m2 defective poxvirus

Abstract

The present invention is in the field of oncolytic viruses. The present invention provides novel poxviruses engineered to be defective in a function encoded by the M2L locus (i.e., m2 function). Such poxviruses lack functional m2 binding activity for at least one or both of the CD80 and CD86 costimulatory antigens. The oncolytic poxvirus is preferably a vaccinia virus with a full or partial deletion of the M2L locus. The present invention also relates to cells and compositions comprising such poxviruses, as well as their use for the treatment of proliferative diseases such as cancer and for disease prevention (vaccination, particularly in the veterinary field). More precisely, the present invention provides an alternative to existing oncolytic viruses primarily used in virotherapy. m2-deficient poxviruses are particularly useful for expressing immunomodulatory polypeptides, such as anti-CTLA-4 antibodies, to stimulate or improve immune responses.

Description

[Technical field]

[0001] TECHNICAL FIELD OF THEINVENTION The present invention is an invention in the field of oncolytic viruses. The present invention provides new poxviruses engineered to be defective in the function encoded by the M2L locus (i.e. m2 function). Such poxviruses lack functional m2 binding activity for at least one or both of the CD80 and CD86 co-stimulatory antigens. The oncolytic poxvirus is preferably a vaccinia virus with a total or partial deletion of the M2L locus. The present invention also relates to cells and compositions comprising such poxviruses, and to the use of such cells and compositions comprising such poxviruses for the treatment of proliferative diseases such as cancer and for the prevention of diseases (vaccination, in particular in the veterinary field). More precisely, the present invention provides an alternative to existing oncolytic viruses that are mainly used in virotherapy. m2-deficient poxviruses are particularly useful for expressing immunomodulatory polypeptides, such as anti-CTLA-4 antibodies, with the aim of stimulating or improving the immune response. [Background technology]

[0002] Background technology Cancer is diagnosed in over 12 million subjects worldwide each year. In developed countries, nearly one in five people die from cancer. Numerous chemotherapy regimens exist but are often ineffective, especially against malignant and metastatic tumors that are established very early in the disease.

[0003] Oncolytic virotherapy has emerged for the past 20 years, based on replication-competent viruses, with the aim of destroying cancer cells (Russell et al., 2012, Nat. Biotechnol. 30(7): 658-70). Numerous preclinical and clinical studies are currently underway to evaluate the therapeutic potential of oncolytic viruses armed with various therapeutic genes in various types of cancer.

[0004] To retain the oncolytic phenotype, therapeutic genes are usually inserted into non-essential genes in the viral genome. Insertion into the J2R locus (tk) has been widely used in the art, to the extent that it also facilitates identification of recombinant viruses 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 also been proposed for insertion, for example, into the Hind F fragment, the 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 the A56R locus (encoding hemagglutinin (HA)).

[0005] Poxviruses, in particular vaccinia viruses (VVs), have provided several promising oncolytic candidates, such as JX594 (Sillajen / Transgene), GL-ONC1 (Genelux), TG6002 (Transgene) and vvDD-CDSR (University of Pittsburg) (De Graaf et al., 2018, doi.org / 10.1016 / j.cytogfr.2018.03.006). These oncolytic VVs are derived from different VV strains with diverse genomic modifications and expression of various therapeutic genes. JX-594 (Wyeth strain), attenuated by deletion of the viral J2R gene (encoding thymidine kinase (tk)) and further armed with GM-CSF, is currently being clinically evaluated 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 in place of the F14.5L, J2R, and A56R loci, respectively, of the parental Lister virus genome. Similarly, TG6002, a VV (Copenhagen strain) defective in J2R (tk) and I4L (the I4L locus encodes ribonucleotide reductase (rr-)) and encoding the FCU1 enzyme that converts nontoxic 5-fluorocytosine (5-FC) to cytotoxic 5-fluorouracile (5-FU), is being evaluated in several clinical trials. The double deletion of tk and rr restricts viral replication to cells with abundant nucleotide pools, and therefore TG6002 cannot replicate in resting cells (Foloppe et al., 2008, Gene Ther. 15: 1361-71; WO2009 / 065546). vvDD-CDSR is currently being assayed in patients with refractory cutaneous and subcutaneous tumors. It is engineered with a double deletion of tk (J2R locus) and the gene encoding vaccinia growth factor (vgf), and is armed with a cytosine deaminase (CD) gene to convert 5-FC to 5-FU, and a somatostatin receptor (SR) gene for in vivo imaging.

[0006] Initially, direct tumor regression was thought to be the only mechanism by which oncolytic viruses exert their antitumor effects. Only recently has it been recognized that the immune system plays a key 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, aiming to block many of the strategies utilized by the host to fight viral infection (Smith and Kotwal, 2002, Crit. Rev. Microbiol. 28(3): 149-85). In addition, tumor cells have also evolved mechanisms of T cell exhaustion, characterized by the upregulation of inhibitory receptors, to evade the host immune system. The best documented are CTLA-4 (short for cytotoxic T-lymphocyte-associated protein-4; also known as CD152) and PD-1 (short for programmed cell death protein 1) and its ligands PD-L1 and PD-L2. These immunosuppressive receptors serve as immune checkpoints that act at different levels of T-cell immunity. CTLA-4 inhibits early steps of T-cell activation in lymph nodes and also stimulates unwanted Tregs, while PD-1 acts at a later stage.

[0007] More specifically, T cell activation involves the interaction of costimulatory ligands, such as CD80 (also called B7-1) and CD86 (also called B7.2), present on the surface of APCs (short for antigen presenting cells), with receptors, such as CD28, CTLA-4, and PDL-1, present on the surface of T cells. CD80 is the ligand for these three cell surface receptors, whereas CD86 binds to CD28 and CTLA-4. The CD28 receptor is constitutively expressed on resting T cells, and ligation of CD28 with the costimulatory CD80 and CD86 ligands delivers a positive stimulatory signal to the T cell, inducing it to proliferate and secrete IL-2, and inhibiting apoptosis by increasing Bcl-X expression (Chen, 2004, Nat. Rev. Immunol. 4: 336-347). In contrast, CTLA-4 or PD-L1 play a role in the negative regulation of T cells after initial T cell activation (in the case of CTLA-4) or at a later stage (in the case of PD-L1). Specifically, CTLA-4 ligates with CD80 and CD86 costimulatory ligands, and acts in cis on activated T cells to counter the costimulatory signal provided by the interaction of CD28 with CD80 and CD86, mediating IL-10 production. Moreover, CTLA-4 is constitutively expressed on a subset of immunosuppressive regulatory T cells (Tregs). On the other hand, it has been demonstrated that ligation of CD80 with PD-L1 on the surface of T regulatory cells increases 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 in GenBank Accession No. L15006.

[0008] There has been growing interest in blocking such immune-suppressive checkpoints as a means of rescuing exhausted antitumor T cells. Numerous antagonistic antibodies have been developed in the past decade (Kahn et al., 2015, J. Oncol. Doi: 10.1155 / 2015 / 847383), several of which have been approved by the FDA, the first of which were directed against CTLA4 (e.g., ipilimumab / Yervoy, Bristol-Myers Squibb) and PD-1 (pembrolizumab / Keytruda, developed by Merck, and nivolumab / Optivo, developed by BMS). Traditional treatments rely on administering antibodies to patients, but vectorization with viral or plasmid vectors is now believed to deliver these antibodies directly to tumor cells (see, e.g., WO2016 / 008976). For example, anti-PD-1-armed tk- and rr-VVs were shown to induce tumor growth control in the MCA-205 mouse model (Kleinpetter et al., 2016, OncoImmunology 5(10): e1220467).

[0009] However, preclinical and even more clinical stage studies can be difficult to conduct due to the complex nature of these immune-interacting molecules and viral vectors and the risk of triggering cascade events.

[0010] Thus, there remains a need for further development of oncolytic viruses, compositions, and methods for delivering therapeutic polypeptides, such as checkpoint-directed antagonist antibodies aimed at enhancing anti-tumor adaptive immune responses in cancer patients. Summary of the Invention

[0011] Technical Problems and Proposed Solutions Contrary to expectations, the inventors found that the supernatant of cells infected with vaccinia virus (VV) interacts with costimulatory CD80 and CD86 ligands, whereas the supernatant of cells infected with attenuated modified vaccinia virus Ankara (MVA) does not have this property. The inventors suspect that the M2 protein, encoded by the VV M2L locus, has the binding property to CD80 and CD86. Prior to the present invention, M2 was reported to be a protein retained in the endoplasmic reticulum that acts as an NfKb pathway inhibitor (Hinthong et al., 2008, Virology 373(2): 248-62) and is involved in virus uncoating (Baoming Liu et al., 2018, J. Virol. 92(7) e02152-17). In addition to VV, we found that M2 orthologues are present in a large number of replication-competent poxviruses.

[0012] The present invention describes the ability of M2 protein to bind to CD80 and CD86 and affect three immunosuppressive pathways, respectively: (i) blocking the interaction of CD80 and CD86 with CD28; (ii) promoting the interaction of CD80 with PD-L1; and (iii) inducing reverse signaling in CD80 / CD86 positive cells.

[0013] In the context of the present invention, we have generated vaccinia viruses defective in m2 function. When armed with immunomodulatory polypeptides such as anti-CTLA-4 antibodies, its expression inhibits CTLA-4-mediated immunosuppressive signals. In the absence of m2, T cell responses are redirected to CD28-mediated immunostimulatory signals, whereas M2L-positive vaccinia viruses are predicted to negatively interfere with such CD28-mediated positive signals by binding m2 to CD80 and CD86 costimulatory ligands.

