The present invention is in the field of oncolytic viruses. The invention provides new poxviruses which are engineered to be defective for the function encoded by the M2L locus (i.e., m2 function). Such poxviruses lack a functional m2 binding activity to at least one or both of CD80 and CD86 co-stimulatory antigens. Said oncolytic poxviruses are preferably vaccinia virus having a total or partial deletion of the M2L locus. The present invention also relates to cells and compositions comprising such poxviruses and their use for treating proliferative diseases such as cancers and for preventing diseases (vaccination, especially in veterinary field). More precisely, the invention provides an alternative to the existing oncolytic viruses which are largely used in virotherapy. The m2-defective poxviruses are particularly useful for the expression of immunomodulatory polypeptides such as anti-CTLA-4 antibodies with the purposes of stimulating or improve immune response.

BACKGROUND ART

Each year, cancer is diagnosed in more than 12 million subjects worldwide. In industrialized countries, approximately one person out five will die of cancer. Although a vast number of chemotherapeutics exists, they are often ineffective, especially against malignant and metastatic tumors that establish at a very early stage of the disease.

Oncolytic virotherapy has been emerging for two decades based on replication-competent viruses to destroy cancer cells (Russell et al. , 2012, Nat. Biotechnol. 30(7): 658-70). Numerous preclinical and clinical studies are presently ongoing to assess in various types of cancers the therapeutic potential of oncolytic viruses armed with a variety of therapeutic genes.

Therapeutic genes are usually inserted in the viral genome within non-essential genes to retain oncolytic phenotype. Insertion in the J2R locus (tk) is widely used in the art insofar as it also facilitates identification of recombinant virus in the presence of BUdR (Mackett et al., 1984 J. of Virol., 49: 857-64; Boyle et al., 1985, Gene 35, 169-177). However, other loci have been also proposed, e.g. into Hind F fragment, into M2L locus (Smith et al., 1993, Vaccine 11 (1) : 43-53 ; Guo et al., 1990, J. Virol. 64: 2399-2406; Bloom et al., 1991 , J. Virol. 65(3): 1530-42; Hodge et al., 1994, Cancer Res. 54: 5552-5; McLaughlin et al., 1996, Cancer Res. 56: 2361-67) and A56R locus (encoding hemagglutinin (HA)).

Poxviruses and especially Vaccinia viruses (VV) have provided several promising oncolytic candidates (De Graaf et al., 2018, doi.org/10.1016/j.cytogfr.2018.03.006), such as JX594 (Sillajen/Transgene), GL-ONC1 (Genelux), TG6002 (Transgene) and vvDD-CDSR (University of Pittsburg). These oncolytic VV originate from different VV strains with diverse genomic modifications and expression of various therapeutic genes. JX-594 (Wyeth strain) attenuated through deletion of the viral J2R gene (which encodes thymidine kinase (tk)) and further armed with GM-CSF is currently under clinical evaluation in a randomized Phase III trial in hepatocellular carcinoma (Parato et al., 2012, Molecular Therapy 20(4): 749-58). GL-ONC1 was generated by inserting three expression cassettes respectively in place of the F14.5L, J2R and A56R loci of the parental viral Lister strain genome. On the same line, TG6002, a J2R (tk) and I4L (I4L locus encodes ribonucleotide reductase (rr-))-defective VV (Copenhagen strain) encoding the FCU1 enzyme that converts the non-toxic 5-fluorocytosine (5-FC) into the cytotoxic 5 fluorouracile (5-FU) is being evaluated in some clinical trials. The tk and rr double deletion restricts the replication of the virus to cells containing a high pool of nucleotides, making TG6002 unable to replicate in resting cells (Foloppe et al. , 2008, Gene Ther. 15: 1361-71 ; W02009/065546). vvDD-CDSR is currently assayed in patients with refractory cutaneous and subcutaneous tumors. It was engineered by double deletion of the tk (J2R locus) and vaccinia growth factor (vgf) encoding genes and armed with both a cytosine deaminase (CD) gene for conversion of 5-FC to 5-FU and a somatostatin receptor (SR) gene for in vivo imaging.

Initially, direct oncolysis was thought to be the sole mechanism through which oncolytic viruses exert their antitumor effect. Only recently, it was appreciated that the immune system plays a critical role in the success of virotherapy (Chaurasiya et al., 2018, Current Opinion in Immunology 51 : 83-90). However, most viruses have developed self-defense mechanisms through a repertoire of proteins involved in immune evasion and immune modulation aimed at blocking many of the strategies employed by the host to combat viral infections (Smith and Kotwal, 2002, Crit. Rev. Microbiol. 28(3): 149-85). Moreover, tumor cells have also evolved a mechanism of T cell exhaustion to escape host’s immune system, which is characterized by the upregulation of inhibitory receptors; CTLA-4 (for cytotoxic T-lymphocyte associated protein-4; also known as CD152) and PD-1 (for programmed cell death protein 1) and its ligands PD-L1 and PD-L2, being the most documented. These immunosuppressive receptors serve as immune checkpoints acting at different levels of T cell immunity. CTLA-4 inhibits early stages of T cell activation in the lymph node and also stimulates undesirable Treg while PD-1 acts at a later stage.

More specifically, activation of T cells involves the interaction of co-stimulatory ligands such as CD80 (also designated B7-1) and CD86 (also designated B7.2), present at the surface of the APC (for Antigen Presenting cell) with receptors present at the surface of T

cells such as CD28, CTLA-4 and PDL-1. CD80 is the ligand for these 3 cell surface receptors whereas CD86 binds CD28 and CTLA-4. CD28 receptor is constitutively expressed on resting T cells and ligation of CD28 with costimulatory CD80 and CD86 ligands delivers a positive stimulatory signal to T cells, induces them to proliferate and secrete IL-2 and inhibits apoptosis through increased expression of Bcl-XL (Chen, 2004, Nat. Rev. Immunol. 4: 336-347). In contrast, CTLA-4 or PD-L1 play a role in negative regulation of T cells either following initial T cell activation (for CTLA-4) or at a later stage (for PD-L1). Specifically, upon ligation with CD80 and CD86 costimulatory ligands, CTLA-4 acts in cis on activated T cells to oppose the co-stimulatory signal provided by interactions of CD28 with CD80 and CD86 and is involved in IL-10 production. In addition, CTLA-4 is constitutively expressed on a subset of immunosuppressive regulatory T cells (Treg). On the other hands, ligation of CD80 to PD-L1 on the surface of the T regulatory cells have been demonstrated to increase the proliferation of these immunosuppressive cells (Yi, 2011 , J Immunol. 186:2739-2749). CTLA4 was identified in 1987 (Brunet et al. , 1987, Nature 328: 267-70) and is encoded by the CTLA4 gene (Dariavach et al., Eur. J. Immunol. 18: 1901-5). The complete CTLA-4 nucleic acid sequence can be found under GenBank Accession No LI 5006.

