus (optionally mutant and / or recombinant) is lower than the complement-mediated virus neutralization rate of the parental vaccinia virus Copenhagen strain (COP) (optionally mutant and / or recombinant), measured under the same conditions. More preferably, the complement-mediated virus neutralization rate of the chimeric poxvirus (optionally mutant and / or recombinant) is lower than the complement-mediated virus neutralization rates of at least two, preferably at least three, more preferably at least four, more preferably at least five, and even more preferably, of six parental poxvirus strains (optionally mutant and / or recombinant), namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR), and modified vaccinia virus Ankara strain (MVA), measured under the same conditions.

[0045] In an eighth aspect, the present invention provides a chimeric poxvirus (optionally mutant and / or recombinant), which has a higher syncytium formation ability in at least one producer cell (preferably a tumor cell) than the syncytium formation ability of at least one of six parental poxvirus strains (optionally mutant and / or recombinant), namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara strain (MVA), measured under the same conditions and at the same time post-infection.

[0046] In a preferred embodiment, the present invention provides a chimeric poxvirus (optionally mutant and / or recombinant), which has a higher syncytium formation ability in at least one producer cell (preferably a tumor cell) than the syncytium formation ability of the parental vaccinia virus Copenhagen strain (COP) (optionally mutant and / or recombinant), measured under the same conditions and at the same time post-infection. More preferably, the syncytium formation ability of said chimeric poxvirus (optionally mutant and / or recombinant) with respect to at least one producer cell (preferably a tumor cell) is higher than the syncytium formation ability of at least two of six parental poxvirus strains (optionally mutant and / or recombinant), namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara strain (MVA), measured under the same conditions and at the same time post-infection. For example, in a preferred embodiment, the syncytium formation ability of said chimeric poxvirus (optionally mutant and / or recombinant) with respect to at least one type of producer cell (preferably a tumor cell) is higher than the syncytium formation ability of at least two, preferably at least three, more preferably at least four, at least five, more preferably of six parental poxvirus strains (optionally mutant and / or recombinant), namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara strain (MVA), measured under the same conditions and at the same time post-infection.

[0047] The present invention also provides chimeric poxviruses (optionally mutant and / or recombinant) that combine the features of the chimeric poxviruses (optionally mutant and / or recombinant) described above in aspects 1-8.

[0048] The present invention also relates to chimeric poxviruses obtained or obtainable by a particular method of directed evolution as described below.

[0049] In a ninth aspect, the present invention provides a method of directed evolution for obtaining chimeric poxviruses with enhanced anti-cancer activity, the method comprising: (i) infecting a first tumor cell line with multiple parental poxvirus strains, wherein the tumor cell line is permissive to each parental poxvirus strain, to obtain a first infected tumor cell line; (ii) amplifying the parental poxvirus strains on the first infected tumor cell line of step (i) for at least 12 hours (preferably at least 24 hours), and at most 3 days, to obtain one or more different chimeric poxviruses by homologous genomic recombination between at least two of the parental poxvirus strains in the supernatant; (iii) at the end of step (ii), recovering the supernatant containing one or more different chimeric poxviruses; (iv) infecting a second tumor cell line with one or more different chimeric poxviruses from the supernatant of step (iii), wherein said second tumor cell line is permissive to each of the parental poxvirus strains of steps (i) and (ii), to obtain a second infected tumor cell line; (v a ) amplifying one or more different chimeric poxviruses of step (iv) on said second infected tumor cell line of step (iv), preferably for at least 12 hours and at most 24 hours, and then recovering the supernatant; (vi) determining for at least one third tumor cell line that the oncolytic activity is higher than the oncolytic activity of at least one, preferably some, more preferably all, of the parent oncolytic poxvirus strains in the first tumor cell line of step (i) and / or the second tumor cell line of step (iv), measured under the same conditions and at the same time post-infection; a ), wherein for a given tumor, a given virus, a given condition and a given time post-infection, the oncolytic potency OP(tumor, virus, condition, time post-infection) is: OP(tumor, virus, condition, time post-infection) = (100 - percentage of surviving tumor cells after infection with virus) is defined as The compound comprises:

[0050] More specifically, the directed evolution method comprises the following steps: (i) infecting a first tumor cell line with multiple parental poxvirus strains, wherein the tumor cell line is permissive to each parental poxvirus strain, to obtain a first infected tumor cell line; (ii) amplifying the parental poxvirus strains on the first infected tumor cell line of step (i) for at least 12 hours (preferably at least 24 hours), and at most 3 days, to obtain one or more different chimeric poxviruses by homologous genomic recombination between at least two of the parental poxvirus strains in the supernatant; (iii') at the end of step (ii), collecting the supernatant containing one or more different chimeric poxviruses and serially diluting it 5 to 20 times to obtain at least two diluted supernatants each containing one or more chimeric poxviruses; (iv') infecting at least two samples of a second tumor cell line with one or more different chimeric poxviruses from each of the diluted supernatants of step (iii') to obtain at least two samples of an infected second tumor cell line, wherein said second tumor cell line is permissive to each of the parent poxvirus strains of steps (i) and (ii); (v' a ) amplifying the one or more different chimeric poxviruses of each of the at least two samples of the second infected tumor cell line of step (iv') on the second infected tumor cell line of step (iv'), preferably for at least 12 hours and at most 24 hours; (v' b ) collecting supernatant from a sample of a second infected tumor cell line infected with a low-dilution supernatant that shows no signs of cytopathic effect and performing 5- to 20-fold serial dilutions; (v' c) steps (iv'), (v') until one or more different chimeric poxviruses that satisfy the selection criteria of step (vi) are obtained. a ) and (v' b ) repeating the steps; and (vi) determining whether at least one third tumor cell line has an oncolytic activity higher than the oncolytic activity of at least one, preferably some, more preferably all, of the parent oncolytic poxvirus strains in the first tumor cell line of step (i) or the second tumor cell line of step (iv'), measured under the same conditions and at the same time post-infection; c ), wherein for a given tumor, a given virus, a given condition and a given time post infection, the oncolytic potency OP(tumor, virus, condition, time post infection) is: OP(tumor, virus, condition, time post-infection) = (100 - percentage of surviving tumor cells after infection with virus) is defined as The compound comprises:

[0051] Even more particularly, the method of directed evolution comprises: (i) infecting a first tumor cell line with multiple parental poxvirus strains, wherein the tumor cell line is permissive to each parental poxvirus strain, to obtain a first infected tumor cell line; (ii) amplifying the parental poxvirus strains on the first infected tumor cell line of step (i) for at least 12 hours (preferably at least 24 hours), and at most 3 days, to obtain one or more different chimeric poxviruses by homologous genomic recombination between at least two of the parental poxvirus strains in the supernatant; (iii'' a ) at the end of step (ii), recovering both the cells and the supernatant containing one or more different chimeric poxviruses; (iii'' b ) a second tumor cell line, ainfecting both the cells and the supernatant containing one or more different chimeric poxviruses of steps (i) and (ii) to obtain a second infected tumor cell line, wherein said second tumor cell line is permissive to each of the parent poxvirus strains of steps (i) and (ii); (iii'' c ) process (iii'' b ) for at least 48 hours (preferably at least 72 hours) and up to 3 days, b amplifying said second infected tumor cell line; (iii'' d ) process (iii'' c collecting the cell and supernatant fractions containing one or more different chimeric poxviruses of (iii'' e ) process (iii'' b ), (iii'' c ) and (iii'' d ) at least once; (iii'' f ) process (iii'' e At the end of step (1), collecting the supernatant containing one or more different chimeric poxviruses and serially diluting the supernatant 5 to 20 times to obtain at least two diluted supernatants; (iv'') subjecting at least two samples of a third tumor cell line to step (iii''). f infecting each of the diluted supernatants of step (i) and (ii) with one or more different chimeric poxviruses to obtain at least two samples of a third infected tumor cell line, wherein said third tumor cell line is permissive to each of the parent poxvirus strains of steps (i) and (ii); (v'' a ) amplifying the one or more different chimeric poxviruses of each of the at least two samples of the third infected tumor cell line of step (iv'') on the third infected tumor cell line of step (iv'') for at least 12 hours and at most 24 hours; (v'' b ) collecting supernatant from a sample of a third infected tumor cell line infected with a low dilution of supernatant that shows no signs of cytopathic effect and performing 5- to 20-fold serial dilutions; (v'' c ) steps (iv''), (v'') until one or more different chimeric poxviruses that satisfy the selection criteria of step (vi) are obtained. a ) and (v'' b ) repeating the steps; and (vi) selecting at least one fourth tumor cell line having an oncolytic activity higher than that of at least one, preferably some, more preferably all of the parent oncolytic poxvirus strains in the tumor cell line of step (iv"), measured under the same conditions and at the same time post-infection; c ), wherein for a given tumor, a given virus, a given condition and a given time post infection, the oncolytic potency OP(tumor, virus, condition, time post infection) is: OP (tumor, virus, condition, time post-infection) = (100 - percentage of surviving tumor cells after infection with the virus), The compound comprises:

[0052] In a particular embodiment, the parental poxvirus strain used in the first step (i) is selected from the group consisting of rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Western Reserve strain (WR), vaccinia virus Wyeth strain (WY), modified vaccinia virus Ankara strain (MVA), raccoon poxvirus Harman strain (RCN), orf virus NZ2 strain (ORF), pseudocowpox TJS strain (PCP), bovine papular stomatitis virus Illinois 721 strain (BPS), myxoma virus Lausanne strain (MYX), squirrelpox virus Kilham strain (SQF), fowlpox virus FP9 strain (FPV), swinepox virus Kasuza strain (SPV), Yaba-like disease virus Davis strain (YLD) and Chotiavirus SP An 232 strain (CTV). More preferably, the parental poxvirus strains used in the first step (i) comprise at least one, preferably at least two, at least three, at least four, at least five, or even all of the six rabbitpoxvirus strains Utrecht (RPX), Brighton (CPX), Cowpoxvirus strain Copenhagen (COP), Western Reserve (WR), Wyeth (WY), and modified vaccinia virus strain Ankara (MVA). The inventors have shown that these six parental poxviruses can recombine with each other, and therefore the parental poxvirus strains used in the first step (i) can be selected from this more limited group. In one embodiment, the parent poxvirus strain used in the first step (i) may consist of, inter alia, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Western Reserve strain (WR), vaccinia virus Wyeth strain (WY) and modified vaccinia virus Ankara strain (MVA).

[0053] In any one of the above directed evolution methods, the first, second, and optionally third permissive tumor cell lines can be different or the same. The permissive tumor cell lines are of higher mammalian origin, and preferably, the permissive tumor cell lines used as the first, second, third, and optionally fourth tumor cell lines in the above methods are selected from the group consisting of A549, CAL-33, HepG2, HCT116, Hela, SK-MEL-1, PANC-1, Hs746T, SK-OV-3, and CV-1, and preferably A549. In a preferred embodiment, the first, second, third, and optionally fourth permissive tumor cell lines are the same, and preferably are all A549 lung cancer cell lines.

[0054] In a further aspect, the present invention also relates to mutant chimeric poxviruses, ie chimeric poxviruses according to the invention that have been engineered by alteration of one or more viral genes (eg deletion of the TK-encoding gene).

[0055] In a further aspect, the present invention also relates to recombinant chimeric poxviruses, ie chimeric poxviruses according to the invention comprising one or more heterologous transgenes.

[0056] In a further aspect, the present invention also relates to recombinant mutant chimeric poxviruses, i.e. chimeric poxviruses according to the invention that have been engineered by alteration of one or more viral genes (e.g. deletion of the TK-encoding gene) and comprise one or more heterologous transgenes.

[0057] In a further aspect, the present invention also provides a method for producing a chimeric (optionally mutant and / or recombinant) poxvirus, comprising the steps of: (i) infecting a producer cell with a chimeric (optionally mutant and / or recombinant) poxvirus according to the invention as disclosed herein; (ii) culturing the infected producer cells under conditions suitable to allow the production of chimeric poxviruses (optionally mutant and / or recombinant); and (iii) recovering the chimeric poxvirus (optionally mutated and / or recombinant) from the producer cell culture. The present invention relates to a method comprising at least

[0058] Optionally, the recovered chimeric (optionally mutant and / or recombinant) poxvirus can be at least partially purified.

[0059] Another aspect of the present invention relates to an isolated nucleic acid encoding the chimeric (optionally mutant and / or recombinant) poxvirus of the present invention.

[0060] In another aspect of the present invention, there is provided a composition comprising the chimeric (optionally mutated and / or recombinant) poxvirus of the present invention and a pharmaceutically acceptable vehicle. In one embodiment, the chimeric (optionally mutated and / or recombinant) poxvirus is preferably formulated for parenteral administration, preferably intravenous or intratumoral.

[0061] Another aspect of the present invention relates to a chimeric (optionally mutated and / or recombinant) poxvirus or composition of the present invention for use as a medicament, preferably for the treatment of a proliferative disease, in a preferred embodiment, said proliferative disease is selected from cancer, tumor and restenosis.

[0062] Another aspect of the present invention relates to a method for treating a disease in a subject in need thereof, comprising administering to said subject a chimeric poxvirus (optionally mutated and / or recombinant) or composition according to the present invention, in one embodiment said disease is a proliferative disease, preferably selected from cancer, tumor and restenosis. [Brief explanation of the drawings]

[0063] For ease of reading, the code references used in the figures and examples are included in Table 1, which is presented in the Examples section. [Figure 1] Figure 1: Oncolytic effect of POXSTG19503 on a panel of tumor cells. Cells (seeded at 3 x 10 cells / well in 6-well culture dishes) were infected at the indicated MOI, and cell viability was determined by trypan blue exclusion after 5 days. Parental COP was used as a reference. Results are expressed as the mean ± SD of triplicates. [Figure 2] Figure 2: Genome analysis. (A) Annotated POXSTG19503 mono-ITR genome. Large gray arrows highlight segments longer than 500 nucleotides that are 100% identical to the parental viral genome, as labeled and coded in the legend at the top right. The 3' ITR is indicated by a black arrow at the end. (B) 40-60 kb regions extracted from the global alignment of the POXSTG19503 core region and the parental genome. Large gray arrows highlight segments longer than 500 nucleotides that are 100% identical to the parental viral genome, as labeled and coded in the legend at the top right. Automatically detected open reading frames are indicated by small dark gray arrows at the bottom of each lane. [Figure 3] Figure 3: Oncolytic effect of POXSTG19508 on A549 (A), HCT116 (B), and HepG2 (C) tumor cells. Cells (seeded at 3 x 10 cells / well in 6-well culture dishes) were infected at the indicated MOI, and cell viability was determined by trypan blue exclusion after 4 days. Six parental poxvirus strains were used as references. Results are expressed as the mean ± SD of triplicates. [Figure 4] Figure 4: Replication in tumor cells and primary human cells. (A) HepG2 tumor cells were infected at an MOI of 10-5 and harvested 3 days post-infection. Human primary hepatocytes were infected at an MOI of 10-4 and harvested 3 days post-infection. 3D Phenion FT skin models were infected with 1.105 PFU (plaque-forming units) and harvested 7 days post-infection. Viral progeny production was determined by plaque titration. Results are expressed as fold viral amplification (corresponding to the output / input ratio). Results are presented as the mean ± SD of triplicates. (B) Ratio between fold viral amplification observed in HepG2 hepatoma cells and hepatocytes 3 days post-infection. Values ​​are presented as the mean of each triplicate determination. [Figure 5] Figure 5: Functionality of FCU1 expressed by the chimeric poxvirus POXSTG19508. Conversion of 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) and release of 5-FU into the cell culture supernatant. A549 tumor cells were infected with the indicated vectors at an MOI of 10-4 and then incubated with 1 mM 5-FC at 6 h postinfection. The relative concentrations of 5-FC and 5-FU in the culture supernatant were measured by HPLC 1–3 days postinfection. Results are expressed as the percentage of released 5-FU relative to the total amount of 5-FC + 5-FU. Values ​​are expressed as the mean ± SD of triplicate determinations. [Figure 6] Figure 6: Production of EEV and total morphological progeny virus at early time points post-infection of A549 monolayers. A549 cells were infected with VVTG17111 or POXSTG19508 at an MOI of 0.1. Supernatants or cell fractions were harvested 16 and 24 hours post-infection. Virus titers are shown for supernatant alone (EEV) and both supernatant and cells (IMV + EEV, total progeny virus) at 16 hours (A) and 24 hours (B) post-infection. (C) EEV / IMV ratio at 16 and 24 hours post-infection. Results are expressed as the mean ± SD of triplicates. [Figure 7] Figure 7: Representative images of comets. The indicated viruses were seeded onto a monolayer of A549 cells. Two days later, plaques were imaged using a fluorescent microscope (GFP). After image acquisition, cells were stained with crystal violet (CV). [Figure 8] Figure 8: EEV morphology and total progeny virus production at early time points after infection of A549 monolayers. A549 cells were infected with the indicated viruses at an MOI of 0.1. Supernatants or cell fractions were harvested 16 and 24 hours post-infection. Virus titers are shown for supernatants alone (EEV) and both supernatants and cells (IMV + EEV, total progeny virus) at 16 hours (A) and 24 hours (B) post-infection. (C) EEV / IMV ratios at 16 and 24 hours post-infection. Values ​​are expressed as the mean ± SD of triplicate determinations. [Figure 9]Figure 9: VACV neutralization assay. Viability of HCT116 tumor cells infected with TG6002 (A) or POXSTG19508 (B) in the presence of patient-derived serum (immune serum) collected 42 days after IV administration of TG6002. Serum-free conditions (control) and serum collected from the same patient before administration of TG6002 (pre-immune serum) were used as negative controls. The 50% effective concentrations (EC50) of the two viruses were determined by infecting HCT116 cells (1 x 104 cells / well) seeded in 96-well plates with serial dilutions of the indicated viruses and assessing cell death 3 days post-infection using a CellTiter Blue cell viability assay. Cell viability was determined by comparison with uninfected cell controls. Results are presented as the mean ± SD of triplicates. (C) Table of EC50 values ​​calculated by fitting a sigmoidal dose-response curve to the results obtained in (A) and (B). [Figure 10] Figure 10: Viral immunostaining in HCT116 xenograft tumors. (A) Immunostaining of tumors performed 2, 7, and 16 days after a single intravenous injection of 1 x 105 PFU of the indicated viruses. Cellular DNA was stained blue with DAPI (medium gray in the photo), and virus was stained green (light gray in the photo). (B) Viral area density in tumors from three mice per group at 2, 7, and 16 days post-injection. Results are expressed as the mean ± SD of three tumors. [Figure 11] Figure 11: Viral immunostaining in B16F10 syngeneic mouse tumors. (A) Immunostaining of tumors performed 3 and 8 days after a single intratumoral injection of 1 x 10 PFU of the indicated viruses. Cellular DNA was stained blue with DAPI (medium gray in the photo), and virus was stained green (light gray in the photo). (B) Viral area density in tumors from three mice per group at 3 and 8 days post-injection. Results are expressed as the mean ± SD of three tumors. [Figure 12]Figure 12: In vivo antitumor efficacy of POXSTG19508 in a colorectal xenograft model. Subcutaneous HCT116 tumors were implanted into the right flank of nude mice. 15 days after implantation, mice received a single intravenous injection of PBS (control), 3 x 104 PFU of TG6002, or POXSTG19508 (indicated by vertical arrows). (A) Tumor growth over time for individual mice (n = 10 per group). Vertical arrows indicate systemic injections. (B) Kaplan-Meier survival analysis. Vertical arrows indicate systemic injections. Significant differences between groups were determined by the log-rank test. NS: not significant. [Figure 13] Figure 13: In vivo antitumor efficacy of POXSTG19508 in a liver cancer xenograft model. Subcutaneous HepG2 tumors were implanted into the right flank of nude mice. Twenty-eight days after implantation, mice received a single intravenous injection of PBS (control), 3 x 10 PFU of TG6002, or POXSTG19508 (indicated by vertical arrows). (A) Tumor growth over time for each mouse (n = 10 per group). Vertical arrows indicate systemic injections. (B) Kaplan-Meier survival analysis. Vertical arrows indicate systemic injections. Significant differences between groups were determined by the log-rank test. [Figure 14] Figure 14: In vivo antitumor efficacy of POXSTG19508 in the CT26 syngeneic mouse tumor model. Subcutaneous CT26 tumors were implanted into the right flank of nude mice. On days 7, 9, and 11 after implantation, mice received daily intratumoral injections of PBS (control), 1 x 10 PFU of TG6002, or POXSTG19508 (indicated by vertical arrows). (A) Tumor growth over time for individual mice (n = 10 per group). (B) Kaplan-Meier survival analysis. Vertical arrows indicate intratumoral injections. Significant differences between groups were determined by the log-rank test. NS: not significant. [Figure 15]Figure 15: In vivo antitumor efficacy of POXSTG19508 in a bilateral flank xenograft mouse model. Subcutaneous HCT116 xenografts were injected intratumorally with 1x106 PFU of TG6002, POXSTG19508, or PBS (control) intratumorally in the right tumor only (n=10 per group). (A) Tumor growth over time for injected tumors. (B) Tumor growth over time for uninjected tumors. (C) Kaplan-Meier survival analysis. Vertical arrows indicate IT injections in right tumors. Significant differences between groups were determined by the log-rank test. NS: not significant. (D) Viral titers in injected and uninjected tumors 13 days after virus injection. Each dot represents one tumor. Horizontal bars represent the mean. [Figure 16] Figure 16: In vivo antitumor efficacy of systemic administration of mIL-12-expressing chimeric poxvirus in a B16F10 syngeneic mouse tumor model. Subcutaneous B16F10 tumors were implanted into the right flank of nude mice. On days 7 and 9 post-implantation, mice received intravenous administration of PBS (control), 1 x 10 PFU of VVTG19328, or POXSTG19847. (A) Tumor growth over time for individual mice (n = 10 per group). (B) Kaplan-Meier survival analysis. Vertical arrows indicate systemic injections. Significant differences between groups were determined by the log-rank test. NS: not significant. [Figure 17] Figure 17: Syncytia formation. HCT116 cells were injected with VVTG17111 or POXSTG19508 at an MOI of 10-2 or 10-3. Cell morphology was observed by light microscopy and fluorescence microscopy (GFP) at 40 hours (MOI 10-2) or 60 hours (MOI 10-3) post-infection. [Figure 18] Figure 18: Specific T cell responses measured by IFNγ ELISpot in mouse splenocytes. Specific T cell responses to both tumor and virus measured by IFNγ ELISpot in splenocytes from CT26 tumor-bearing mice treated intratumorally with VVTG17111, POXSTG19508, or vehicle as a negative control. Each bar represents the mean + / - SEM of quadruplicate spot counts from 10 splenocytes isolated from each spleen. *P <0.001. [Figure 19]Figure 19: Sensitivity to neutralization by complement. TG6002 and POXSTG19508 were incubated with human serum for 1 hour, then inoculated onto Vero cells and incubated for 3 days to allow plaque formation. The number of plaques seen is expressed as a percentage of the number of plaques seen with heat-inactivated human serum. Heat-inactivated serum was used as a negative control. Data represent the mean ± SD of three experiments. Specific Description of the Invention

[0064] General definition Terms used herein generally have their ordinary meaning in the art unless otherwise specified. Certain terms are described below or elsewhere in this specification to provide further guidance to those skilled in the art in describing the products and methods of the present invention and how to use them. Additionally, alternative language and synonyms may be used for any of the terms described herein. Synonyms for particular terms are provided. The description of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of terms described herein, is for illustrative purposes only and does not in any way limit the scope or meaning of the invention or any exemplified term.

[0065] As used throughout this application, the terms "a" and "an" are used to mean "at least one," "at least a first," "one or more," or "a plurality" of the referenced component or step. For example, the term "a chimeric poxvirus" encompasses a single chimeric poxvirus as well as multiple chimeric poxviruses, including mixtures of different chimeric poxviruses.

[0066] The term "one or more" refers to one or more than one (e.g., two, three, four, etc.).

[0067] The term "at least" refers either to the numerical value considered to be the minimum followed by the expression "at least," or to a numerical value greater than that minimum.

[0068] As used herein, the term "and / or" includes the meaning of "and", "or" and "all or any other combination of the elements connected by that term".

[0069] The terms "about" or "approximately," as used herein, mean within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

[0070] The term "different" means "not identical" or "distinguished as not identical." For example, two different proteins have different amino acid sequences, different shapes, etc.

[0071] As used herein, and when used to define products and compositions, the words "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, unrecited ingredients or method steps. The phrase "consisting essentially of" means excluding other ingredients or steps of substantial importance. Thus, a composition consisting essentially of the recited ingredients does not exclude minor ingredients, contaminants, and pharmaceutically acceptable carriers. "Consisting of" is intended to mean excluding other ingredients or steps beyond trace elements. Wherever the term "comprising" (or any derivative thereof, such as "comprise" and "comprises") is used in this specification, the invention also relates to the same embodiments wherein "comprising" (or any derivative thereof, such as "comprise" and "comprises") is replaced with "consisting essentially of" or "consisting of."

[0072] The terms "protein," "polypeptide," and "peptide" are used interchangeably and refer to a polymer of amino acid residues comprising at least nine or more amino acids linked via peptide bonds. The polymer may be linear, branched, or cyclic, may comprise naturally occurring amino acids and / or amino acid analogs, and may contain inserted non-amino acids. As a general designation, if an amino acid polymer has more than 50 amino acid residues, it is preferably referred to as a "polypeptide" or "protein," and if it is 50 amino acids or less in length, it is referred to as a "peptide." Proteins, polypeptides, and peptides are defined by their amino acid sequences.

[0073] In the context of the present invention, the terms "nucleic acid," "nucleic acid molecule," "polynucleotide," and "nucleotide sequence" are used interchangeably and define polymers of any length, either polydeoxyribonucleotides (DNA) (e.g., cDNA, genomic DNA, plasmids, vectors, viral genomes, isolated DNA, probes, primers, and any mixtures thereof) or polyribonucleotides (RNA) (e.g., mRNA, antisense RNA, siRNA) or mixed polyribopolydeoxyribonucleotides. These encompass polynucleotides that are single- or double-stranded, linear or circular, natural or synthetic, modified or unmodified. Furthermore, polynucleotides may comprise non-naturally occurring nucleotides or have inserted non-nucleotide components.

[0074] The term "nucleotide" refers to any of a variety of compounds consisting of a sugar, usually ribose or deoxyribose, a purine or pyrimidine base, and one or more phosphate groups. The term "nucleotide" refers to both ribonucleotides and deoxyribonucleotides.

[0075] Generally, the terms "identity" or "identical" in the context of a viral sample refer to an amino acid-to-amino acid or nucleotide-to-nucleotide correspondence between a viral polypeptide and another reference polypeptide, or between a viral nucleic acid sequence and another reference nucleic acid sequence, 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 and the length of each gap that need to be introduced for optimal alignment of the two sequences overall. In the art, various computer programs and mathematical algorithms are available for determining the percentage identity between amino acid sequences after optimal global alignment, such as Mafft, ClustalW 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). Such global alignment computer programs and mathematical algorithms are selected according to the general knowledge of those skilled in the art to be more bioinformatically or biologically relevant and perform optimal global alignment. Optimal global alignment programs for determining the identity between nucleotide sequences can also be used in specialized databases (for example, Genbank, Wisconsin Sequence Analysis Package, BESTFIT, FASTA and GAP programs).