[0014] Importantly and surprisingly, the poxviruses described herein do not synthesize functional m2 protein in infected cells and are therefore expected to stimulate or improve immune responses to antigens, particularly lymphocyte-mediated responses. In contrast, in conventional poxviruses (M2L positive), the produced viral m2 protein binds to CD80 and CD86 costimulatory ligands, thus blocking the positive pathway mediated by CD28. Furthermore, the poxviruses described herein lack proteins involved in viral immune evasion and therefore exhibit improved propensity to be accepted by the host immune system. This feature represents a competitive advantage over M2 positive poxviruses. The present invention provides a unique product that combines oncolytic and immunostimulatory activities, e.g., to kill dividing cells and to overcome cancer-associated immune exhaustion, thus improving the therapeutic potential of oncolytic viruses.

[0015] This technical problem is solved by the provisions of the embodiments 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, which are presented for the purpose of disclosure.

[0016] Summary of the Invention The present disclosure relates to poxviruses, particularly oncolytic poxviruses engineered to be defective in the m2 protein encoded by the M2L locus, and methods of making and using such viruses.As disclosed herein, poxviruses defective in the m2 function encoded by the M2L locus have been made and isolated, optionally in combination with other functional inactivation of the tk-encoding locus and / or the rr-encoding locus.Also contemplated are m2-defective vaccinia viruses engineered to express anti-CTLA4 antibodies.

[0017] According to a first aspect of the present invention, there is provided a modified poxvirus whose genome comprises an M2L locus which in its native (wild-type) context encodes a functional m2 poxvirus protein and has been modified to be defective in said m2 function, wherein said functional M2 poxvirus protein is capable of binding to CD80 or CD86 costimulatory ligands or is capable of binding to both CD80 and CD86 costimulatory ligands, and wherein said defective m2 function is incapable of binding to said CD80 and CD86 costimulatory ligands.

[0018] In one embodiment, the modified poxvirus is made or obtained from the subfamily Chordopoxvirinae, preferably selected from the group of genera consisting of Avipoxvirus, Capripoxvirus, Leporipoxvirus, Molluscipoxvirus, Orthopoxvirus, Parapoxvirus, Suipoxvirus, Cervidpoxvirus, and Yatapoxvirus. In a preferred embodiment, the modified poxvirus is a member of the Orthopoxvirus genus, 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, and is particularly preferably a modified Vaccinia virus.

[0019] In one embodiment, the inability to bind to the CD80 and CD86 costimulatory ligands results from a genetic lesion in the M2L locus or from an abnormal interaction that impairs m2 function either directly or indirectly. The genetic lesion comprises a partial or total deletion and / or one or more non-silent mutations (that alter amino acid residues) either in the m2 coding sequence or in the regulatory elements that control M2L expression, and preferably leads to synthesis of defective m2 protein or to lack of m2 synthesis. The genetic lesion is preferably a partial or total deletion of the M2L locus.

[0020] In one embodiment, the modified poxvirus is further modified in a region other than the M2L locus, in particular at the J2R locus (resulting in a modified poxvirus defective in both m2 and tk functions) or at the I4L / F4L locus (resulting in a modified poxvirus defective in both m2 and rr functions). Preferably, the modified poxvirus is further modified at the J2R and I4L / F4L loci, resulting in a modified poxvirus defective in m2, tk and rr activities.

[0021] In one embodiment, the modified poxvirus is oncolytic.

[0022] In one embodiment, the modified poxvirus is recombinant. The modified poxvirus is preferably engineered to express at least one polypeptide selected from the group consisting of an antigenic polypeptide, a polypeptide with nucleoside / nucleotide regulatory function, and an immunomodulatory polypeptide. The immunomodulatory polypeptide is preferably selected from the group consisting of a cytokine, a chemokine, a ligand, and an antibody, or any combination thereof. In a preferred embodiment, the modified poxvirus is defective in m2 activity, tk activity, and rr activity and encodes an anti-CTLA-4 antibody. In another preferred embodiment, the modified poxvirus is defective in m2 activity, tk activity, and rr activity and encodes an anti-PD-L1 antibody.

[0023] According to another aspect, there is provided a method for producing a 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 to allow production of virus; (d) recovering the produced virus from the culture of the producer cell line; and optionally (e) purifying the recovered virus.

[0024] According to a further aspect, there is provided a composition comprising a therapeutically effective amount of a modified poxvirus and a pharma- ceutically acceptable vehicle. The composition is desirably administered in an amount of about 10 3 ~about 10 12 pfu, advantageously about 10 4 pfu~about 10 11 pfu, preferably about 10 5 pfu~about 10 10 pfu, more preferably about 10 6 pfu~about 10 9pfu modified poxvirus. The composition is preferably formulated for intravenous or intratumoral administration.

[0025] In yet another aspect, the composition is for use in treating or preventing a proliferative disease selected from the group consisting of cancer and diseases associated with 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 kidney cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, gastric cancer, bile duct cancer, endometrial cancer, pancreatic cancer, and ovarian cancer. The modified poxvirus and composition are for use as a sole therapy or together with one or more additional therapies, preferably 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, and transplantation.

[0026] In yet another aspect, the modified poxvirus or composition is for use to stimulate or improve an immune response. [The present invention 1001] A modified poxvirus whose genome contains an M2L locus that encodes a functional m2 poxvirus protein in its native (wild-type) context and has been modified to have defective m2 function, wherein the functional M2 poxvirus protein is capable of binding to CD80 or CD86 costimulatory ligands, or is capable of binding to both CD80 and CD86 costimulatory ligands, and wherein the defective m2 function is incapable of binding to the CD80 and CD86 costimulatory ligands. [The present invention 1002] 1001. A modified poxvirus of the present invention, produced or obtained from the subfamily Chordopoxvirinae, preferably selected from the group of genera consisting of Avipoxvirus, Capripoxvirus, Leporipoxvirus, Molluscipoxvirus, Orthopoxvirus, Parapoxvirus, Suipoxvirus, Cervidpoxvirus and Yatapoxvirus. [The present invention 1003] The modified poxvirus of the present invention 1002 is a member of the Orthopoxvirus genus and 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. [The present invention 1004] The modified poxvirus of the present invention 1003 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., and particularly preferably WR, Copenhagen, and Wyeth strains. [The present invention 1005] The modified poxvirus of the present invention 1002, which is a member of the genus Leporipoxvirus, preferably a Myxomavirus. [The present invention 1006] Any of the modified poxviruses of the present invention 1001 to 1005, wherein the inability to bind to CD80 and CD86 costimulatory ligands results from genetic damage within the M2L locus or from an abnormal interaction that impairs m2 function either directly or indirectly. [The present invention 1007] The modified poxvirus of the present invention 1006, wherein the genetic lesion comprises a partial or total deletion and / or one or more non-silent mutations either within the m2 coding sequence or in the regulatory elements controlling M2L expression, preferably leading to the synthesis of defective m2 protein or to the absence of m2 synthesis. [The present invention 1008] 1007. The modified poxvirus of the present invention, wherein the genetic lesion is a partial or complete deletion of the M2L locus. [The present invention 1009] The modified poxvirus of any of the present inventions 1001 to 1008, which is further modified in a region other than the M2L locus. [The present invention 1010] The modified poxvirus of the present invention 1009, which is further modified at the J2R locus, resulting in a modified poxvirus defective in both m2 and tk functions. [The present invention 1011] The modified poxvirus of the invention 1009 or 1010, further modified in the I4L and / or F4L locus, resulting in a modified poxvirus defective in both m2 and rr functions. [The present invention 1012] The modified poxvirus of any of claims 1009 to 1011, further modified in the J2R and I4L / F4L loci, thereby resulting in a modified poxvirus defective in m2 activity, tk activity, and rr activity. [The present invention 1013] The modified poxvirus of any one of claims 1001 to 1012, which is oncolytic. [The present invention 1014] The modified poxvirus of any one of the present inventions 1001 to 1013, which is a recombinant poxvirus. [The present invention 1015] The modified poxvirus of the present invention 1014, which has been engineered to express at least one polypeptide selected from the group consisting of an antigenic polypeptide, a polypeptide having a nucleoside / nucleotide pool modulating function, and an immunomodulatory polypeptide. [The present invention 1016] The modified poxvirus of the present invention 1015, wherein the immunomodulatory polypeptide is selected from the group consisting of a cytokine, a chemokine, a ligand, and an antibody, or any combination thereof. [The present invention 1017] The modified poxvirus of the present invention, wherein the antibody specifically binds to an immune checkpoint protein, preferably an immune checkpoint protein selected from the group consisting of CD3, 4-1BB, GITR, OX40, CD27, CD40, PD1, PDL1, CTLA4, Tim-3, BTLA, Lag-3, and Tigit. [The present invention 1018] The modified poxvirus of the present invention 1017, which expresses an antagonistic antibody that specifically binds to PD-L1 or CTLA4. [The present invention 1019] A modified poxvirus of the invention 1018, defective in m2 activity, tk activity and rr activity and encoding an anti-CTLA-4 antibody, preferably ipilimumab or tremelimumab. [The present invention 1020] The modified poxvirus of the present invention 1018, having defects in m2 activity, tk activity, and rr activity, and encoding an anti-PD-L1 antibody, preferably atezolizumab, durvalumab, or avelumab. [The present invention 1021] A method for producing a modified poxvirus of any one of the present inventions 1001 to 1020, 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 appropriate conditions to allow for the production of virus; (d) recovering the produced virus from the culture of the producer cell line; and Optionally, (e) purifying the recovered virus. A method comprising: [The present invention 1022] A composition comprising a therapeutically effective amount of any of the modified poxviruses of the present inventions 1001 to 1020 and a pharma- ceutically acceptable vehicle. [The present invention 1023] about 10 3 ~about 10 12 pfu, advantageously about 10 4 pfu~about 1011 pfu, preferably about 10 5 pfu~about 10 10 pfu, more preferably about 10 6 pfu~about 10 9 pfu of modified poxvirus, particularly about 10 6 、5x10 6 、10 7 、5x10 7 、10 8 , or 5x10 8 A composition of the invention 1022 comprising individual doses of pfu. [The present invention 1024] The composition of invention 1022 or 1023, which is formulated for intravenous or intratumoral administration. [The present invention 1025] Any of the compositions of the present inventions 1022 to 1024 for use in treating or preventing a proliferative disease selected from the group consisting of cancer, and diseases associated with increased osteoclast activity, such as rheumatoid arthritis and osteoporosis, and cardiovascular diseases, such as restenosis. [The present invention 1026] The composition of the present invention 1025, wherein the cancer is selected from the group consisting of renal cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, gastric cancer, bile duct cancer, endometrial cancer, pancreatic cancer, and ovarian cancer. [The present invention 1027] For use in stimulating or improving the immune response, in particular · for stimulating or improving lymphocyte-mediated immune responses (especially to antigenic polypeptides); · to stimulate or improve APC activity; To stimulate or improve anti-tumor responses; · to stimulate or improve the CD28 signaling pathway; to improve the therapeutic efficacy provided by the modified poxviruses herein in a treated subject or group of treated subjects; and / or To reduce the toxicity provided by the modified poxviruses herein in a treated subject or group of treated subjects, Any of the compositions according to claims 1022 to 1024 for use. [The present invention 1028] Any of the compositions of inventions 1022 to 1027, for use as the sole therapy or, preferably, together with one or more additional therapies 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, and transplantation. [Brief description of the drawings]