There has been increasing interest in blocking such immunosuppressive checkpoints as a means of rescuing exhausted antitumor T cells. A vast number of antagonistic antibodies have been developed during the last decade (Kahn et al., 2015, J. Oncol. Doi: 10.1155/2015/847383) and several have been approved by the FDA of which the first were against CTLA4 (e.g. Ipilimumab / Yervoy, Bristol-Myers Squibb) and PD-1 (pembrolizumab / Keytruda developed by Merck and Nivolumab / Optivo developed by BMS). While conventional treatments rely on the administration of the antibodies to the patients, vectorization by virus or plasmid vectors is now being considered to deliver these antibodies directly to tumor cells (see e.g. WO2016/008976). For example, a tk- and rr- VV armed with anti-PD-1 was shown to induce tumor growth control in MCA-205 mouse model (Kleinpetter et al., 2016, Oncolmmunology 5(10): e1220467).

However, due to the complex nature of these immunity-interacting molecules and virus vectors and the risk of triggering cascade events, preclinical and even more clinical studies may be difficult to implement.

Therefore, there is still a need to further develop oncolytic viruses, compositions and methods for delivering therapeutic polypeptides such as checkpoint-directed antagonist antibodies for enhancing anti-tumoral adaptative immune responses in cancerous patients.

TECHNICAL PROBLEM AND PROPOSED SOLUTION

Unexpectedly, the inventors have identified that supernatants of cells infected with vaccinia virus (VV) interact with the co-stimulatory CD80 and CD86 ligands whereas supernatants of cells infected with the attenuated Modified Vaccinia virus Ankara (MVA) lack this property. The inventors have assigned the CD80 and CD86 binding properties to the M2 protein encoded by the VV M2L locus. Before the invention, M2 was reported as a protein retained in endoplasmic reticulum acting as an inhibitor of the NfKb pathway (Hinthong et al., 2008, Virology 373(2): 248-62) and involved in uncoating of the virus (Baoming Liu et al., 2018, J. Virol. 92(7) e02152-17). Further to VV, the inventors have identified the existence of M2 orthologs in numerous replicative poxviruses.

The present invention illustrates the capacity of the M2 protein of binding to CD80 and CD86 and impacting three immunosuppressive pathways; respectively i) it blocks the CD80 and CD86 interactions with CD28; ii) it promotes the interaction of CD80 with PD-L1 ; and iii) it triggers a reverse signalling to the CD80/CD86 positive cells.

In the context of the present invention, the inventors have generated a vaccinia virus that is defective for m2 function. When armed with an immunomodulatory polypeptide such as an anti-CTLA-4 antibody, its expression inhibits the CTLA-4-mediated immunosuppressive signals and it is expected that the absence of m2 permits to redirect the T cell response to the CD28-mediated immunostimulatory signals whereas a M2L-positive vaccinia virus would negatively interfere with such CD28-mediated positive signals due to the m2 binding to CD80 and CD86 co-stimulatory ligands.

Importantly and surprisingly poxviruses described herein are expected to stimulate or improve immune response, especially the lymphocyte-mediated response, against an antigen due to the absence of synthesis of a functional m2 protein in the infected cells whereas in a conventional poxvirus (M2L-positive), the produced viral m2 protein would bind CD80 and CD86 co-stimulatory ligands and, thus, prevent CD28-mediated positive pathways. Moreover, poxviruses described herein display an enhanced propensity to be accepted by the host’s immune system since they lack a protein involved in immune evasion of the virus; which feature provides a competitive advantage over M2-positive poxviruses. The present invention offers a unique product combining oncolysis for killing dividing cells and immunostimulatory activities, e.g. for breaking cancer-associated immune exhaustion, thus improving therapeutic capacities of the oncolytic virus.

This technical problem is solved by the provision of the embodiments as defined in the claims. Other and further aspects, features and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.

SUMMARY OF THE INVENTION

The disclosure relates to poxviruses, especially oncolytic poxviruses, that have been engineered to be defective for the m2 protein encoded by the M2L locus, and methods of generating and using such viruses. As disclosed herein, poxviruses defective for the m2 function encoded by the M2L locus, optionally in combination with other functional inactivation(s) of the tk-encoding locus and/or rr-encoding locus were generated and isolated. m2-defective vaccinia virus engineered to express an anti-CTLA4 antibody are also comtemplated.

According to a first aspect of the present invention, there is provided a modified poxvirus which genome comprises in the native (wild-type) context a M2L locus encoding a functional m2 poxviral protein and which is modified to be defective for the said m2 function; wherein said functional m2 poxviral protein is able to bind CD80 or CD86 co-stimulatory ligands or both CD80 and CD86 co-stimulatory ligands and wherein said defective m2 function is unable to bind said CD80 and CD86 co-stimulatory ligands.

In one embodiment, the modified poxvirus is generated or obtained from a

Chordopoxvirinae, preferably selected from the group of genus consisting of Avipoxvirus, Capripoxvirus, Lepori poxvirus, Molluscipoxvirus, Orthopoxvirus, Parapoxvirus, Suipoxvirus, Cervidpoxvirus and Yatapoxvirus. In a preferred embodiment, said modified poxvirus is a member of the Orthopoxvirus, preferably selected from the group consisting of vaccinia virus (VV), cowpox (CPXV), raccoonpox (RCN), rabbitpox, Monkeypox, Horsepox, Volepox, Skunkpox, variola virus (or smallpox) and Camelpox; with a specific preference for a modified vaccinia virus.