[0076] As used herein, the term "host cell" should be understood broadly without limitation with respect to a specific organ in a tissue, organ, or isolated cell. Such cells may be of a specific type, such as a cultured cell line, a healthy cell (preferably a primary cell), and a dividing cell, or a group of different types of cells. In the context of the present invention, the term "host cell" includes prokaryotic cells, lower eukaryotic cells such as yeast, and other eukaryotic cells such as insect cells, plant cells, and mammalian (e.g., human or non-human) cells, as well as cells capable of infection and replication of the chimeric poxvirus of the present invention (these cells are referred to as "permissive cells"). When used to produce the chimeric poxvirus of the present invention, permissive cells are also referred to as "producer cells," and are host cells that permit infection and replication of the chimeric poxvirus of the present invention.

[0077] The terms "chimeric virus" or "viral chimera" are used interchangeably and according to their usual meaning in virology to refer to hybrid viruses created by joining nucleic acid fragments from two or more different viral strains, referred to as "parent viruses." Chimeric viruses can be obtained by the process of viral directed evolution, in which a mixture of several parent viruses is contacted with a desired producer cell to generate a pool of chimeric viruses, allowing recombination events to occur between the genomes of the several parent viruses.

[0078] In the context of the present invention, the term "chimeric gene" defines a hybrid gene formed by the recombination of part or all of a gene fragment from two or more different viral strains.

[0079] The term "wild-type virus" refers to a parental or chimeric virus that has not been manipulated by modifying one or more genes of the viral genome and does not contain a heterologous transgene.

[0080] The term "mutant virus" refers to a parental or chimeric virus that has been engineered by modifying one or more viral genes (eg, deletion of the TK-encoding gene).

[0081] The term "recombinant virus" refers to a parental or chimeric virus comprising one or more heterologous transgenes.

[0082] The terms "recombinant mutant virus" or "mutant recombinant virus" are used interchangeably and refer to a parental or chimeric virus that has been engineered by altering one or more viral genes (e.g., deletion of the TK-encoding gene) and comprises one or more heterologous transgenes.

[0083] The term "oncolytic," as used herein, refers to the ability of a virus to replicate in dividing cells (e.g., proliferating cells such as cancer cells) with the intent of slowing their growth and / or lysing them, either in vitro or in vivo. Oncolytic viruses are characterized by their "oncolytic potential." For a given tumor, a given virus, a given condition, and a given time post-infection, the oncolytic potential OP(tumor, virus, condition, time post-infection) is: OP(tumor, virus, condition, time post-infection) = (100 - percentage of surviving tumor cells after virus infection) It is defined as follows.

[0084] For a given tumor and virus, the value of oncolytic OP (tumor, virus, condition, time after infection) may vary depending on conditions (e.g., MOI, culture medium, tumor cell density, temperature, etc., especially MOI) and time after infection. When comparing the oncolytic OP (tumor, virus, condition, time after infection) of two viruses, the comparison is performed under the same conditions and the same time after infection. However, for a given tumor and two given viruses, if the difference in oncolytic potency between the two viruses is sufficient (e.g., at least 3-fold) or significant, the level of oncolytic potency generally remains the same under any suitable conditions and time after infection, so any suitable conditions (see examples below) and time after infection may be used for comparison (provided that these conditions are the same for the two viruses). For a given tumor and virus, oncolytic potency is generally determined by injecting the virus into tumor cells at an MOI of 10. -5 ~10 -2 The β-amyloid level can be determined in vitro 3 to 5 days after infection with β-amyloid.

[0085] Oncolytic potency is expressed as a percentage and represents the percentage of a particular tumor cell lysed by a given virus under specific conditions at a specific time post-infection. For example, 5 days post-infection, an MOI of 10 -5 The Copenhagen strain of vaccinia virus exhibited 24% oncolytic activity against A549 tumor cells cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) at 37°C and 5% CO2, meaning that 24% of the A549 tumor cells were lysed by COP (Figure 1). The higher this percentage, the greater the oncolytic activity of a given virus against a given tumor.

[0086] The terms "replication" and "viral replication" refer to the replication of viral genomes in target host cells (e.g., tumor or healthy cells) or the synthesis of viral proteins in target host cells. The stages of a virus's life cycle include, but are not limited to, viral attachment to the host cell surface, penetration or entry into the host cell (e.g., via receptor-mediated endocytosis or membrane fusion), uncoating (a process in which the viral capsid is removed and degraded by viral or host enzymes, releasing viral genomic nucleic acid), genome replication, synthesis of viral messenger RNA (mRNA), synthesis of viral proteins, and assembly of viral ribonucleoprotein complexes for genome replication, assembly of viral particles, post-translational modification of viral proteins, and release from the host cell by lysis or budding, and acquisition of a phospholipid envelope containing embedded viral glycoproteins. Intracellular viral replication is occurring when the viral titer (measured both intracellularly and extracellularly) increases to a value greater than 1. In permissive tumor cell lines, the level of viral replication can be low (e.g., a viral titer increase of >1 and <20,000 at 48 hours post-infection), moderate (e.g., a viral titer increase of 20,000-40,000 at 48 hours post-infection), or high (e.g., a viral titer increase of >40,000 at 48 hours post-infection).

[0087] For a given tumor or healthy cell (preferably primary cell), viral replication is generally achieved by injecting the virus into the tumor or healthy cell (preferably primary cell) at an MOI of 10. -5 ~10 -2 The β-amyloid level can be determined in vitro 2 to 8 days after infection with β-amyloid.

[0088] The "therapeutic index" refers to the ratio of viral replication in tumor and corresponding healthy cells (i.e., healthy cells in the same organ). For a given organ's healthy and tumor cells, a given virus, a given condition, and a given time post-infection, the therapeutic index TI(organ's healthy cells, organ's tumor, virus, condition, time post-infection) is given as follows: TI (organ healthy cells, organ tumor, virus, condition, time post infection) = (viral replication in organ tumor cells / viral replication in organ healthy cells) It is defined as follows.

[0089] For a given organ and virus, the therapeutic index may vary depending on conditions (e.g., MOI, culture medium, tumor cell density, temperature, etc., especially MOI) and post-infection time, and when comparing the therapeutic index of two viruses, the comparison should be performed under the same conditions and the same post-infection time. However, similar to the oncolytic ability, if the difference in therapeutic index between two viruses is sufficient, the therapeutic index hierarchy generally remains the same under any suitable conditions and post-infection time, so any suitable conditions (see the example below) and post-infection time may be used for comparison (provided that these conditions are the same for the two viruses). For a given organ and virus, the therapeutic index is generally determined by injecting the virus into tumor or healthy cells (preferably primary cells) at an MOI of 10. -5 ~10 -2 The β-amyloid level can be determined in vitro 2 to 8 days after infection with β-amyloid.

[0090] This therapeutic index is improved by increasing replication activity on tumor cells and / or decreasing replication on corresponding healthy cells (preferably primary cells).

[0091] The term "extracellular enveloped virus (EEV) secretion capacity", abbreviated as "EEV-SC", refers to the percentage of EEV particles among all (EEV+IMV) particles for a given tumor, a given virus, a given condition, and a given time post-infection; EEV-SC (tumor, virus, condition, time post-infection) = number of EEV particles / number of (EEV + IMV) particles It is defined as follows.

[0092] For a given tumor and virus, EEV-SC may vary depending on conditions (e.g., MOI, culture medium, tumor cell density, temperature, etc., especially MOI) and post-infection time. When comparing EEV-SC of two viruses, the comparison should be performed under the same conditions and post-infection time. However, as with tumor lytic activity, if the difference in EEV-SC between two viruses is sufficient, the EEV-SC hierarchy generally remains the same under any appropriate conditions and post-infection time, so any appropriate conditions (see examples below) and post-infection time may be used for comparison (provided that these conditions are identical for the two viruses). For a given organ and virus, EEV-SC is generally obtained by injecting the virus into tumor cells at an MOI of 10. -4 ~10 -1 The β-amyloid level can be determined in vitro 16 to 24 hours after infection with β-amyloid.

[0093] The term "syncytium formation" refers to the ability of a virus to induce fusion between infected and neighboring cells, forming a multinucleated, enlarged cell called a "syncytium." This allows the virus to spread more rapidly and is no longer limited to the initially infected cell. As a result, viruses that do not induce syncytium formation infect more tumor cells than viruses that do not induce syncytium formation (Burton et al., 2019, Mol Ther Oncolytics, 15, 131-139, Krabbe et al., 2018, Cancers, 10, 216).

[0094] The term "spreading capacity" refers to the ability of a virus to spread between cells (e.g., tumor cells) or between tumors (e.g., spreading from a first tumor at a first location to a second tumor at a distal second location). Spreading capacity is known to be related to the formation of EEVs and / or syncytia; the more EEVs and / or syncytia a virus can produce, the higher its spreading capacity. Spreading capacity can be assessed by various techniques well known to those skilled in the art, including the viral comet assay, which can assess comet tail formation (e.g., comet number, comet tail size). In some cases, an increase in comet number or comet tail size can indicate an increase in the amount of EEV relative to the IMV form of the virus strain. The assessment of comet tail formation for a given tumor and virus generally involves injecting the virus into tumor cells at an MOI of 10. -4 ~10 -1 It can be determined in vitro 16-24 hours after infection with

[0095] The terms "virus neutralization rate" and "neutralization rate" are used interchangeably and measure the inhibition of viral oncolytic activity induced by antiviral antibodies. For a given virus, a given tumor, a given poxvirus-specific antibody, a given condition, and a given time post-infection, NT (virus, tumor, poxvirus-specific antibody, condition, time post-infection) = EC 50 (Poxvirus-specific antibodies present) / EC 50 (No poxvirus-specific antibodies) where the EC 50 , or 50% effective concentration, is known to those skilled in the art as the concentration of a drug (here, a chimeric poxvirus) that induces a response halfway between baseline and maximum, or the concentration required to obtain 50% efficacy (here, 50% tumor cell viability).

[0096] For a given virus, tumor, and poxvirus-specific antibody, the neutralization rate may vary depending on conditions (e.g., MOI, culture medium, tumor cell density, temperature, etc., especially MOI) and post-infection time. When comparing the neutralization rates of two viruses, the comparison should be performed under the same conditions and post-infection time. However, as with tumor lytic activity, if the difference in neutralization rate between two viruses is sufficient, the neutralization rate hierarchy generally remains the same under any appropriate conditions and post-infection time, so any appropriate conditions (see examples below) and post-infection time may be used for comparison (provided that these conditions are the same for the two viruses). For a given organ and virus, the neutralization rate is generally calculated by injecting the virus into tumor cells at an MOI of 3 × 10. -5 It can be determined in vitro 3-5 days after infection with .

[0097] The term "complement-mediated virus neutralization rate" is used to measure complement-induced inhibition of the oncolytic potential of a virus. For a given virus and given conditions: CMV-NT (virus, condition) = virus titer (active serum) / virus titer (heat-inactivated serum) where virus titer is assessed by plaque assay.

[0098] "Active serum" refers to serum that contains active complement components. While serum typically contains active complement components, these components can be altered and inactivated by prolonged storage or heat. "Heat-inactivated serum" refers to serum that has undergone a heat treatment (typically at 56°C for 30 minutes) to inactivate the complement components present in the serum.

[0099] Plaque assays are well known in the art and those skilled in the art would know how to perform them based on their common general knowledge. Examples of such assays are disclosed in the Materials and Methods section of the Examples below.

[0100] For a given virus, the complement-mediated virus neutralization rate may vary depending on conditions (e.g., MOI, culture medium, cell density, temperature, etc., especially MOI), and when comparing the complement-mediated virus neutralization rates of two viruses, the comparison should be performed under the same conditions. However, as with tumor lytic activity, if the difference in complement-mediated virus neutralization rate between two viruses is sufficient, the hierarchy of complement-mediated virus neutralization rate generally remains the same under any appropriate conditions, so any appropriate conditions (see examples below) may be used for comparison (provided that the conditions are the same for the two viruses). For a given virus, the complement-mediated virus neutralization rate is generally measured in the presence of activated serum or heat-inactivated serum at a dose of 10 4 ~10 8 PFU / mL of virus can be determined in vitro.

[0101] The term "treatment" (and any form of treatment, such as "treating," "treat," etc.) as used herein ultimately encompasses treatment (e.g., in a subject diagnosed with a condition) in conjunction with conventional therapeutic modalities. The outcome of treatment is to slow, cure, ameliorate, or control the progression of the target condition. For example, a subject is said to be successfully treated for cancer if, after administration of a chimeric poxvirus, a mutant chimeric poxvirus, a recombinant chimeric poxvirus, or a composition thereof as described herein, alone or in combination, the subject shows an observable improvement in their clinical condition.

[0102] The term "administrating" (or any form of administration, such as "administrated"), as used herein, refers to the delivery of a therapeutic agent, such as a chimeric poxvirus (mutant and / or recombinant) described herein, to a subject.

[0103] As used herein, the term "proliferative disease" encompasses any disease or condition resulting from uncontrolled cell growth and proliferation, including cancer and some cardiovascular diseases (such as restenosis due to proliferation of smooth muscle cells in the walls of blood vessels). The term "cancer" can be used interchangeably with terms such as "tumor," "tumor," "malignant tumor," and "neoplasm." These terms are meant to include any type of tissue, organ, or cell, and any stage of malignancy (e.g., pre-cancerous to stage IV).

[0104] The term "subject" generally refers to an organism for which the products and methods of the present invention are 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 diagnosed with or at risk of having a proliferative disease, such as cancer. The terms "subject" and "patient" can be used interchangeably when referring to a human organism and include males and females. The subject of treatment may be a neonate, infant, young adult, adult, or elderly.

[0105] The terms "combination treatment," "combination therapy," "combined treatment," or "combinatorial treatment" can be used interchangeably and refer to the treatment of a subject with a chimeric poxvirus as described herein and at least one additional therapeutic modality. The additional therapeutic modality may be selected from the group consisting of surgery, radiation therapy, chemotherapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, cytokine therapy, targeted cancer therapy, gene therapy, photodynamic therapy, transplantation, etc. The combination treatment may include a third or further therapeutic modality. For combination treatment, the optimal concentration of each component of the combination may be determined by one skilled in the art.

[0106] "Chimeric poxvirus" or "poxvirus chimera" (wild type) The present invention relates to chimeric poxviruses with improved properties, such as the POXSTG19503 chimeric poxvirus produced by the present inventors, which, compared to their parent strains, exhibit enhanced oncolytic properties and therapeutic index in vitro, and better spreading ability in vivo due to improved EEV secretion and syncytium formation abilities.

[0107] The terms "chimeric poxvirus" and "poxvirus chimera" are used interchangeably and according to their usual meaning in virology to refer to a hybrid poxvirus created by joining nucleic acid fragments from two or more different poxvirus strains.

[0108] The terms "poxvirus," "poxvirus particle," "poxvirus vector," and "poxvirus virion" are used interchangeably and are understood to mean a vehicle comprising at least one element of a wild-type poxvirus genome. The poxvirus particle is preferably infectious (i.e., capable of infecting and entering a host cell or subject). The term encompasses both naturally occurring poxviruses as well as genetically modified (e.g., engineered) poxviruses.

[0109] Poxviruses are characterized by a 200 kb double-stranded DNA genome that encodes many viral enzymes and factors that allow the virus to replicate independently of the host cell machinery. The majority of poxvirus particles are intracellular (IMV stands for intracellular mature virion), have a single lipid envelope, and remain in the cytoplasm of infected cells until lysis. Extracellular forms are enveloped particles with an additional membrane that bud from infected cells (e.g., EEV stands for extracellular enveloped virus).

[0110] The nucleic acid sequence of poxviruses consists of a core sequence and two inverted terminal repeats (ITRs). The terms "core," "core region," or "core sequence" are used interchangeably and refer to the poxvirus nucleic acid region that is the major viral nucleic acid sequence flanked by the two ITRs. The length of the core region varies among poxvirus strains. The term "inverted terminal repeats" or "ITRs" refers to nucleic acid regions that are duplicated and inverted at both the 5' and 3' ends of the viral genome. ITRs are composed of non-coding repeat patterns (e.g., short tandem repeats, microsatellites, minisatellites, etc.) at their ends, which can vary between two viruses. Poxviruses contain two ITRs, one located at the 5' end and the other at the 3' end of the viral nucleic acid sequence, each a reverse complement of the other. The length of the ITRs varies among poxvirus strains.

[0111] The terms "rabbitpox virus," "rabbitpox virus particle," "rabbitpox virus vector," and "rabbitpox virus virion" are used interchangeably. This term encompasses wild-type, mutant, recombinant, and recombinant mutant rabbitpox viruses.

[0112] The term "rabbitpox virus Utrecht strain" (also referred to as "RPX" or "RPXV") is used according to its common and ordinary meaning and refers to identically and similarly named virus strains, as well as functional fragments and homologs thereof. RPX is available from culture collections such as the American Type Culture Collection (ATCC) (e.g., VR-1591™). The term includes wild-type, mutant, recombinant, and recombinant mutant-emerging forms of the rabbitpox virus Utrecht strain that maintain Utrecht activity. Their genomes preferably share sequence identity (e.g., about 97%, 98%, 99%, or 100%) with the rabbitpox virus Utrecht strain genome. The genome of RPX used in the examples comprises SEQ ID NO: 2, which represents the core region (composed of nucleotides 1 to 183029) and one of the two ITRs (composed of nucleotides 183030 to 186491). RPX comprising SEQ ID NO: 2 is particularly preferred.

[0113] The terms "cowpox virus," "cowpox virus particle," "cowpox virus vector," and "cowpox virus virion" are used interchangeably. This term encompasses wild-type, mutant, recombinant, and recombinant mutant cowpox viruses.

[0114] The term "Cowpox virus Brighton strain" (also referred to as "CPX" or "CPXV") is used according to its common and ordinary meaning and refers to identically and similarly named virus strains, as well as functional fragments and homologs thereof. This term includes wild-type, mutant, recombinant, and recombinant mutant forms of the cowpox virus Brighton strain or its variants that maintain Brighton activity. Their genomes preferably share sequence identity (e.g., about 97%, 98%, 99%, or 100%) with the cowpox virus Brighton strain genome. The genome of CPX used in the examples comprises SEQ ID NO: 3, which represents the core region (composed of nucleotides 1 to 206014) and one of the two ITRs (composed of nucleotides 206015 to 212521). CPX comprising SEQ ID NO: 3 is particularly preferred.

[0115] The terms "vaccinia virus," "vaccinia virus particle," "vaccinia virus vector," and "vaccinia virus virion" (also referred to as "VACV" or "VV") are used interchangeably. Vaccinia viruses are available from culture collections such as the ATCC (e.g., VR-1354, VR-2056, VR-2034, VR-2035, VR-2010). These terms encompass wild-type, mutant, recombinant, and recombinant mutant VACV viruses.

[0116] The term "Copenhagen" or "COP" is used according to its common and ordinary meaning and refers to virus strains of the same or similar name, as well as functional fragments and homologs thereof. This term includes wild-type, mutant, recombinant, and recombinant variant forms of vaccinia virus COP strains or mutants thereof that maintain COP activity. Their genomes preferably share sequence identity (e.g., about 97%, 98%, 99%, or 100%) with the genome of the vaccinia virus COP strain. The genome of the COP used in the examples comprises SEQ ID NO: 4, which represents the core region (composed of nucleotides 1 to 167702) and one of the two ITRs (composed of nucleotides 167703 to 175860). A COP comprising SEQ ID NO: 4 is particularly preferred.

[0117] The term "Wyeth" or "WY" is used according to its common and ordinary meaning and refers to virus strains of the same or similar name, as well as their functional fragments and homologs. WY is available from culture collections such as ATCC (e.g., VR-1536™). The term includes wild-type, mutant, recombinant, and recombinant mutant forms of the vaccinia virus Wyeth strain or its variants that maintain Wyeth activity. Their genomes preferably share sequence identity (e.g., about 97%, 98%, 99%, or 100%) with the vaccinia virus Wyeth strain genome. The WY genome used in the examples comprises SEQ ID NO: 5, which represents the core region (nucleotides 1 to 166358) and one of the two ITRs (nucleotides 166359 to 182664). WY comprising SEQ ID NO: 5 is particularly preferred.

[0118] The term "Western Reserve" or "WR" is used according to its common and ordinary meaning and refers to virus strains of the same or similar name, as well as their functional fragments and homologs. The WR is available from culture collections such as ATCC (e.g., VR-1354™). The term includes wild-type, mutant, recombinant, and recombinant mutant forms of the vaccinia virus Western Reserve strain or its variants that maintain Western Reserve activity. Their genomes preferably share sequence identity (e.g., about 97%, 98%, 99%, or 100%) with the vaccinia virus Western Reserve strain genome. The genome of the WR used in the examples comprises SEQ ID NO: 6, which represents the core region (composed of nucleotides 1 to 174481) and one of the two ITRs (composed of nucleotides 174482 to 181419). A WR comprising SEQ ID NO: 6 is particularly preferred.

[0119] The term "modified vaccinia virus" or "MVA" is used according to its common and ordinary meaning and refers to identically and similarly named virus strains, as well as their functional fragments and homologs. Such MVA is available from culture collections such as the American College of Clinical Oncology (ATCC) (e.g., VR-1508™). The term includes wild-type, mutant, recombinant, and recombinant mutant forms of vaccinia virus MVA strains or variants thereof that maintain MVA activity. Their genomes preferably share sequence identity (e.g., about 97%, 98%, 99%, or 100%) with the genome of a vaccinia virus MVA strain. The genome of the MVA used in the examples comprises SEQ ID NO: 7, which represents the core region (consisting of nucleotides 1 to 159456) and one of the two ITRs (consisting of nucleotides 159457 to 163444). This vaccinia virus MVA strain genome expresses the eGFP gene under the control of the p11k7.5 promoter (MVATG15938) and has been previously constructed and characterized (Erbs et al., 2008, Cancer Gene Ther. 2008, 15, 18-28). Particularly preferred is the MVA comprising SEQ ID NO: 7.

[0120] Chimeric poxviruses with improved anti-cancer activity (higher cancer cell killing ability and better tumor selectivity) are defined by the structure of their genome (nucleic acid features), or by functional features, or as being obtained or obtainable by specific methods of directed evolution as described below, or by a combination of these features, as defined below.

[0121] Chimeric poxviruses defined by nucleic acid characteristics The sequences of the core region (composed of nucleotides 1 to 175910) and one of the two ITRs (composed of nucleotides 175911 to 185577) of the chimeric poxvirus POXSTG19503 obtained by the present inventors were determined and found to correspond to SEQ ID NO:1.

[0122] Thus, in a first aspect, the present invention provides a method for the preparation of a nucleic acid sequence having at least 96,6%, preferably at least 96,7%, at least 96,8%, at least 96,9%, at least 97%, at least 97,1%, at least 97,2%, at least 97,3%, 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%, 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%, at least 99,91%, at least 99,92%, at least 99,93%, at least 99,94%, at least 99,95%, at least 99,96%, at least 99,97%, at least 99,98%, at least 99,99%, or even 100% sequence identity.

[0123] In one embodiment, the chimeric poxvirus of the present invention is the chimeric poxvirus POXSTG19503 clone 7, deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) on October 20, 2022 under the accession number CNCM I-5913.

[0124] In the present disclosure, the chimeric poxvirus deposited under accession number CNCM I-5913 is also referred to as POXSTG19503.

[0125] The chimeric poxvirus of the present invention is preferably obtained by shuffling nucleic acid sequences from six parental poxvirus strains: rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Western Reserve strain (WR), vaccinia virus Wyeth strain (WY), and modified vaccinia virus Ankara strain (MVA), and more preferably comprises nucleic acid sequences from at least two parental poxvirus strains. The nucleic acid segments from the at least two parental poxvirus strains contain essential genes required for replication. The chimeric poxvirus may also comprise nucleic acid sequences from at least three, at least four, at least five, or even six parental poxvirus strains.