[0027] [Figure 1A]Illustrated are CD80 / CTLA4 (1A) and CD86 / CTLA4 (1B) competitive ELISA assays performed with supernatants collected from uninfected avian DF1 cells (dotted line), or avian DF1 cells infected with wild-type VV (diamonds) or Yervoy (inverted triangles). Binding of His-tagged B7-Fc protein to immobilized CTLA4-Fc was performed with an anti-His tag-HRP conjugated antibody. [Figure 1B] See legend to Figure 1A. [Diagram 2] Illustrated is a CD80 / CTLA4 competition ELISA performed with supernatants collected from HeLa cells infected with MVA (MVA), Copenhagen vaccinia virus strain (Cop VV), Western Reserve strain (WR VV), Wyeth strain (Wyeth VV), raccoon pox (RCN), rabbit pox (RPX), cowpox (CPX), fowl pox (FPV), and pseudocowpox (PCPV), as well as supernatants from uninfected HeLa cells (negative control). [Diagram 3] Western blots are shown in which supernatants of uninfected CEF cells (cell supernatants), MVA-infected CEF cells (MVA supernatants), or Copenhagen vaccinia virus-infected CEF cells (VV supernatants) were either directly collected or concentrated 20-fold (x20) and subjected to non-reducing SDS-PAGE and probed with human CD86 Fc fragment fusion (hCD86-Fc), human CD80 Fc fragment fusion (hCD80-Fc), and human CTLA4 Fc fragment fusion (hCTLA4-Fc). Detection was performed with an anti-Fc binding antibody. [Figure 4]Figure 1 illustrates a competitive ELISA in which the interaction of biotinylated CD80 and biotinylated CD86 with their cognate receptors, CD28 / CD86, CD28 / CD80, CTLA4 / CD80, and PDL1 / CD80, respectively, was tested. Supernatants collected from MVA-infected CEF cells (MVA) and Copenhagen vaccinia virus strain-infected CEF cells (VV) were compared with supernatants from uninfected CEF cells (CEF) (negative control) and Yervoy antibody (10 μg / ml). Reactivity of recombinant human PD1 (hPD1), human CD80 (hCD80), and human CTLA4 (hCTLA4), all at 10 μg / ml, was used as a positive control competing with the PDL1 / CD80 interaction. Detection of bound biotinylated B7 protein was performed with HRP-conjugated streptavidin. [Figure 5A] FIG. 1 illustrates the experimental approach used to identify "interference factors (IFs)" by affinity chromatography with immobilized CD86-Fc fusions. [Figure 5B] The sequence of IF captured in VV-infected CEF cells is shown. [Figure 6] Illustrated is a CD80 / CTLA4 competition ELISA performed with supernatants collected from negative control, uninfected HeLa or DF1 cells (HeLa or DF1), or HeLa or DF1 cells infected with the double deleted (tk-rr-) Copenhagen vaccinia virus (VVTG18277) or triple deleted (tk-rr-m2-) Copenhagen vaccinia virus (COPTG19289). Binding of his-tagged CD80-Fc protein to immobilized CTLA4-Fc was monitored using an anti-His tag-HRP conjugated antibody. [Figure 7] Illustrated is the oncolytic activity of tk-rr-m2-vaccinia virus (COPTG19289) and its tk-rr-counterpart (VVTG18277) 4 days after infection of LOVO (A) and HCT116 (B) cells at various MOIs (10-1 to 10-4). Mock-treated cells were used as a negative control. [Figure 8]Illustrates luciferase expression in C57BL / 6 mice implanted subcutaneously with B16F10 tumors. VVTG18277 virus and COPTG19289 (107 pfu) were injected intratumorally on days 0, 3, 6, 10, and 14, and tumor sampling was collected on days 1, 2, 6, 9, 13, and 16 to assess luciferase activity per gram of tumor (RLU / g tumor). Time points included 3 mice. [Figure 9] Figure 1 illustrates antitumor activity in Balb / c mice subcutaneously implanted with CT26 tumors. 107 pfu of VVTG18277 (squares), COPTG19289 (triangles), or mock (circles) were injected intratumorally on D0, D3, D6, D10, and D14 (10 mice / group). Tumor growth was followed twice weekly (mice were killed when tumor volume reached 2000 mm3). [Figure 10] Antitumor activity in Swiss nude mice implanted subcutaneously with HT116 tumors is illustrated. On D10, when tumors reached 100-200 mm3, mice (10 mice / group) received a single intravenous injection of 105 (A) or 107 (B) pfu of VVTG18277 (circles), COPTG19289 (squares), or mock (diamonds). Tumor growth was followed twice weekly. [Figure 11]Figure 1 illustrates the effect of supernatants from cells infected with M2-defective poxviruses on mixed lymphocyte reactions (MLR). PBMCs were purified from two different donors and cultured in the presence of supernatants obtained from CEFs infected (MOI 0.05) with COPTG19289 (tk-, rr-, and m2-), VVTG18058 (tk-rr-), or MVAN33 (wild type). Culture supernatants were collected 48 hours after infection and concentrated approximately 20-fold. These concentrated supernatants were added to PBMC cultures (20 μL in 200 μL) undiluted or diluted 10- or 100-fold to a final "supernatant concentration" of 2-fold, 0.2-fold, and 0.02-fold, respectively. The amount of IL-2 secreted into the PBMC culture medium was measured by ELISA. IL-2 measurements were performed in triplicate for each sample tested. Measurements were normalized by dividing the average IL-2 concentration of the three replicates of a given sample by the average IL-2 concentration of the three replicates of PBMC incubated with medium. [Figure 12] Figure 1 illustrates the effect on tumor burden provided by M2-deficient COPTG19289 in a humanized mouse model. NOD / Shi-scid / IL-2Rγ null immunodeficient mice (NCG) were humanized with CD34+ human stem cells and implanted with human colorectal cancer cells HCT-116 (5x106 cells were injected SC into one flank of the mice; equivalent to D0). Twelve days after implantation (D12), mice received a single IV injection of COPTG19289 (TD) or the m2+ counterpart VVTG18058 (DD) at a dose of 106 pfu (A) or 105 pfu (B). Vehicle-treated mice were used as negative controls. Tumor growth was monitored for 60 days after cell implantation. The mean tumor growth of each group was expressed in mm3 as a function of days after cell injection. [Figure 13]Figure 1 illustrates the effect on survival provided by the M2-deficient COPTG19289 in the humanized NCG-CD34+ mouse model described above. Twelve days after tumor implantation (D12), mice were given a single IV injection of COPTG19289 (TD) or the m2+ counterpart VVTG18058 (DD) at a dose of 106 pfu (A) or 105 pfu (B). Vehicle-treated mice were used as negative controls. Mouse survival was monitored for 90 days after cell implantation. Percent survival of each group is shown as a function of days after cell injection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Detailed Description General definition A number of definitions are provided herein to aid in the understanding of the present invention.However, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.All references cited herein are incorporated by reference in their entirety.

[0029] 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 components or steps, unless the context clearly indicates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0030] The term "one or more" refers to one or more than one (e.g., 2, 3, 4, 5, etc.).

[0031] The term "and / or" wherever used in this specification includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0032] As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

[0033] As used herein, when used to define products, compositions, and methods, the terms "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are open-ended and do not exclude additional, unrecited elements or method steps. Thus, a polypeptide "comprises" an amino acid sequence when that amino acid sequence is capable of becoming a part of the final amino acid sequence of that polypeptide. "Consisting of" means excluding other ingredients or steps of essential importance. Thus, a composition consisting of the recited components does not exclude trace contaminants and pharma- ceutically acceptable carriers. A polypeptide "consisting of" an amino acid sequence refers to the presence of such amino acid sequence, and optionally only a few additional and non-essential amino acid residues. However, it is preferred that the polypeptide does not contain any amino acids other than the recited amino acid sequence. In this description, the term "comprising" (especially when referring to a specific sequence) can be replaced with "consisting of" if necessary.

[0034] Within the context of the present invention, the terms "nucleic acid", "nucleic acid molecule", "polynucleotide", and "nucleotide sequence" are used interchangeably to define polymers of any length of 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 polyribo-polydeoxyribonucleotides. These terms encompass polynucleotides that are single- or double-stranded, linear or circular, natural or synthetic, modified or unmodified.

[0035] The term "polypeptide" should be understood as a polymer of at least nine amino acid residues linked by peptide bonds, regardless of size and the presence or absence of post-translational components (e.g., glycosylation). No limit is imposed on the maximum number of amino acids contained in a polypeptide. As a general indication, the term refers to short polymers (typically referred to in the art as peptides) and longer polymers (typically referred to in the art as polypeptides or proteins). The term encompasses, inter alia, native polypeptides, modified polypeptides (also referred to as derivatives, analogs, variants, or mutants), polypeptide fragments, polypeptide multimers (e.g., dimers), and fusion polypeptides. The term also refers to recombinant polypeptides expressed from a polynucleotide sequence encoding the polypeptide. Typically, this involves translation of the encoding nucleic acid into an mRNA sequence and its translation by the ribosomal machinery of the cell to which the polynucleotide sequence has been delivered.