In one embodiment, the inability to bind said CD80 and CD86 co-stimulatory ligands originates from a genetic lesion within the M2L locus or from an abnormal interaction impairing the m2 function either directly or indirectly. Said genetic lesion(s) include partial or total deletion and/or one or more non-silent mutation(s) (that translates in a change of amino acid residue(s)) either within the m2-coding sequence or in the regulatory elements controlling M2L expression, preferably leading to the synthesis of a defective m2 protein or to the lack of m2 synthesis. Said genetic lesion is preferably a partial or entire deletion of the M2L locus.

In one embodiment, the modified poxvirus is further modified in a region other than

M2L locus; in particular in the J2R locus (resulting in a modified poxvirus defective for both m2 and tk functions) or in the I4L/F4L locus/loci (resulting in a modified poxvirus defective for both m2 and rr functions). Preferably, the modified poxvirus is further modified in the J2R and I4L/F4L loci, resulting in a modified poxvirus defective for m2, tk and rr activities.

In one embodiment, the modified poxvirus is oncolytic.

In one embodiment, said modified poxvirus is recombinant. Said modified poxvirus is preferably engineered to express at least one polypeptide selected from the group consisting of antigenic polypeptides, polypeptides having nucleos/tide pool modulating function and immunomodulatory polypeptides. Said immunomodulatory polypeptide is desirably selected from the group consisting of cytokines, chemokines, ligands and antibodies or any combination thereof. In a preferred embodiment, said modified poxvirus is defective for m2, tk and rr activities and encodes an anti-CTLA-4 antibody. In another preferred embodiment, said modified poxvirus is defective for m2, tk and rr activities and encodes an anti-PD-L1 antibody.

According to another aspect, there is provided a method for producing the modified poxvirus comprising the steps of a) preparing a producer cell line, b) transfecting or infecting the prepared producer cell line with the modified poxvirus, c) culturing the transfected or infected producer cell line under suitable conditions so as to allow the production of the virus, d) recovering the produced virus from the culture of said producer cell line and optionally e) purifying said recovered virus.

According to a further aspect, there is provided a composition comprising a therapeutically effective amount of the modified poxvirus and a pharmaceutically acceptable vehicle. The composition desirably comprises from approximately 103 to approximately 1012 pfu, advantageously from approximately 104 pfu to approximately 1011 pfu, preferably from approximately 105 pfu to approximately 101° pfu; and more preferably from approximately 106 pfu to approximately 109 pfu of the modified poxvirus. The composition is preferably formulated for intravenous or intratumoral administration.

In still another aspect, the composition is for use for treating or preventing a proliferative disease selected from the group consisting of cancers as well as diseases associated to an increased osteoclast activity such as rheumatoid arthritis and osteoporosis and cardiovascular diseases such as restenosis. The cancer to be treated or prevented is preferably selected from the group consisting of renal cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, gastric cancer, bile duct carcinoma, endometrial cancer, pancreatic cancer and ovarian cancer. The modified poxvirus and composition is for use as stand-alone therapy or in conjunction with one or more additional therapies, preferably selected from the group consisting of surgery, radiotherapy, chemotherapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy, cytokine therapy, targeted cancer therapy, gene therapy, photodynamic therapy and transplantation.

In still another aspect, the modified poxvirus or composition is for use for stimulating or improving an immune response.

DESCRIPTION OF THE FIGURES

Figure 1 illustrates CD80/CTLA4 (1A) and CD86/CTLA4 (1 B) competition ELISA assays carried out with the supernatants collected from avian DF1 cells either uninfected (dotted line) or infected with wild type VV (diamond) or Yervoy (inverted triangle). Binding of His-tagged B7-Fc proteins to immobilized CTLA4-Fc was performed using an anti-His tag-HRP conjugated antibody.

Figure 2 illustrates CD80/CTLA4 competition ELISA carried out with the supernatants collected from HeLa cells infected with MVA (MVA), vaccinia virus of Copenhagen strain (Cop VV), Western Reserve strain (WR VV), Wyeth strain (Wyeth VV), raccoonpox (RCN), rabbitpox (RPX), cowpox (CPX), fowlpox (FPV) and pseudocowpox (PCPV) and the supernatant of uninfected HeLa cells (negative control).

Figure 3 illustrates western blot performed in non-reducing SDS-PAGE with supernatants of CEF cells either uninfected (Sup. cells) or infected with MVA (Sup. MVA) or Copenhagen vaccinia virus (Sup.VV) collected directly or 20-fold concentrated (x20) and probed with fusions of human CD86 with Fc fragment (hCD86-Fc), human CD80 with Fc fragment (hCD80-Fc) and human CTLA4 with Fc fragment (hCTLA4-Fc). Detection was performed with an anti-Fc conjugated antibody.

Figure 4 illustrates competition ELISA testing the interaction of biotinylated-CD80 and biotinylated-CD86 with their cognate receptors, CD28/CD86, CD28/CD80, CTLA4/CD80 and PDL1/CD80 respectively. Supernatants collected from CEF cells infected with MVA (MVA) and vaccinia virus of Copenhagen strain (VV) are compared to the supernatant of uninfected CEF cells (CEF) (negative control) and Yervoy antibody (10pg/ml). Reactivity of recombinant human PD1 (hPD1), human CD80 (hCD80) and human CTLA4 (hCTLA4) all at 10pg/ml are used as positive control for competing with the PDL1/CD80 interaction. Detection of the bound biotinylated B7 proteins was performed using HRP conjugated streptavidin.

Figure 5A illustrates the experimental approach used to identify the“interference factor (IF)” by affinity chromatography with immobilized CD86-Fc fusion and Figure 5B provides the sequence of the IF captured in VV-infected CEF cells.

Figure 6 illustrates CD80/CTLA4 competition ELISA carried out with the supernatants collected from uninfected HeLa or DF1 cells (HeLa or DF1) as negative controls or infected with a double deleted (tk- rr-) Copenhagen vaccinia virus (VVTG18277) or a triple deleted (tk-rr- m2-) Copenhagen vaccinia virus (COPTG19289). Binding of his-tagged CD80-Fc proteins to immobilized CTLA4-Fc was monitored using an anti-His tag- HRP conjugated antibody.