[0126] In particular, chimeric poxviruses (a) a nucleic acid sequence consisting of nucleotides 562 to 4701 of SEQ ID NO: 2 or a sequence having at least 99% identity to nucleotides 562 to 4701 of SEQ ID NO: 2, a nucleic acid sequence consisting of nucleotides 54042 to 59851 of SEQ ID NO: 2 or a sequence having at least 99% identity to nucleotides 54042 to 59851 of SEQ ID NO: 2, a nucleic acid sequence consisting of nucleotides 83610 to 88879 of SEQ ID NO: 2 or a sequence having at least 99% identity to nucleotides 83610 to 88879 of SEQ ID NO: 2, a nucleic acid sequence consisting of nucleotides 127290 to 130589 of SEQ ID NO: 2 or a sequence having at least 99% identity to nucleotides 127290 to 130589 of SEQ ID NO: 2, a nucleic acid sequence consisting of nucleotides 137520 to 154979 of SEQ ID NO: 2 comprising glutamic acid at position 151 or a sequence having at least 99% identity to nucleotides 137520 to 154979 of SEQ ID NO: 2 comprising glutamic acid at position 151, and a nucleic acid sequence consisting of nucleotides 157002 to 162091 of SEQ ID NO:2 or a sequence having at least 99% identity to nucleotides 157002 to 162091 of SEQ ID NO:2 at least one rabbitpox virus Utrecht strain (RPX)-derived nucleic acid sequence selected from: (b) a nucleic acid sequence consisting of nucleotides 14242 to 51241 of SEQ ID NO: 3 or a sequence having at least 99% identity to nucleotides 14242 to 51241 of SEQ ID NO: 3, and a nucleic acid sequence consisting of nucleotides 59852 to 72141 of SEQ ID NO: 3 or a sequence having at least 99% identity to nucleotides 59852 to 72141 of SEQ ID NO: 3 at least one nucleic acid sequence derived from cowpox virus Brighton strain (CPX) selected from: (c) a nucleic acid sequence consisting of nucleotides 7612 to 8521 of SEQ ID NO: 4 or a sequence having at least 99% identity to nucleotides 7612 to 8521 of SEQ ID NO: 4 at least one Copenhagen (COP)-derived nucleic acid sequence selected from: (d) a nucleic acid sequence consisting of nucleotides 76630 to 78639 of SEQ ID NO: 5 or a sequence having at least 99% identity to nucleotides 76630 to 78639 of SEQ ID NO: 5; a nucleic acid sequence consisting of nucleotides 81060 to 83529 of SEQ ID NO: 5 or a sequence having at least 99% identity to nucleotides 81060 to 83529 of SEQ ID NO: 5; a nucleic acid sequence consisting of nucleotides 116250 to 118459 of SEQ ID NO: 5 or a sequence having at least 99% identity to nucleotides 116250 to 118459 of SEQ ID NO: 5, a nucleic acid sequence consisting of nucleotides 162290 to 164599 of SEQ ID NO: 5 or a sequence having at least 99% identity to nucleotides 162290 to 164599 of SEQ ID NO: 5; a nucleic acid sequence consisting of nucleotides 176100 to 179909 of SEQ ID NO: 5 or a sequence having at least 99% identity to nucleotides 176100 to 179909 of SEQ ID NO: 5, and a nucleic acid sequence consisting of nucleotides 181920 to 184099 of SEQ ID NO:5 or a sequence having at least 99% identity to nucleotides 181920 to 184099 of SEQ ID NO:5 at least one Wyeth (WY)-derived nucleic acid sequence selected from: (e) a nucleic acid sequence consisting of nucleotides 169190 to 171579 of SEQ ID NO: 6 or a sequence having at least 99% identity to nucleotides 169190 to 171579 of SEQ ID NO: 6, and a nucleic acid sequence consisting of nucleotides 173730 to 176099 of SEQ ID NO: 6 or a sequence having at least 99% identity to nucleotides 173730 to 176099 of SEQ ID NO: 6 at least one Western Reserve (WR)-derived nucleic acid sequence selected from: (f) a nucleic acid sequence consisting of nucleotides 88880 to 90899 of SEQ ID NO: 7 or a sequence having at least 99% identity to nucleotides 88880 to 90899 of SEQ ID NO: 7, a nucleic acid sequence consisting of nucleotides 91460 to 93839 of SEQ ID NO: 7 or a sequence having at least 99% identity to nucleotides 91460 to 93839 of SEQ ID NO: 7; a nucleic acid sequence consisting of nucleotides 95530 to 116249 of SEQ ID NO: 7 or a sequence having at least 99% identity to nucleotides 95530 to 116249 of SEQ ID NO: 7; a nucleic acid sequence consisting of nucleotides 118460 to 127289 of SEQ ID NO: 7 or a sequence having at least 99% identity to nucleotides 118460 to 127289 of SEQ ID NO: 7, and a nucleic acid sequence consisting of nucleotides 134800 to 137519 of SEQ ID NO: 7 or a sequence having at least 99% identity to nucleotides 134800 to 137519 of SEQ ID NO: 7 at least one modified vaccinia virus Ankara (MVA)-derived nucleic acid sequence selected from: (g) Any combination of (a) to (f) may comprise:

[0127] In a preferred embodiment of the present invention, the chimeric poxvirus comprises a glutamic acid (151E) at position 151 of the protein encoded by the A34R gene. This amino acid at position 151 of the A34R gene is known to be associated with increased production and spread of whole progeny viruses and extracellular enveloped viruses (EEV), potentially immunoinvasive forms (Thirunavukarasu et al., 2013, Mol. Ther. Vol. 21 no. 5, pp. 1024-1033). The sequence comprising glutamic acid at position 151 of the protein encoded by the A34R gene may be inherited from rabbitpox virus, preferably the parent rabbitpox virus Utrecht strain (RPX). In a further preferred embodiment of the present invention, the chimeric poxvirus comprises a valine (19V) at position 19 of the protein encoded by the A34R gene. The sequence comprising valine at position 19 of the protein encoded by the A34R gene may be inherited from the parent modified vaccinia virus Ankara (MVA). In a more preferred embodiment of the present invention, the chimeric poxvirus comprises an A34R chimeric gene, wherein said A34R chimeric gene encodes a protein comprising valine at position 19 (19V) and glutamic acid at position 151 (151E). The sequence comprising valine at position 19 (19V) and glutamic acid at position 151 of the protein encoded by the A34R gene may be inherited from the parent modified vaccinia virus Ankara (MVA) and the parent rabbitpox virus Utrecht strain (RPX), respectively. In an even more preferred embodiment, the protein encoded by the A34R chimeric gene has at least 85%, preferably at least 90%, more preferably at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 11 and has a glutamic acid at position 151 (151E) and, optionally, a valine at position 19 (19V).

[0128] In a further preferred embodiment of the present invention, the chimeric poxvirus is partially or completely deficient in the A56R locus. Deletion of the A56R locus, resulting in alterations in the hemagglutinin-encoding gene, is associated with the induction of syncytia formation in infected cells by bypassing the inhibition of cell-cell fusion (Turner et al. 2008, Virology 380, 226-233). The chimeric poxvirus is partially or completely deficient in the A56R locus, resulting in the expression of an ineffective protein encoded by the A56R gene, a deletion or mutation of the N-terminal domain (e.g., at least 34 amino acids, preferably at least 36 amino acids, at least 38 amino acids, more preferably at least 40 amino acids, at least 42 amino acids, or at least 44 amino acids) of the protein encoded by the A56R gene, or a mutation at positions 34 and / or 103 of the protein encoded by the A56R gene, compared to that expressed by the parent Copenhagen vaccinia virus (COP) sequence. In particular, the chimeric poxvirus may comprise a deletion of 44 amino acid residues in the N-terminal domain of the protein expressed by the A56R gene compared to that expressed by the parental vaccinia virus Copenhagen strain (COP) sequence. In a more particular embodiment, the chimeric poxvirus may comprise the sequence of the protein encoded by the A56R gene inherited from rabbitpox virus, preferably the parental rabbitpox virus Utrecht strain (RPX). In an even more preferred embodiment, the protein encoded by the A56R gene has at least 85%, preferably at least 90%, more preferably at least 95% or 100% identity with the amino acid sequence set forth in SEQ ID NO: 12.

[0129] Alternatively or in combination, said chimeric poxvirus according to the invention may comprise a gene encoding a zinc ring finger protein, said gene being inherited from a rabbitpox virus, preferably the parent rabbitpox virus Utrecht strain (RPX).

[0130] Alternatively or in combination, said chimeric poxvirus according to the invention preferably does not comprise a gene encoding an ankyrin repeat protein inherited from rabbitpox virus, preferably from the parent rabbitpox virus Utrecht strain (RPX).

[0131] Alternatively or in combination, said chimeric poxvirus according to the invention may preferably not comprise a gene encoding a chemokine-binding protein inherited from rabbitpox virus, preferably the parent rabbitpox virus Utrecht strain (RPX).

[0132] Alternatively or in combination therewith, said chimeric poxvirus according to the invention preferably comprises: - comprising a gene encoding a zinc ring finger protein, said gene being inheritable from a rabbitpox virus, preferably the parent rabbitpox virus Utrecht strain (RPX), and does not contain genes encoding ankyrin repeat proteins and chemokine-binding proteins inherited from rabbitpox virus, preferably the parent rabbitpox virus Utrecht strain (RPX);

[0133] Chimeric poxviruses defined by functional characteristics The chimeric poxvirus POXSTG19503 obtained by the inventors is characterized by several advantageous functional properties, including higher oncolytic potential and replication in some tumor cells than the parental strain COP (see Figures 1 and 4A), and lower replication in healthy cells (preferably primary cells) (see Figure 4A), and therefore a higher therapeutic index (see Figure 4B).

[0134] Furthermore, the TK-mutant (POXSTG19508) was found to exhibit higher oncolytic activity than all parental TK-mutants (see Figure 3), higher replication in cancer cells and lower replication in healthy cells (preferably primary cells), and therefore a higher therapeutic index than the TK-COP parental strain (see Figure 4), express a higher amount of the transgene than the TK-COP parental strain (see Figure 5), produce a higher proportion of EEVs than the TK-COP parental strain (see Figure 6) and than RPX and IHDJ-WT (see Figure 8), induce syncytia formation (see Figure 17), increase the number and tail size in the comet assay (see Figure 7), and exhibit better spreading in vivo (see Figure 10). The TK-mutant POXSTG19508 was also found to be less sensitive to poxvirus-specific antibody neutralization (see Figure 9), less sensitive to complement-mediated virus neutralization (see Figure 19), more efficient in various in vivo animal models (see Figures 11-15), and induce better T cell responses against tumors (see Figure 18).

[0135] Similar results were obtained with the TK-RR-mutant (POXSTG19730, data not shown).

[0136] Each of these improved functional characteristics is of interest for the oncolytic treatment of proliferative diseases, particularly cancer.

[0137] Chimeric poxvirus with high oncolytic activity Thus, in another aspect, the present invention provides a chimeric poxvirus (optionally mutant and / or recombinant), which for at least one tumor has an oncolytic potency higher than the oncolytic potency of at least one of the oncolytic parent poxvirus strains, namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post infection, such that for a given tumor, a given virus, given conditions and a given time post infection, the oncolytic potency OP(tumor, virus, condition, time post infection) is: OP(tumor, virus, condition, time post-infection) = (100 - percentage of surviving tumor cells after infection with virus) It is defined as follows.

[0138] In a preferred embodiment, for at least one tumor, the oncolytic activity of the chimeric poxvirus is higher than that of the parental COP or parental RPX, measured under the same conditions and at the same time post-infection. More preferably, for at least one tumor, the oncolytic activity of the chimeric poxvirus is higher than that of at least two of the oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, measured under the same conditions and at the same time post-infection. For example, for at least one tumor, the oncolytic activity of the chimeric poxvirus is higher than that of the parental COP and parental CPX, measured under the same conditions and at the same time post-infection. In a more preferred embodiment, for at least one tumor, the oncolytic activity of the chimeric poxvirus is higher than that of at least three, more preferably at least four, and even more preferably each of the five oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, measured under the same conditions and at the same time post-infection.

[0139] For a given tumor and virus, oncolytic potency is generally determined by injecting the virus into tumor cells at an MOI of 10. -5~10 -2 The results can be determined 3 to 5 days after infection with

[0140] In a particular embodiment of this aspect, the present invention provides chimeric poxviruses (optionally mutant and / or recombinant) that exhibit a higher oncolytic activity than at least one of the oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, measured under the same conditions and at the same time post-infection, for at least one tumor cell line selected from A549, MIA Paca-2, U-87-MG, B16F10, and HepG2. In a preferred embodiment, the oncolytic activity of the chimeric poxvirus is higher than the oncolytic activity of the parental COP or RPX, measured under the same conditions and at the same time post-infection, for at least one tumor cell line selected from A549, MIA Paca-2, U-87-MG, B16F10, and HepG2. More preferably, the oncolytic activity of the chimeric poxvirus is higher than that of at least two of the oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, measured under the same conditions and at the same time post-infection, for at least one tumor cell line selected from A549, MIA Paca-2, U-87-MG, B16F10, and HepG2. For example, the oncolytic activity of the chimeric poxvirus is higher than that of the parental COP and parental RPX, measured under the same conditions and at the same time post-infection, for at least one tumor cell line selected from A549, MIA Paca-2, U-87-MG, B16F10, and HepG2. In a more preferred embodiment, the oncolytic activity of the chimeric poxvirus for at least one tumor cell line selected from A549, MIA Paca-2, U-87-MG, B16F10 and HepG2 is higher than the oncolytic activity of at least three, more preferably at least four, and even more preferably each of the five oncolytic parental poxvirus strains RPX, CPX, COP, WY and WR, measured under the same conditions and at the same time post-infection.

[0141] In a more specific embodiment of this aspect, a) In A549, the oncolytic activity of the chimeric poxvirus is at least 69% different from that of at least one of the oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, measured under the same conditions and at the same time post-infection. Preferably, the oncolytic activity of the chimeric poxvirus is at least 69% different from that of the parental COP (J2R locus deleted or not) or parental RPX, measured under the same conditions and at the same time post-infection. More preferably, the oncolytic activity of the chimeric poxvirus is at least 69% different from that of at least two of the oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, measured under the same conditions and at the same time post-infection. For example, the oncolytic activity of the chimeric poxvirus is at least 69% different from that of the parental COP and parental RPX, measured under the same conditions and at the same time post-infection. In a more preferred embodiment, the oncolytic potency of said chimeric poxvirus is at least 69% different from the oncolytic potency of at least three, more preferably at least four, and even more preferably all five of the parental oncolytic poxvirus strains RPX, CPX, COP, WY and WR, measured under the same conditions and at the same time post-infection; and / or b) In MIA PaCa-2 mice, the oncolytic activity of the chimeric poxvirus is at least 37% different from that of at least one of the oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, measured under the same conditions and at the same time post-infection. Preferably, the oncolytic activity of the chimeric poxvirus is at least 37% different from that of the parental COP (J2R locus deleted or not) or parental RPX, measured under the same conditions and at the same time post-infection. More preferably, the oncolytic activity of the chimeric poxvirus is at least 37% different from that of at least two of the oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, measured under the same conditions and at the same time post-infection. For example, the oncolytic activity of the chimeric poxvirus is at least 37% different from that of the parental COP and parental RPX, measured under the same conditions and at the same time post-infection. In a more preferred embodiment, the oncolytic potency of said chimeric poxvirus is at least 37% different from the oncolytic potency of at least three, more preferably at least four, and even more preferably all five of the parental oncolytic poxvirus strains RPX, CPX, COP, WY and WR, measured under the same conditions and at the same time post-infection; and / or c) In U-87 MG, the oncolytic activity of the chimeric poxvirus is at least 37% different from that of at least one of the oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, measured under the same conditions and at the same time post-infection. Preferably, the oncolytic activity of the chimeric poxvirus is at least 37% different from that of the parental COP or parental RPX, measured under the same conditions and at the same time post-infection. More preferably, the oncolytic activity of the chimeric poxvirus is at least 37% different from that of at least two of the oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, measured under the same conditions and at the same time post-infection. For example, the oncolytic activity of the chimeric poxvirus is at least 37% different from that of the parental COP and parental RPX, measured under the same conditions and at the same time post-infection. In a more preferred embodiment, the oncolytic potency of said chimeric poxvirus is at least 37% different from the oncolytic potency of at least three, more preferably at least four, and even more preferably all five of the parental oncolytic poxvirus strains RPX, CPX, COP, WY and WR, measured under the same conditions and at the same time post-infection; and / or d) In B16F10, the oncolytic activity of the chimeric poxvirus is at least 20% different from that of at least one of the oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, measured under the same conditions and at the same time post-infection. Preferably, the oncolytic activity of the chimeric poxvirus is at least 20% different from that of the parental COP or parental RPX, measured under the same conditions and at the same time post-infection. More preferably, the oncolytic activity of the chimeric poxvirus is at least 20% different from that of at least two of the oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, measured under the same conditions and at the same time post-infection. For example, the oncolytic activity of the chimeric poxvirus is at least 20% different from that of the parental COP and parental RPX, measured under the same conditions and at the same time post-infection. In a more preferred embodiment, the oncolytic potency of said chimeric poxvirus differs by at least 20% from the oncolytic potency of at least three, more preferably at least four, and even more preferably all five of the parental oncolytic poxvirus strains RPX, CPX, COP, WY and WR, measured under the same conditions and at the same time post-infection; and / or e) In HepG2, the oncolytic activity of the chimeric poxvirus is at least 49% different from that of at least one of the oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, measured under the same conditions and at the same time post-infection. Preferably, the oncolytic activity of the chimeric poxvirus is at least 49% different from that of the parental COP or parental RPX, measured under the same conditions and at the same time post-infection. More preferably, the oncolytic activity of the chimeric poxvirus is at least 49% different from that of at least two of the oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, measured under the same conditions and at the same time post-infection. For example, the oncolytic activity of the chimeric poxvirus is at least 49% different from that of the parental COP and parental RPX, measured under the same conditions and at the same time post-infection. In a more preferred embodiment, the difference between the oncolytic potency of the chimeric poxvirus and the oncolytic potency of at least three, more preferably at least four, and even more preferably all five of the parental oncolytic poxvirus strains RPX, CPX, COP, WY and WR, measured under the same conditions and at the same time post-infection, is at least 49%.

[0142] Preferably, in this embodiment, oncolytic potency is measured at 5 days post-infection at an MOI of 10 for A549, U-87-MG, and HepG2. -5 In MIA PaCa-2, MOI was 10. -4 So, B16F10 has an MOI of 10. -3 More preferably, in this embodiment, the tumor cell line is cultured at 37°C, 5% CO2, in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS).

[0143] Chimeric poxviruses with low replication in healthy cells (preferably primary cells) In another aspect, the present invention provides a chimeric poxvirus (optionally mutant and / or recombinant), wherein the viral replication of said chimeric poxvirus in at least one type of healthy cell (preferably primary cell) is lower than the viral replication of at least one of five oncolytic parental poxvirus strains: rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), and vaccinia virus Western Reserve strain (WR). Preferably, the viral replication of said chimeric poxvirus in at least one type of healthy cell (preferably primary cell) is lower than the viral replication of the parental vaccinia virus Copenhagen strain (COP). More preferably, for at least one healthy cell (preferably primary cell), the viral replication of said chimeric poxvirus in the healthy cell (preferably primary cell) is lower than the viral replication of at least two, preferably at least three, more preferably at least four, and even more preferably each of five oncolytic parental poxvirus strains, namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR).

[0144] For a given tumor or healthy cell (preferably primary cell), viral replication is generally achieved by injecting the virus into the tumor or healthy cell (preferably primary cell) at an MOI of 10. -5 ~10 -2 The β-amyloid level can be determined in vitro 2 to 8 days after infection with β-amyloid.

[0145] In a particular embodiment of this aspect, the present invention provides a chimeric poxvirus (optionally mutant and / or recombinant), wherein for at least one healthy cell (preferably primary cell) selected from skin cells and liver cells, viral replication of said chimeric poxvirus in the healthy cells is lower than viral replication of at least one of the five oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR. Preferably, for at least one healthy cell (preferably primary cell) selected from skin cells and liver cells, viral replication of said chimeric poxvirus in the healthy cells is lower than viral replication of the parental COP. More preferably, for at least one healthy cell (preferably primary cell) selected from skin cells and liver cells, viral replication of said chimeric poxvirus in the healthy cells (preferably primary cell) is lower than viral replication of at least two, preferably at least three, more preferably at least four, and even more preferably each of the five oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR.

[0146] In a more specific embodiment of this aspect, viral replication of said chimeric poxvirus is a) in at least one healthy cell line (preferably a primary cell line) selected from skin cells and liver cells, the viral replication of at least one of the five oncolytic parental poxvirus strains (optionally mutant and / or recombinant) RPX, CPX, COP, WY and WR, preferably the viral replication of the parental COP, more preferably at least 1.9-fold lower than the viral replication of at least two, more preferably at least three, more preferably at least four, even more preferably each of the five oncolytic parental poxvirus strains (optionally mutant and / or recombinant) RPX, CPX, COP, WY and WR; and / or b) in hepatocytes, it is at least 5.1 times lower than the viral replication of at least one of the five oncolytic parental poxvirus strains (optionally mutated and / or recombinant) RPX, CPX, COP, WY and WR, preferably lower than the viral replication of the parental COP, more preferably lower than the viral replication of at least two, more preferably lower than at least three, more preferably lower than at least four, and even more preferably lower than the viral replication of each of the five oncolytic parental poxvirus strains (optionally mutated and / or recombinant) RPX, CPX, COP, WY and WR.

[0147] Preferably, in this embodiment, viral replication is at an MOI of 10 in HepG2. -4 at 3 days post-infection in the human skin model or at 10 5 In vitro evaluation is performed 7 days after infection with PFU. More preferably, in this embodiment, tumor cell lines are cultured at 37°C, 5% CO2, in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS).

[0148] Chimeric poxvirus with a high therapeutic index In another aspect, the present invention provides a chimeric poxvirus (optionally mutant and / or recombinant) having, for at least one organ, a therapeutic index of said chimeric poxvirus higher than the therapeutic index of at least one of five oncolytic parental poxvirus strains, namely rabbitpox Utrecht (RPX), cowpox Brighton (CPX), vaccinia Copenhagen (COP), vaccinia Wyeth (WY) and vaccinia Western Reserve (WR), measured under the same conditions and at the same time post infection, and wherein for a given organ, a given tumor, a given virus, a given condition and a given time post infection, the therapeutic index TI(organ, tumor, virus, condition, time post infection) is: TI (organ, tumor, virus, condition, time post infection) = (viral replication in tumor cells of organ / viral replication in healthy cells of organ) It is defined as follows.

[0149] In a preferred embodiment, the therapeutic index of the chimeric poxvirus is higher for at least one organ than that of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection. More preferably, the therapeutic index of the chimeric poxvirus is higher for at least one organ than that of at least two, more preferably at least three, more preferably at least four, and even more preferably each of five oncolytic parental poxvirus strains, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), and vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post-infection.

[0150] For a given organ and virus, the therapeutic index is generally determined by injecting the virus into tumor or healthy cells (preferably primary cells) at an MOI of 10. -5 ~10 -2 The β-amyloid level can be determined in vitro 2 to 8 days after infection with β-amyloid.

[0151] The therapeutic index is related to both oncolytic potency and replication in healthy cells (preferably primary cells). A higher therapeutic index can be due to higher oncolytic potency, lower replication, or both, in healthy cells (preferably primary cells).

[0152] In certain embodiments of this aspect, the hepatic therapeutic index of said chimeric poxvirus (optionally mutant and / or recombinant) is higher than the hepatic therapeutic index of at least one of the parent oncolytic poxvirus strains RPX, CPX, COP, WY and WR (optionally mutant and / or recombinant) measured under the same conditions and at the same time post infection, such that for a given virus, the hepatic therapeutic index TI(liver, HepG2, virus, condition, time post infection) is: TI(liver, HepG2, virus, condition, time post infection) = (viral replication in HepG2 tumor cells / viral replication in healthy liver cells) It is defined as follows.

[0153] Preferably, the hepatic therapeutic index of said chimeric poxvirus (optionally mutant and / or recombinant) is higher than that of the parental COP strain measured under the same conditions and at the same time post-infection. In a more preferred embodiment, the hepatic therapeutic index of said chimeric poxvirus is higher than that of at least two, preferably at least three, more preferably at least four, and even more preferably all five of the parental oncolytic poxvirus strains RPX, CPX, COP, WY, and WR, measured under the same conditions and at the same time post-infection.

[0154] Preferably, the hepatic therapeutic index of the chimeric poxvirus is at least 5-fold higher, more preferably at least 10-fold higher, more preferably at least 15-fold higher, even more preferably at least 20-fold higher than the hepatic therapeutic index of at least one of the parental oncolytic poxvirus strains RPX, CPX, COP, WY and WR (optionally mutant and / or recombinant) measured under the same conditions and at the same time post-infection, preferably the hepatic therapeutic index of the parental COP measured under the same conditions and at the same time post-infection, more preferably the hepatic therapeutic index of at least two, preferably at least three, more preferably at least four, even more preferably all five of the parental oncolytic poxvirus strains RPX, CPX, COP, WY and WR measured under the same conditions and at the same time post-infection.

[0155] More preferably, in this embodiment, the hepatic therapeutic index is 10 or less when the chimeric poxvirus is administered to HepG2 tumor cells at an MOI of 10 or less, respectively. -5 and healthy hepatocytes (preferably primary hepatocytes) at an MOI of 10. -4and the replication of the chimeric poxvirus in HepG2 tumor cells and healthy hepatocytes is measured in vitro 3 days after infection. Even more preferably, in this embodiment, the HepG2 tumor cell line is cultured at 37°C, 5% CO2, in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS), and the healthy hepatocytes are cultured at 37°C, 5% CO2, in basal hepatocyte medium (BIOPREDICS catalog reference MIL600) and hepatocyte culture medium supplement (BIOPREDICS catalog reference ADD222C).

[0156] Chimeric poxvirus with high EEV secretion ability Poxviruses have two distinct types of infectious virus particles capable of initiating infection: intracellular mature virus (IMV) and extracellular enveloped virus (EEV). EEV is typically produced early after infection of susceptible cells and is released from the cells prior to lysis. Therefore, the EEV form rapidly spreads throughout the body within the infected host, avoiding immune-mediated clearance from the blood. Kirn et al. (2008, Cancer Res. 68(7):2071-5) compared the oncolytic potential of low- and high-EEV-producing vaccinia strains. EEV-enhanced vaccinia strains showed significantly improved antitumor efficacy and demonstrated improved intratumoral spread after systemic delivery. Furthermore, EEV-enhanced strains also demonstrated improved spread via the blood between injected and uninjected distant tumors. Importantly, EEV-enhanced strains also exhibited reduced clearance by circulating neutralizing antibodies.

[0157] In another aspect, the present invention provides a chimeric poxvirus (optionally mutant and / or recombinant), wherein the extracellular enveloped virus (EEV) secretion capacity (SC) (abbreviated as EEV-SC) of said chimeric poxvirus is measured for at least one producer cell (preferably a tumor cell) under the same conditions and at the same time post-infection with six parental poxvirus strains, namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (VPX), and cowpox virus Brighton strain (CPX). and the EEV-SC is higher than the EEV-SC of at least one of the following: the Covid-19 strain (COP), the Wyeth strain (WY), the Western Reserve strain (WR), and the modified Ankara strain (MVA), for a given virus, a given producer cell, under given conditions, and at a given time post-infection with the virus, where the EEV-SC is the ratio of extracellular enveloped virus (EEV) to all forms of virus (the extracellular enveloped virus (EEV) form and the intracellular mature virus (IMV) form of the virus), EEV-SC (virus, producer cells, conditions, time post-infection) = number of EEV particles / number of (EEV + IMV) particles It is defined as follows.

[0158] In a preferred embodiment, the present invention provides a chimeric poxvirus (optionally mutant and / or recombinant) that, for at least one type of producer cell (preferably tumor cell), has a higher EEV-SC than that of the parental vaccinia virus Copenhagen strain (COP) or parental rabbitpox virus Utrecht strain (RPX), measured under the same conditions and at the same time post-infection. More preferably, for at least one type of producer cell (preferably tumor cell), the chimeric poxvirus has a higher EEV-SC than that of at least two of six parental poxvirus strains, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR), and modified vaccinia virus Ankara strain (MVA), measured under the same conditions and at the same time post-infection. For example, for at least one type of producer cell (preferably tumor cell), the EEV-SC of the chimeric poxvirus is higher than the EEV-SC of the parental vaccinia virus Copenhagen strain (COP) and the parental rabbitpox virus Utrecht strain (RPX) measured under the same conditions and at the same time post-infection. In a more preferred embodiment, for at least one type of producer cell (preferably tumor cell), the EEV-SC of the chimeric poxvirus is higher than the EEV-SC of at least three, preferably at least four, more preferably at least five, and even more preferably of six parental poxvirus strains, namely, the rabbitpox virus Utrecht strain (RPX), the cowpox virus Brighton strain (CPX), the vaccinia virus Copenhagen strain (COP), the vaccinia virus Wyeth strain (WY), the vaccinia virus Western Reserve strain (WR), and the modified vaccinia virus Ankara strain (MVA), measured under the same conditions and at the same time post-infection.

[0159] The IHD-J vaccinia virus strain is known as a high EEV-producing strain of vaccinia virus. Therefore, alternatively or in combination with this, for at least one producer cell (preferably a tumor cell), the EEV-SC of the chimeric poxvirus (optionally a mutant and / or recombinant) is higher than the EEV-SC of the IHD-J vaccinia virus strain measured under the same conditions and at the same post-infection time.

[0160] For a given tumor and virus, EEV-SC generally injects the virus into tumor cells at an MOI of 10. -4 ~10 -1 The β-amyloid level can be determined in vitro 16 to 24 hours after infection with β-amyloid.

[0161] In a more specific embodiment of this aspect, the chimeric poxvirus EEV-SC comprises: a) The chimeric poxvirus was inoculated into the A549 tumor cell line at an MOI of 10 -2 at least 4%, preferably at least 5%, more preferably at least 6% 16 hours after infection at an MOI of 0.1, preferably at an MOI of 0.1; and / or b) Infecting the chimeric poxvirus into the A549 tumor cell line at an MOI of 10 -2 24 hours after infection at an MOI of 0.1, preferably at least 5%, at least 6%, at least 7%, at least 8%, preferably at least 9%, more preferably at least 10%, and even more preferably at least 11%.