[0036] The term "identity" refers to amino acid-to-amino acid or nucleotide-to-nucleotide correspondence between two polypeptide or nucleic acid sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment and the length of each gap. To determine the percent identity between amino acid sequences, various computer programs and mathematical algorithms are available in the art, such as the Blast program available at NCBI or ALIGN in Atlas of Protein Sequence and Structure (Dayhoffed, 1981, Suppl., 3: 482-9), or the Needleman and Wunsh algorithm (J.Mol. Biol. 48,443-453, 1970). Programs for determining identity between nucleotide sequences are also available in specialized databases (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 necessary to achieve maximum alignment across the sequences being compared. For illustrative purposes, "at least 70%" means 70% or more (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%). In contrast, "at least 80% identity" means 80% or more (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%" means 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).

[0037] As used herein, the term "isolated" refers to a component (e.g., a polypeptide, nucleic acid molecule, virus, vector, etc.) that has been removed from its natural environment (i.e., separated from at least one other component with which it is naturally associated or found in nature). For example, a nucleotide sequence is isolated if it is separated from sequences with which it is normally associated in nature (e.g., dissociated from a genome), but may be associated with heterologous sequences.

[0038] The terms "obtained from," "originating," or "originate," and any equivalents, are used to identify the original source of a component (e.g., a polypeptide, a nucleic acid molecule, a virus, a vector, etc.), but are not intended to limit the manner in which the component was made, which may be, for example, by chemical synthesis or by recombinant means.

[0039] The term "host cell" as used herein should be understood broadly without any restriction regarding a specific organization in a tissue, organ, or isolated cell. Such cells may be unique types of cells or may be a group of different types of cells, such as cultured cell lines, primary cells, and dividing cells. In the context of the present invention, the term "host cell" preferably refers to eukaryotic cells, such as mammalian (e.g., human or non-human) cells, as well as cells capable of producing the poxviruses described herein. The term also includes cells that can be or have been recipients of poxviruses, as well as the progeny of such cells.

[0040] The term "subject" generally refers to an organism for which any of the poxviruses, compositions, and methods described herein are needed or may be beneficial. Typically, the organism is a mammal, particularly a mammal selected from the group consisting of farm animals, livestock, 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 cancer. The terms "subject" and "patient" can be used interchangeably when referring to a human organism, and include males and females. The subject to be treated may be a neonate, infant, young adult, adult, or geriatric.

[0041] As used herein, the term "treatment" (and any form of treatment, such as "treating", "treat" and the like) encompasses prophylaxis (e.g., preventative measures in subjects at risk of having the pathological condition being treated) and / or therapy (e.g., in subjects diagnosed with the pathological condition), ultimately associated with conventional therapeutic modalities. The outcome of treatment is to slow, cure, ameliorate, or manage the progression of the targeted pathological condition. For example, a subject has been successfully treated for cancer if, after administration of a poxvirus as described herein, the subject shows an observable improvement in the clinical condition.

[0042] As used herein, the term "administering" (or any form of administration, such as "administered") refers to the delivery of a therapeutic agent, such as a poxvirus described herein, to a subject.

[0043] The term "combination" or "association" as used herein refers to any possible combination of various components (e.g., poxvirus and one or more substances effective in anticancer therapy). Such combinations include mixtures of the components as well as separate combinations for simultaneous or sequential administration. The present invention encompasses combinations that include equimolar concentrations of each component as well as combinations with very different concentrations. It is understood that the optimal concentration of each component of the combination can be determined by one skilled in the art.

[0044] m2 defective poxvirus In one aspect, the present invention provides a modified poxvirus whose genome comprises an M2L locus that, in a native (wild-type) context, encodes a functional m2 poxvirus protein and has been modified to be defective in said m2 function, wherein said functional M2 poxvirus protein is capable of binding to CD80 or CD86 costimulatory ligands, or is capable of binding to both CD80 and CD86 costimulatory ligands, and said defective m2 function is incapable of binding to said CD80 and CD86 costimulatory ligands.

[0045] The term "poxvirus" or "pox virus" as used herein refers to any currently identified or later identified Poxviridae virus that is infectious for one or more mammalian cells (e.g., human cells) and whose genome contains, in the native (wild-type) context, a functional M2L locus that encodes the so-called M2 protein. The term "virus", when used in the context of a poxvirus or any other virus referred to herein, encompasses the viral genome as well as the viral particle (capsid and / or enveloped genome).

[0046] Poxviruses are a widespread family of DNA viruses that contain a double-stranded genome. Like most viruses, poxviruses have developed self-defense mechanisms through a repertoire of proteins involved in immune evasion and immune regulation aimed at blocking many of the strategies utilized by the host to fight viral infection (Smith and Kotwal, 2002, Crit. Rev. Microbiol. 28(3): 149-85). Typically, poxvirus genomes encode more than 20 host response modifiers that allow the virus to manipulate the host immune response, thus facilitating viral replication, spread, and transmission. These include growth factors, anti-apoptotic proteins, inhibitors of the NFkB pathway and interferon signaling, and major histocompatibility complex (MHC) down-regulators.

[0047] For general guidance, wild-type vaccinia virus (VV) genomes contain the M2L locus, whose coding sequence encodes a protein called m2 that is produced early in the viral life cycle. It is likely to be secreted or localized to the reticulum endoplasmic (RE) and 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 is not required for in vitro viral replication (Smith, 1993, Vaccine 11: 43-53). Moreover, its function of downregulating cellular NF-κB transcription factors via inhibition of Erk1 phosphorylation is now well documented (Gedey et al., 2006, J. Virol. 80: 8676-85). This therefore suggests that m2 is involved in the host antiviral response during poxvirus infection. The VV "M2L" locus is located in the 5'-third part of the wild-type VV genome. Specifically, the coding sequence is located between positions 27324 and 27986 of the Copenhagen (Cop) VV genome. The gene product encoded by Cop M2L is a 220 amino acid protein (having the amino acid sequence shown in SEQ ID NO:1; also disclosed in Uniprot under accession number P21092), which consists of a 203 amino acid residue mature polypeptide containing 8 Cys residues and an N-terminal 17 amino acid residue long signal peptide, also containing one Cys residue.

[0048] The poxvirus genome is a double-stranded DNA of about 200 kb in the native context, capable of encoding nearly 200 proteins with different functions, including the M2L locus. The genome sequence and the encoded open reading frames (ORFs) are known. The modified poxviruses of the present invention comprise at least a genome that has been modified by the hand of man to be defective in the m2 function encoded by the native M2L locus, and may further comprise one or more additional modifications, such as those described herein.

[0049] Identification of the presence of the M2L locus within poxvirus genomes Using the information provided herein and general knowledge in the art, it is within the skill of the art to determine whether a given poxvirus contains, in its native context, an M2L locus that encodes a functional m2 protein. The particular choice of assay technique is not critical, and it is within the skill of the art to combine these conventional methodologies to determine whether a candidate poxvirus contains an M2L locus that encodes a functional m2 protein.

[0050] In one embodiment, the M2L locus in a given poxvirus can be identified by hybridization or PCR methods using the information provided herein and by designing appropriate probes or primers to screen the poxvirus genome sequence. For general guidance, hybridization assays are typically based on oligonucleotide probes derived from known nucleotide (nt) sequence information presented herein for the M2L locus to be detected, using nucleic acid extracted from cells infected with or containing such candidate poxvirus under conditions suitable for hybridization. Oligonucleotide probes are short pieces of single-stranded RNA or DNA (usually 10-30 nucleotides long) designed to be complementary (i.e., at least 80% identical) to the target M2L sequence. The probes are preferably labeled to allow detection (e.g., radioactively, fluorescently, or enzymatically labeled probes). Hybridization is typically performed under stringent conditions that only form specific hybrids.

[0051] In yet another or alternative embodiment, the presence of the M2L locus in a given poxvirus can be identified based on the amino acid sequence of the encoded gene product. For example, the presence of the M2L locus can be identified by translation analysis of genomic sequences in available databases and blast searches of the encoded open reading frame (ORF) amino acid sequences against known poxvirus m2 proteins, e.g., Cop VV m2 (SEQ ID NO:1) or myxoma virus gp-120-like protein (SEQ ID NO:2), to search for the presence of encoded ORFs that exhibit at least 40%, desirably at least 50%, preferably at least 70%, more preferably at least 80%, and with an absolute preference of at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0052] Alternatively, or in addition, the amino acid sequence of the ORF encoded by the poxvirus genome can be aligned against available databases. A candidate poxvirus is considered to contain the M2L locus if it encodes a so-called m2 polypeptide family that gives the same results after searching in a domain database (e.g., Gene3D, PANTHER, Pfam, PIRSF, PRINTS, ProDom, PROSITE, SMART, SUPERFAMILY, or TIGRFAMs) as that of the m2 VV protein (referred to in Uniprot by accession number P21092; also disclosed herein as SEQ ID NO:1). Thus, a candidate poxvirus is identified as containing the M2L locus if it encodes a polypeptide that is assigned to the PFAM motif number PF04887 or Interpro motif number IPR006971 signature in Uniprot when submitted to Blast analysis using the databases cited above.

[0053] Functionality of the encoded m2 protein As used herein, functional m2 protein refers to the protein being capable of binding to CD80 and / or CD86 costimulatory ligands in vitro or in vivo. The ability of a poxvirus to encode a functional m2 polypeptide can be assessed by routine techniques. Standard assays to assess 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), RIA, flow cytometry, and ELISA. The particular choice of assay technique is not critical, and it is within the skill of the art to adapt any of these conventional methodologies to ascertain whether a candidate m2 protein binds to CD80 and / or CD86 costimulatory ligands.