Figure 7 illustrates oncolytic activity of the tk- rr- m2- vaccinia virus (COPTG19289) and its tk- rr- counterpart (VVTG18277) four days after infection of LOVO (A) and HCT116 (B) cells at various MOI (from 10 1 to 104). MOCK-treated cells are used as negative control.

Figure 8 illustrates luciferase expression in C57BL/6 mice subcutaneously implanted with B16F10 tumors. VVTG18277 virus and COPTG19289 (107 pfu) were injected intratumorally at day 0, 3, 6, 10 and 14 and tumor samplings were collected at day 1 , 2, 6, 9, 13 and 16 for evaluation of luciferase activity par gram of tumor (RLU/g tumor). Three mice were included by time point.

Figure 9 illustrates antitumoral activity in Balb/c mice subcutaneously implanted with CT26 tumors. 107 pfu of VVTG18277 (square), COPTG19289 (triangle) or Mock (circle) were injected intratumorally at DO, D3, D6, D10 and D14 (10 mice/group). Tumor growth was followed twice a week (mice were killed when the tumor volume reached 2000 mm3).

Figure 10 illustrates antitumoral activity in Swiss Nude mice subcutaneously implanted with HT116 tumors. Mice (10 mice/group) received a single intravenously injection at D10 when tumor reached 100 to 200mm3 of either 105 (A) or 107 (B) pfu of VVTG18277 (circle), COPTG19289 (square) or Mock (diamond). Tumor growth was followed twice a week.

Figure 11 illustrates the effect of supernatant of cells infected by M2 defective poxvirus on mixed lymphocyte reaction (MLR). PBMC were purified from two different donors and cultured in the presence of supernatants obtained from CEF infected (MOI 0.05) with COPTG19289 (tk-, rr- and m2-), VVTG18058 (tk- rr-) or MVAN33 (wild type). Culture supernatants were harvested 48h post-infection and concentrated about 20-fold. These concentrated supernatants were added to the PBMC culture (20pL in 200pL) either undiluted or diluted 10 or 100-fold to yield a final“supernatant concentration” of 2, 0.2 and 0.02 -fold, respectively. The amount of IL-2 secreted in the culture medium of PBMC was measured by ELISA. IL-2 measurement was made in triplicate for each sample tested. The measures were normalized by dividing the mean of IL-2 concentration of the

three replicates of a given sample by the mean of IL-2 concentration of the three replicates of PBMC incubated with medium.

Figure 12 illustrates the effect on tumor volume provided by the M2 defective COPTG19289 in a humanized mouse model. NOD/Shi-scid/IL-2Rynull immunodeficient mice (NCG) were humanized with CD34+ human stem cells and engrafted with human colorectal carcinoma cells HCT-1 16 (5x106 cells injected SC in one mouse’s flank; representing DO). Twelve days post implantation (D12), mice received a single IV injection of either COPTG 19289 (TD) or the m2+ counterpart VVTG 18058 (DD) at doses of 106 pfu (A) or 105 pfu (B). Vehicle-treated mice were used as negative controls. Tumor growth were monitored over 60 days post cell implantation. Mean tumor growth in mm3 is represented for each group as a function of the number of days post cell injection.

Figure 13 illustrates the effect on survival provided by the M2 defective COPTG19289 in the humanized NCG-CD34+ mouse model described above. Twelve days post tumor implantation (D12), mice received a single IV injection of either COPTG19289 (TD) or the m2+ counterpart VVTG18058 (DD) at doses of 106 pfu (A) or 105 pfu (B). Vehicle-treated mice were used as negative controls. Mice survival were monitored over 90 days post cell implantation. Survival (percent) is given for each group as a function of the number of days post cell injection.

DETAILED DESCRIPTION

GENERAL DEFINITIONS

A number of definitions are provided here that will assist in the understanding of the invention. However, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All references cited herein are incorporated by reference in their entirety.

As used throughout the entire application, the terms "a" and "an" are used in the sense that they mean "at least one", "at least a first", "one or more" or "a plurality" of the referenced components or steps, unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

The term“one or more” refers to either one or a number above one (e.g. 2, 3, 4, 5, etc).

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

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

As used herein, when used to define products, compositions and methods, the term

"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 elements or method steps. Thus, a polypeptide "comprises" an amino acid sequence when the amino acid sequence might be part of the final amino acid sequence of the polypeptide. "Consisting of" means excluding other components or steps of any essential significance. Thus, a composition consisting of the recited components would not exclude trace contaminants and pharmaceutically acceptable carriers. A polypeptide "consisting of” an amino acid sequence refers to the presence of such an amino acid sequence with optionally only a few additional and non-essential amino acid residues. It is nevertheless preferred that the polypeptide does not contain any amino acids but the recited amino acid sequence. In the present description, the term“comprising” (especially when referring to a specific sequence) may be replaced with “consisting of”, if required.

Within the context of the present invention, the terms“nucleic acid”,“nucleic acid molecule”,“polynucleotide” and“nucleotide sequence” are used interchangeably and define a polymer of any length of either polydeoxyribonucleotides (DNA) (e.g. cDNA, genomic DNA, plasmids, vectors, viral genomes, isolated DNA, probes, primers and any mixture thereof) or polyribonucleotides (RNA) (e.g. mRNA, antisense RNA, SiRNA) or mixed polyribo-polydeoxyribonucleotides. They encompass single or double-stranded, linear or circular, natural or synthetic, modified or unmodified polynucleotides.

The term“polypeptide” is to be understood to be a polymer of at least nine amino acid residues bonded via peptide bonds regardless of its size and the presence or not of post-translational components (e.g. glycosylation). No limitation is placed on the maximum number of amino acids comprised in a polypeptide. As a general indication, the term refers to both short polymers (typically designated in the art as peptide) and to longer polymers (typically designated in the art as polypeptide or protein). This term encompasses native polypeptides, modified polypeptides (also designated derivatives, analogs, variants or mutants), polypeptide fragments, polypeptide multimers (e.g. dimers), fusion polypeptides among others. The term also refers to a recombinant polypeptide expressed from a polynucleotide sequence which encodes said polypeptide. Typically, this involves translation of the encoding nucleic acid into a mRNA sequence and translation thereof by the ribosomal machinery of the cell to which the polynucleotide sequence is delivered.