[0162] In a more specific embodiment of this aspect, the chimeric poxvirus EEV-SC comprises: a) The chimeric poxvirus was inoculated into the A549 tumor cell line at an MOI of 10 -2 4% to 9%, more preferably 6% to 8%, 16 hours after infection at an MOI of 0.1 or less, preferably 0.1; and / or b) Infecting the chimeric poxvirus into the A549 tumor cell line at an MOI of 10 -224 hours after infection at an MOI of 0.1, the concentration is 5% to 15%, more preferably 10% to 13%.

[0163] Highly dispersible chimeric poxvirus Preferably, for at least one tumor, the spreading capacity of the chimeric poxvirus (optionally mutant and / or recombinant) is higher than that of at least one of six parental poxvirus strains, i.e., rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR), and modified vaccinia virus Ankara strain (MVA), measured under the same conditions and at the same time post-infection, where spreading capacity is defined as the ability of a virus to spread between cells (e.g., tumor cells) or between tumors (e.g., from an injected tumor to a distant tumor) for a given virus, producer cell, condition, and time post-infection. In a preferred embodiment, for at least one tumor, the spreading capacity of the chimeric poxvirus is higher than that of parental COP or parental RPX, measured under the same conditions and at the same time post-infection. More preferably, for at least one tumor, the diffusion capacity of the chimeric poxvirus is higher than that of at least two of the six parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, measured under the same conditions and at the same time post-infection. For example, for at least one tumor, the diffusion capacity of the chimeric poxvirus is higher than that of the parental COP and parental RPX, measured under the same conditions and at the same time post-infection. In a more preferred embodiment, for at least one tumor, the diffusion capacity of the chimeric poxvirus is higher than that of at least three, preferably at least four, more preferably at least five, or even more preferably each of the six parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA.

[0164] For a given tumor and virus, the spreading capacity is generally determined by injecting the virus into tumor cells at an MOI of 10. -4 ~10-1 The β-amyloid level can be determined in vitro 16 to 24 hours after infection with β-amyloid.

[0165] Chimeric poxviruses with low neutralization rates in the presence of poxvirus-specific antibodies In another aspect, the present invention provides chimeric poxviruses (optionally mutant and / or recombinant), wherein the neutralization rate of said chimeric poxvirus for at least one poxvirus-specific antibody and tumor is lower than the neutralization rate of at least one of six parental poxvirus strains, namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara strain (MVA), measured under the same conditions and at the same time post-infection, and wherein the neutralization rate (NT(virus, tumor, condition, time post-infection)) for a given virus, a given tumor, a given poxvirus-specific antibody, a given condition and a given time post-infection measures the inhibition of the oncolytic activity of the virus induced by the antibody, NT (virus, tumor, poxvirus-specific antibody, condition, time after infection) = EC50 (with poxvirus-specific antibody) / EC50 (without poxvirus-specific antibody) It is defined as follows.

[0166] In a preferred embodiment, the neutralization rate of the chimeric poxvirus is lower than that of the parental COP under the same conditions and at the same time post-infection for at least one poxvirus-specific antibody and tumor. More preferably, the neutralization rate of the chimeric poxvirus is lower than that of at least two, preferably at least three, more preferably at least four, more preferably at least five, and even more preferably each of the six parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA for at least one poxvirus-specific antibody and tumor.

[0167] For a given tumor and virus, virus neutralization rates are generally determined by injecting the virus into tumor cells at an MOI of 3 x 10 -5 The antibody titer can be determined in vitro 3 to 5 days after infection with 10 to 3. If the anti-poxvirus antibody is derived from human serum, the serum may be diluted 10 to 1000 times.

[0168] Chimeric poxviruses with low complement-mediated virus neutralization rates In another aspect, the present invention provides a chimeric poxvirus, wherein the complement-mediated virus neutralization rate of the chimeric poxvirus is lower than the complement-mediated virus neutralization rate of at least one of six parental poxvirus strains, i.e., rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR), and modified vaccinia virus Ankara strain (MVA), measured under the same conditions; and the complement-mediated virus neutralization rate (CMV-NT(virus, condition)) for a given virus and for a given condition measures the complement-induced inhibition of the oncolytic activity of the virus, CMV-NT (virus, condition) = virus titer (active serum) / virus titer (heat-inactivated serum) It is defined as follows.

[0169] In a preferred embodiment, the complement-mediated virus neutralization rate of the chimeric poxvirus is lower than that of the parental COP measured under the same conditions. More preferably, the complement-mediated virus neutralization rate of the chimeric poxvirus is lower than that of at least two, preferably at least three, more preferably at least four, more preferably at least five, and even more preferably each of the six parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA.

[0170] For a given virus, complement-mediated virus neutralization rates are generally higher than those observed in the presence of human serum (active or heat-inactivated) at a dose of 10 4 ~10 8PFU / mL of virus can be determined in vitro.

[0171] Chimeric poxviruses with high syncytium-forming ability In another aspect, the present invention provides a chimeric poxvirus, wherein the syncytium formation ability of the chimeric poxvirus in at least one producer cell (preferably a tumor cell) is higher than the syncytium formation ability of at least one of six parental poxvirus strains, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR), and modified vaccinia virus Ankara strain (MVA), measured under the same conditions and at the same time post-infection.

[0172] In a preferred embodiment, the present invention provides a chimeric poxvirus, the syncytium formation ability of which is higher in at least one type of producer cell (preferably tumor cell) than that of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection. More preferably, the syncytium formation ability of which is higher in at least one type of producer cell (preferably tumor cell) than that of at least two of six parental poxvirus strains, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR), and modified vaccinia virus Ankara strain (MVA), measured under the same conditions and at the same time post-infection. For example, in a preferred embodiment, the syncytium formation ability of the chimeric poxvirus is higher for at least one type of producer cell (preferably a tumor cell) than the syncytium formation ability of at least two, preferably at least three, more preferably at least four, or at least five, more preferably of six parental poxvirus strains, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara strain (MVA), measured under the same conditions and at the same time post-infection.

[0173] For a given tumor and virus, syncytium formation is generally determined by injecting the virus into tumor cells at an MOI of 10. -5 It can be determined in vitro 16-96 hours after infection with 1-1.

[0174] Chimeric poxviruses combining several functional features of interest Particularly preferred are chimeric poxviruses according to the invention that combine several of the functional features described above. Thus, the chimeric poxviruses of the invention (optionally mutant and / or recombinant) may comprise any combination of the functional features described herein.

[0175] The chimeric poxviruses of the present invention are in particular - high oncolytic potency as disclosed in the corresponding section above (preferably compared to the parental vaccinia virus Copenhagen strain (COP), the parental rabbitpox virus Utrecht strain (RPX), the parental COP and RPX, or all parental poxvirus strains); - low viral replication in healthy cells (preferably primary cells) as disclosed in the corresponding section above (preferably compared to the parental vaccinia virus Copenhagen strain (COP) or all parental poxvirus strains); a high EEV-SC, a high syncytium formation ability, a high spreading ability, a low neutralization rate in the presence of poxvirus-specific antibodies and / or a low complement-mediated virus neutralization rate (preferably compared to the parental vaccinia virus Copenhagen strain (COP), the parental rabbitpox virus Utrecht strain (RPX), the vaccinia virus IHD-J strain or all parental poxvirus strains) as disclosed in the corresponding sections above The composition may comprise any combination of:

[0176] In particular, the chimeric poxvirus of the present invention high oncolytic potential as disclosed in the corresponding section above and low viral replication in healthy cells (preferably primary cells) as disclosed in the corresponding section above; - high oncolytic potential as disclosed in the corresponding section above and (high EEV-SC, high syncytium formation ability, high spreading ability, low neutralization rate in the presence of poxvirus-specific antibodies and / or low complement-mediated virus neutralization rate as disclosed in the corresponding section above); - low viral replication in healthy cells (preferably primary cells) as disclosed in the corresponding sections above and (high EEV-SC, high syncytium formation ability, high spreading ability, low neutralization rate in the presence of poxvirus-specific antibodies and / or low complement-mediated viral neutralization rate as disclosed in the corresponding sections above); or high oncolytic potential as disclosed in the corresponding section above, low viral replication in healthy cells (preferably primary cells) as disclosed in the corresponding section above, and (high EEV-SC, high syncytium formation ability, high spreading ability, low neutralization rate in the presence of poxvirus-specific antibodies and / or low complement-mediated viral neutralization rate as disclosed in the corresponding section above) may comprise:

[0177] Particularly preferred chimeric poxviruses according to the invention comprise the following functional characteristics: - the oncolytic potency of the chimeric poxvirus is greater for at least one tumor than the oncolytic potency of the parental vaccinia virus Copenhagen strain (COP) or the parental rabbitpox virus Utrecht strain (RPX) measured under the same conditions and at the same time post-infection; - in at least one type of healthy cell (preferably primary cell), the viral replication of the chimeric poxvirus in the healthy cell (preferably primary cell) is lower than the viral replication of the parental vaccinia virus Copenhagen strain (COP); - for at least one producer cell (preferably a tumor cell), the EEV-SC of the chimeric poxvirus is higher than the EEV-SC of the parent rabbitpox virus Utrecht strain (RPX) or the vaccinia virus IHD-J strain measured under the same conditions and at the same time post-infection; optionally, the neutralization rate of the chimeric poxvirus is lower for at least one poxvirus-specific antibody and tumor than the neutralization rate of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection; optionally, the complement-mediated virus neutralization rate of the chimeric poxvirus is lower than the complement-mediated virus neutralization rate of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions; and Optionally, the syncytium formation ability of the chimeric poxvirus is higher in at least one producer cell (preferably a tumor cell) than the syncytium formation ability of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection.

[0178] Further particularly preferred chimeric poxviruses according to the invention comprise the following functional characteristics: - the oncolytic potency of the chimeric poxvirus is greater for at least one tumor than the oncolytic potency of the parental vaccinia virus Copenhagen strain (COP) or the parental rabbitpox virus Utrecht strain (RPX) measured under the same conditions and at the same time post-infection; - in at least one type of healthy cell (preferably primary cell), the viral replication of the chimeric poxvirus in the healthy cell (preferably primary cell) is lower than the viral replication of the parental vaccinia virus Copenhagen strain (COP); the syncytium formation ability of the chimeric poxvirus is higher in at least one producer cell (preferably a tumor cell) than the syncytium formation ability of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection; optionally, for at least one producer cell (preferably a tumor cell), the EEV-SC of said chimeric poxvirus is higher than the EEV-SC of the parent rabbitpox virus Utrecht strain (RPX) or vaccinia virus IHD-J strain measured under the same conditions and at the same time post-infection; optionally, the neutralization rate of the chimeric poxvirus is lower for at least one poxvirus-specific antibody and tumor than the neutralization rate of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection; and Optionally, the complement-mediated virus neutralization rate of the chimeric poxvirus is lower than the complement-mediated virus neutralization rate of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions.

[0179] More particularly, preferred chimeric poxviruses according to the invention comprise the following functional characteristics: - the oncolytic potency of the chimeric poxvirus is greater for at least one tumor than the oncolytic potency of the parental vaccinia virus Copenhagen strain (COP) or the parental rabbitpox virus Utrecht strain (RPX) measured under the same conditions and at the same time post-infection; - in at least one type of healthy cell (preferably primary cell), the viral replication of the chimeric poxvirus in the healthy cell (preferably primary cell) is lower than the viral replication of the parental vaccinia virus Copenhagen strain (COP); - for at least one producer cell (preferably a tumor cell), the EEV-SC of the chimeric poxvirus is higher than the EEV-SC of the parent rabbitpox virus Utrecht strain (RPX) or the vaccinia virus IHD-J strain measured under the same conditions and at the same time post-infection; the syncytium formation ability of the chimeric poxvirus is higher in at least one producer cell (preferably a tumor cell) than the syncytium formation ability of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection; optionally, the neutralization rate of the chimeric poxvirus is lower for at least one poxvirus-specific antibody and tumor than the neutralization rate of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection; and Optionally, the complement-mediated virus neutralization rate of the chimeric poxvirus is lower than the complement-mediated virus neutralization rate of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions.

[0180] The chimeric poxvirus of the present invention may alternatively comprise a high therapeutic index as disclosed in the corresponding sections above, as well as (a high EEV-SC, a high syncytium formation ability, a high spreading ability, a low neutralization rate in the presence of poxvirus-specific antibodies and / or a low complement-mediated virus neutralization rate as disclosed in the corresponding sections above). Particularly preferred chimeric poxviruses according to the present invention comprise the following functional characteristics: the therapeutic index of said chimeric poxvirus is higher than the therapeutic index of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection for at least one organ; - for at least one producer cell (preferably a tumor cell), the EEV-SC of the chimeric poxvirus is higher than the EEV-SC of the parent rabbitpox virus Utrecht strain (RPX) or the vaccinia virus IHD-J strain measured under the same conditions and at the same time post-infection; optionally, the neutralization rate of the chimeric poxvirus is lower for at least one poxvirus-specific antibody and tumor than the neutralization rate of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection; optionally, the complement-mediated virus neutralization rate of the chimeric poxvirus is lower than the complement-mediated virus neutralization rate of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions; and Optionally, the syncytium formation ability of the chimeric poxvirus is higher in at least one producer cell (preferably a tumor cell) than the syncytium formation ability of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection.

[0181] Further particularly preferred chimeric poxviruses according to the invention comprise the following functional characteristics: the therapeutic index of said chimeric poxvirus is higher than the therapeutic index of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection for at least one organ; the syncytium formation ability of the chimeric poxvirus is higher in at least one producer cell (preferably a tumor cell) than the syncytium formation ability of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection; optionally, for at least one producer cell (preferably a tumor cell), the EEV-SC of said chimeric poxvirus is higher than the EEV-SC of the parent rabbitpox virus Utrecht strain (RPX) or vaccinia virus IHD-J strain measured under the same conditions and at the same time post-infection; optionally, the neutralization rate of the chimeric poxvirus is lower for at least one poxvirus-specific antibody and tumor than the neutralization rate of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection; and Optionally, the complement-mediated virus neutralization rate of the chimeric poxvirus is lower than the complement-mediated virus neutralization rate of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions.

[0182] More particularly, preferred chimeric poxviruses according to the invention comprise the following functional characteristics: the therapeutic index of said chimeric poxvirus is higher than the therapeutic index of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection for at least one organ; - for at least one producer cell (preferably a tumor cell), the EEV-SC of the chimeric poxvirus is higher than the EEV-SC of the parent rabbitpox virus Utrecht strain (RPX) or the vaccinia virus IHD-J strain measured under the same conditions and at the same time post-infection; the syncytium formation ability of the chimeric poxvirus is higher in at least one producer cell (preferably a tumor cell) than the syncytium formation ability of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection; optionally, the neutralization rate of the chimeric poxvirus is lower for at least one poxvirus-specific antibody and tumor than the neutralization rate of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection; and Optionally, the complement-mediated virus neutralization rate of the chimeric poxvirus is lower than the complement-mediated virus neutralization rate of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions.

[0183] Chimeric poxviruses obtained or obtainable by directed evolution methods for selecting chimeric poxviruses with high oncolytic activity In another aspect, the present invention provides chimeric poxviruses obtained or obtainable by any embodiment of the directed evolution method described below.

[0184] Chimeric poxviruses combining nucleic acid, functional, and / or method features As explained above, the chimeric poxviruses of the invention can comprise any combination of the nucleic acids, functional features and / or method features described herein.

[0185] In particular, preferred chimeric poxviruses of the present invention are a nucleic acid sequence having a sequence identity of at least 96.6% (or any other percentage of identity disclosed in the corresponding section above) with SEQ ID NO:1; and One of the following functional features or a combination of functional features: a) high oncolytic potency as disclosed in the corresponding section above (preferably compared to the parental vaccinia virus Copenhagen strain (COP), the parental rabbitpox virus Utrecht strain (RPX) or all parental poxvirus strains); b) low viral replication in healthy cells (preferably primary cells) as disclosed in the corresponding section above (preferably compared to the parental vaccinia virus Copenhagen strain (COP) or all parental poxvirus strains); c) a high EEV-SC, a high syncytium formation ability, a high spreading ability, a low neutralization rate in the presence of poxvirus-specific antibodies and / or a low complement-mediated virus neutralization rate (preferably compared to the parental vaccinia virus Copenhagen strain (COP), the parental rabbitpox virus Utrecht strain (RPX), the vaccinia virus IHD-J strain or all parental poxvirus strains) as disclosed in the corresponding sections above; d) a) and b); e) a) and c); f) b) and c); and g) a) and b) and c) may comprise:

[0186] In particular, the chimeric poxvirus of the present invention a nucleic acid sequence having a sequence identity of at least 96.6% (or any other percentage of identity disclosed in the corresponding section above) with SEQ ID NO:1; and One of the following functional features or a combination of functional features: a) the oncolytic potential of said chimeric poxvirus is greater for at least one type of tumor than the oncolytic potential of the parental vaccinia virus Copenhagen strain (COP) or the parental rabbitpox virus Utrecht strain (RPX) measured under the same conditions and at the same time post-infection; b) for at least one type of healthy cell (preferably primary cell), the viral replication of said chimeric poxvirus in healthy cells (preferably primary cells) is lower than the viral replication of the parental vaccinia virus Copenhagen strain (COP); c) for at least one producer cell (preferably a tumor cell), the EEV-SC of said chimeric poxvirus is higher than the EEV-SC of the parent rabbitpox virus Utrecht strain (RPX) or the vaccinia virus IHD-J strain measured under the same conditions and at the same time post-infection; d) the neutralization rate of said chimeric poxvirus is lower than the neutralization rate of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection with at least one poxvirus-specific antibody and tumor; e) the complement-mediated virus neutralization rate of the chimeric poxvirus is lower than the complement-mediated virus neutralization rate of the parent vaccinia virus Copenhagen strain (COP) measured under the same conditions; f) the syncytium formation ability of the chimeric poxvirus is higher in at least one producer cell (preferably a tumor cell) than the syncytium formation ability of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection; g) a) and b); h) a) and c); i) a) and d); j) a) and e); k) a) and f); l)b) and c); m) b) and d); n)b) and e); o)b) and f); p)c) and d); q)c) and e); r)c) and f); s) a) and b) and c); t) a) and b) and d); u) a) and b) and e); v) a) and b) and f); w) a) and c) and d); x) a) and c) and f); y) b) and c) and d); z) b) and c) and f); aa) a) and b) and c) and d); bb) a) and b) and c) and e); cc) a) and b) and c) and f); and dd) any combination thereof may comprise:

[0187] For all of the above embodiments combining at least 96.6% sequence identity with SEQ ID NO: 1 (or any other percentage of identity disclosed in the corresponding section above) with one or more functional features, the poxvirus of the invention may further comprise nucleic acid sequences from at least two, at least three, at least four, at least five or all of the six parental poxvirus strains, namely, Rabbitpox virus Utrecht strain (RPX), Cowpox virus Brighton strain (CPX), Vaccinia virus Copenhagen strain (COP), Vaccinia virus Western Reserve strain (WR), Vaccinia virus Wyeth strain (WY) and Modified Vaccinia virus Ankara strain (MVA), preferably one or more specific fragments of the parental poxvirus strains defined in the section above regarding chimeric poxviruses defined by nucleic acid features. For all of the above embodiments combining nucleic acid features with one or more functional features, the poxvirus of the invention may have been obtained by one of the methods of directed evolution disclosed in the next section.

[0188] Chimeric poxvirus of strain POXSTG19503 with accession number CNCM-I-5913 comprising functional characteristics According to a particular embodiment, the chimeric poxvirus of the invention is strain POXSTG19503 with accession number CNCM-I-5913 and has one or a combination of the following functional characteristics: a) high oncolytic potency as disclosed in the corresponding section above (preferably compared to the parental vaccinia virus Copenhagen strain (COP), the parental rabbitpox virus Utrecht strain (RPX) or all parental poxvirus strains); b) low viral replication in healthy cells (preferably primary cells) as disclosed in the corresponding section above (preferably compared to the parental vaccinia virus Copenhagen strain (COP) or all parental poxvirus strains); c) a high EEV-SC, a high syncytium formation ability, a high spreading ability, a low neutralization rate in the presence of poxvirus-specific antibodies and / or a low complement-mediated virus neutralization rate (preferably compared to the parental vaccinia virus Copenhagen strain (COP), the parental rabbitpox virus Utrecht strain (RPX), the vaccinia virus IHD-J strain or all parental poxvirus strains) as disclosed in the corresponding sections above; d) a) and b); e) a) and c); f) b) and c); and g) a) and b) and c) may comprise:

[0189] In particular, a preferred chimeric poxvirus of the invention is the strain POXSTG19503, accession number CNCM-I-5913, which has one or a combination of the following functional characteristics: a) the oncolytic potential of said chimeric poxvirus is greater for at least one type of tumor than the oncolytic potential of the parental vaccinia virus Copenhagen strain (COP) or the parental rabbitpox virus Utrecht strain (RPX) measured under the same conditions and at the same time post-infection; b) for at least one type of healthy cell (preferably primary cell), the viral replication of said chimeric poxvirus in healthy cells (preferably primary cells) is lower than the viral replication of the parental vaccinia virus Copenhagen strain (COP); c) for at least one producer cell (preferably a tumor cell), the EEV-SC of said chimeric poxvirus is higher than the EEV-SC of the parent rabbitpox virus Utrecht strain (RPX) or the vaccinia virus IHD-J strain measured under the same conditions and at the same time post-infection; d) the neutralization rate of said chimeric poxvirus is lower than the neutralization rate of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection with at least one poxvirus-specific antibody and tumor; e) the complement-mediated virus neutralization rate of the chimeric poxvirus is lower than the complement-mediated virus neutralization rate of the parent vaccinia virus Copenhagen strain (COP) measured under the same conditions; f) the syncytium formation ability of the chimeric poxvirus is higher in at least one producer cell (preferably a tumor cell) than the syncytium formation ability of the parental vaccinia virus Copenhagen strain (COP) measured under the same conditions and at the same time post-infection; g) a) and b); h) a) and c); i) a) and d); j) a) and e); k) a) and f); l)b) and c); m) b) and d); n)b) and e); o)b) and f); p)c) and d); q)c) and e); r)c) and f); s) a) and b) and c); t) a) and b) and d); u) a) and b) and e); v) a) and b) and f); w) a) and c) and d); x) a) and c) and f); y) b) and c) and d); z) b) and c) and f); aa) a) and b) and c) and d); bb) a) and b) and c) and e); cc) a) and b) and c) and f); and dd) any combination thereof may comprise:

[0190] For all of the above embodiments combining one or more functional features with a chimeric poxvirus of the strain POXSTG19703 with accession number CNCM-I-5913, the poxvirus of the invention may further comprise nucleic acid sequences from at least two, at least three, at least four, at least five or all of the six parental poxvirus strains, namely, Rabbitpox virus Utrecht strain (RPX), Cowpox virus Brighton strain (CPX), Vaccinia virus Copenhagen strain (COP), Vaccinia virus Western Reserve strain (WR), Vaccinia virus Wyeth strain (WY) and Modified Vaccinia virus Ankara strain (MVA), preferably one or more specific fragments of the parental poxvirus strains defined in the section above with respect to the chimeric poxvirus defined by the strain POXSTG19503 with accession number CNCM-I-5913. For all of the above embodiments combining one or more functional features with the chimeric poxvirus of strain POXSTG19503 with accession number CNCM-I-5913, the poxvirus of the invention may further be obtained by one of the methods of directed evolution disclosed in the following sections.