[0054] For example, the supernatant of cells infected with a candidate poxvirus can be used to probe CD80 or CD86 immobilized on a plate (ELISA) or displayed on the cell surface (FACS). Sandwich competitive ELISA assays (see the Examples section) are particularly suitable due to the fact that it is not necessary to make tagged recombinant proteins to obtain results. For example, an ELISA plate can be coated with a ligand of interest (e.g., CD86-Fc) and then the sample to be tested (e.g., supernatant of cells infected with a poxvirus) can be added. If the sample contains M2 polypeptide, it will bind to the coated ligand. A labeled detection ligand is then usually added (e.g., His-tagged CTLA4-Fc is recognized by an anti-His tag antibody conjugated to HRP (short for horseradish peroxidase) to be detected, for example, by the action of an enzyme that converts the labeled substance into a colored product that can be measured using a plate reader). A decrease in color detection in the presence of a candidate sample compared to no sample or a negative control sample indicates that the sample contains an M2 polypeptide that competes with the detection ligand for binding to the coated ligand. The reverse can also be followed, for example, using CTLA-4-Fc as the coated ligand and CD80-Fc-His tag as the detection ligand.

[0055] As used herein, "defective in m2 function" is intended to mean the inability of the m2 protein to bind CD80 and / or CD86 costimulatory ligands in vitro or in vivo. This inability may result from genetic lesions within the native M2L locus that prevent the normal binding activity of the encoded m2 protein. Thus, functional inactivation may result from one or more mutations at the M2L locus. Such mutations are preferably selected from the group consisting of insertions, deletions, and base changes in the coding sequence or regulatory sequences controlling expression of the m2 protein. Alternatively, functional inactivation may occur due to an abnormal interaction of the m2 protein with one or more other gene products that bind to or otherwise prevent the functional activity of the m2 protein.

[0056] For general guidance, the inventors have indeed identified M2L loci (encoding a functional m2 protein or its orthologues) in a wide variety of poxviruses, as described below, and more specifically in seven strains of vaccinia virus, seven strains of myxoma virus, four strains of monkeypox, multiple strains of cowpox virus, eight strains of variola virus, and a variety of other poxviruses, including, but not limited to, horsepox, Taterapox, camelpox, raccoonpox, Shankpox, Yokapox, Rabbit fibroma virus, Murmansk pox, Eptesipox, Deerpox, Tanapox, Cotia virus, and Volepox. For illustrative purposes, as depicted in Table 1, the encoded M2 protein orthologues of horsepox, variola, monkeypox, camelpox, and cowpox show greater than 90% identity with the reference Cop m2 protein (represented by SEQ ID NO:1), while the encoded M2 protein orthologues of myxoma, skunk, chochia, and volepox viruses show 50%, 74%, 70%, and 72% sequence identity, respectively, with the CopVV m2 protein.

[0057] Table 1 shows a summary of the GenBank accession numbers of various poxvirus genomic sequences that contain the M2L locus in the native context and an indication of the amino acid identity of the m2 protein relative to the Cop m2 protein (Uniprot accession number P21092 and SEQ ID NO:1).

[0058] [Table 1]

[0059] For ease of understanding, the gene nomenclature used herein to designate poxvirus M2L locus and encoded m2 protein is the gene nomenclature of vaccinia virus (more specifically, the gene nomenclature of the Copenhagen strain). This is also used herein for other poxviruses that contain functionally equivalent M2L genes and m2 proteins to those mentioned herein, unless otherwise specified. In practice, the nomenclature of genes and their respective gene products may vary depending on the family, genus, and strain of poxvirus, but the correspondence between vaccinia virus and other poxviruses is generally available in the literature. For illustrative purposes, the corresponding VV M2L gene is called M154L in the myxoma genome, CPXV040 or P2L in the cowpox genome, O2L in the monkeypox genome, RPXV023 in the rabbitpox genome, and O2L or Q2 in the smallpox virus genome.

[0060] However, the genomes of some poxviruses, such as attenuated vaccinia virus MVA (modified vaccinia virus Ankara) and pseudopox poxvirus (PCPV), lack the M2L locus in the native context due to large genomic deletions that occurred during the attenuation process (Antoine et al., 1998, Virology 244(2) 365-96). In the context of the present invention, the term "poxvirus" does not include poxviruses that have an M2L locus (or a corresponding one) that contains a genomic deletion or mutation in the native context and thus lack an m2 polypeptide or encode a non-functional m2 protein, such as pseudopox poxvirus (PCPV), MVA, and NYVAC virus.

[0061] In one embodiment, the modified poxvirus of the invention is made or obtained from the subfamily Chordopoxvirinae, preferably selected from the group of genera consisting of Avipoxvirus, Capripoxvirus, Leporipoxvirus, Morsipoxvirus, Orthopoxvirus, Parapoxvirus, Suipoxvirus, Cerbidopoxvirus and Yatapoxvirus. The genomic sequences of these poxviruses are available in the art, in particular in specialized databases such as GenBank or Refseq.

[0062] In a preferred embodiment, the modified poxvirus is made or obtained from the orthopoxvirus genus. Any orthopoxvirus genus can be used, but 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. Vaccinia virus is particularly preferred. Any vaccinia virus strain is suitable in the context of the present invention (except MVA), including but not limited to Western Reserve (WR), Copenhagen (Cop), Lister, LIVP, Wyeth, Tashkent, Tian Tan, Brighton, Ankara, LC16M8, LC16M0 strains, etc. Lister, WR, Copenhagen, and Wyeth strains are particularly preferred. The genome sequences are available in the literature as well as in GenBank (e.g., accession numbers AY678276 (Lister), M35027 (Cop), AF0956891 (Tian Tan), and AY243312.1 (WR). The viruses are also available from virus collections (e.g., ATCC VR-1354 for WR, ATCC VR-1536 for Wyeth, and ATCC VR-1549 for Lister).

[0063] In another embodiment, the modified poxvirus is made or obtained from the genus Leporipoxvirus, preferably Myxomavirus, whose genome sequence is disclosed in GenBank under the accession number NP_051868.1. The M2L ortholog locus in Myxomavirus is called the M154L locus, which has the amino acid sequence shown in SEQ ID NO:2 and encodes a so-called gp120-like protein that shows 50% identity with the m2 protein encoded by Cop (SEQ ID NO:1).

[0064] Defective m2 function As noted above, the inability of m2 protein to bind CD80 and / or CD86 costimulatory ligands may result from genetic lesions within the M2L locus, or from aberrant interactions that impair m2 function either directly or indirectly. Specifically, "defective m2 function" refers to at least a 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% reduced ability, or complete inability to bind CD80 (e.g., human) and CD86 (e.g., human) as compared to native m2 protein (e.g., as found in the supernatant of cells infected with an m2-positive poxvirus) as measured by conventional assays such as competitive ELISA assays.

[0065] Modified poxviruses can be engineered to be defective in m2 function by a number of methods 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 suppresses the expression of m2 protein by the virus. Such genetic lesions comprise partial or total deletions and / or one or more non-silent mutations (resulting in changes of amino acid residues) either in the m2 coding sequence or in the regulatory elements that control M2L expression. Said genetic lesions preferably lead to synthesis of defective m2 protein (not ensuring the activity of the native protein described above) or to lack of m2 synthesis (no protein at all). For example, said genetic lesions are partial or total deletions of the M2L locus, e.g. partial deletions extending from upstream of the m2 coding sequence 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 into the coding sequence), frameshift mutation (altering the reading frame), insertion mutation (by insertion of one or more nucleotides that disrupt the coding sequence), or deletion or substitution of one or more residues involved in or responsible for CD80 and / or CD86 binding function, or any combination thereof. Similarly, exogenous nucleic acid can be introduced into the coding sequence to disrupt the m2 open reading frame. Similarly, gene promoters can be deleted or mutated, thus inhibiting M2L expression. Based on the present disclosure, one skilled in the art will easily ascertain whether a particular modification functionally inactivates m2 by comparing wild-type and mutant m2 proteins for their ability to bind CD80 and / or CD86, as described in the Examples section.

[0066] Other poxvirus modifications In one embodiment, the modified poxvirus of the present invention is further modified in a region other than the M2L locus. A variety of further modifications may be contemplated in the context of the present invention.

[0067] The one or more additional modifications encompassed by the present invention affect, for example, oncolytic activity (e.g., improved replication in dividing cells), safety (e.g., tumor selectivity), and / or virus-induced immunity, as compared to a poxvirus lacking such modifications. Exemplary modifications preferably relate to viral genes involved in DNA metabolism, host virulence, or the IFN pathway (see, for example, Guse et al., 2011, Expert Opinion Biol. Ther.11(5):595-608).

[0068] A particularly suitable gene to be disrupted is the locus encoding thymidine kinase (tk) (J2R; GenBank accession number AAA48082). The tk enzyme is involved in deoxyribonucleoside synthesis. Tk is required for viral replication in normal cells when these cells generally have low concentrations of nucleotides, whereas it is not required in dividing cells that contain high concentrations of nucleotides. Furthermore, viruses defective in tk are known to have high selectivity for tumor cells. In one embodiment, the modified poxvirus is further modified in the J2R locus (preferably a modification that suppresses the expression of the viral tk protein), resulting in a modified poxvirus defective in both m2 and tk functions (m2-tk-poxvirus). Partial or complete deletion of the J2R locus as well as insertion of foreign nucleic acid into the J2R locus to inactivate tk function are contemplated in the context of the present invention. Such modified m2-tk-poxviruses are desirably oncolytic.

[0069] Alternatively or in combination, the modified poxvirus may be further modified in the I4L and / or F4L loci (preferably a modification that suppresses the expression of the viral ribonucleotide reductase (rr) protein), resulting in a modified poxvirus defective in both m2 and rr functions (m2 and rr defective poxvirus). In the natural context, this enzyme catalyzes the reduction of ribonucleotides to deoxyribonucleotides, a crucial step in DNA biosynthesis. This viral enzyme has a similar subunit structure to the mammalian enzyme and is composed of two heterologous subunits, R1, encoded by the I4L locus, and R2, encoded by the F4L locus. The sequences of the I4L and F4L genes of various poxviruses and their location in the genome are available in public databases (see, for example, WO2009 / 065546). In the context of the present invention, to provide a rr-defective poxvirus, the poxvirus can be modified in the I4L gene (encoding the r1 large subunit) or the F4L gene (encoding the r2 small subunit) or both, for example by partial or complete deletion of said I4L and / or F4L loci. Such modified m2-rr- poxviruses are desirably oncolytic.