The term“identity” refers to an amino acid to amino acid or nucleotide to nucleotide correspondence between two polypeptide or nucleic acid sequences. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps which need to be introduced for optimal alignment and the length of each gap. Various computer programs and mathematical algorithms are available in the art to determine the percentage of identity between amino acid sequences, such as for example the Blast program available at NCBI or ALIGN in Atlas of Protein Sequence and Structure (Dayhoffed, 1981 , Suppl., 3: 482-9), or the algorithm of Needleman and Wunsh (J.Mol. Biol. 48,443-453, 1970). Programs for determining identity between nucleotide sequences are also available in specialized data base (e.g. Genbank, the Wisconsin Sequence Analysis Package, BESTFIT, FASTA and GAP programs). Those skilled in the art can determine appropriate parameters for measuring alignment including any algorithms needed to achieve maximum alignment over the sequences to be compared. For illustrative purposes,“at least 70%” means 70% or above (including 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% whereas“at least 80% identity” means 80% or above (including 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% and“at least 90%” 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%).

As used herein, the term“isolated” refers to a component (e.g. polypeptide, nucleic acid molecule, virus, vector, etc.), that is removed from its natural environment (i.e. separated from at least one other component(s) with which it is naturally associated or found in nature). For example, a nucleotide sequence is isolated when it is separated of sequences normally associated with it in nature (e.g. dissociated from a genome) but it can be associated with heterologous sequences.

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

As used herein, the term “host cell” should be understood broadly without any limitation concerning particular organization in tissue, organ, or isolated cells. Such cells may be of a unique type of cells or a group of different types of cells such as cultured cell lines, primary cells and dividing cells. In the context of the invention, the term“host cells” preferably refers to eukaryotic cells such as mammalian (e.g. human or non-human) cells as well as

cells capable of producing the poxvirus described herein. This term also includes cells which can be or has been the recipient of the poxvirus as well as progeny of such cells.

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

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

The term“administering” (or any form of administration such as“administered”) as used herein refers to the delivery to a subject of a therapeutic agent such as the poxvirus described herein.

The term“combination” or“association” as used herein refers to any arrangement possible of various components (e.g. a poxvirus and one or more substance effective in anticancer therapy). Such an arrangement includes mixture of said components as well as separate combinations for concomitant or sequential administrations. The present invention encompasses combinations comprising equal molar concentrations of each component as well as combinations with very different concentrations. It is appreciated that optimal concentration of each component of the combination can be determined by the artisan skilled in the art.

M2-defective poxvirus

In one aspect, the present invention provides a modified poxvirus which genome comprises in the native (wild-type) context a M2L locus encoding a functional m2 poxviral protein and which is modified to be defective for the said m2 function; wherein said functional m2 poxviral protein is able to bind CD80 or CD86 co-stimulatory ligands or both CD80 and

CD86 co-stimulatory ligands and wherein said defective m2 function is unable to bind said CD80 and CD86 co-stimulatory ligands..

As used herein, the term “poxvirus” or“poxviral” refers to any Poxviridae virus identified at present time or being identified afterwards that is infectious for one or more mammalian cells (e.g. human cells) and which genome comprises in the native (i.e. wild-type) context a M2L locus encoding a functional so-called M2 protein. The term“virus” as used in the context of poxvirus or any other virus mentioned herein encompasses the viral genome as well as the viral particle (encapsided and/or enveloped genome).

Poxviruses are a broad family of DNA viruses containing a double-stranded genome. Like most viruses, poxviruses have developed self-defence mechanisms through a repertoire of proteins involved in immune evasion and immune modulation aimed at blocking many of the strategies employed by the host to combat viral infections (Smith and Kotwal, 2002, Crit. Rev. Microbiol. 28(3): 149-85). Typically, the poxvirus genome encodes more than 20 host response modifiers that allow the virus to manipulate host immune responses and, thus, facilitate virus replication, spread, and transmission. These include growth factors, anti-apoptotic proteins, inhibitors of the NFkB pathway and interferon signalling, and down-regulators of the major histocompatibility complex (MHC).

For general guidance, the wild type vaccinia virus (VV) genome comprises a M2L locus which coding sequence encodes a protein called m2 produced during the early stage of the virus life cycle. It is either secreted or localized in the reticulum endoplasmic (RE) and likely glycosylated (Hinthong et al., 2008, Virology 373: 248-262). Although its function is still under investigation, it is involved in core uncoating and viral DNA replication (Liu et al., 2018, J. Virol., doi/10.1128/JVI.02152-17) but it is dispensable for in vitro viral replication (Smith, 1993, Vaccine 11 : 43-53). In addition, its function of downregulating the cellular NF-KB transcription factor via Erk1 phosphorylation inhibition is now well established (Gedey et al., 2006, J. Virol. 80: 8676-85) suggesting that m2 is thus involved in the host’s antiviral response during poxviral infection. The VV“M2L” locus is present in the 5’ third part of the wild-type VV genome; specifically, the coding sequence is located between position 27324 and position 27986 of the Copenhagen (Cop) VV genome. The Cop M2L-encoded gene product is a protein of 220 amino acids (having the amino acid sequence shown in SEQ ID NO: 1 ; also disclosed in Uniprot under P21092 accession number) and composed of a mature polypeptide long of 203 amino acid residues including 8 Cys residues and a N-term 17 amino acid residue long signal peptide also having one Cys residue.

The poxvirus genome in the native context is a double-stranded DNA of approximately 200kb and has the potential of encoding nearly 200 proteins with different functions including a M2L locus. The genomic sequence and the encoded open reading frames (ORFs) are well known. The modified poxvirus of the invention comprises a genome which has been modified

by the man’s hands to be at least defective for the m2 function encoded by a native M2L locus and may further comprise one or more additional modifications such as those described herein.

Identification of the presence of a M2L locus within a poxviral genome

Determination if a given poxvirus comprises or not in the native context a M2L locus encoding a functional m2 protein is within the reach of the skilled artisan using the information given herein and the general knowledge in the art. The particular choice of assay technology is not critical and it is within the reach of those skilled in the art to adapt any of these conventional methodologies to the determination if a candidate poxvirus comprises a M2L locus encoding a functional m2 protein.