[0191] A directed evolution approach to select chimeric poxviruses with high oncolytic activity In another aspect, the present invention provides a method for directed evolution to obtain chimeric poxviruses with high oncolytic potency, the method comprising: (i) infecting a first tumor cell line with multiple parental poxvirus strains, wherein the tumor cell line is permissive to each parental poxvirus strain, to obtain a first infected tumor cell line; (ii) amplifying the parental poxvirus strains on the first infected tumor cell line of step (i) for at least 12 hours (preferably at least 24 hours), and at most 3 days, to obtain one or more different chimeric poxviruses by homologous genomic recombination between at least two of the parental poxvirus strains in the supernatant; (iii) at the end of step (ii), recovering the supernatant containing one or more different chimeric poxviruses; (iv) infecting a second tumor cell line with one or more different chimeric poxviruses from the supernatant of step (iii), wherein said second tumor cell line is permissive to each of the parental poxvirus strains of steps (i) and (ii), to obtain a second infected tumor cell line; (v a ) amplifying one or more different chimeric poxviruses of step (iv) on said second infected tumor cell line of step (iv), preferably for at least 12 hours and at most 24 hours, and then recovering the supernatant; (vi) determining for at least one third tumor cell line that the oncolytic activity is higher than the oncolytic activity of at least one, preferably some, more preferably all, of the parent oncolytic poxvirus strains in the first tumor cell line of step (i) and / or the second tumor cell line of step (iv), measured under the same conditions and at the same time post-infection; a ), wherein for a given tumor, a given virus, a given condition and a given time post-infection, the oncolytic potency OP(tumor, virus, condition, time post-infection) is: OP(tumor, virus, condition, time post-infection) = (100 - percentage of surviving tumor cells after infection with virus) is defined as The compound comprises:

[0192] More specifically, the directed evolution method comprises: below, (i) infecting a first tumor cell line with multiple parental poxvirus strains, wherein the tumor cell line is permissive to each parental poxvirus strain, to obtain a first infected tumor cell line; (ii) amplifying the parental poxvirus strains on the first infected tumor cell line of step (i) for at least 12 hours (preferably at least 24 hours), and at most 3 days, to obtain one or more different chimeric poxviruses by homologous genomic recombination between at least two of the parental poxvirus strains in the supernatant; (iii') at the end of step (ii), collecting the supernatant containing one or more different chimeric poxviruses and serially diluting it 5 to 20 times to obtain at least two diluted supernatants each containing one or more chimeric poxviruses; (iv') infecting at least two samples of a second tumor cell line with one or more different chimeric poxviruses from each of the diluted supernatants of step (iii') to obtain at least two samples of an infected second tumor cell line, wherein said second tumor cell line is permissive to each of the parent poxvirus strains of steps (i) and (ii); (v' a ) amplifying the one or more different chimeric poxviruses of each of the at least two samples of the second infected tumor cell line of step (iv') on the second infected tumor cell line of step (iv'), preferably for at least 12 hours and at most 24 hours; (v' b ) collecting supernatant from a sample of a second infected tumor cell line infected with a low-dilution supernatant that shows no signs of cytopathic effect and performing 5- to 20-fold serial dilutions; (v' c ) steps (iv'), (v') until one or more different chimeric poxviruses that satisfy the selection criteria of step (vi) are obtained. a ) and (v' b ) repeating the steps; and (vi) determining whether at least one third tumor cell line has an oncolytic activity higher than the oncolytic activity of at least one, preferably some, more preferably all, of the parent oncolytic poxvirus strains in the first tumor cell line of step (i) or the second tumor cell line of step (iv'), measured under the same conditions and at the same time post-infection; c ), wherein for a given tumor, a given virus, a given condition and a given time post infection, the oncolytic potency OP(tumor, virus, condition, time post infection) is: OP(tumor, virus, condition, time post-infection) = (100 - percentage of surviving tumor cells after infection with virus) is defined as The compound comprises:

[0193] Even more particularly, the method of directed evolution comprises: (i) infecting a first tumor cell line with multiple parental poxvirus strains, wherein the tumor cell line is permissive to each parental poxvirus strain, to obtain a first infected tumor cell line; (ii) amplifying the parental poxvirus strains on the first infected tumor cell line of step (i) for at least 12 hours (preferably at least 24 hours), and at most 3 days, to obtain one or more different chimeric poxviruses by homologous genomic recombination between at least two of the parental poxvirus strains in the supernatant; (iii'' a ) at the end of step (ii), recovering both the cells and the supernatant containing one or more different chimeric poxviruses; (iii'' b ) a second tumor cell line, a infecting both the cells and the supernatant containing one or more different chimeric poxviruses of steps (i) and (ii) to obtain a second infected tumor cell line, wherein said second tumor cell line is permissive to each of the parent poxvirus strains of steps (i) and (ii); (iii'' c ) process (iii'' b ) for at least 48 hours (preferably at least 72 hours) and up to 3 days, b amplifying said second infected tumor cell line; (iii'' d ) process (iii'' c collecting the cell and supernatant fractions containing one or more different chimeric poxviruses of (iii'' e ) process (iii'' b ), (iii'' c ) and (iii'' d ) at least once; (iii'' f ) process (iii'' eAt the end of step (1), collecting the supernatant containing one or more different chimeric poxviruses and serially diluting the supernatant 5 to 20 times to obtain at least two diluted supernatants; (iv'') subjecting at least two samples of a third tumor cell line to step (iii''). f infecting each of the diluted supernatants of step (i) and (ii) with one or more different chimeric poxviruses to obtain at least two samples of a third infected tumor cell line, wherein said third tumor cell line is permissive to each of the parent poxvirus strains of steps (i) and (ii); (v'' a ) amplifying the one or more different chimeric poxviruses of each of the at least two samples of the third infected tumor cell line of step (iv'') on the third infected tumor cell line of step (iv'') for at least 12 hours and at most 24 hours; (v'' b ) collecting supernatant from a sample of a third infected tumor cell line infected with a low dilution of supernatant that shows no signs of cytopathic effect and performing 5- to 20-fold serial dilutions; (v'' c ) steps (iv''), (v'') until one or more different chimeric poxviruses that satisfy the selection criteria of step (vi) are obtained. a ) and (v'' b ) repeating the steps; and (vi) selecting at least one fourth tumor cell line having an oncolytic activity higher than that of at least one, preferably some, more preferably all of the parent oncolytic poxvirus strains in the tumor cell line of step (iv"), measured under the same conditions and at the same time post-infection; c ), wherein for a given tumor, a given virus, a given condition and a given time post infection, the oncolytic potency OP(tumor, virus, condition, time post infection) is: OP (tumor, virus, condition, time post-infection) = (100 - percentage of surviving tumor cells after infection with the virus), The compound comprises:

[0194] In the directed evolution method, different parental poxvirus strains are pooled and the blend is used to infect a first permissive tumor cell line. The resulting mixture is then passaged several times in a second permissive tumor cell line. In this general manner, this method facilitates recombination between different poxvirus strains and the generation of new chimeric poxviruses. The newly generated chimeric poxviruses are then selected for their lytic potential and, optionally, other functional characteristics.

[0195] Preferably, in the above directed evolution method, at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six, for example, 2 to 50, 3 to 40, 4 to 30, 5 to 25, 6 to 20, 6 to 19, 6 to 18, 6 to 17, or 6 to 16 different parental poxvirus strains are used to infect the permissive tumor cells in step (i).

[0196] If the number of different poxviruses used as parental poxvirus strains in the first step (i) is 16 or less, they are preferably selected from the group consisting of rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Western Reserve strain (WR), vaccinia virus Wyeth strain (WY), modified vaccinia virus Ankara strain (MVA), raccoon poxvirus Harman strain (RCN), orf virus NZ2 strain (ORF), pseudocowpox TJS strain (PCP), bovine papular stomatitis virus Illinois 721 strain (BPS), myxoma virus Lausanne strain (MYX), squirrelpox virus Kilham strain (SQF), fowlpox virus FP9 strain (FPV), swinepox virus Kasuza strain (SPV), Yaba-like disease virus Davis strain (YLD), and Chotiavirus SP An 232 strain (CTV). Preferably, the parental poxvirus strain is selected from the group consisting of 1.5 x 10 3 PFU (plaque-forming unit) ~ 1.5 x 10 6 PFU, more preferably 1 × 10 4 PFU ~ 1 × 10 6 PFU, and even more preferably 1.5 x 104 PFU~1.5×0 5 It is used in a dose of PFU.

[0197] In a preferred embodiment, the parental poxvirus strain in the first step (i) is preferably an orthopoxvirus strain selected from the group consisting of rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Western Reserve strain (WR), vaccinia virus Wyeth strain (WY), and modified vaccinia virus Ankara strain (MVA) (when six or fewer different orthopoxviruses are used as parental poxvirus strains). Preferably, the parental orthopoxvirus strain is 1.5 x 10 3 PFU ~ 1.5 × 10 6 PFU, more preferably 1 × 10 4 PFU ~ 1 × 10 6 PFU, and even more preferably 1.5 x 10 4 PFU ~ 1.5 × 10 5 It is used in a dose of PFU.

[0198] Preferably, in the above directed evolution method, the permissive tumor cell line used to generate the chimeric poxvirus of the invention is a mammalian cell, which is permissive for poxvirus infection and replication.

[0199] In the above method, all tumor cell lines are permissive for replication of the parent poxvirus strain used in step (i). The permissive tumor cell lines used as the first, second, third, and optionally fourth tumor cell lines in the above method can be the same or different, and preferably, the same tumor cell line is used in all steps of the method. Examples of permissive tumor cell lines include A549, CAL-33, HepG2, HCT116, Hela, SK-MEL-1, PANC-1, Hs746T, SK-OV-3, and CV-1. In a preferred embodiment, the first, second, third, and optionally fourth permissive tumor cell lines used to generate the chimeric poxvirus are A549.

[0200] Steps (i), (iv), (iv'), (iii'' b ) and (iv'') comprise the infection of permissive tumor cell lines with several different poxvirus strains and the amplification of said poxvirus strains. In such cases, genetic exchange, also called "shuffling", occurs between the various strains, which can lead to the generation of chimeric poxviruses. When permissive tumor cell lines are used, genetic exchange, including point mutations and recombination events, occurs frequently between different viral strains. As a result, the amplification step allows the enrichment of the resulting chimeric poxviruses with enhanced oncolytic potential in the tumor cell line used for amplification.

[0201] In a preferred embodiment, the method of directed evolution according to the present invention comprises in step (i) infecting an A549 tumor cell line with rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Western Reserve strain (WR), vaccinia virus Wyeth strain (WY), modified vaccinia virus Ankara strain (MVA), raccoon poxvirus Harman strain (RCN), orf virus NZ2 strain (ORF), pseudocowpox TJS strain (PCP), bovine papular stomatitis virus Illinois 721 strain (BPS), myxoma virus Lausanne strain (MYX), squirrelpox virus Kilham strain (SQF), fowlpox virus FP9 strain (FPV), swinepox virus Kasuza strain (SPV), Yaba-like disease virus Davis strain (YLD), and Chotiavirus SP An 232 strain (CTV). More preferably, step (i) of the method comprises injecting 1.5×10 4 PFU of rabbitpox virus Utrecht strain (RPX), 1.5 × 10 4 PFU of cowpox virus Brighton strain (CPX), 1.5 × 10 4 PFU of vaccinia virus Copenhagen strain (COP), 1.5 × 10 4 PFU of vaccinia virus Western Reserve strain (WR), 1.5 × 10 4 PFU of vaccinia virus Wyeth strain (WY), 1.5 × 10 4PFU of modified vaccinia virus Ankara strain (MVA), 1.5 × 10 4 PFU of raccoon poxvirus Harman strain (RCN), 1.5 × 10 4 PFU of orf virus strain NZ2 (ORF), 1.5 × 10 4 PFU of pseudocowpox TJS strain (PCP), 1.5 × 10 4 PFU of bovine papular stomatitis virus Illinois 721 strain (BPS), 1.5 × 10 4 PFU of myxoma virus Lausanne strain (MYX), 1.5 × 10 4 PFU of squirrelpox virus Kilham strain (SQF), 1.5 × 10 4 PFU of fowlpox virus FP9 strain (FPV), 1.5 × 10 4 PFU of swinepox virus Kasuza strain (SPV), 1.5 × 10 4 PFU of Yaba-like disease virus Davis strain (YLD), and 1.5 × 10 4 The method includes infecting the cells with PFU of Chothiavirus SP An 232 strain (CTV). The above embodiment corresponds to the method used in the Examples for the chimeric poxvirus POXSTG19503 produced.

[0202] However, in the case of POXSTG19503, only the orthopoxvirus parent strains were recombined with each other. As a result, only the orthopoxvirus parent strains are usable. Thus, in another preferred embodiment, the directed evolution method comprises, in step (i), infecting an A549 tumor cell line with rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Western Reserve strain (WR), vaccinia virus Wyeth strain (WY), and modified vaccinia virus Ankara strain (MVA), and in step (iii), infecting the A549 tumor cell line with one or more different chimeric poxviruses. More preferably, step (i) of the method comprises infecting the A549 tumor cell line with 1.5 x 10 4 PFU of rabbitpox virus Utrecht strain (RPX), 1.5 × 10 4 PFU of cowpox virus Brighton strain (CPX), 1.5 × 10 4PFU of vaccinia virus Copenhagen strain (COP), 1.5 × 10 4 PFU of vaccinia virus Western Reserve strain (WR), 1.5 × 10 4 PFU of vaccinia virus Wyeth strain (WY) and 1.5 × 10 4 The study involved infection with PFU of modified vaccinia virus Ankara strain (MVA).

[0203] Steps (iii), (v) a ), (iii'), (v' b ), (v' c ), (iii'' a ), (iii'' d ), (iii'' f ), (v'' b ) and (v'' c Step (v) comprises the recovery of the supernatant comprising one or more oncolytic chimeric poxviruses. a ), (v' b ), (v' c ), (v'' b ) and (v'' c In step (v), the supernatant is harvested 12 to 24 hours post-infection. At this time post-infection, the tumor-permissive cell line has generally not yet been lysed by the one or more chimeric oncolytic poxviruses, and therefore the one or more chimeric oncolytic poxviruses present in the harvested supernatant should primarily comprise EEV particles. a ), (v' b ), (v' c ), (v'' b ) and (v'' c The specific collection of supernatants at 12 to 24 hours after infection with ) ensures that not only highly oncolytic chimeric poxviruses are selected, but also chimeric poxviruses with high EEV-SC (high EEV-SC correlates with high diffusion capacity and low neutralization rate, resulting in high diffusion capacity and low neutralization rate). Therefore, step (v') c ) or (v'' cSimilarly, by repeating the process (infection with the supernatant, amplification for 12–24 hours, and collection of the supernatant) several times, highly oncolytic chimeric poxviruses with high EEV-SC (and consequently high spreading ability and low neutralization rate) are enriched.

[0204] Steps (iii'), (v' b ), (v' c ), (iii'' f ), (v'' b ) and (v'' c In step (a), the collected supernatant is serially diluted 5 to 20 times, i.e., a portion of the supernatant is diluted 5 to 20 times, and this portion of the 5 to 20-fold diluted supernatant is then diluted 5 to 20 times to obtain a supernatant diluted 25 to 400 times. The serial dilution value is preferably 1 to 5, more preferably 2 to 4, for example, 2, 3, and 4. Preferably, the dilution factor between each serial dilution is 6 to 18 times, more preferably 7 to 15 times, and more preferably 8 to 12 times. In particular, 10-fold serial dilutions can be used. Preferably, the dilution factor between the undiluted supernatant and the most diluted supernatant in the serial dilutions is 100 to 10,000, preferably 200 to 8,000, 300 to 6,000, 400 to 4,000, 500 to 2,000, or 750 to 1,500.

[0205] In one embodiment, step (iii'' e ) is repeated 1 to 30 times, preferably 1 to 25 times, more preferably 1 to 20 times, more preferably 1 to 15 times, more preferably 1 to 10 times, more preferably 1 to 5 times, and even more preferably 1 to 3 times.

[0206] In one embodiment, step (v' c ) and (v'' c ) is repeated 1 to 200 times, preferably 2 to 150 times, more preferably 3 to 100 times, more preferably 4 to 50 times, more preferably 5 to 40 times, more preferably 6 to 30 times, more preferably 6 to 20 times, more preferably 7 to 15 times, and more preferably 8 to 12 times.

[0207] In any one of the above directed evolution methods, the selection of one or more different chimeric poxviruses in step (vi) may comprise obtaining a viral clone. In a preferred embodiment, the obtaining of said viral clone may be the result of dilution, isolation, and amplification of one or more different chimeric poxviruses in step (v). Alternatively, or in combination, the selection in step (vi) may comprise testing the oncolytic ability of the different chimeric poxviruses in step (v) on one or more tumor cell lines.

[0208] In one embodiment, the method of directed evolution comprises steps (i), (ii), (iii), (iii'), and (iii'') to increase genetic diversity. a ), (iii'' b ), (iii'' c ), (iii'' d ), (iii'' e ), (iii'' f ), (iv), (iv'), (iv''), (v), (v' a ), (v' b ), (v' c ), (v'' a ), (v'' b ), (v'' c The method further comprises the use of at least one mutagen in one or more of (i) and (ii). For example, the mutagen may be selected from a physical agent, a chemical agent, and a biological agent. More preferably, the physical agent is selected from the group consisting of ultraviolet light, ionizing radiation, and radioactive decay; the chemical agent is selected from the group consisting of urea, nitrosourea, reactive oxygen species, deaminating agents, polycyclic aromatic hydrocarbons, alkylating agents, aromatic amines, alkaloids, bromine, sodium azide, and benzene; and the biological agent is selected from the group consisting of DNA base analogs and transposons. Other physical, chemical, and biological mutagens known to those skilled in the art can be used in the context of the present invention.

[0209] Mutant chimeric poxvirus Various oncolytic poxvirus mutants with improved properties (in particular lower replication in healthy cells, preferably primary cells, and therefore higher therapeutic index) are known in the art; therefore, mutants of the chimeric poxviruses of the present invention with similar changes in one or more viral genes may be advantageous for use in the treatment of proliferative diseases such as cancer.

[0210] Thus, in another aspect, the present invention relates to mutant chimeric poxviruses, ie chimeric poxviruses according to the invention which have been modified by altering one or more poxvirus genes.

[0211] In one embodiment, the modification of the mutant chimeric poxvirus of the present invention preferably results in the synthesis (or lack of synthesis) of a defective protein that cannot ensure the activity of a protein produced by an unmodified gene under normal conditions. Exemplary modifications have been disclosed in the literature for the purpose of altering viral genes involved in DNA metabolism, host pathogenicity, and the IFN pathway (e.g., Guse et al., 2011, Expert Opinion Biol. Ther. 11(5): 595-608). Modifications for altering viral gene loci include deletion, mutation, and / or substitution of one or more nucleotides (contiguous or noncontiguous) within a viral gene or its regulatory element. Modifications can be performed using conventional techniques and by many methods known to those skilled in the art.

[0212] In the context of the present invention, mutant chimeric poxviruses can be deficient at a specific locus by many methods, including the substitution, deletion, and / or insertion of one or more nucleotides present at this locus. For example, the insertion of a polynucleotide at a locus can disrupt the open reading frame (ORF) encoded by the nucleic acid sequence at the locus. Partial or complete deletion of a specific locus is also suitable for creating mutant chimeric poxviruses that are deficient at a specific locus.

[0213] Mutant chimeric poxviruses may in particular be partially or completely defective in one or more specific genetic loci.

[0214] Mutant chimeric poxviruses with deletions at the J2R locus Preferably, the mutant chimeric poxvirus is defective in the J2R locus, where the thymidine kinase (TK)-encoding gene (similar to the J2R gene of COP) is located. The term "defective in the J2R locus" means that the chimeric poxvirus of the present invention encodes a non-functional thymidine kinase enzyme or does not encode any thymidine kinase enzyme. Preferably, the chimeric poxvirus of the present invention does not encode any thymidine kinase enzyme. The TK enzyme is involved in the synthesis of deoxyribonucleotides. TK is required for viral replication in healthy cells (preferably primary cells) because these cells generally have low nucleotide concentrations, but not in dividing cells (e.g., tumor cells) where nucleotide concentrations are high. Therefore, altering the expression (e.g., deletion of the J2R locus) or function of the TK enzyme can improve the tumor selectivity of the chimeric poxvirus by reducing replication of the chimeric poxvirus in non-tumor cells. In a preferred embodiment, deletion of the TK-encoding gene has no or only a minor effect on the lytic activity of the virus. As a result, mutant chimeric poxviruses with a deletion in the J2R locus may replicate less well in healthy cells (preferably primary cells) and therefore have an improved therapeutic index.

[0215] In this case, the mutant chimeric poxvirus preferably has 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%, at least 99.9%, at least 99.91%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99%, or even 100% identity to SEQ ID NO: 8. SEQ ID NO: 8 is the sequence of the core region and one of the ITRs of POXSTG19508, which corresponds to POXSTG19503 except for the insertion of the GFP::FCU1 sequence at the J2R locus.

[0216] In a further embodiment, the mutant chimeric poxvirus is strain POXSTG19503, accession number CNCM-I-5913, which is deleted in the J2R locus.

[0217] Preferably, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus according to the present invention for at least one type of tumor is higher than that of at least one of the mutant oncolytic parent poxvirus strains having a deletion in the J2R locus, i.e., rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), and vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post-infection. Preferably, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus for at least one type of tumor is higher than that of the mutant parent vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus or the mutant parent rabbitpox virus Utrecht strain (RPX) having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. More preferably, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus for at least one type of tumor is higher than that of at least two of the mutant oncolytic parent poxvirus strains, all of which are deleted in the J2R locus, i.e., rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), and vaccinia virus Western Reserve strain (WR), when measured under the same conditions and at the same time post-infection. For example, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus for at least one type of tumor is higher than that of the mutant parent vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus and the mutant parent rabbitpox virus Utrecht strain (RPX) having a deletion in the J2R locus, when measured under the same conditions and at the same time post-infection.In a more preferred embodiment, for at least one type of tumor, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is higher than the oncolytic activity of at least three, more preferably at least four, and even more preferably all five of the mutant parent oncolytic poxvirus strains, all of which have deletions in the J2R locus, namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post-infection.

[0218] For a given tumor and virus, oncolytic potency is generally determined by injecting the virus into tumor cells at an MOI of 10. -5 ~10 -2 The β-amyloid level can be determined in vitro 3 to 5 days after infection with β-amyloid.

[0219] In a specific aspect of this embodiment, the oncolytic potency of the mutant chimeric poxvirus having a deletion in the J2R locus is greater than the oncolytic potency of at least one of the mutant oncolytic parent poxvirus strains having a deletion in the J2R locus, i.e., rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), and vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post-infection in at least one tumor cell line selected from A549, HCT116, and HepG2. Preferably, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus against at least one tumor selected from A549, HCT116, and HepG2 is higher than the oncolytic activity of the mutant parental vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus or the mutant parental rabbitpox virus Utrecht strain (RPX) having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. More preferably, for at least one tumor cell line selected from A549, HCT116, and HepG2, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is higher than the oncolytic activity of at least two of the mutant oncolytic parent poxvirus strains, all of which have deletions in the J2R locus, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), and vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post-infection. For example, for at least one tumor cell line selected from A549, HCT116, and HepG2, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is higher than the oncolytic activity of the mutant parent vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus and the mutant parent rabbitpox virus Utrecht strain (RPX) having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection.In a more preferred embodiment, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus for at least one tumor cell line selected from A549, HCT116 and HepG2 is higher than the oncolytic activity of at least three, more preferably at least four, and even more preferably all five of the mutant parent oncolytic poxvirus strains, all of which have deletions in the J2R locus, namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post-infection.

[0220] Preferably, in this embodiment, the oncolytic potency is at an MOI of 10. -5 ~10 -4 , preferably 10 -5 or 10 -4 More preferably, in this embodiment, tumor cell lines are cultured at 37°C, 5% CO2, in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS).

[0221] In a more detailed aspect of this embodiment, a) A549 MOI 10 -5In the present invention, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 39% different from that of at least one of the mutant oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection. Preferably, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 39% different from that of the mutant parental COP having a deletion in the J2R locus or the mutant parental RPX having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. More preferably, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 39% different from that of at least two of the mutant oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection. For example, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 39% different from that of a mutant parental COP having a deletion in the J2R locus and a mutant parental RPX having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. In a more preferred embodiment, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 39% different from that of at least three, more preferably at least four, and even more preferably all five of the mutant parental oncolytic poxvirus strains RPX, CPX, COP, WY, and WR, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection; and / or b) A549 MOI 10 -4In the present invention, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 13% different from that of at least one of the mutant oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection. Preferably, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 20% different from that of the mutant parental COP having a deletion in the J2R locus or the mutant parental RPX having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. More preferably, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 13% different from that of at least two of the mutant oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection. For example, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 20% different from that of the mutant parental COP having a deletion in the J2R locus and the mutant parental RPX having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. In a more preferred embodiment, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 13% different from that of at least three, more preferably at least four, and even more preferably all five of the mutant parental oncolytic poxvirus strains RPX, CPX, COP, WY, and WR, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection; and / or c) HCT116 at an MOI of 10 -5In the present invention, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 80% different from that of at least one of the mutant oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, all of which are deleted in the J2R locus. Preferably, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 80% different from that of the mutant parental COP having a deletion in the J2R locus or the mutant parental RPX having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. More preferably, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 80% different from that of at least two of the mutant oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection. For example, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 80% different from that of a mutant parental COP having a deletion in the J2R locus and a mutant parental RPX having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. In a more preferred embodiment, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 80% different from that of at least three, more preferably at least four, and even more preferably all five of the mutant parental oncolytic poxvirus strains RPX, CPX, COP, WY, and WR, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection; and / or d) HCT116 at an MOI of 10 -4In the present invention, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 33% different from that of at least one of the mutant oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection. Preferably, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 33% different from that of the mutant parental COP having a deletion in the J2R locus or the mutant parental RPX having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. More preferably, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 33% different from that of at least two of the mutant oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection. For example, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 33% different from that of a mutant parental COP having a deletion in the J2R locus and a mutant parental RPX having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. In a more preferred embodiment, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 33% different from that of at least three, more preferably at least four, and even more preferably all five of the mutant parental oncolytic poxvirus strains RPX, CPX, COP, WY, and WR, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection; and / or e) HepG2, MOI 10 -5In the present invention, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 66% different from that of at least one of the mutant oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection. Preferably, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 66% different from that of the mutant parental COP having a deletion in the J2R locus or the mutant parental RPX having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. More preferably, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 66% different from that of at least two of the mutant oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection. For example, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 66% different from that of a mutant parental COP having a deletion in the J2R locus and a mutant parental RPX having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. In a more preferred embodiment, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 66% different from that of at least three, more preferably at least four, and even more preferably all five of the mutant parental oncolytic poxvirus strains RPX, CPX, COP, WY, and WR, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection; and / or f) HepG2, MOI 10 -4In the present invention, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 29% different from that of at least one of the mutant oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection. Preferably, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 29% different from that of the mutant parental COP having a deletion in the J2R locus or the mutant parental RPX having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. More preferably, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 29% different from that of at least two of the mutant oncolytic parental poxvirus strains RPX, CPX, COP, WY, and WR, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection. For example, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 29% different from that of the mutant parental COP having a deletion in the J2R locus and the mutant parental RPX having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. In a more preferred embodiment, the oncolytic activity of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 29% different from that of at least three, more preferably at least four, and even more preferably all five of the mutant parental oncolytic poxvirus strains RPX, CPX, COP, WY, and WR, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection.

[0222] Preferably, in this embodiment, oncolytic potency is assessed 4 days post-infection.

[0223] Alternatively, or in combination, for at least one type of healthy cell (preferably primary cell), the viral replication of the mutant chimeric poxvirus defective in the J2R locus in the healthy cell (preferably primary cell) is lower than the viral replication of at least one of five mutant oncolytic parental poxvirus strains, all of which are defective in the J2R locus, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), and vaccinia virus Western Reserve strain (WR). Preferably, for at least one type of healthy cell (preferably primary cell), the viral replication of the mutant chimeric poxvirus defective in the J2R locus in the healthy cell (preferably primary cell) is lower than the viral replication of the mutant parental vaccinia virus Copenhagen strain (COP) defective in the J2R locus. In a preferred embodiment, for at least one type of healthy cell (preferably a primary cell), the viral replication of the mutant chimeric poxvirus defective in the J2R locus in the healthy cell (preferably a primary cell) is lower than the viral replication of at least two, more preferably at least three, more preferably at least four, and even more preferably all five of the mutant oncolytic parental poxvirus strains, all of which are defective in the J2R locus, namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR).

[0224] For a given tumor or healthy cell (preferably primary cell), viral replication is generally achieved by injecting the virus into the tumor or healthy cell (preferably primary cell) at an MOI of 10. -5 ~10 -2 The β-amyloid level can be determined in vitro 2 to 8 days after infection with β-amyloid.

[0225] In a specific aspect of this embodiment, the viral replication of the mutant chimeric poxvirus defective in the J2R locus in healthy cells (preferably primary cells) selected from skin cells and hepatocytes is lower than the viral replication of at least one of five mutant oncolytic parental poxvirus strains, all of which are defective in the J2R locus, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), and vaccinia virus Western Reserve strain (WR). Preferably, for at least one healthy cell (preferably primary cell) selected from skin cells and hepatocytes, the viral replication of the mutant chimeric poxvirus defective in the J2R locus in said healthy cell (preferably primary cell) selected from skin cells and hepatocytes is lower than the viral replication of the mutant parental vaccinia virus Copenhagen strain (COP) defective in the J2R locus. In a preferred embodiment, in at least one healthy cell (preferably primary cell) selected from skin cells and liver cells, the viral replication of a mutant chimeric poxvirus deleted in the J2R locus in said healthy cell (preferably primary cell) selected from skin cells and liver cells is lower than the viral replication of at least two, preferably at least three, more preferably at least four, and even more preferably all five of the mutant oncolytic parent poxvirus strains all deleted in the J2R locus, namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR).