[0070] Modified poxviruses that are further modified at the J2R and I4L / F4L loci (M2L, J2R, and I4L loci; M2L, J2R, and F4L loci, or triple defective viruses with modifications at the M2L, J2R, I4L, and F4L loci) are also provided, resulting in modified poxviruses that are defective in m2, tk, and rr activities (m2-, tk-rr- poxviruses). Such modified tk-rr- and m2- poxviruses are desirably oncolytic.

[0071] In preferred embodiments, such double and triple defective poxviruses preferably originate from the Orthopoxvirus or Leporipoxvirus genera as described above for m2 defective poxviruses. Oncolytic vaccinia viruses other than MVA are particularly preferred, with Lister, WR, Copenhagen, and Wyeth strains being particularly preferred. VVs defective in tk and m2 activity, and VVs defective in tk, rr, and m2 activity are particularly preferred for use in stimulating or improving immune responses (e.g., lymphocyte-mediated responses to antigens or epitopes thereof) or for treating proliferative diseases as described herein.

[0072] Other suitable further modifications include modifications that suppress the expression of one or more viral gene products selected from the group consisting of viral hemagglutinin (A56R); serine protease inhibitor (B13R / B14R), complement 4b binding protein (C3L), genes encoding VGF and interferon regulatory genes (B8R or B18R). Another suitable modification includes inactivation of the F2L locus, which suppresses the expression of viral dUTPase (deoxyuridine triphosphate degrading enzyme), which is involved in maintaining the fidelity of DNA replication and providing a precursor for TMP production by thymidylate synthase (WO2009 / 065547).

[0073] As with M2L, the gene nomenclature used herein is that of the CopVV strain, which is also used herein for homologous genes in other poxviruses unless otherwise specified, and the correspondence between Copenhagen and other poxviruses is available to those skilled in the art.

[0074] In yet another embodiment, the modified poxvirus of the present invention is oncolytic. The term "oncolytic" as used herein refers to the ability of a poxvirus to selectively replicate in dividing cells (e.g., proliferating cells, such as cancer cells) in vitro or in vivo, targeting the slowing down and / or lysis of said dividing cells, but not or only minimally replicate in non-dividing (e.g., normal or healthy) cells. "Replication" (or any form of replication, such as "replicate" and "replicating") refers to viral duplication, which may occur at the nucleic acid level or, preferably, at the level of infectious viral particles. The term "infectivity" (or any form of infectivity, such as infect, infecting) refers to the ability of the virus to infect and enter a host cell or subject. Typically, an oncolytic poxvirus contains a viral genome (capsid and / or enveloped genome) packaged within a viral particle, although in the context of the present invention the term may also encompass the viral genome (e.g., genomic DNA) or portions thereof.

[0075] Recombinant m2 defective poxvirus In one embodiment, the modified poxvirus of the invention is recombinant.

[0076] The term "recombinant" as used herein indicates that the poxvirus has been engineered to express at least one foreign nucleic acid (also called recombinant gene, transgene or nucleic acid). In the context of the present invention, the "foreign nucleic acid" inserted into the poxvirus genome is not found in or expressed by the native poxvirus genome. Nevertheless, the foreign nucleic acid may be homologous or heterologous to the subject into which the recombinant poxvirus is introduced. More specifically, the foreign nucleic acid may be of human or non-human origin (e.g., of bacterial, yeast or viral origin, excluding poxviruses). Advantageously, said recombinant nucleic acid is a nucleic acid sequence that encodes a polypeptide or is capable of at least partially (by hybridization) binding to a complementary cellular nucleic acid (e.g., DNA, RNA, miRNA) present in diseased cells, with the goal of inhibiting a gene involved in said disease. Such a recombinant nucleic acid may be a native gene or a part thereof (e.g., cDNA), or any variant thereof obtained by mutation, deletion, substitution and / or addition of one or more nucleotides.

[0077] In one embodiment, the recombinant nucleic acid encodes a polypeptide of therapeutic or prophylactic interest (i.e., a polypeptide of therapeutic interest) that, when properly administered to a subject, leads to a beneficial effect on the course or symptoms of the pathological condition to be treated. Numerous polypeptides of therapeutic interest can be envisaged. In a preferred embodiment, the modified poxviruses described herein are engineered to express at least one polypeptide selected from the group consisting of antigenic polypeptides (e.g., tumor-associated antigens or vaccine antigens), polypeptides with nucleoside / nucleotide regulatory functions, and immunomodulatory polypeptides. Recombinant modified poxviruses that code for detectable gene products may also be useful in the context of the present invention. As used herein, "engineered" refers to the insertion of one or more foreign nucleic acids into the appropriate locus of the viral genome (e.g., in place of the J2R locus) and under the control of appropriate regulatory elements so that the foreign nucleic acids are expressed in the host cell or organism.

[0078] Immunomodulatory Polypeptides In one embodiment, the modified poxviruses described herein are engineered to express at least one immunomodulatory polypeptide. The term "immunomodulatory polypeptide" refers to a polypeptide that targets a component of a signaling pathway that can be directly or indirectly involved in regulating the immune response. "Modulating" an immune response refers to any change in the cells of the immune system or the activity of such cells (e.g., T cells). Such modulation includes stimulation or suppression of the immune system, manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other change that may occur within the immune system. Preferably, such polypeptides are capable of at least partially downregulating inhibitory pathways (antagonists) and / or at least partially upregulating stimulatory pathways, in particular immune pathways that exist between antigen-presenting cells (APCs) or cancer cells and effector T cells (agonists).

[0079] The immunomodulatory polypeptides expressed by the modified poxviruses described herein can act at any stage of T cell-mediated immunity, including clonal selection of antigen-specific cells, T cell activation, proliferation, trafficking to antigenic and inflammatory sites, execution of direct effector functions, and signaling through cytokines and membrane ligands, each of which is regulated by balancing stimulatory and inhibitory signals that fine-tune the response.

[0080] Suitable immunomodulatory polypeptides and the method of using them are described in the literature.Exemplary immunomodulatory polypeptides include, but are not limited to, cytokines, chemokines, ligands and antibodies, or any combination thereof.The present invention encompasses modified, preferably oncolytic poxviruses that code multiple immunomodulatory polypeptides (e.g., one cytokine and one antibody; one cytokine and one ligand; two cytokines; two immune checkpoint antibodies; one cytokine, one ligand and one antibody; one antibody and two cytokines, etc.).

[0081] In one embodiment, the immunomodulatory polypeptide expressed by the modified poxvirus described herein is a cytokine, preferably selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-36, IFNa, IFNg, and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0082] In another embodiment, the immunomodulatory polypeptide expressed by the modified poxvirus described herein is a chemokine, preferably selected from the group consisting of MIPIα, IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19, and CCL21.

[0083] In yet another embodiment, the immunomodulatory polypeptides expressed by the modified poxviruses described herein can be independently selected from the group consisting of peptides (e.g., peptide ligands), soluble domains of natural receptors, and antibodies. In the context of the present invention, antibodies that specifically bind to immune checkpoint proteins, preferably immune checkpoint proteins selected from the group consisting of CD3, 4-1BB, GITR, OX40, CD27, CD40, PD1, PDL1, CTLA4, Tim-3, BTLA, Lag-3, and Tigit, are particularly suitable.

[0084] The term "specifically binds" refers to the capacity for binding specificity and affinity to a particular target or epitope, even in the presence of a heterogeneous population of other proteins and biologics. Thus, under designated assay conditions, the antibody binds preferentially to its target and does not bind in significant amounts to other components present in the test sample or subject. Preferably, such an antibody binds to a target that is less than 1x10 -6 Equilibrium dissociation constant equal to or less than M (e.g., at least 0.5x10 -6 , 1x10 -7 , 1x10 -8 , 1x10 -9 , 1x10 -10 etc.) and exhibit high affinity binding to its target. Standard assays for assessing the binding ability of an antibody to its target are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometry.

[0085] In the context of the present invention, "antibody" ("Ab") is used in the broadest sense and encompasses natural and human engineered antibodies, including synthetic, monoclonal, polyclonal, and full-length antibodies and fragments, variants, or fusions thereof, provided that such fragments, variants, or fusions retain the binding properties with the target protein. Such antibodies may be of any origin: human or non-human (e.g., rodent or camelid antibodies) or chimeric. Non-human antibodies may be humanized by recombinant methods to reduce immunogenicity in humans. Antibodies may be from any of the known isotypes (e.g., IgA, IgG, and IgM) and any subclass of IgG (IgG1, IgG2, IgG3, IgG4). Furthermore, antibodies may be glycosylated, partially glycosylated, or non-glycosylated. Unless otherwise indicated by the context, the term "antibody" also includes antigen-binding fragments of any of the above antibodies, including monovalent or bivalent fragments and single-chain antibodies. The term antibody also includes multispecific (e.g., bispecific) antibodies, so long as they exhibit the same binding specificity as the parent antibody. Screening the binding properties of candidate antibodies is within the skill of the art.