In one embodiment, a M2L locus can be identified in a given poxvirus by hybridization or PCR techniques using the information given herein and designing appropriate probes or primers to screen the poxviral genomic sequence. For general guidance, hybridization assays are typically based on oligonucleotide probes derived from the known nucleotide (nt) sequence information set forth herein for M2L locus to be detected with nucleic acids extracted from cells infected or containing such a candidate poxvirus, under conditions suitable for hybridization. Oligonucleotide probe is a short piece of single-stranded RNA or DNA (usually 10 to 30 nucleotides long) that is designed to be complementary (i.e. at least 80% identity) to the target M2L sequence. Probes are preferably labeled to permit detection (e.g. a radioactively, fluorescently or enzymatically-labeled probes). Hybridization is usually performed under stringent conditions allowing only specific hybrids to be formed.

In still another or alternative embodiment, the presence of a M2L locus in the genome of a given poxvirus can be identified based on the amino acid sequence of the encoded gene product. For example, the presence of a M2L locus can be identified by translational analysis of the genomic sequence and blasting the amino acid sequences of the encoded open reading frames (ORFs) in available databases against the known poxviral m2 proteins such as the Cop VV m2 (SEQ ID NO: 1) or the myxoma virus gp-120 like protein (SEQ ID NO: 2) to search for the presence of an encoded ORF displaying at least 40%, desirably at least 50%, preferably, at least 70%, more preferably at least 80% and as an absolute preference at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or in SEQ ID NO: 2.

Alternatively or in addition, the amino acid sequences of the ORFs encoded by the poxviral genome can be aligned against available databases. The candidate poxvirus is considered as comprising a M2L locus if it encodes a so-called m2 polypeptide family which gives an outcome after search in domain databases (e.g, Gene3D, PANTHER, Pfam, PIRSF,

PRINTS, ProDom, PROSITE, SMART, SUPERFAMILY or TIGRFAMs) which is the same as the outcome of the m2 VV protein (referenced in Uniprot under accession number P21092; also disclosed herein as SEQ ID NO: 1). Therefore, a candidate poxvirus is identified as comprising a M2L locus if it encodes a polypeptide which, when submitted to a Blast analysis using the above-cited databases, is assigned in Uniprot a PFAM motif n°PF04887 or an Interpro motif n° IPR006971 signature.

Functionality of the encoded m2 protein.

A functional m2 protein as used herein refers to the capacity of said protein of binding CD80 and/or CD86 co-stimulatory ligands either in vitro or in vivo. The ability of a poxvirus to encode a functional m2 polypeptide can be evaluated by routine techniques. Standard assays to evaluate the binding ability of a protein to its target are known in the art, including for example, Biacore™, calorimetry, fluorometry, Bio-Layer Interferometry, Immunoblot (e.g. Western blot), RIAs, flow cytometry and ELISAs. The particular choice of assay technology is not critical and it is within the reach of those skilled in the art to adapt any of these conventional methodologies to determine if a candidate m2 protein binds to CD80 and/or CD86 co-stimulatory ligands.

For example, supernatants of cells infected with the candidate poxvirus can be used to probe CD80 or CD86 either immobilized on plate (ELISA) or displayed on cell surface (FACS). Sandwich competition ELISA assays (see the Example section) are particularly appropriate due to the fact that there is no need to generate a tagged recombinant protein to get a result. For example, ELISA plates may be coated with a ligand of interest (e.g. CD86-Fc) before adding the sample to be tested (e.g. a supernatant of cells infected with a poxvirus). If the sample comprises a M2 polypeptide, it will bind to the coated ligand. Then, a detection ligand is added which is usually labelled to be detected, e.g. by the action of an enzyme that converts the labelling substance into a coloured product which can be measured using a plate reader (e.g. CTLA4-Fc with a Histag recognized by anti-Histag antibodies coupled to HRP (for horseradish peroxidase). A reduction of chromogenic detection in the presence of a candidate sample as compared to no sample or a negative control sample is indicative that the sample contains a M2 polypeptide competing with the detection ligand for binding to the coated ligand. One may also proceed vice versa, e.g. by using CTLA-4-Fc as coated ligand and CD80-Fc-Histag as detection ligand.

“Defective for m2 function” as used herein is intended to mean the inability of a m2 protein to bind CD80 and/or CD86 co-stimulatory ligands either in vitro or in vivo. This inability may originate from a genetic lesion within the native M2L locus that prevents the normal binding activity of the encoded m2 protein. Thus, functional inactivation could result from one or more mutation(s) in the M2L locus. Such a mutation is preferably selected from the group consisting of insertions, deletions, and base changes in either the coding sequence or in the regulatory sequences controlling expression of the m2 protein. Alternatively, functional inactivation may occur by the abnormal interaction of the m2 protein with one or more other gene products which bind to or otherwise prevent the functional activity of said m2 protein.

For general guidance, the inventors have indeed identified a M2L locus (encoding a functional m2 protein or ortholog thereof) in a vast variety of poxviruses as described hereinafter; more specifically in seven strains of vaccinia virus, in seven strains of myxoma virus, in 4 strains of Monkeypox, in multiple strains of cowpox virus, in eight strains of variola virus as well as in a variety of other poxviruses including, but not limited to, Horsepox, Taterapox, Camelpox, Raccoonpox, Shunkpox, Yokapox, Rabbit fibroma virus, Murmansk pox, Eptesipox, Deerpox, Tanapox, Cotia virus and Volepox. For illustrative purposes, the encoded M2 protein orthologs of Horsepox, Variola virus, Monkeypox, Camelpox, cowpox display more than 90% identity with the reference Cop m2 protein (as represented by SEQ ID NO: 1) and those of myxoma, Skunk, Cotia and Volepox viruses shows respectively 50%, 74%, 70% and 72% sequence identity with the CopVV m2 protein, as illustrated in Table 1.

Table 1 provides an overview of the Genbank’s accession numbers for the genomic sequences of various poxviruses comprising a M2L locus in the native context and an indication of the amino acid identity of their m2 protein with respect to Cop m2 protein (Uniprot’s accession number P21092 and SEQ ID NO: 1).