[0226] In a more specific embodiment of this aspect, viral replication of said mutant chimeric poxvirus having a deletion in the J2R locus is a) in at least one healthy cell line (preferably a primary cell line) selected from skin cells and liver cells, the viral replication is at least 1.6-fold lower than that of at least one of the five mutant oncolytic parental poxvirus strains RPX, CPX, COP, WY and WR, all of which are defective in the J2R locus, preferably lower than that of the mutant oncolytic parental COP, all of which are defective in the J2R locus, more preferably lower than that of at least two, more preferably lower than that of at least three, more preferably lower than that of at least four, and even more preferably lower than that of each of the five mutant oncolytic parental poxvirus strains RPX, CPX, COP, WY and WR, all of which are defective in the J2R locus; and / or b) in hepatocytes, the viral replication is at least 4.5 times lower than that of at least one of the five mutant oncolytic parental poxvirus strains RPX, CPX, COP, WY and WR, all of which are defective in the J2R locus, preferably lower than that of the mutant oncolytic parental COP, all of which are defective in the J2R locus, more preferably lower than that of at least two, more preferably lower than that of at least three, more preferably lower than that of at least four, and even more preferably lower than that of each of the five mutant oncolytic parental poxvirus strains RPX, CPX, COP, WY and WR, all of which are defective in the J2R locus;

[0227] Preferably, in this embodiment, viral replication is at an MOI of 10 in HepG2. -4 at 3 days post-infection in the human skin model or at 10 5 PFU are assessed in vitro 7 days after infection. More preferably, in this embodiment, tumor cell lines are cultured at 37°C, 5% CO2, in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS).

[0228] Alternatively or in combination, the therapeutic index of the mutant chimeric poxvirus having a deletion in the J2R locus for at least one organ is higher than the therapeutic index of at least one of the mutant parent oncolytic poxvirus strains, all of which are deleted in the J2R locus, i.e., rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), and vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post-infection. Preferably, the therapeutic index of the mutant chimeric poxvirus having a deletion in the J2R locus for at least one organ is higher than the therapeutic index of the mutant parent vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. In a more preferred embodiment, the therapeutic index of the mutant chimeric poxvirus having a deletion in the J2R locus for at least one organ is higher than the therapeutic index of at least two, more preferably at least three, more preferably at least four, and even more preferably all five of the mutant parent oncolytic poxvirus strains having deletions in the J2R locus, namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR), all measured under the same conditions and at the same time post-infection.

[0229] For a given organ and virus, the therapeutic index is generally determined by injecting the virus into tumor or healthy cells (preferably primary cells) at an MOI of 10. -5 ~10 -2 The β-amyloid level can be determined in vitro 2 to 8 days after infection with β-amyloid.

[0230] In a specific aspect of this embodiment, the hepatic therapeutic index of the mutant chimeric poxvirus having a deletion in the J2R locus is higher than the hepatic therapeutic index of at least one of the mutant parent oncolytic poxvirus strains, all of which have deletions in the J2R locus, i.e., rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), and vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post-infection. Preferably, the hepatic therapeutic index of the mutant chimeric poxvirus having a deletion in the J2R locus is higher than the hepatic therapeutic index of the mutant parent vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. In a more preferred embodiment, the hepatic therapeutic index of the mutant chimeric poxvirus having a deletion in the J2R locus is higher than the hepatic therapeutic index of at least two, more preferably at least three, more preferably at least four, and even more preferably all five of the mutant parent oncolytic poxvirus strains having deletions in the J2R locus, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post-infection.

[0231] Preferably, the hepatic therapeutic index of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 9 times higher than that of at least one of the mutant parent oncolytic poxvirus strains, all of which are deleted in the J2R locus, i.e., rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), and vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post-infection. Preferably, the hepatic therapeutic index of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 9 times higher than that of the mutant parent vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. In a more preferred embodiment, the hepatic therapeutic index of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 9 times higher than the hepatic therapeutic index of at least two, preferably at least three, more preferably at least four, and even more preferably all five of the mutant parent oncolytic poxvirus strains having deletions in the J2R locus, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR), all of which are measured under the same conditions and at the same time post-infection.

[0232] More preferably, in this embodiment, the hepatic therapeutic index is 10 or less when the mutant chimeric poxvirus is administered to HepG2 tumor cells at an MOI of 10 or less. -5 and healthy hepatocytes (preferably primary hepatocytes) at an MOI of 10. -4and the replication of the mutant chimeric poxvirus in HepG2 tumor cells and healthy hepatocytes (preferably primary hepatocytes) is measured in vitro 3 days after infection. Even more preferably, in this embodiment, the HepG2 tumor cell line is cultured at 37°C, 5% CO2, in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS), and the healthy hepatocytes are cultured at 37°C, 5% CO2, in basal hepatocyte medium (see BIOPREDICS catalog MIL600) and hepatocyte culture medium additive (see BIOPREDICS catalog ADD222C). Optionally, the mutant chimeric poxvirus with a deletion in the J2R locus has a hepatic therapeutic index higher than the corresponding hepatic therapeutic index of a wild-type chimeric poxvirus without a deletion in the J2R locus.

[0233] Alternatively or in combination, for at least one producer cell (preferably a tumor cell), the EEV-SC of the mutant chimeric poxvirus defective in the J2R locus is higher than the EEV-SC of at least one of the mutant parental poxvirus strains, all of which are defective in the J2R locus, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara strain (MVA), measured under the same conditions and at the same time post-infection. Preferably, for at least one type of producer cell (preferably a tumor cell), the EEV-SC of the mutant chimeric poxvirus having a deletion in the J2R locus is higher than the EEV-SC of at least one of the mutant parental COP having a deletion in the J2R locus or the mutant parental rabbitpoxvirus Utrecht strain (RPX) having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. In a preferred embodiment, for at least one producer cell (preferably a tumor cell), the EEV-SC of the mutant chimeric poxvirus having a deletion in the J2R locus is higher than the EEV-SC of at least two of the mutant parental poxvirus strains, all of which have deletions in the J2R locus, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR), and modified vaccinia virus Ankara strain (MVA), measured under the same conditions and at the same time post-infection. For example, for at least one type of producer cell (preferably a tumor cell), the EEV-SC of the mutant chimeric poxvirus having a deletion in the J2R locus is higher than the EEV-SC of at least one of the mutant parental vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus and the mutant parental rabbitpox virus Utrecht strain (RPX) having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection.In a more preferred embodiment, for at least one type of producer cell (preferably a tumor cell), the EEV-SC of the mutant chimeric poxvirus having a deletion in the J2R locus is higher than the EEV-SC of at least three, preferably at least four, more preferably at least five, and even more preferably all six of the mutant parental poxvirus strains, all of which have deletions in the J2R locus, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara strain (MVA), measured under the same conditions and at the same time post-infection.

[0234] For a given tumor and virus, EEV-SC generally injects the virus into tumor cells at an MOI of 10. -4 ~10 -1 The β-amyloid level can be determined in vitro 16 to 24 hours after infection with β-amyloid.

[0235] In a specific aspect of this embodiment, the EEV-SC of the mutant chimeric poxvirus having a deletion in the J2R locus in the A549 tumor cell line is higher than the EEV-SC of at least one of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which have deletions in the J2R locus, measured under the same conditions and at the same time post-infection. Preferably, the EEV-SC of the mutant chimeric poxvirus having a deletion in the J2R locus in the A549 tumor cell line is higher than the EEV-SC of at least one of the mutant parental COP having a deletion in the J2R locus or the mutant parental RPX having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. In a preferred embodiment, the EEV-SC of the mutant chimeric poxvirus having a deletion in the J2R locus in the A549 tumor cell line is higher than the EEV-SC of at least two of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which have deletions in the J2R locus, measured under the same conditions and at the same time post-infection. For example, the EEV-SC of the mutant chimeric poxvirus having a deletion in the J2R locus in the A549 tumor cell line is higher than the EEV-SC of at least one of the mutant parental COP having a deletion in the J2R locus and the mutant parental RPX having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. In a more preferred embodiment, in the A549 tumor cell line, the EEV-SC of the mutant chimeric poxvirus having a deletion in the J2R locus is higher than the EEV-SC of at least three, preferably at least four, more preferably at least five, and even more preferably all six of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which have deletions in the J2R locus, measured under the same conditions and at the same time post-infection.

[0236] Preferably, the mutant chimeric poxvirus EEV-SC, which is defective in the J2R locus, comprises: a) higher than EEV-SC of at least one of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR and MVA, all of which are deleted in the J2R locus, measured 16 hours after injection in the A549 tumor cell line under the same conditions and at the same time post-infection, preferably higher than EEV-SC of at least one of the mutant parental COP strains deleted in the J2R locus or the mutant parental RPX strains deleted in the J2R locus, more preferably higher than EEV-SC of all of the mutant parental poxvirus strains RPX, CPX, COP, WY and WR, all of which are deleted in the J2R locus. and MVA, even more preferably than EEV-SC of one of the mutant parental COP and mutant parental RPX, all of which are deleted in the J2R locus, and even more preferably than EEV-SC of at least three, preferably at least four, more preferably at least five, and even more preferably all six of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are deleted in the J2R locus; and / or b) at least one EEV-SC of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR and MVA, all of which are deleted in the J2R locus, measured 24 hours after injection in the A549 tumor cell line under the same conditions and at the same time post-infection, preferably at least one EEV-SC of the mutant parental COP, all of which are deleted in the J2R locus, or at least one EEV-SC of the mutant parental RPX, all of which are deleted in the J2R locus, more preferably at least one EEV-SC of the mutant parental COP, all of which are deleted in the J2R locus, or at least one EEV-SC of the mutant parental RPX, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection, It is at least three times higher than EEV-SC of at least two of WY, WR and MVA, even more preferably than EEV-SC of one of the mutant parental COP having a defect in the J2R locus and the mutant parental RPX having a defect in the J2R locus, and even more preferably than EEV-SC of at least three, preferably at least four, more preferably at least five, and even more preferably all six of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR and MVA, all of which have defects in the J2R locus.

[0237] For at least one type of producer cell (preferably a tumor cell), the EEV-SC of the mutant chimeric poxvirus having a deletion in the J2R locus may further be higher than the EEV-SC of the vaccinia virus IHD-J strain measured under the same conditions and at the same time post-infection.

[0238] Preferably, the mutant chimeric poxvirus EEV-SC, which is deleted in the J2R locus, comprises at least: a) 2-fold higher than EEV-SC of the vaccinia virus IHD-J strain measured under the same conditions and at the same time post-infection in the A549 tumor cell line 16 hours after injection; and / or b) 24 hours after injection in the A549 tumor cell line, 3 times higher than EEV-SC of the vaccinia virus IHD-J strain measured under the same conditions and at the same time post-infection.

[0239] In a more specific embodiment of this aspect, the mutant chimeric poxvirus EEV-SC, which is defective in the J2R locus, comprises: a) A549 tumor cell line, MOI 10 -2 at least 4%, preferably at least 5%, more preferably at least 6% 16 hours after infection with the mutant chimeric poxvirus containing a deletion in the J2R locus at an MOI of 1 to 1, preferably 0.1; and / or b) A549 tumor cell line, MOI 10 -2 At least 5%, at least 6%, at least 7%, at least 8%, preferably at least 9%, more preferably at least 10%, and even more preferably at least 11% 24 hours after infection with the mutant chimeric poxvirus having a deletion in the J2R locus at an MOI of 1 or less, preferably 0.1.

[0240] In a more specific embodiment of this aspect, the mutant chimeric poxvirus EEV-SC, which is defective in the J2R locus, comprises: a) A549 tumor cell line, MOI 10 -24% to 9%, more preferably 6% to 8%, 16 hours after infection with the mutant chimeric poxvirus having a deletion in the J2R locus at an MOI of 1 to 1, preferably 0.1; and / or b) A549 tumor cell line, MOI 10 -2 The cell viability is 5% to 15%, more preferably 10% to 13%, 24 hours after infection with the mutant chimeric poxvirus having a deletion in the J2R locus at an MOI of 1 to 1, preferably 0.1.

[0241] Alternatively or in combination, for at least one tumor, the spreading capacity of the mutant chimeric poxvirus having a deletion in the J2R locus is higher than the spreading capacity of at least one of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection. Preferably, for at least one tumor, the spreading capacity of the mutant chimeric poxvirus having a deletion in the J2R locus is higher than the spreading capacity of the mutant parental COP having a deletion in the J2R locus or the mutant parental RPX having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. More preferably, for at least one tumor, the spreading capacity of the mutant chimeric poxvirus having a deletion in the J2R locus is higher than the spreading capacity of at least two of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection. For example, for at least one type of tumor, the spreading ability of the mutant chimeric poxvirus defective in the J2R locus is higher than that of the mutant parental COP defective in the J2R locus and the mutant parental RPX defective in the J2R locus, measured under the same conditions and at the same time post-infection. In a preferred embodiment, for at least one type of tumor, the spreading ability of the mutant chimeric poxvirus defective in the J2R locus is higher than that of at least three, preferably at least four, more preferably at least five, and even more preferably all six of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are defective in the J2R locus, measured under the same conditions and at the same time post-infection.

[0242] Alternatively or in combination, the neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus with respect to at least one poxvirus-specific antibody and tumor is lower than the neutralization rate of at least one of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which have a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. Preferably, the neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus with respect to at least one poxvirus-specific antibody and tumor is lower than the neutralization rate of the mutant parental COP having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection. More preferably, for at least one poxvirus-specific antibody and tumor, the neutralization rate of the mutant chimeric poxvirus defective in the J2R locus is lower than the neutralization rate of at least two, preferably at least three, more preferably at least four, even more preferably at least five, and even more preferably all six of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are defective in the J2R locus, measured under the same conditions and at the same time post-infection.

[0243] For a given tumor and virus, virus neutralization rates are generally determined by injecting the virus into tumor cells at an MOI of 3 x 10 -5 It can be determined in vitro 3-5 days after infection with .

[0244] In this embodiment, the neutralization rate of the mutant chimeric poxvirus defective in the J2R locus with respect to the at least one vaccinia virus-specific antibody and HCT116 is lower than the neutralization rate of at least one of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are defective in the J2R locus, measured under the same conditions and at the same time post-infection. Preferably, the neutralization rate of the mutant chimeric poxvirus defective in the J2R locus with respect to the at least one vaccinia virus-specific antibody and HCT116 is lower than the neutralization rate of the mutant parental COP defective in the J2R locus, measured under the same conditions and at the same time post-infection. More preferably, for at least one vaccinia virus-specific antibody and HCT116, the neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is lower than the neutralization rate of at least two, preferably at least three, more preferably at least four, even more preferably at least five, and even more preferably all six of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which have deletions in the J2R locus, measured under the same conditions and at the same time post-infection.

[0245] In a specific aspect of this embodiment, with at least one vaccinia virus-specific antibody and HCT116, the neutralization rate of the mutant chimeric poxvirus, all of which are deleted in the J2R locus, is at least 60-fold, more preferably at least 70-fold, lower than the neutralization rate of at least one of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are deleted in the J2R locus, preferably the neutralization rate of the mutant parental COP, all of which are deleted in the J2R locus, and more preferably the neutralization rate of at least two, preferably at least three, more preferably at least four, even more preferably at least five, and even more preferably all six of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection.

[0246] In a more specific aspect of this embodiment, with at least one vaccinia virus-specific antibody and HCT116, the neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is 30 to 100 times lower, even more preferably 40 to 90 times lower, and even more preferably 55 to 75 times lower than the neutralization rate of at least one of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection, preferably the neutralization rate of the mutant parental COP, all of which are deleted in the J2R locus, and more preferably the neutralization rate of at least two, preferably at least three, more preferably at least four, even more preferably at least five, and even more preferably all six of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are deleted in the J2R locus.

[0247] Alternatively or in combination, the complement-mediated virus neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is lower than the complement-mediated virus neutralization rate of at least one of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are deleted in the J2R locus, measured under the same conditions. Preferably, the complement-mediated virus neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is lower than the complement-mediated virus neutralization rate of the mutant parental COP, all of which are deleted in the J2R locus, measured under the same conditions. More preferably, the complement-mediated virus neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is lower than the complement-mediated virus neutralization rate of at least two, preferably at least three, more preferably at least four, even more preferably at least five, and even more preferably all six of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are deleted in the J2R locus, measured under the same conditions.

[0248] For a given virus, complement-mediated virus neutralization rates are generally higher than those observed in the presence of activated or heat-inactivated serum at a dose of 10 4 ~10 8PFU / mL of virus can be determined in vitro.

[0249] In a specific aspect of this embodiment, the complement-mediated virus neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is at least 5-fold, more preferably at least 6-fold, and even more preferably at least 6-8-fold lower than the complement-mediated virus neutralization rate of at least one of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are deleted in the J2R locus, measured under the same conditions, preferably the complement-mediated virus neutralization rate of the mutant parental COP, all of which are deleted in the J2R locus, and more preferably the complement-mediated virus neutralization rate of at least two, preferably at least three, more preferably at least four, even more preferably at least five, and even more preferably all six of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are deleted in the J2R locus.

[0250] In a more specific aspect of this embodiment, the complement-mediated virus neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is 5 to 10 times, and even more preferably 6 to 8 times, lower than the complement-mediated virus neutralization rate of at least one of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are deleted in the J2R locus, measured under the same conditions, preferably lower than the complement-mediated virus neutralization rate of the mutant parental COP, all of which are deleted in the J2R locus, and more preferably lower than the complement-mediated virus neutralization rate of at least two, preferably at least three, more preferably at least four, even more preferably at least five, and even more preferably all six of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are deleted in the J2R locus.

[0251] Alternatively or in combination, the syncytium formation ability of the mutant chimeric poxvirus defective in the J2R locus is higher for at least one producer cell (preferably a tumor cell) than the syncytium formation ability of at least one of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are defective in the J2R locus, measured under the same conditions and at the same time post-infection. Preferably, the syncytium formation ability of the mutant chimeric poxvirus defective in the J2R locus is higher for at least one producer cell (preferably a tumor cell) than the syncytium formation ability of the mutant parental COP defective in the J2R locus, measured under the same conditions and at the same time post-infection. More preferably, the syncytium formation ability of the mutant chimeric poxvirus defective in the J2R locus with respect to at least one type of producer cell (preferably a tumor cell) is higher than the syncytium formation ability of at least two of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are defective in the J2R locus, measured under the same conditions and at the same time post-infection. For example, the syncytium formation ability of the mutant chimeric poxvirus defective in the J2R locus with respect to at least one type of producer cell is higher than the syncytium formation ability of at least three, preferably at least four, more preferably at least five, and even more preferably all six of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are defective in the J2R locus, measured under the same conditions and at the same time post-infection.

[0252] In a specific aspect of this embodiment, the syncytium formation ability of the mutant chimeric poxvirus deleted in the J2R locus in HCT116 is higher than the syncytium formation ability of at least one of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection. Preferably, the syncytium formation ability of the mutant chimeric poxvirus deleted in the J2R locus in HCT116 is higher than the syncytium formation ability of the mutant parental COP, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection. More preferably, the syncytium formation ability of the mutant chimeric poxvirus deleted in the J2R locus in HCT116 is higher than the syncytium formation ability of at least two of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are deleted in the J2R locus, measured under the same conditions and at the same time post-infection. For example, with respect to at least one type of producer cell, the syncytium formation ability of a mutant chimeric poxvirus defective in the J2R locus is higher than the syncytium formation ability of at least three, preferably at least four, more preferably at least five, and even more preferably all six of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA, all of which are defective in the J2R locus, measured under the same conditions and at the same time post-infection.

[0253] The mutant chimeric poxviruses according to the present invention that are defective in the J2R locus can combine several of the above functional characteristics, including: a high oncolytic potential as disclosed above (preferably compared to a mutant parental vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus, a mutant parental rabbitpox virus Utrecht strain (RPX) having a deletion in the J2R locus, a mutant parental COP having a deletion in the J2R locus and a mutant parental RPX having a deletion in the J2R locus, or all mutant parental poxvirus strains having a deletion in the J2R locus); - reduced viral replication in healthy cells (preferably primary cells) as disclosed above (preferably compared to the mutant parental vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus or to all mutant parental poxvirus strains having a deletion in the J2R locus); a high EEV-SC, a high syncytium formation ability, a high spreading ability, a low neutralization rate in the presence of poxvirus-specific antibodies, and / or a low complement-mediated virus neutralization rate, as disclosed above (preferably compared to a mutant parental vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus, a mutant parental rabbitpox virus Utrecht strain (RPX) having a deletion in the J2R locus, a vaccinia virus IHD-J strain, or any mutant parental poxvirus strain having a deletion in the J2R locus).

[0254] In particular, mutant chimeric poxviruses with deletions in the J2R locus high oncolytic potential as disclosed above and low viral replication in healthy cells (preferably primary cells) as disclosed above; - high oncolytic potential as disclosed above and (high EEV-SC, high syncytium formation ability, high spreading ability, low neutralization rate in the presence of poxvirus-specific antibodies, and / or low complement-mediated virus neutralization rate as disclosed above); - low viral replication in healthy cells (preferably primary cells) as disclosed above and (high EEV-SC, high syncytium formation ability, high spreading ability, low neutralization rate in the presence of poxvirus-specific antibodies, and / or low complement-mediated viral neutralization rate as disclosed above); or high oncolytic potential as disclosed above, low viral replication in healthy cells (preferably primary cells) as disclosed above, and (high EEV-SC, high syncytium formation ability, high spreading ability, low neutralization rate in the presence of poxvirus-specific antibodies, and / or low complement-mediated viral neutralization rate as disclosed above) may comprise:

[0255] Particularly preferred mutant chimeric poxviruses according to the present invention that are defective in the J2R locus comprise the following functional characteristics: the oncolytic activity of said mutant chimeric poxvirus having a deletion in the J2R locus is greater for at least one tumor than the oncolytic activity of the mutant parental vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus or the mutant parental rabbitpox virus Utrecht strain (RPX) having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection; - the viral replication of said mutant chimeric poxvirus defective in the J2R locus in at least one type of healthy cell (preferably primary cell) is lower than the viral replication of the mutant parental vaccinia virus Copenhagen strain (COP) defective in the J2R locus; - for at least one type of producer cell (preferably a tumor cell), the EEV-SC of said mutant chimeric poxvirus having a deletion in the J2R locus is higher than the EEV-SC of the mutant parent rabbitpox virus Utrecht strain (RPX) or vaccinia virus IHD-J strain having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection; optionally, the neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is lower with respect to at least one poxvirus-specific antibody and tumor than the neutralization rate of the mutant parent vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus measured under the same conditions and at the same time post-infection; optionally, the complement-mediated virus neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is lower than the complement-mediated virus neutralization rate of the parental vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus measured under the same conditions; and Optionally, the syncytium formation ability of the mutant chimeric poxvirus defective in the J2R locus is higher in at least one type of producer cell (preferably a tumor cell) than the syncytium formation ability of the parental vaccinia virus Copenhagen strain (COP) defective in the J2R locus, measured under the same conditions and at the same time post-infection.

[0256] Further particularly preferred mutant chimeric poxviruses according to the invention that are defective in the J2R locus comprise the following functional characteristics: the oncolytic activity of said mutant chimeric poxvirus having a deletion in the J2R locus is greater for at least one tumor than the oncolytic activity of the mutant parental vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus or the mutant parental rabbitpox virus Utrecht strain (RPX) having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection; - the viral replication of said mutant chimeric poxvirus defective in the J2R locus in at least one type of healthy cell (preferably primary cell) is lower than the viral replication of the mutant parental vaccinia virus Copenhagen strain (COP) defective in the J2R locus; the syncytium formation ability of said mutant chimeric poxvirus defective in the J2R locus is higher in at least one type of producer cell (preferably a tumor cell) than the syncytium formation ability of the parental vaccinia virus Copenhagen strain (COP) defective in the J2R locus, measured under the same conditions and at the same time post-infection; optionally, for at least one type of producer cell (preferably a tumor cell), the EEV-SC of said mutant chimeric poxvirus having a deletion in the J2R locus is higher than the EEV-SC of the mutant parent rabbitpox virus Utrecht strain (RPX) or vaccinia virus IHD-J strain having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection; optionally, the neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is lower with respect to at least one poxvirus-specific antibody and tumor than the neutralization rate of the mutant parent vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus measured under the same conditions and at the same time post-infection; and In some cases, the complement-mediated virus neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is lower than the complement-mediated virus neutralization rate of the parental vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus measured under the same conditions.

[0257] More particularly, preferred chimeric mutant poxviruses defective in the J2R locus according to the present invention comprise the following functional characteristics: the oncolytic activity of said mutant chimeric poxvirus having a deletion in the J2R locus is greater for at least one tumor than the oncolytic activity of the mutant parental vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus or the mutant parental rabbitpox virus Utrecht strain (RPX) having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection; - the viral replication of said mutant chimeric poxvirus defective in the J2R locus in at least one type of healthy cell (preferably primary cell) is lower than the viral replication of the mutant parental vaccinia virus Copenhagen strain (COP) defective in the J2R locus; - for at least one type of producer cell (preferably a tumor cell), the EEV-SC of said mutant chimeric poxvirus having a deletion in the J2R locus is higher than the EEV-SC of the mutant parent rabbitpox virus Utrecht strain (RPX) or vaccinia virus IHD-J strain having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection; the syncytium formation ability of said mutant chimeric poxvirus defective in the J2R locus is higher in at least one type of producer cell (preferably a tumor cell) than the syncytium formation ability of the parental vaccinia virus Copenhagen strain (COP) defective in the J2R locus, measured under the same conditions and at the same time post-infection; optionally, the neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is lower with respect to at least one poxvirus-specific antibody and tumor than the neutralization rate of the mutant parent vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus measured under the same conditions and at the same time post-infection; and In some cases, the complement-mediated virus neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is lower than the complement-mediated virus neutralization rate of the parental vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus measured under the same conditions.

[0258] The mutant chimeric poxviruses of the present invention having a deletion in the J2R locus may alternatively comprise a high therapeutic index as disclosed above and (high EEV-SC, high syncytium formation ability, high spreading ability, low neutralization rate in the presence of poxvirus-specific antibodies and / or low complement-mediated virus neutralization rate as disclosed above). Particularly preferred mutant chimeric poxviruses of the present invention having a deletion in the J2R locus comprise the following functional characteristics: the therapeutic index of said mutant chimeric poxvirus having a deletion in the J2R locus is higher than the therapeutic index of the mutant parental vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus for at least one organ, measured under the same conditions and at the same time post-infection; - for at least one type of producer cell (preferably a tumor cell), the EEV-SC of said mutant chimeric poxvirus having a deletion in the J2R locus is higher than the EEV-SC of the mutant parent rabbitpox virus Utrecht strain (RPX) or vaccinia virus IHD-J strain having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection; optionally, the neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is lower with respect to at least one poxvirus-specific antibody and tumor than the neutralization rate of the mutant parent vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus measured under the same conditions and at the same time post-infection; optionally, the complement-mediated virus neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is lower than the complement-mediated virus neutralization rate of the parental vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus measured under the same conditions; and Optionally, the syncytium formation ability of the mutant chimeric poxvirus defective in the J2R locus is higher in at least one type of producer cell (preferably a tumor cell) than the syncytium formation ability of the parental vaccinia virus Copenhagen strain (COP) defective in the J2R locus, measured under the same conditions and at the same time post-infection.