[0086] For illustrative purposes, a full-length antibody is a glycoprotein with at least two heavy (H) chains and two light (L) chains connected to each other by disulfide bonds. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region made up of three CH1, CH2 and CH3 domains (finally with a hinge between CH1 and CH2). Each light chain contains a light chain variable region (VL) and a light chain constant region containing one CL domain. The VH and VL regions contain three hypervariable regions, named complementarity determining regions (CDRs), interspersed with four conserved regions, named framework regions (FRs), in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDR regions of the heavy and light chains determine the binding specificity. As used herein, a "humanized antibody" refers to a non-human (e.g., mouse, camel, rat, etc.) antibody whose protein sequence has been modified to increase similarity to a human antibody (i.e., one naturally produced in humans). The humanization process is well known in the art and is typically carried out by substituting one or more residues in the FR regions to resemble human immunoglobulin sequences, while the majority of residues in the variable regions (especially the CDRs) are unaltered and correspond to those of the non-human immunoglobulin. A "chimeric antibody" contains one or more elements of one species and one or more elements of another species. For example, a non-human antibody contains at least a portion of the constant region (Fc) of a human immunoglobulin.

[0087] Representative examples of antigen-binding fragments are known in the art, including Fab, Fab', F(ab')2, dAb, Fd, Fv, scFv, ds-scFv, and diabodies. A particularly useful antibody fragment is a single-chain antibody (scFv), which contains the two domains VL and VH of the Fv fragment, ultimately fused together by a linker into one protein chain.

[0088] In one embodiment, the antibody expressed by the modified poxvirus described herein is a monoclonal or single chain antibody that specifically binds to a molecule on the surface of a T cell, preferably to an immunoinhibitory receptor involved in the regulation of T cell activation. In addition to the binding ability, such an antibody is further capable of inhibiting the biological activity of said immunoinhibitory receptor.

[0089] A particularly preferred embodiment is directed to modified, preferably oncolytic, poxviruses expressing antagonistic antibodies that specifically bind to PD-L1 or CTLA4, and preferably inhibit the biological activity of such receptors, in particular by inhibiting their interaction with the costimulatory CD80 and / or CD86 ligands.

[0090] In a preferred embodiment, the antagonist antibody expressed by the recombinant modified poxvirus described herein is an anti-CTLA-4 antibody that specifically binds to mammalian CTLA-4 (e.g., human CTLA-4) and inhibits its ability to deliver immunosuppressive signals (e.g., by blocking the binding of CTLA-4 to the CD80 and CD86 ligands).

[0091] Conventional poxviruses contain an M2L locus within their genomes, and expressed anti-CTLA-4 antibodies act to inhibit CTLA-4-mediated immunosuppressive signals, whereas in situ produced M2 protein interacts with CD80 and CD86 ligands, thus reducing or inhibiting CD28-mediated costimulatory signals. In contrast, the engineered (i.e., m2-deficient) poxviruses described herein that express anti-CTLA-4, which lack m2 function, are capable of both inhibiting CTLA-4-mediated immunosuppressive signals and redirecting immune responses to CD28-mediated costimulatory signals.

[0092] Many anti-CTLA-4 antibodies are available in the art (see, e.g., those described in, inter alia, US8,491,895, WO2000 / 037504, WO2007 / 113648, WO2012 / 122444, and WO2016 / 196237), some of which have been approved by the FDA or have undergone advanced clinical development over the past decade. Representative examples of anti-CTLA-4 antibodies that can be used in the present disclosure are, for example, ipilimumab, sold by Bristol Myer Squibb as Yervoy® (see, e.g., US 6,984,720; US 8,017,114), MK-1308 (Merck), AGEN-1884 (Agenus Inc.; WO 2016 / 196237), and tremelimumab (AstraZeneca; US 7,109,003 and US 8,143,379), as well as single chain anti-CTLA4 antibodies (see, e.g., WO 97 / 20574 and WO 2007 / 123737).

[0093] Preferred embodiments are directed to (i) modified (preferably oncolytic) poxviruses, preferably oncolytic vaccinia viruses encoding anti-CTLA-4 antibodies and defective in both m2 and tk functions (due to inactivating mutations in both the M2L locus and the J2R locus); (ii) modified (preferably oncolytic) poxviruses, preferably oncolytic vaccinia viruses encoding anti-CTLA-4 antibodies and defective in m2 and rr activity (due to inactivating mutations in both the M2L locus and the I4L and / or F4L genes), and (iii) modified (preferably oncolytic) poxviruses, preferably oncolytic vaccinia viruses encoding anti-CTLA-4 antibodies and defective in m2, tk, and rr activity (due to inactivating mutations in the M2L, J2R, and I4L / F4L loci).

[0094] In certain embodiments, the anti-CTLA-4 antibody is ipilimumab.

[0095] In certain embodiments, the anti-CTLA-4 antibody is tremelimumab.

[0096] Another preferred example of an immunomodulatory polypeptide suitable for expression by the modified poxviruses described herein represents an antibody that specifically binds to and inhibits the biological activity of PDL-1 (programmed death ligand-1). Formation of the PD-1 / PD-L1 receptor / ligand complex inhibits CD8+ T cells and thus the immune response. PD-L1 is one of two cell surface glycoprotein ligands of PD-1 (the other is PD-L2), which downregulates T cell activation and cytokine secretion upon binding to PD-1. The complete human PD-L1 sequence can be found at GenBank accession number Q9NZQ7.

[0097] Antagonist anti-PD-L1 antibodies are available in the art from a variety of providers, such as Merck, Sigma Aldrich, and Abcam, and some have been approved by the FDA or are undergoing advanced late-stage clinical development. Representative examples of anti-PD-L1 antibodies that can be used in the present disclosure include, for example, BMS-936559 (Bristol Myer Currently under development by Squibb. Also known as MDX-1105; WO2013 / 173223), atezolizumab (currently under development by Roche; also known as TECENTRIQ®; US8,217,149), durvalumab (AstraZeneca; also known as EVIFINZI™; WO2011 / 066389), MPDL3280A (currently under development by Genentech / Roche), and avelumab (developed by Merck and Pfizer under the trade name Bavencio; WO2013 / 079174), STI-1014 (Sorrento; WO2013 / 181634), and CX-072 (Cytomx; WO2016 / 149201). The corresponding nucleotide sequences can be cloned or isolated according to standard techniques based on the information disclosed in the available literature.

[0098] Preferred embodiments are directed to (i) modified (preferably oncolytic) poxviruses, preferably oncolytic vaccinia viruses encoding an anti-PD-L1 antibody and defective in both m2 and tk function (due to inactivating mutations in both the M2L locus and the J2R locus); (ii) modified (preferably oncolytic) poxviruses, preferably oncolytic vaccinia viruses encoding an anti-PD-L1 antibody and defective in m2 and rr activity (due to inactivating mutations in both the M2L locus and the I4L and / or F4L genes); and (iii) modified (preferably oncolytic) poxviruses, preferably oncolytic vaccinia viruses encoding an anti-PD-L1 antibody and defective in m2, tk, and rr activity (due to inactivating mutations in the M2L, J2R, and I4L / F4L loci).

[0099] In certain embodiments, the anti-PD-L1 antibody is atezolizumab.

[0100] In one particular embodiment, the anti-PD-L1 antibody is durvalumab.

[0101] In certain embodiments, the anti-PD-L1 antibody is avelumab.

[0102] Other embodiments are directed to (i) modified, preferably oncolytic poxviruses, preferably oncolytic vaccinia viruses encoding an anti-CTLA-4 antibody and an anti-PD-L1 antibody, defective in both m2 and tk function (due to inactivating mutations in both the M2L locus and the J2R locus); (ii) modified, preferably oncolytic poxviruses, preferably oncolytic vaccinia viruses encoding an anti-CTLA-4 antibody and an anti-PD-L1 antibody, defective in m2 and rr activity (due to inactivating mutations in both the M2L locus and the I4L and / or F4L genes), and (iii) modified, preferably oncolytic poxviruses, preferably oncolytic vaccinia viruses encoding an anti-CTLA-4 antibody and an anti-PD-L1 antibody, defective in m2, tk, and rr activity (due to inactivating mutations in the M2L, J2R, and I4L / F4L loci).

[0103] In certain embodiments, the anti-CTLA-4 antibody is ipilimumab and the anti-PD-L1 antibody is avelumab.

[0104] Antigenic Polypeptides The term "antigenicity" refers to the ability to induce or stimulate a measurable immune response in a subject into which a recombinant poxvirus described herein encoding a polypeptide deemed antigenic has been introduced. The stimulated or induced immune response against the antigenic polypeptide expressed by said recombinant poxvirus may be humoral and / or cellular (e.g., production of antibodies, cytokines, and / or chemokines involved in the activation of effector immune cells). The stimulated or induced immune response usually contributes to a protective effect in the administered subject. A wide variety of direct or indirect biological assays are available in the art to assess the antigenicity of a polypeptide in vivo (in an animal or human subject) or in vitro (e.g., in a biological sample). For example, the ability of a particular antigen to stimulate innate immunity can be performed by measuring, for example, NK / NKT cells (e.g., typically activation levels) and the cascade of producing IFN-related cytokines and / or chemokines, TLR activation (in the case of Toll-like receptors), and other innate immune markers (Scott-Algara et al., 2010 PLOS One 5(1), e8761; Zhou et al., 2006, Blood 107, 2461-2469; Chan, 2008, Eur. J. Immunol. 38, 2964-2968). The ability of a particular antigen to stimulate a cellular immune response can be determined, for example, by quantification of cytokines produced by activated T cells, including cytokines derived from CD4+ and CD8+ T cells, using routine bioassays (e.g., ELISpot, multiparameter flow cytometry, ICS (for intracellular cytokine staining), cytokine profile analysis using multiplex techniques, or characterization and / or quantification of T cells by ELISA), as described herein, by measurement of the proliferative capacity of T cells (e.g., [ 3This can be performed by assaying the cytotoxic potential of antigen-specific T lymphocytes in sensitized subjects, by [H]thymidine incorporation assays (T cell proliferation assays), by identifying lymphocyte subpopulations by flow cytometry, as well as by immunization of appropriate animal models.

[0105] The term antigenic polypeptide is intended to encompass native antigens as well as fragments (e.g., epitopes, immunogenic domains, etc.) and variants thereof, provided that such fragments or variants can be the target of an immune response. A preferred antigenic polypeptide for use herein is a tumor-associated antigen. It is within the skill of the art to select one or more antigenic polypeptides suitable for treating a particular pathological condition.