For sake of clarity, the gene nomenclature used herein to designate the poxviral M2L locus and the encoded m2 protein is that of vaccinia virus (and more specifically that of Copenhagen strain). It is also used herein for other poxviruses containing functionally equivalent M2L genes and m2 proteins to those referred herein unless otherwise indicated. Indeed, gene and respective gene product nomenclature may be different according to the poxvirus families, genus and strains but correspondences between vaccinia virus and other poxviruses are generally available in the literature. For illustrative purposes, equivalents of the VV M2L gene is designated M154L in myxoma’s genome, CPXV040 or P2L in cowpox genome, 02L in Monkeypox genome, RPXV023 in rabbitpox genome and 02L or Q2L in variola virus genome.

However, the genome of a few poxviruses, such as the attenuated vaccinia virus MVA (Modified vaccinia virus Ankara) and the pseudocowpox virus (PCPV), in the native context, lacks a M2L locus (Antoine et al., 1998, Virology 244(2) 365-96) due to the large genomic deletions having occurred during the attenuation process. In the context of the invention, the term “poxvirus” does not include poxviruses which in the native context have genomic deletion(s) or mutation(s) encompassing M2L locus (or equivalent) which thus, lack a m2 polypeptide or encode a non-functional m2 protein such as Pseudocowpox virus (PCPV), MVA and NYVAC virus.

In one embodiment, the modified poxvirus of the present invention is generated or obtained from a Chordopoxvirinae, preferably selected from the group of genus consisting of Avipoxvirus, Capripoxvirus, Lepori poxvirus, Molluscipoxvirus, Orthopoxvirus, Parapoxvirus, Suipoxvirus, Cervidpoxvirus and Yatapoxvirus. Genomic sequences of these poxviruses are available in the art, notably in specialized databases such as Genbank or Refseq.

In a preferred embodiment, the modified poxvirus is generated or obtained from an Orthopoxvirus. Although any orthopox may be used, it is preferably selected from the group consisting of vaccinia virus (VV), cowpox (CPXV), raccoonpox (RCN), rabbitpox, Monkeypox, Horsepox, Volepox, Skunkpox, variola virus (or smallpox) and Camelpox. Particularly

preferred is a vaccinia virus. Any vaccinia virus strain is appropriate in the context of the present invention (except MVA) including, without limitation, Western Reserve (WR), Copenhagen (Cop), Lister, LIVP, Wyeth, Tashkent, Tian Tan, Brighton, Ankara, LC16M8, LC16M0 strains, etc., with a specific preference for Lister, WR, Copenhagen and Wyeth strains. Genomic sequences thereof are available in the literature and Genbank (e.g. under accession numbers AY678276 (Lister), M35027 (Cop), AF095689 1 (Tian Tan) and AY243312.1 (WR). These viruses can also be obtained from virus collections (e.g. ATCC VR-1354 for WR, ATCC VR-1536 for Wyeth and ATCC VR-1549 for Lister).

In another embodiment, the modified poxvirus is generated or obtained from the Leporipoxvirus genus, with a preference for myxoma virus (which genomic sequences are disclosed in Genbank under accession number NP_051868.1). The M2L ortholog locus in myxoma virus is designated M154L locus and encodes a so-called gp120-like protein having the amino acid sequence shown in SEQ ID NO: 2 and displaying 50% identity with Cop-encoded m2 protein (SEQ ID NO: 1).

Defective m2 function

As described above, the unability of a m2 protein to bind CD80 and/or CD86 co stimulatory ligands may originate from a genetic lesion in the M2L locus or from an abnormal interaction impairing the m2 function either directly or indirectly. Specifically, a "defective m2 function" refers to a reduced capacity by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or even total inability to bind CD80 (e.g. human) and CD86 (e.g. human) as compared to a native m2 protein (e.g. as found in supernatants of cells infected with a m2-positive poxvirus) as measured by a conventional assay such as competition ELISA assay.

A modified poxvirus may be engineered so as to be defective for m2 function by a number of ways known to those skilled in the art using conventional molecular techniques. In a preferred embodiment, the modified poxvirus comprises at least one genetic lesion in the native M2L locus that results in supressed expression of the m2 protein by the virus. Such genetic lesion(s) include partial or total deletion and/or one or more non-silent mutation(s) (that translates in a change of amino acid residue(s)) either within the m2-coding sequence or in the regulatory elements controlling M2L expression. Said genetic lesion(s) preferably lead(s) to the synthesis of a defective m2 protein (unable to ensure the activity of the native protein as described above) or to the lack of m2 synthesis (no protein at all). For example, said genetic lesion is a partial or entire deletion of the M2L locus, e.g. a partial deletion extending from upstream the m2 coding sequences to at least 100 codons of the m2 coding

sequence. Alternatively, or in combination, the M2L locus can be modified by point mutation (e.g. introduction of a STOP codon within the coding sequence), frameshift mutation (so as to modify the reading frame), insertional mutation (by insertion of one or more nucleotide(s) that disrupt the coding sequence) or by deletion or substitution of one or more residues involved in or responsible for the CD80 and/or CD86 binding function or any combination thereof. Also, a foreign nucleic acid can be introduced within the coding sequence to disrupt the m2 open reading frame. Also, the gene promoter can be deleted or mutated, thus inhibiting M2L expression. A person skilled in the art, based on the present disclosure would readily determine if a particular modification functionally inactivates m2, by comparing the wild-type and the mutated m2 protein for their ability to bind CD80 and/or CD86 as illustrated in the Example section.

Other Poxvirus modifications

In one embodiment, the modified poxvirus of the present invention is further modified in a region other than M2L locus. Various additional modifications can be contemplated in the context of the invention.

WE CLAIMS

1. A modified poxvirus which genome comprises in the native (wild-type) context a M2L locus encoding a functional m2 poxviral protein and which is modified to be defective for the said m2 function; wherein said functional M2 poxviral protein is able to bind

CD80 or CD86 co-stimulatory ligands or both CD80 and CD86 co-stimulatory ligands and wherein said defective m2 function is unable to bind said CD80 and CD86 co stimulatory ligands.