[0259] Further preferred mutant chimeric poxviruses according to the present invention that are defective in the J2R locus comprise the following functional characteristics: the therapeutic index of said mutant chimeric poxvirus having a deletion in the J2R locus is higher than the therapeutic index of the mutant parental vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus for at least one organ, measured under the same conditions and at the same time post-infection; the syncytium formation ability of said mutant chimeric poxvirus defective in the J2R locus is higher in at least one type of producer cell (preferably a tumor cell) than the syncytium formation ability of the parental vaccinia virus Copenhagen strain (COP) defective in the J2R locus, measured under the same conditions and at the same time post-infection; optionally, for at least one type of producer cell (preferably a tumor cell), the EEV-SC of said mutant chimeric poxvirus having a deletion in the J2R locus is higher than the EEV-SC of the mutant parent rabbitpox virus Utrecht strain (RPX) or vaccinia virus IHD-J strain having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection; optionally, the neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is lower with respect to at least one poxvirus-specific antibody and tumor than the neutralization rate of the mutant parent vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus measured under the same conditions and at the same time post-infection; and In some cases, the complement-mediated virus neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is lower than the complement-mediated virus neutralization rate of the parental vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus measured under the same conditions.

[0260] More particularly preferred mutant chimeric poxviruses defective in the J2R locus according to the present invention comprise the following functional characteristics: the therapeutic index of said mutant chimeric poxvirus having a deletion in the J2R locus is higher than the therapeutic index of the mutant parental vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus for at least one organ, measured under the same conditions and at the same time post-infection; - for at least one type of producer cell (preferably a tumor cell), the EEV-SC of said mutant chimeric poxvirus having a deletion in the J2R locus is higher than the EEV-SC of the mutant parent rabbitpox virus Utrecht strain (RPX) or vaccinia virus IHD-J strain having a deletion in the J2R locus, measured under the same conditions and at the same time post-infection; the syncytium formation ability of said mutant chimeric poxvirus defective in the J2R locus is higher in at least one type of producer cell (preferably a tumor cell) than the syncytium formation ability of the parental vaccinia virus Copenhagen strain (COP) defective in the J2R locus, measured under the same conditions and at the same time post-infection; optionally, the neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is lower with respect to at least one poxvirus-specific antibody and tumor than the neutralization rate of the mutant parent vaccinia virus Copenhagen strain (COP) having a deletion in the J2R locus measured under the same conditions and at the same time post-infection; and In some cases, the complement-mediated virus neutralization rate of the mutant chimeric poxvirus having a deletion in the J2R locus is lower than the complement-mediated virus neutralization rate of the parental Copenhagen vaccinia virus strain (COP) having a deletion in the 2R locus.

[0261] Any mutant chimeric poxvirus having a deletion in the J2R locus that combines several of the functional characteristics as described above may further have 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%, at least 99.91%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99% or even 100% identity to SEQ ID NO:8.

[0262] Mutant chimeric poxviruses with defects in the I4L and / or F4L loci In another embodiment, the mutant chimeric poxvirus of the present invention has a defect in at least one of the ribonucleotide reductase (RR) loci, which contain the RR-encoding genes: the I4L-encoding gene (similar to the I4L gene in COP) and the F4L-encoding gene (similar to the F4L gene in COP). In nature, the ribonucleotide reductase enzyme catalyzes the reduction of ribonucleotides to deoxyribonucleotides, a key step in DNA biosynthesis. The viral enzyme has a subunit structure similar to that of mammalian enzymes and is composed of two heterologous subunits, termed I4L and F4L. In the context of the present invention, the defect may be in either the locus encoding the R1 large subunit or the locus encoding the R2 small subunit, or both.

[0263] In one embodiment, the mutant chimeric poxvirus of the present invention is defective in the I4L locus. Alternatively, the mutant chimeric poxvirus of the present invention is defective in the F4L locus or in both the I4L and F4L loci.

[0264] Mutant chimeric poxviruses with deletions of the J2R locus and deletions of the I4L and / or F4L loci In a preferred embodiment, the mutant chimeric poxvirus of the present invention can be defective in the J2R locus and one or both of the I4L and F4L loci. Such double-deleted mutant chimeric poxviruses are defective in both TK activity and RR activity (e.g., as described in WO2009 / 065546 and Foloppe et al., 2008, Gene Ther., 15: 1361-1371). Thus, the double-deleted mutant chimeric poxviruses of J2R and one or both of I4L and F4L are particularly advantageous due to their low replication in healthy cells (preferably primary cells) and a higher therapeutic index than the corresponding wild-type chimeric poxvirus.

[0265] Preferably, the mutant chimeric poxvirus has 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%, at least 99.91%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99%, or even 100% identity to SEQ ID NO: 9. SEQ ID NO: 9 is the sequence of the core region and one of the ITRs of POXSTG19730, which corresponds to POXSTG19503 except that a GFP::FCU1 sequence has been inserted into the J2R locus and an mCherry gene under the control of the pH5R promoter has been inserted into the I4L locus.

[0266] More preferably, the mutant chimeric poxvirus has 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%, at least 99.91%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99%, or even 100% identity to SEQ ID NO: 10. SEQ ID NO: 10 is the sequence of the core region and one of the ITRs of POXSTG20150, which corresponds to POXSTG19503 after deletion of the J2R gene sequence encoding the TK protein and the I4L gene sequence encoding the large subunit of the RR protein.

[0267] Even more preferably, the mutant chimeric poxvirus is strain POXSTG19503, accession number CNCM-I-5913, which has a deletion in the J2R locus and a deletion in the I4L and / or F4L loci.

[0268] Alternatively or in combination, the mutant chimeric poxvirus defective in J2R and one or both of the I4L and F4L loci (preferably the I4L locus) preferably comprises the following functional feature or combination of functional features: a) for at least one tumor, the oncolytic activity of the mutant chimeric poxvirus according to the present invention, which is deficient in the J2R locus and / or the I4L and F4L loci, is higher than the oncolytic activity of at least one (preferably the mutant oncolytic parent COP, which is deficient in the J2R locus and / or the I4L and F4L loci), at least two, at least three, at least four or all five of the mutant oncolytic parent poxvirus strains, which are deficient in the J2R locus and / or the I4L and F4L loci, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post-infection; b) in at least one healthy cell (preferably primary cell), the viral replication of the mutant chimeric poxvirus defective in the J2R locus and one or both of the I4L and F4L loci in the healthy cell (preferably primary cell) is lower than the viral replication of at least one (preferably the mutant oncolytic parental COP defective in the J2R locus and one or both of the I4L and F4L loci), at least two, at least three, at least four, or all five of the mutant oncolytic parental poxvirus strains defective in the J2R locus and one or both of the I4L and F4L loci, namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), and vaccinia virus Western Reserve strain (WR); c) for at least one producer cell (preferably a tumor cell), the EEV-SC of the mutant chimeric poxvirus defective in the J2R locus and / or the I4L and F4L loci is higher than the EEV-SC of at least one (preferably the mutant oncolytic parental COP defective in the J2R locus and / or the I4L and F4L loci), at least two, at least three, at least four, at least five or all six of the mutant parental poxvirus strains defective in the J2R locus and / or the I4L and F4L loci, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara (MVA), all measured under the same conditions and at the same time post-infection; d) for at least one tumor, the spreading ability of the mutant chimeric poxvirus defective in the J2R locus and / or the I4L and F4L loci is higher than the spreading ability of at least one (preferably the mutant oncolytic parental COP defective in the J2R locus and / or the I4L and F4L loci), at least two, at least three, at least four, at least five or all six of the mutant parental poxvirus strains defective in the J2R locus and / or the I4L and F4L loci, namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara (MVA), all measured under the same conditions and at the same time post-infection; e) the neutralization rate of the mutant chimeric poxvirus defective in the J2R locus and / or the I4L and F4L loci is lower than the neutralization rate of at least one (preferably the mutant oncolytic parental COP defective in the J2R locus and / or the I4L and F4L loci), at least two, at least three, at least four, at least five or all six of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR and MVA defective in the J2R locus and / or the I4L and F4L loci, all measured under the same conditions and at the same time post-infection; f) the complement-mediated virus neutralization rate of the mutant chimeric poxvirus defective in the J2R locus and / or the I4L and F4L loci is lower than the complement-mediated virus neutralization rates of at least one (preferably a mutant oncolytic parental COP defective in the J2R locus and / or the I4L and F4L loci), at least two, at least three, at least four, at least five or six of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR and MVA, all of which are defective in the J2R locus and / or the I4L and F4L loci, measured under the same conditions; g) the syncytium formation ability of the mutant chimeric poxvirus defective in the J2R locus and / or the I4L and F4L loci is higher in at least one producer cell (preferably a tumor cell) than the syncytium formation ability of at least one (preferably the mutant oncolytic parental COP defective in the J2R locus and / or the I4L and F4L loci), at least two, at least three, at least four, at least five, or at least six of the mutant parental poxvirus strains defective in the J2R locus and / or the I4L and F4L loci, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR), and modified vaccinia virus Ankara (MVA), all of which are measured under the same conditions and at the same time post-infection; h) a) and b); i) a) and (c) or d) or e) or f) or g)); j) a) and b) and (c) or d) or e) or f) or g)), preferably a) and b) and c), or a) and b) and g); k) a) and b) and c) and (e) or f) or g)), preferably a) and b) and c) and f); or a) and b) and c) and e); l) a) and b) and g) and (e) or f)); m) a) and b) and c) and e) and (f) or g)); n) a) and b) and c) and f) and g); o) a) and b) and e) and f) and g); p) a) and b) and c) and e) and f) and g), and q) Any combination thereof.

[0269] Alternatively, the mutant chimeric poxvirus, which is deficient in the J2R gene and in one or both of the I4L gene and the F4L locus (preferably the I4L locus), preferably comprises the following functional characteristics or combinations of functional characteristics: a) the therapeutic index of said mutant chimeric poxvirus defective in the J2R locus and / or the I4L and F4L loci is higher in at least one organ than the therapeutic index of at least one (preferably the mutant oncolytic parent COP defective in the J2R locus and / or the I4L and F4L loci), at least two, at least three, at least four, at least five or all six of the mutant parent oncolytic poxvirus strains defective in the J2R locus and / or the I4L and F4L loci, namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR), all of which are measured under the same conditions and at the same time post-infection; b) for at least one producer cell (preferably a tumor cell), the EEV-SC of the mutant chimeric poxvirus defective in the J2R locus and / or the I4L and F4L loci is higher than the EEV-SC of at least one (preferably the mutant oncolytic parental COP defective in the J2R locus and / or the I4L and F4L loci), at least two, at least three, at least four, at least five or six of the mutant parental poxvirus strains defective in the J2R locus and / or the I4L and F4L loci, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara (MVA), all measured under the same conditions and at the same time post-infection; c) for at least one tumor, the spreading ability of the mutant chimeric poxvirus defective in the J2R locus and / or the I4L and F4L loci is higher than the spreading ability of at least one (preferably the mutant oncolytic parental COP defective in the J2R locus and / or the I4L and F4L loci), at least two, at least three, at least four, at least five or six of the mutant parental poxvirus strains defective in the J2R locus and / or the I4L and F4L loci, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara (MVA), all of which are measured under the same conditions and at the same time post-infection; d) the neutralization rate of the mutant chimeric poxvirus defective in the J2R locus and / or the I4L and F4L loci is lower for at least one poxvirus-specific antibody and tumor than the neutralization rate of at least one (preferably the mutant oncolytic parental COP defective in the J2R locus and / or the I4L and F4L loci), at least two, at least three, at least four, at least five or all six of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR and MVA defective in the J2R locus and / or the I4L and F4L loci, all measured under the same conditions and at the same time post-infection; e) the complement-mediated virus neutralization rate of the mutant chimeric poxvirus defective in the J2R locus and / or the I4L and F4L loci is lower than the complement-mediated virus neutralization rates of at least one (preferably a mutant oncolytic parental COP defective in the J2R locus and / or the I4L and F4L loci), at least two, at least three, at least four, at least five or six of the mutant parental poxvirus strains RPX, CPX, COP, WY, WR and MVA, all of which are defective in the J2R locus and / or the I4L and F4L loci, measured under the same conditions; f) the syncytium formation ability of the mutant chimeric poxvirus defective in the J2R locus and / or the I4L and F4L loci is higher in at least one producer cell (preferably a tumor cell) than the syncytium formation ability of at least one (preferably the mutant oncolytic parental COP defective in the J2R locus and / or the I4L and F4L loci), at least two, at least three, at least four, at least five or all six of the mutant parental poxvirus strains defective in the J2R locus and / or the I4L and F4L loci, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara (MVA), all of which are measured under the same conditions and at the same time post-infection; g) a) and (b) or c) or d) or e) or f)), preferably a) and b); h) a) and b) and (d) or e) or f)), preferably a) and b) and d), or a) and b) and e), or a) and b) and f); i) a) and d) and (e) or f)); j) a) and e) and f); k) a) and b) and d) and (e) or f)); l) a) and b) and d) and e); m) a) and b) and e) and f); n) a) and d) and e) and f); o) a) and b) and d) and e) and f); and p) any combination thereof.

[0270] Further viral genes that may be altered in the mutant chimeric poxviruses of the present invention Alternatively or in combination with a deletion in the J2R locus, one or both of the I4L and F4L loci, or one or both of the J2R locus and the I4L and F4L loci, the mutant chimeric poxvirus of the present invention may further have a deletion in the M2L locus (a modification that results in suppression of expression of the viral m2 protein, such as a deletion in the M2L locus, is preferred).

[0271] In one embodiment, the mutant chimeric poxvirus is defective in the J2R locus (preferably a modification that results in the suppression of expression of the viral TK protein) and in the M2L locus (preferably a modification that results in the suppression of expression of the viral m2 protein), resulting in a mutant chimeric poxvirus that is defective in both m2 and TK functions (m2-tk-mutant chimeric poxvirus). Partial or complete deletion of the M2L locus and / or J2R locus and insertion of foreign nucleic acid at the M2L locus and / or J2R locus are contemplated in the context of the present invention to inactivate m2 and tk functions.

[0272] In another embodiment, the mutant chimeric poxvirus is deficient in one or both of the I4L and F4L loci (preferably a modification that results in the suppression of expression of the viral ribonucleotide reductase (RR) protein) and the M2L locus (preferably a modification that results in the suppression of expression of the viral m2 protein), resulting in a chimeric poxvirus that is deficient in both m2 and rr functions (an m2- and rr-deficient mutant chimeric poxvirus). In the context of the present invention, the mutant chimeric poxvirus can be modified in either or both of the I4L gene (encoding the R1 large subunit) and the F4L gene (encoding the R2 small subunit) to provide an RR-deficient mutant chimeric poxvirus. For example, partial or complete deletion of the I4L and / or F4L loci / loci.

[0273] In another embodiment, the mutant chimeric poxvirus is defective in the J2R locus, one or both of the I4L and F4L loci, and the M2L locus (the M2L, J2R, and I4L locus; triple-deficient viruses in which the M2L, J2R, and F4L loci or the M2L, J2R, I4L, and F4L loci have been modified), resulting in a mutant chimeric poxvirus that is defective in M2 activity, TK activity, and RR activity (m2-, tk-, rr-mutant chimeric poxvirus).

[0274] Alternatively, or in combination with alterations in one or more of the J2R locus, the I4L and / or F4L locus, and the M2 locus, the mutant chimeric poxvirus of the invention may be deficient in dUTPase by alteration of the dUTPase-encoding gene (similar to the F2L gene in COP).

[0275] Alternatively, or in combination, other strategies can be employed to further enhance viral tumor specificity. Representative examples of suitable modifications include disruption of hemagglutinin-encoding genes (similar to the A56R gene in COP), possibly combined with J2R deletion (Zhang et al., 2007, Cancer Res. 67:10038-46). Disruption of interferon-regulatory genes (similar to the B8R or B18R gene in COP) or caspase-1 inhibitors (similar to the B13R gene in COP) may also be advantageous. Another suitable modification consists of disruption of the gene encoding viral dUTPase, which is involved in both maintaining the fidelity of DNA replication and providing the precursor for TMP production by thymidylate synthase (Broyles et al., 1993, Virol. 195:863-5).

[0276] Recombinant chimeric poxviruses encoding heterologous nucleic acids of interest The chimeric poxviruses of the invention (wild-type or mutant as described above) may further comprise one or more heterologous nucleic acids of interest inserted into their genome. Thus, the present invention also provides recombinant (wild-type or mutant as described above) chimeric poxviruses further comprising one or more heterologous nucleic acids of interest inserted into their genome.

[0277] According to the present invention, the heterologous nucleic acid of interest may be derived from a prokaryote (including the kingdoms Bacteria and Archaea), an anukaryote (including viruses), or a eukaryote (including the kingdoms Protista, Fungi, Plantae, and Animalia). Advantageously, said nucleic acid of interest encodes all or part of a polypeptide. A polypeptide is understood to be any translation product of a polynucleotide, regardless of size, whether glycosylated or not, and includes peptides and proteins.

[0278] In one embodiment, the nucleic acid of interest encodes a therapeutic polypeptide that, when properly administered to a subject, is expected to provide biological activity or to induce a beneficial effect on the course or symptoms of the pathology being treated. Numerous nucleic acids of interest are contemplated in the context of the present invention, including those that encode polypeptides capable of complementing a protein missing or deleted in a subject, those that function to limit or eliminate harmful cells from the body through toxic effects, or those that encode immunity-conferring polypeptides. These may be naturally occurring or may be derived from the latter by mutation, deletion, substitution, and / or addition of one or more nucleotides. Representative examples of suitable polypeptides for therapeutic purposes include, but are not limited to, polypeptides capable of enhancing antitumor efficacy (e.g., immunostimulatory polypeptides), antigens for inducing or activating immune humoral and / or cellular responses, suicide polypeptides capable of enhancing the oncolytic properties of the chimeric poxvirus of the present invention, or permeases for increasing cellular nucleoside or nucleotide pools, among others.

[0279] The present invention also encompasses chimeric poxviruses that express 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.

[0280] Immunostimulatory polypeptides Certain embodiments of the present invention are directed to recombinant (wild-type or mutant) chimeric poxviruses comprising immunostimulatory polypeptides. As used herein, "immunostimulatory polypeptide" refers to a polypeptide or protein that has the ability to stimulate the immune system in a specific or non-specific manner. A vast number of proteins are known in the art for their ability to exert immunostimulatory effects. Examples of immunostimulatory proteins suitable in the context of the present invention include, but are not limited to, immune checkpoint inhibitors, such as, but not limited to, anti-PD1, anti-PDL1, anti-PDL-2, anti-CTLA4, anti-Tim3, anti-LAG3, anti-BTLA; cytokines such as alpha, beta, or gamma interferons, interleukins, or tumor necrosis factors; agents that affect the regulation of cell surface receptors, such as inhibitors of epidermal growth factor receptors (particularly cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab, gefitinib ... agents that affect angiogenesis, such as inhibitors of vascular endothelial growth factor (especially bevacizumab or ranibizumab); agents that stimulate stem cells to produce granulocytes, macrophages (e.g., granulocyte-macrophage colony-stimulating factor and B7 protein). In a preferred embodiment, the nucleic acid of interest is a cytokine, more preferably an interleukin, even more preferably IL-12.

[0281] In a preferred embodiment of the present invention, the recombinant chimeric poxvirus mutant is defective in the J2R locus and further encodes an interleukin, hi a more preferred embodiment, the recombinant chimeric poxvirus mutant is defective in the J2R locus and further encodes IL-12.

[0282] In a preferred embodiment of the present invention, the recombinant chimeric mutant poxvirus is defective in the J2R locus and one or both of the F4L and I4L loci and further encodes an interleukin, hi a more preferred embodiment, the recombinant chimeric poxvirus is defective in the J2R locus and one or both of the F4L and I4L loci and further encodes IL-12.

[0283] antigen Another embodiment of the present invention is directed to a recombinant (wild-type or mutant) chimeric poxvirus encoding an antigen. The term "antigen" generally refers to a substance that is recognized and selectively bound by an antibody or T-cell antigen receptor to elicit an immune response. The term antigen is intended to encompass native antigens as well as fragments (e.g., epitopes, immunogenic domains, etc.) and analogs thereof, so long as such fragments or analogs can be targets of an immune response. Suitable antigens in the context of the present invention are preferably polypeptides (e.g., peptides, polypeptides, post-translationally modified polypeptides, etc.) that contain one or more B-cell epitopes or one or more T-cell epitopes, or both B-cell and T-cell epitopes, and that can elicit an immune response, preferably a humoral or cellular response that may be specific for the antigen. Generally, one or more antigens associated with the disease to be treated are selected. Preferred antigens for use herein are cancer antigens and antigens of tumor-inducing pathogens.

[0284] In certain embodiments, the antigen encoded by the recombinant chimeric poxvirus is a cancer antigen (also called a tumor-associated antigen) that is associated with and / or functions as a marker for cancer. Cancer antigens encompass various categories of polypeptides, including those that are normally silent (i.e., not expressed) in healthy cells (preferably primary cells), those that are expressed at low levels or only at specific differentiation stages, and those that are transiently expressed, such as embryonic and fetal antigens, as well as those resulting from mutations in cellular genes, such as oncogenes (e.g., activated ras oncogenes), protooncogenes (e.g., ErbB family members), or proteins resulting from chromosomal translocations. Cancer antigens also encompass antigens encoded by pathogenic organisms (bacteria, viruses, parasites, fungi, viroids, or prions) that can induce malignant conditions in subjects (e.g., chronically infected subjects), such as RNA and DNA tumor viruses (e.g., HPV, HCV, EBV, etc.) and bacteria (e.g., Helicobacter pylori). Cancer antigens can also be neoantigens that are specific to a patient's tumor and used in personalized medicine (EP2018 / 066668).

[0285] Some non-limiting examples of cancer antigens include, but are not limited to, MART-1 / Melan-A, gp100, 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 tumor antigens, GAGE ​​family tumor antigens, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family (e.g., MUC1, MUC16, etc.; see, e.g., U.S. Pat. No. 6,054,438; WO98 / 04727; or WO98 / 37095), HER2 / neu, p21ras, alpha-fetoprotein, E-cadherin, catenin family, and HPV-16 and HPV-18. These include viral antigens such as E6 and E7 antigens.

[0286] Other antigens suitable for use in the present invention include marker antigens (such as β-galactosidase, luciferase, green fluorescent protein, etc.).

[0287] suicide polypeptide In one embodiment, the recombinant (wild-type or mutant) chimeric poxvirus of the present invention can encode at least a suicide polypeptide. The term "suicide polypeptide" refers to a polypeptide that can convert a drug precursor (also called a "prodrug") into a cytotoxic compound. Examples of suicide polypeptides and corresponding prodrugs suitable for use herein are disclosed in the table below.

[0288] [Table 1]

[0289] In this embodiment, the oncolytic activity of the recombinant chimeric poxvirus encoding a suicide polypeptide for at least one tumor is higher than the oncolytic activity of at least one of the oncolytic parental recombinant poxvirus strains encoding a suicide polypeptide, i.e., rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), and vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post-infection. In a preferred embodiment, the oncolytic activity of the recombinant parental vaccinia virus Copenhagen strain (COP) encoding a suicide gene or the recombinant parental rabbitpox virus Utrecht strain (RPX) encoding a suicide gene is measured under the same conditions and at the same time post-infection for at least one tumor. More preferably, the oncolytic ability of the recombinant chimeric poxvirus encoding a suicide gene for at least one tumor is higher than that of at least two of the recombinant oncolytic parental poxvirus strains encoding a suicide gene, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post-infection. For example, the oncolytic ability of the recombinant chimeric poxvirus encoding a suicide gene for at least one tumor is higher than that of the recombinant parental vaccinia virus Copenhagen strain (COP) encoding a suicide gene and the recombinant parental rabbitpox virus Utrecht strain (RPX) encoding a suicide gene, measured under the same conditions and at the same time post-infection.In a more preferred embodiment, the oncolytic potency of the recombinant chimeric poxvirus encoding a suicide gene for at least one tumor is higher than the oncolytic potency of at least three, more preferably at least four, and even more preferably each of five oncolytic recombinant parental poxvirus strains encoding suicide genes, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR), measured under the same conditions and at the same time post-infection.

[0290] Alternatively or in combination, the present invention provides a recombinant chimeric poxvirus encoding a suicide polypeptide, wherein, for at least one healthy cell (preferably a primary cell), the viral replication of said chimeric recombinant poxvirus encoding a suicide polypeptide in the healthy cell is lower than that of at least one of five oncolytic recombinant parental poxvirus strains encoding a suicide polypeptide, i.e., RPX, CPX, COP, WY and WR, preferably lower than that of a recombinant parental COP encoding a suicide gene or a recombinant parental RPX encoding a suicide gene, more preferably lower than that of at least two of the recombinant oncolytic parental poxvirus strains encoding a suicide gene (e.g., a recombinant parental COP encoding a suicide gene and a recombinant parental RPX encoding a suicide gene), more preferably lower than that of at least three of the five oncolytic recombinant parental poxvirus strains encoding a suicide gene, more preferably lower than that of at least four, and even more preferably lower than that of each of them.

[0291] Alternatively, or in combination, the present invention provides a recombinant chimeric poxvirus encoding a suicide polypeptide, wherein the therapeutic index of said recombinant chimeric poxvirus encoding a suicide polypeptide, for at least one organ, is higher than the therapeutic index of at least one of the oncolytic recombinant parental poxvirus strains RPX, CPX, COP, WY and WR encoding the suicide polypeptide, measured under the same conditions and at the same time post-infection; preferably higher than the therapeutic index of the recombinant parental COP encoding a suicide gene or the recombinant parental RPX encoding a suicide gene, measured under the same conditions and at the same time post-infection; more preferably higher than the therapeutic index of at least two of the recombinant oncolytic parental poxvirus strains RPX, CPX, COP, WY and WR encoding a suicide gene (e.g., the recombinant parental COP encoding a suicide gene and the recombinant parental RPX encoding a suicide gene), measured under the same conditions and at the same time post-infection; and more preferably higher than the therapeutic index of at least three, more preferably at least four, and even more preferably each of the five oncolytic recombinant parental poxvirus strains RPX, CPX, COP, WY and WR encoding a suicide gene, measured under the same conditions and at the same time post-infection.

[0292] Alternatively, or in combination, the present invention provides a recombinant chimeric poxvirus encoding a suicide polypeptide, wherein the EEV-SC of said recombinant chimeric poxvirus encoding a suicide polypeptide is higher in at least one producer cell (preferably a tumor cell) than the EEV-SC of at least one of the recombinant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA encoding a suicide polypeptide, measured under the same conditions and at the same time post-infection; preferably higher than the EEV-SC of at least two of the recombinant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA encoding a suicide gene (e.g., the recombinant parental COP encoding a suicide gene and the recombinant parental RPX encoding a suicide gene), measured under the same conditions and at the same time post-infection; and even more preferably higher than the EEV-SC of at least three, preferably at least four, more preferably at least five, and even more preferably, of the six recombinant parental poxvirus strains RPX, CPX, COP, WY, WR, and MVA encoding a suicide gene, measured under the same conditions and at the same...

Claims

1. at least 96.6%, preferably at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, 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%, 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 1. A chimeric poxvirus comprising a nucleic acid sequence having 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%, at least 99.91%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99%, or even 100% sequence identity.

2. The chimeric poxvirus of claim 1, comprising nucleic acid fragments derived from rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara strain (MVA).