2. The modified poxvirus of claim 1 , wherein the modified poxvirus is generated or obtained from a Chordopoxvirinae, preferably selected from the group of genus consisting of Avipoxvirus, Capripoxvirus, Leporipoxvirus, Molluscipoxvirus, Orthopoxvirus, Parapoxvirus, Suipoxvirus, Cervidpoxvirus and Yatapoxvirus.

3. The modified poxvirus of claim 2, wherein the modified poxvirus is a member of the

Orthopoxvirus, preferably selected from the group consisting of vaccinia virus (VV), cowpox (CPXV), raccoonpox (RCN), rabbitpox, Monkeypox, Horsepox, Volepox, Skunkpox, variola virus (or smallpox) and Camelpox.

4. The modified poxvirus of claim 3, wherein the modified poxvirus is a vaccinia virus preferably selected from the group consisting of Western Reserve (WR), Copenhagen (Cop), Lister, LIVP, Wyeth, Tashkent, Tian Tan, Brighton, Ankara, LC16M8, LC16M0 strains, etc., with a specific preference for WR, Copenhagen and Wyeth strains.

5. The modified poxvirus of claim 2, wherein the modified poxvirus is a member of the

Leporipoxvirus genus, with a preference for myxoma virus.

6. The modified poxvirus of any one of claims 1 to 5, wherein the inability to bind said CD80 and CD86 co-stimulatory ligands originates from a genetic lesion within the M2L locus or from an abnormal interaction impairing the m2 function either directly or indirectly.

7. The modified poxvirus of claim 6, wherein said genetic lesion(s) include partial or total deletion and/or one or more non-silent mutation(s) either within the m2-coding sequence or in the regulatory elements controlling M2L expression, preferably leading to the synthesis of a defective m2 protein or to the lack of m2 synthesis.

8. The modified poxvirus of claim 7, wherein said genetic lesion is a partial or entire deletion of the M2L locus.

9. The modified poxvirus of any one of claims 1 to 8, wherein the modified poxvirus is further modified in a region other than M2L locus.

10. The modified poxvirus of claim 9, wherein the modified poxvirus is further modified in the J2R locus resulting in a modified poxvirus defective for both m2 and tk functions.

11. The modified poxvirus of claim 9 or 10, wherein the modified poxvirus is further modified in the I4L and/or F4L locus/loci, resulting in a modified poxvirus defective for both m2 and rr functions.

12. The modified poxvirus of any one of claims 9 to 11 , wherein the modified poxvirus is further modified in the J2R and I4L/F4L loci, resulting in a modified poxvirus defective for m2, tk and rr activities.

13. The modified poxvirus of any one of claims 1 to 12, wherein the modified poxvirus is oncolytic.

14. The modified poxvirus of any one of claims 1 to 13, wherein the modified poxvirus is recombinant.

15. The modified poxvirus of claim 14, wherein the modified poxvirus is engineered to express at least one polypeptide selected from the group consisting of antigenic polypeptides, polypeptides having nucleos/tide pool modulating function and immunomodulatory polypeptides.

16. The modified poxvirus of claim 15, wherein said immunomodulatory polypeptide is selected from the group consisting of cytokines, chemokines, ligands and antibodies or any combination thereof.

17. The modified poxvirus of claim 16, wherein said antibody specifically binds an immune checkpoint protein, preferably selected from the group consisting of CD3, 4-1 BB,

GITR, 0X40, CD27, CD40, PD1 , PDL1 , CTLA4, Tim- 3, BTLA, Lag-3 and Tigit.

18. The modified poxvirus of claim 17, wherein the modified poxvirus expresses an antagonist antibody that specifically binds to PD-L1 or CTLA4.

19. The modified poxvirus of claim 18, wherein the modified poxvirus is defective for m2, tk and rr activities and encodes an anti-CTLA-4 antibody, with a preference for ipilimumab or tremelimumab.

20. The modified poxvirus of claim 18, wherein the modified poxvirus is defective for m2, tk and rr activities and encodes an anti-PD-L1 antibody, with a preference for atezolizumab, durvalumab or avelumab.

21. A method for producing the modified poxvirus of any one of claims 1 to 20 comprising the steps of a) preparing a producer cell line, b) transfecting or infecting the prepared producer cell line with the modified poxvirus, c) culturing the transfected or infected producer cell line under suitable conditions so as to allow the production of the virus, d) recovering the produced virus from the culture of said producer cell line and optionally e) purifying said recovered virus.

22. A composition comprising a therapeutically effective amount of the modified poxvirus of any one of claims 1 to 20 and a pharmaceutically acceptable vehicle

23. The composition of claim 22 comprising from approximately 103 to approximately 1012 pfu, advantageously from approximately 104 pfu to approximately 1011 pfu, preferably from approximately 105 pfu to approximately 1010 pfu; and more preferably from approximately 106 pfu to approximately 109 pfu of the modified poxvirus and notably individual doses of approximately 106, 5x106, 107, 5x107, 108 or 5x108 pfu.

24. The composition of claim 22 or 23 which is formulated for intravenous or intratumoral administration.

25. The composition of any one of claims 22 to 24, for use for treating or preventing a proliferative disease selected from the group consisting of cancers as well as diseases associated to an increased osteoclast activity such as rheumatoid arthritis and osteoporosis and cardiovascular diseases such as restenosis.

26. The composition of claim 25, wherein said cancer is selected from the group consisting of renal cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, gastric cancer, bile duct carcinoma, endometrial cancer, pancreatic cancer and ovarian cancer.

27. The composition of any one of claims 22 to 24, for use for stimulating or improving an immune response, and especially:

• for stimulating or improving a lymphocyte-mediated immune response (especially against an antigenic polypeptide);

• for stimulating or improving the activity of APC;

• for stimulating or improving an anti-tumoral response;

• for stimulating or improving the CD28-signalling pathway;

• for improving the therapeutic efficacy provided by the modified poxvirus described herein in a treated subject or a group of treated subjects; and/or

• for reducing the toxicity provided by the modified poxvirus described herein in a treated subject or a group of treated subjects.

28. The composition of any one of claims 22 to 27, for use as stand-alone therapy or in conjunction with one or more additional therapies, preferably selected from the group consisting of surgery, radiotherapy, chemotherapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy, cytokine therapy, targeted cancer therapy, gene therapy, photodynamic therapy and transplantation.