3. The chimeric poxvirus of claim 1 or 2, which comprises glutamic acid at position 151 of the A34R gene.

4. (a) i. a nucleic acid sequence consisting of nucleotides 562-4701 of SEQ ID NO:2 or a sequence having at least 99% identity to nucleotides 562-4701 of SEQ ID NO:2; ii. a nucleic acid sequence consisting of nucleotides 54042 to 59851 of SEQ ID NO:2 or a sequence having at least 99% identity to nucleotides 54042 to 59851 of SEQ ID NO:2; iii. a nucleic acid sequence consisting of nucleotides 83610 to 88879 of SEQ ID NO: 2 or a sequence having at least 99% identity to nucleotides 83610 to 88879 of SEQ ID NO: 2; iv. a nucleic acid sequence consisting of nucleotides 127290 to 130589 of SEQ ID NO:2 or a sequence having at least 99% identity to nucleotides 127290 to 130589 of SEQ ID NO:2; v. a nucleic acid sequence consisting of nucleotides 137520 to 154979 of SEQ ID NO: 2 comprising glutamic acid at position 151 or a sequence having at least 99% identity to nucleotides 137520 to 154979 of SEQ ID NO: 2 comprising glutamic acid at position 151; vi. A nucleic acid sequence consisting of nucleotides 157002 to 162091 of SEQ ID NO:2 or a sequence having at least 99% identity to nucleotides 157002 to 162091 of SEQ ID NO:

2. at least one rabbitpox virus Utrecht strain (RPX)-derived nucleic acid sequence selected from: (b) i. a nucleic acid sequence consisting of nucleotides 14242 to 51241 of SEQ ID NO:3 or a sequence having at least 99% identity to nucleotides 14242 to 51241 of SEQ ID NO:3, and ii. A nucleic acid sequence consisting of nucleotides 59852 to 72141 of SEQ ID NO:3 or a sequence having at least 99% identity to nucleotides 59852 to 72141 of SEQ ID NO:

3. at least one nucleic acid sequence derived from cowpox virus Brighton strain (CPX) selected from: (c) i. a nucleic acid sequence consisting of nucleotides 7612 to 8521 of SEQ ID NO:4 or a sequence having at least 99% identity to nucleotides 7612 to 8521 of SEQ ID NO:4 at least one Copenhagen (COP)-derived nucleic acid sequence selected from: (d) i. a nucleic acid sequence consisting of nucleotides 76630 to 78639 of SEQ ID NO:5 or a sequence having at least 99% identity to nucleotides 76630 to 78639 of SEQ ID NO:5; ii. a nucleic acid sequence consisting of nucleotides 81060 to 83529 of SEQ ID NO: 5 or a sequence having at least 99% identity to nucleotides 81060 to 83529 of SEQ ID NO: 5; iii. a nucleic acid sequence consisting of nucleotides 116250 to 118459 of SEQ ID NO: 5 or a sequence having at least 99% identity to nucleotides 116250 to 118459 of SEQ ID NO: 5; iv. a nucleic acid sequence consisting of nucleotides 162290 to 164599 of SEQ ID NO: 5 or a sequence having at least 99% identity to nucleotides 162290 to 164599 of SEQ ID NO: 5; v. a nucleic acid sequence consisting of nucleotides 176100 to 179909 of SEQ ID NO:5 or a sequence having at least 99% identity to nucleotides 176100 to 179909 of SEQ ID NO:5; vi. A nucleic acid sequence consisting of nucleotides 181920 to 184099 of SEQ ID NO:5 or a sequence having at least 99% identity to nucleotides 181920 to 184099 of SEQ ID NO:5 at least one Wyeth (WY)-derived nucleic acid sequence selected from: (e) i. a nucleic acid sequence consisting of nucleotides 169190 to 171579 of SEQ ID NO:6 or a sequence having at least 99% identity to nucleotides 169190 to 171579 of SEQ ID NO:6; ii. A nucleic acid sequence consisting of nucleotides 173730 to 176099 of SEQ ID NO:6 or a sequence having at least 99% identity to nucleotides 173730 to 176099 of SEQ ID NO:

6. at least one Western Reserve (WR)-derived nucleic acid sequence selected from: (f) i. a nucleic acid sequence consisting of nucleotides 88880 to 90899 of SEQ ID NO:7 or a sequence having at least 99% identity to nucleotides 88880 to 90899 of SEQ ID NO:7; ii. a nucleic acid sequence consisting of nucleotides 91460 to 93839 of SEQ ID NO: 7 or a sequence having at least 99% identity to nucleotides 91460 to 93839 of SEQ ID NO: 7; iii. a nucleic acid sequence consisting of nucleotides 95530 to 116249 of SEQ ID NO: 7 or a sequence having at least 99% identity to nucleotides 95530 to 116249 of SEQ ID NO: 7; iv. a nucleic acid sequence consisting of nucleotides 118460 to 127289 of SEQ ID NO: 7 or a sequence having at least 99% identity to nucleotides 118460 to 127289 of SEQ ID NO: 7, and v. A nucleic acid sequence consisting of nucleotides 134800 to 137519 of SEQ ID NO:7 or a sequence having at least 99% identity to nucleotides 134800 to 137519 of SEQ ID NO:7 at least one modified vaccinia virus Ankara (MVA)-derived nucleic acid sequence selected from: (g) Any combination of (a) to (f) The chimeric poxvirus according to any one of claims 1 to 3, comprising:

5. for at least one tumor, the oncolytic potency of said chimeric poxvirus is higher than the oncolytic potency of at least one of the five wild-type parental oncolytic poxvirus strains RPX, CPX, COP, WY and WR, preferably the wild-type parental COP or parental RPX, preferably at least two of the five wild-type parental oncolytic poxvirus strains RPX, CPX, COP, WY and WR, more preferably the wild-type parental COP and parental RPX, more preferably at least three, more preferably at least four, and even more preferably, of the five wild-type parental oncolytic poxvirus strains RPX, CPX, COP, WY and WR, measured under the same conditions and at the same time post-infection, and for a given tumor, a given virus, a given condition and a given time post-infection, the oncolytic potency OP(tumor, virus, condition, time post-infection) is: OP (tumor, virus, condition, time post infection) = (100 - percentage of surviving tumor cells after infection with virus) The chimeric poxvirus according to any one of claims 1 to 4, wherein said chimeric poxvirus is defined as:

6. The chimeric poxvirus of claim 5, wherein the tumor is selected from A549, MIA Paca-2, U-87-MG, B16F10 and HepG2 tumor cell lines.

7. 7. The chimeric poxvirus according to any one of claims 1 to 6, wherein for at least one healthy cell (preferably primary cell), the viral replication of said chimeric poxvirus in healthy cells (preferably primary cells) is lower than the viral replication of at least one of five oncolytic parental poxvirus strains (optionally mutant and / or recombinant), namely Rabbitpox virus Utrecht strain (RPX), Cowpox virus Brighton strain (CPX), Vaccinia virus Copenhagen strain (COP), Vaccinia virus Wyeth strain (WY) and Vaccinia virus Western Reserve strain (WR), preferably the wild-type parental COP, preferably at least two, more preferably at least three, more preferably at least four, even more preferably of the five wild-type parental oncolytic poxvirus strains RPX, CPX, COP, WY and WR.

8. The chimeric poxvirus of claim 7, wherein the healthy cells (preferably primary cells) are selected from skin cells and liver cells.

9. for at least one organ, the therapeutic index of said chimeric poxvirus is higher than the therapeutic index of at least one of five oncolytic parental poxvirus strains, namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR), preferably the wild-type parental COP, preferably at least two, more preferably at least three, more preferably at least four, even more preferably of the five oncolytic wild-type parental poxvirus strains RPX, CPX, COP, WY and WR, measured under the same conditions and at the same time post-infection, and the organ-specific therapeutic index TI (organ, tumor, virus, condition, time post-infection) for a given organ, a given tumor, a given virus, a given condition and a given time post-infection is: TI (organ, tumor, virus, condition, time post infection) = (viral replication in tumor cells of organ / viral replication in healthy cells of organ) The chimeric poxvirus according to any one of claims 1 to 8, wherein said chimeric poxvirus is defined as:

10. The chimeric poxvirus of claim 9, wherein the organ is the liver, the healthy cells of the organ are healthy (preferably primary) liver cells, and the tumor cells of the organ are HepG2 tumor cells.

11. The extracellular enveloped virus (EEV) secretion capacity (SC) (EEV-SC) of the chimeric poxvirus was measured for at least one producer cell (preferably a tumor cell) under the same conditions and at the same time post-infection with at least one of six parental poxvirus strains, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR), and modified vaccinia virus Ankara strain (MVA), preferably the parent wild-type COP. or higher than the EEV-SC of the parent wild-type RPX, preferably at least two of the six wild-type parent poxvirus strains RPX, CPX, COP, WY, WR and MVA, more preferably the parent wild-type COP and the parent wild-type RPX, more preferably at least three, more preferably at least four, even more preferably at least five of the six wild-type parent poxvirus strains RPX, CPX, COP, WY, WR and MVA, or their respective EEV-SCs, for a given virus, a given producer cell, a given condition and a given time post infection, such that EEV-SC (virus, producer cell, condition, time post infection) is: EEV-SC (virus, producer cells, conditions, time post-infection) = number of EEV particles / number of (EEV + IMV) particles The chimeric poxvirus according to any one of claims 1 to 10, defined as:

12. The chimeric poxvirus of claim 11, wherein the producer cell is the A549 tumor cell line.

13. The chimeric poxvirus according to any one of claims 1 to 12, wherein the extracellular enveloped virus (EEV) secretion capacity (SC) (EEV-SC) of the chimeric poxvirus is higher than the EEV-SC of the wild-type vaccinia virus IHD-J strain measured under the same conditions and at the same time post-infection in at least one producer cell (preferably a tumor cell).

14. The chimeric poxvirus of claim 13, wherein the producer cell is the A549 tumor cell line.

15. The spreading ability of the chimeric poxvirus was measured for at least one tumor under the same conditions and at the same time post-infection using at least one of six parental poxvirus strains, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara strain (MVA), preferably the parental wild-type COP or is higher than the spreading capacity of the parent RPX, preferably at least two of the six parent poxvirus strains RPX, CPX, COP, WY, WR and MVA, more preferably the parents COP and RPX, even more preferably at least three, more preferably at least four, more preferably at least five of the six wild-type parent poxvirus strains selected from RPX, CPX, COP, WY, WR and MVA, or their respective spreading capacities.

16. The neutralization rates of the chimeric poxviruses were measured for at least one poxvirus-specific antibody and tumor under the same conditions and at the same time post-infection for six parental poxvirus strains (optionally mutant and / or recombinant), namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara strain (M). and the neutralization rate (NT(virus, tumor, condition, time post infection)) is lower than the neutralization rate of at least one of the six wild-type parental poxvirus strains, preferably the parent wild-type COP, preferably at least two, more preferably at least three, more preferably at least four, more preferably at least five of the six wild-type parental poxvirus strains selected from RPX, CPX, COP, WY, WR and MVA, or their respective wild-type parental poxvirus strains, for a given virus, a given tumor, a given poxvirus-specific antibody, a given condition and a given time post infection. NT (virus, tumor, poxvirus-specific antibody, condition, time post-infection) = EC50 (with poxvirus-specific antibody) / EC50 (without poxvirus-specific antibody). The chimeric poxvirus according to any one of claims 1 to 15, defined as:

17. (i) infecting a first tumor cell line with multiple parental poxvirus strains, wherein said tumor cell line is permissive to each parental poxvirus strain, to obtain a first infected tumor cell line; (ii) amplifying the parental poxvirus strains on the first infected tumor cell line of step (i) for at least 12 hours (preferably at least 24 hours), at most 3 days, to obtain one or more different chimeric poxviruses by homologous genomic recombination between at least two of the parental poxvirus strains in the supernatant; (iii) at the end of step (ii), recovering the supernatant containing one or more different chimeric poxviruses; (iv) infecting a second tumor cell line with one or more different chimeric poxviruses of the supernatant of step (iii), wherein said second tumor cell line is permissive to each of the parental poxvirus strains of steps (i) and (ii), to obtain a second infected tumor cell line; (v ａ ) amplifying one or more different chimeric poxviruses of step (iv) on said second infected tumor cell line of step (iv), preferably for at least 12 hours and at most 24 hours, and then recovering the supernatant; (vi) determining for at least one third tumor cell line that the oncolytic activity is higher than the oncolytic activity of at least one, preferably some, more preferably all of the parent oncolytic poxvirus strains in the first tumor cell line of step (i) and / or the second tumor cell line of step (iv), measured under the same conditions and at the same time post-infection; ａ ), wherein for a given tumor, a given virus, a given condition and a given time post infection, the oncolytic power OP(tumor, virus, condition, time post infection) is: OP (tumor, virus, condition, time post infection) = (100 - percentage of surviving tumor cells after infection with virus) is defined as 17. The chimeric poxvirus according to any one of claims 1 to 16, obtained or obtainable by a method of directed evolution, comprising:

18. The parent poxvirus strain used in the first step (i) is selected from the group consisting of rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Western Reserve strain (WR), vaccinia virus Wyeth strain (WY), modified vaccinia virus Ankara strain (MVA), raccoon poxvirus Harman strain (RCN), orf virus NZ2 strain (ORF), pseudocowpox TJS strain (PCP), bovine papular stomatitis virus Illinois 721 strain (BPS), myxoma virus Lausanne strain (MYX), squirrelpox virus Kilham strain (SQF), fowlpox virus FP9 strain (FPV), swinepox virus Kasuza strain (SPV), Yaba-like disease virus Davis strain (YLD) and Chotiavirus SP An 232 strain (CTV), more preferably selected from the group consisting of rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Western Reserve strain (WR), vaccinia virus Wyeth strain (WY) and modified vaccinia virus Ankara strain (MVA).

19. 18. The chimeric poxvirus of claim 17, wherein the permissive tumor cell line for the method of directed evolution is of higher mammalian origin, preferably the permissive tumor cell line is selected from the group consisting of A549, CAL-33, HepG2, HCT116, Hela, SK-MEL-1, PANC-1, Hs746T, SK-OV-3 and CV-1, preferably A549.

20. A mutant chimeric poxvirus comprising the chimeric poxvirus according to any one of claims 1 to 19, which has been modified by altering one or more poxvirus genes.

21. The mutant chimeric poxvirus of claim 20, wherein the mutant chimeric poxvirus has a deletion in the J2R locus.

22. 22. The mutant chimeric poxvirus of claim 21, comprising a nucleic acid sequence having 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%, at least 99.91%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99%, or even 100% sequence identity to SEQ ID NO:

8.

23. At least one of five mutant parent oncolytic poxvirus strains RPX, CPX, COP, WY and WR, each having a deletion in the J2R locus, in which the oncolytic activity of the mutant chimeric poxvirus is measured for at least one tumor under the same conditions and at the same time post-infection, preferably a mutant parent COP or a mutant parent RPX, each having a deletion in the J2R locus, preferably a mutant parent oncolytic poxvirus strains RPX, CPX, COP, WY and WR, each having a deletion in the J2R locus. is greater than the oncolytic potency of at least two, more preferably the mutant parental COP and parental RPX strains defective in the J2R locus, more preferably at least three, more preferably at least four, and even more preferably, of the five mutant parental oncolytic poxvirus strains RPX, CPX, COP, WY and WR, each of which are defective in the J2R locus, and for a given tumor, a given virus, a given condition, and a given time post infection, the oncolytic potency OP(tumor, virus, condition, time post infection) is: OP (tumor, virus, condition, time post infection) = (100 - percentage of surviving tumor cells after infection with virus) The mutant chimeric poxvirus according to claim 21 or 22, defined as:

24. 24. The mutant chimeric poxvirus of claim 23, wherein the tumor is selected from A549, HCT116 and HepG2 tumor cell lines.

25. 25. The mutant chimeric poxvirus according to any one of claims 21 to 24, wherein for at least one healthy cell (preferably primary cell), the viral replication of said mutant chimeric poxvirus in healthy cells (preferably primary cell) is lower than the viral replication of at least one of five mutant oncolytic parental poxvirus strains deleted in the J2R locus, namely rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY) and vaccinia virus Western Reserve strain (WR), preferably the mutant parental COP deleted in the J2R locus, preferably at least two, more preferably at least three, more preferably at least four, and even more preferably of the five mutant parental oncolytic poxvirus strains deleted in the J2R locus RPX, CPX, COP, WY and WR.

26. The mutant chimeric poxvirus of claim 25, wherein the healthy cells (preferably primary cells) are selected from skin cells and liver cells.

27. Preferably, the therapeutic index of the chimeric poxvirus having a deletion in the J2R locus is measured for at least one organ under the same conditions and at the same time post-infection using at least one of five mutant oncolytic parent poxvirus strains having a deletion in the J2R locus, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), and vaccinia virus Western Reserve strain (WR). or a mutant parental COP defective in the J2R locus, preferably at least two, more preferably at least three, more preferably at least four, and even more preferably, of the five oncolytic mutant parental poxvirus strains RPX, CPX, COP, WY, and WR defective in the J2R locus, and for a given organ, a given tumor, a given virus, a given condition, and a given time post infection, the organ-specific therapeutic index TI (organ, tumor, virus, condition, time post infection) is: TI (organ, tumor, virus, condition, time post infection) = (viral replication in tumor cells of organ / viral replication in healthy cells of organ) The mutant chimeric poxvirus according to any one of claims 21 to 26, defined as:

28. 28. The mutant chimeric poxvirus of claim 27, wherein the organ is the liver, the healthy cells of the organ are healthy (preferably primary) hepatocytes, and the tumor cells of the organ are HepG2 tumor cells.

29. The extracellular enveloped virus (EEV) secretion capacity (SC) (EEV-SC) of the mutant chimeric poxvirus was measured for at least one producer cell (preferably a tumor cell) under the same conditions and at the same time post-infection with at least one of six mutant parental poxvirus strains defective in J2R, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR), and modified vaccinia virus Ankara strain (MVA), preferably a mutant parental COP defective in J2R or a mutant parental COP defective in J2R. or higher than the EEV-SC of a mutant parental RPX having a defect in J2R, preferably at least two of the six mutant parental poxvirus strains RPX, CPX, COP, WY, WR and MVA, more preferably a mutant parental COP and mutant parental RPX having a defect in J2R, more preferably at least three, more preferably at least four, even more preferably at least five of the six mutant parental poxvirus strains RPX, CPX, COP, WY, WR and MVA, which are defective in J2R, or their respective EEV-SC, for a given virus, a given producer cell, a given condition and a given time post infection, EEV-SC (virus, producer cells, conditions, time post-infection) = number of EEV particles / number of (EEV + IMV) particles The mutant chimeric poxvirus according to any one of claims 21 to 28, defined as:

30. 30. The mutant chimeric poxvirus of claim 29, wherein the producer cell is the A549 tumor cell line.

31. The mutant chimeric poxvirus according to any one of claims 21 to 30, wherein the extracellular enveloped virus (EEV) secretion capacity (SC) (EEV-SC) of the mutant chimeric poxvirus is higher than the EEV-SC of the wild-type vaccinia virus IHD-J strain measured under the same conditions and at the same time post-infection in at least one type of producer cell (preferably a tumor cell).

32. The mutant chimeric poxvirus of claim 31 , wherein the producer cell is the A549 tumor cell line.

33. The spreading ability of the mutant chimeric poxvirus was measured for at least one tumor under the same conditions and at the same time post-infection using at least one of six mutant parental poxvirus strains having a deletion in the J2R locus, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR), and modified vaccinia virus Ankara strain (MVA), preferably the mutant parental COP or mutant parental RPX, and preferably the J2R locus.

33. The mutant chimeric poxvirus according to any one of claims 21 to 32, which has a higher spreading capacity than at least two of the six mutant poxvirus strains RPX, CPX, COP, WY, WR and MVA that are defective in the R locus, more preferably at least three, more preferably at least four, more preferably at least five of the six mutant parental poxvirus strains selected from the mutant parental COP and RPX that are defective in the J2R locus, and even more preferably RPX, CPX, COP, WY, WR and MVA that are defective in the J2R locus, or each of them.

34. at least one of six mutant parental poxvirus strains deleted in the J2R locus, namely, rabbitpox virus Utrecht strain (RPX), cowpox virus Brighton strain (CPX), vaccinia virus Copenhagen strain (COP), vaccinia virus Wyeth strain (WY), vaccinia virus Western Reserve strain (WR) and modified vaccinia virus Ankara strain (MVA), in which the neutralization rate of the mutant chimeric poxvirus is measured for at least one poxvirus-specific antibody and tumor under the same conditions and at the same time post-infection; Preferably, the neutralization rate (NT(virus, tumor, condition, time post infection)) is lower than the neutralization rate of at least two, more preferably at least three, more preferably at least four, more preferably at least five of six mutant parent poxvirus strains selected from a mutant parent COP defective in the J2R locus, preferably RPX, CPX, COP, WY, WR and MVA, each of which is defective in the J2R locus, for a given virus, a given tumor, a given poxvirus-specific antibody, a given condition and a given time post infection. NT (virus, tumor, poxvirus-specific antibody, condition, time post-infection) = EC50 (with poxvirus-specific antibody) / EC50 (without poxvirus-specific antibody). The mutant chimeric poxvirus according to any one of claims 21 to 33, defined as:

35. The mutant chimeric poxvirus according to any one of claims 20 to 34, which is deficient in one or both of the I4L locus and the F4L locus.

36. 36. The mutant chimeric poxvirus of claim 35, wherein the poxvirus comprises a nucleic acid sequence having 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%, at least 99.91%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99%, or even 100% sequence identity to SEQ ID NO:

9.

37. The mutant chimeric poxvirus according to any one of claims 20 to 36, which has a deletion in the M2L locus.

38. A recombinant chimeric poxvirus or recombinant mutant chimeric poxvirus consisting of a chimeric poxvirus according to any one of claims 1 to 19 or a mutant chimeric poxvirus according to any one of claims 20 to 37, comprising one or more heterologous nucleic acids of interest inserted into its genome.

39. The recombinant chimeric poxvirus or recombinant mutant chimeric poxvirus according to claim 38, wherein the one or more nucleic acids of interest are selected from an immune checkpoint inhibitor, a cytokine, an agent that affects the regulation of cell surface receptors, an agent that affects angiogenesis, an agent that stimulates stem cells to produce granulocytes and / or macrophages, preferably wherein the nucleic acid of interest is a cytokine, more preferably an interleukin, even more preferably IL-12.

40. 1. A method for producing a chimeric poxvirus, a mutant chimeric poxvirus, a recombinant chimeric poxvirus, or a recombinant mutant chimeric poxvirus, comprising: (i) producing cells with the chimeric poxvirus of any one of claims 1 to 19, the mutant chimeric poxvirus of any one of claims 20 to 37, or the recombinant chimeric poxvirus or recombinant mutant chimeric poxvirus of any one of claims 38 and 39; (ii) culturing the infected producer cells under conditions suitable to allow the production of a chimeric poxvirus, a mutant chimeric poxvirus, a recombinant chimeric poxvirus, or a recombinant mutant chimeric poxvirus; and (iii) recovering the chimeric poxvirus, mutant chimeric poxvirus, recombinant chimeric poxvirus, or recombinant mutant chimeric poxvirus from the producer cell culture. The method comprising:

41. An isolated nucleic acid encoding a chimeric poxvirus according to any one of claims 1 to 19, a mutant chimeric poxvirus according to any one of claims 20 to 37, or a recombinant chimeric poxvirus or recombinant mutant chimeric poxvirus according to any one of claims 38 and 39.

42. A composition comprising a chimeric poxvirus according to any one of claims 1 to 19, a mutant chimeric poxvirus according to any one of claims 20 to 37, or a recombinant chimeric poxvirus or recombinant mutant chimeric poxvirus according to any one of claims 38 and 39, and a pharmaceutically acceptable vehicle.

43. The composition comprises: ５ ~5 x 10 ９ 43. The composition of claim 42, comprising a PFU dose of a chimeric poxvirus, a mutant chimeric poxvirus, a recombinant chimeric poxvirus, or a recombinant mutant chimeric poxvirus.

44. The composition of claim 42 or 43, wherein the chimeric poxvirus, mutant chimeric poxvirus, recombinant chimeric poxvirus or recombinant mutant chimeric poxvirus is formulated for parenteral route of administration, preferably intravenous route or intratumoral route.

45. A chimeric poxvirus according to any one of claims 1 to 19, a mutant chimeric poxvirus according to any one of claims 20 to 37, or a recombinant chimeric poxvirus or recombinant mutant chimeric poxvirus according to any one of claims 38 and 39, or a composition according to any one of claims 42 to 44, for use as a medicament.

46. A chimeric poxvirus according to any one of claims 1 to 19, a mutant chimeric poxvirus according to any one of claims 20 to 37, or a recombinant chimeric poxvirus or recombinant mutant chimeric poxvirus according to any one of claims 38 and 39, or a composition according to any one of claims 42 to 44, for use in the treatment of a proliferative disease.

47. The chimeric poxvirus, mutant chimeric poxvirus, recombinant chimeric poxvirus, recombinant mutant chimeric poxvirus or composition for use according to claim 46, wherein the proliferative disease is selected from cancer, tumor and restenosis, preferably the proliferative disease is selected from cancer.

48. 48. The chimeric poxvirus, mutant chimeric poxvirus, recombinant chimeric poxvirus, recombinant mutant chimeric poxvirus or composition for use according to claim 47, wherein the cancer is selected from lung cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer, colorectal cancer, liver cancer, gastric cancer, pancreatic cancer, melanoma, ovarian cancer and glioblastoma, in particular metastatic cancer.

49. The chimeric poxvirus, mutant chimeric poxvirus, recombinant chimeric poxvirus, recombinant mutant chimeric poxvirus or composition for use according to claim 47 or 48, wherein the cancer is refractory or resistant to a therapy based on at least one oncolytic virus, more preferably to a therapy based on at least one oncolytic poxvirus.

50. 50. The chimeric poxvirus, mutant chimeric poxvirus, recombinant chimeric poxvirus, recombinant mutant chimeric poxvirus or composition for use according to any one of claims 47 to 49, wherein said chimeric poxvirus or composition is administered in combination with one or more substances effective in anti-cancer therapy.

51. 10. A method for treating a disease in a subject in need thereof, comprising administering to said subject a chimeric poxvirus according to any one of claims 1 to 19, a mutant chimeric poxvirus according to any one of claims 20 to 37, or a recombinant chimeric poxvirus or recombinant mutant chimeric poxvirus according to any one of claims 38 and 39, or a composition according to any one of claims 42 to 44.

52. 52. The method of claim 51, wherein the proliferative disease is selected from cancer, tumor and restenosis, preferably the proliferative disease is selected from cancer.

53. 53. The method of claim 52, wherein the cancer is selected from lung cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer, colorectal cancer, liver cancer, gastric cancer, pancreatic cancer, melanoma, ovarian cancer and glioblastoma, in particular metastatic cancer.

54. 54. The method of any one of claims 51 to 53, wherein the cancer is refractory or resistant to at least one oncolytic virus-based therapy, more preferably to at least one oncolytic poxvirus-based therapy.

55. 55. The method of any one of claims 52 to 54, further comprising the administration of one or more substances effective in anti-cancer therapy.

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