[0106] In one embodiment, the antigenic polypeptide encoded by recombinant modified poxvirus is a cancer antigen (also called tumor-associated antigen or TAA) that is associated with cancer and / or serves as a cancer marker.Cancer antigens include various categories of polypeptides, such as the polypeptides that are normally silent (i.e., not expressed) in healthy cells, the polypeptides that are expressed at low levels or only at certain differentiation stages, and the polypeptides that are expressed temporarily, such as embryonic and fetal antigens, and the polypeptides that are caused by mutations in cellular genes, such as oncogenes (e.g., activated ras oncogene), proto-oncogenes (e.g., ErbB family), or the proteins that are caused by chromosomal translocation.

[0107] Numerous tumor-associated antigens are known in the art.Exemplary tumor antigens include, but are not limited to, in particular, colorectal-associated antigen (CRC), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), BAGE, GAGE ​​or MAGE antigen family, p53, mucin antigen (e.g., MUC1), HER2 / neu, p21ras, hTERT, Hsp70, iNOS, tyrosine kinase, mesothelin, c-erbB-2, alpha-fetoprotein, AM-1, and any immunogenic epitope or variant thereof.

[0108] Tumor-associated antigens may also include neo-epitopes / antigens that appear during the carcinogenesis process in cancer cells and contain one or more mutations of amino acid residues compared to the corresponding wild-type antigens. Typically, they are found in cancer cells or tissues obtained from a patient, but not in normal cells or tissues obtained from the patient or from a healthy individual.

[0109] Tumor-associated antigens can also include antigens encoded by pathogenic organisms capable of inducing a malignant condition in a subject (e.g., a chronically infected subject), such as RNA and DNA tumor viruses (e.g., human papillomavirus (HPV), hepatitis C virus (HCV), hepatitis B virus (HBV), Epstein-Barr virus (EBV), etc.) and bacteria (e.g., Helicobacter pilori).

[0110] In another embodiment, the antigenic polypeptide encoded by the recombinant modified poxvirus is a vaccine antigen that aims to therapeutically or prophylactically prevent infectious diseases when delivered to a human or animal subject. Numerous vaccine antigens are known in the art. Exemplary vaccine antigens include, but are not limited to, cellular antigens, viral antigens, bacterial antigens, or parasitic antigens. Cellular antigens include mucin 1 (MUC1) glycoprotein. Viral antigens include, for example, antigens derived from hepatitis viruses A, B, C, D, and E, immunodeficiency viruses (e.g., HIV), herpes viruses, cytomegaloviruses, varicella zoster, papilloma viruses, Epstein-Barr viruses, influenza viruses, parainfluenza viruses, coxsakie viruses, picornaviruses, rotaviruses, respiratory syncytial viruses, rhinoviruses, rubella viruses, papoviruses, mumps viruses, measles viruses, and rabies viruses. Some non-limiting examples of HIV antigens include gp120gp40, gp160, p24, gag, pol, env, vif, vpr, vpu, tat, rev, neftat, nef. Some non-limiting examples of human herpesvirus antigens include gH, gL, gM, gB, gC, gK, gE, or gD, or immediate early proteins, such as ICP27, ICP47, ICP4, ICP36 from HSV1 or HSV2. Some non-limiting examples of cytomegalovirus antigens include gB. Some non-limiting examples of antigens from Epstein-Barr virus (EBV) include gp350. Some non-limiting examples of varicella zoster virus antigens include gp1, 11, 111, and IE63. Some non-limiting examples of Hepatitis C virus antigens include envE1 or E2 proteins, core protein, NS2, NS3, NS4a, NS4b, NS5a, NS5b, p7. Some non-limiting examples of human papillomavirus (HPV) antigens include L1, L2, E1, E2, E3, E4, E5, E6, E7.Antigens from other viral pathogens, such as respiratory syncytial virus (e.g., F and G proteins), parainfluenza virus, measles virus, mumps virus, flavivirus (e.g., yellow fever virus, dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus), and influenza virus cells (e.g., HA, NP, NA, or M proteins), can also be used in accordance with the present invention. Bacterial antigens include, for example, antigens from TB, leprosy-causing mycobacteria, pneumocci, aerobic gram-negative bacilli, mycoplasma, staphyloccocus, streptococcus, salmonella, chlamydia, neisseriae, etc. Parasitic antigenic polypeptides include, for example, antigens from malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasis, and filariasis.

[0111] Nucleoside Pool Modulators In one embodiment, the modified poxvirus described herein carries in its genome one or more recombinant genes with nucleoside pool modulator function.Representative examples include, but are not limited to, cytidine deaminase, particularly yeast cytidine deaminase (CDD1) or human cytidine deaminase (hCD) (see WO2018 / 122088); polypeptides acting on metabolic and immune pathways (e.g., adenosine deaminase, particularly human adenosine deaminase huADA1 or huADA2; see EP17306012.0); polypeptides acting on apoptosis pathways; endonucleases (such as restriction enzymes, CRISPR / Cas9), and target-specific RNA (e.g., miRNA, shRNA, siRNA).

[0112] Detectable gene products Typically, such polypeptides are detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means, and thus may identify the recombinant poxvirus within a host cell or subject. Non-limiting examples of suitable detectable gene products include mCherry, Emerald, firefly luciferase, and green fluorescent protein (GFP and its enhanced version e-GFP), which are detectable by fluorescent means, and β-galactosidase, which is detectable by colorimetric means.

[0113] Expression of recombinant genes Nucleotide sequences encoding therapeutically interesting polypeptides, such as those cited above, may be easily obtained by standard molecular biology techniques (e.g., PCR amplification, cDNA cloning, chemical synthesis) using sequence data accessible in the art and the information provided herein. For example, methods for cloning antibodies, fragments and analogs thereof are known in the art (see, e.g., Harlow and Lane, 1988, Antibodies-A laboratory manual; Cold Spring Harbor Laboratory, Cold Spring Harbor NY). Nucleic acid molecules encoding antibodies may be isolated from hybridomas in production (e.g., Cole et al. Monoclonal antibodies and Cancer Therapy; Alan Liss pp77-96), immunoglobulin gene libraries, or any available source, or nucleotide sequences may be generated by chemical synthesis.

[0114] Furthermore, recombinant nucleic acids can be optimized to provide high-level expression in a particular host cell or subject. It has been observed in practice that the codon usage patterns of organisms are highly non-random, and codon usage can vary significantly between different hosts. For example, a therapeutic gene may originate from bacteria, viruses, or lower eukaryotes, and therefore have a codon usage pattern that is inappropriate for efficient expression in higher eukaryotic cells (e.g., humans). Typically, codon optimization is performed by replacing one or more "native" (e.g., bacterial, viral, or yeast) codons that correspond to the rarely used codons in the host organism with one or more codons that are more commonly used and code for the same amino acid. It is not necessary to replace all native codons that correspond to the rarely used codons, as even partial replacement can increase expression.

[0115] In addition to optimizing codon usage, expression in host cells or subjects can be further improved by further modification of recombinant nucleic acid sequence.For example, various modifications can be envisaged to prevent clustering of rare, non-optimal codons in concentrated regions, and / or suppress or modify "negative" sequence elements that are expected to negatively affect expression levels.Such negative sequence elements include, but are not limited to, regions with very high (>80%) or very low (<30%) GC content; AT-rich or GC-rich sequence regions; unstable direct or inverted repeats; RA secondary structures; and / or internal cryptic regulatory elements, such as internal TATA boxes, chi sites, ribosome entry sites, and / or splicing donor / acceptor sites.

[0116] According to the present invention, each of the one or more recombinant nucleic acid molecules is operably linked to a suitable regulatory element for expression in a host cell or subject. As used herein, the term "regulatory element" or "control sequence" refers to any element that enables, contributes to, or regulates the expression of an encoding nucleic acid in a given host cell or subject, including the replication, duplication, transcription, splicing, translation, stability, and / or transport of the nucleic acid or its derivatives (i.e., mRNA). As used herein, "operably linked" means that the elements that are linked are positioned to function for their intended purpose. For example, a promoter is operably linked to a nucleic acid molecule if it causes transcription from the transcription start to the terminator of said nucleic acid molecule in a permissive host cell.

[0117] It is understood by those skilled in the art that the selection of the control sequence may depend on factors such as the nucleic acid itself, the virus into which the nucleic acid is inserted, the host cell or subject, the desired expression level, etc. Promoters are particularly important. In the context of the present invention, promoters may constitutively direct the expression of nucleic acid molecules in many types of host cells, may be specific to a certain host cell (e.g., liver-specific control sequences), and may be regulated in response to a specific event or exogenous factor (e.g., by temperature, nutrient additives, hormones, etc.) or according to the stage of the virus cycle (e.g., late or early). In order to optimize virus production and to avoid potential toxicity of the expressed polypeptide, promoters that are repressed during the production process in response to a specific event or exogenous factor may also be used.

[0118] Poxvirus promoters are particularly suitable for expressing recombinant genes by modified poxviruses as described herein.Representative examples include, but are not limited to, vaccinia 7.5K, H5R, 11K7.5 (Erbs et al., 2008, Cancer Gene Ther. 15(1): 18-28), TK, p28, p11, pB2R, pA35R and K1L promoters, as well as synthetic promoters, such as those described in Chakrabarti et al. (1997, Biotechniques 23: 1094-7; Hammond et al., 1997, J. Virol Methods 66: 135-8; and Kumar and Boyle, 1990, Virology 179: 151-8), and early / late chimeric promoters.

[0119] Those skilled in the art will understand that the regulatory elements controlling the expression of the nucleic acid molecule inserted into the poxvirus genome may further comprise additional elements for correct initiation, regulation and / or termination of transcription (e.g., polyA transcription termination sequences), for mRNA transport (e.g., nuclear localization signal sequences), for processing (e.g., splicing signals), and for stability (e.g., introns and non-coding 5' and 3' sequences), for translation (e.g., initiator Met, tripartite leader sequences, IRES ribosome binding sites, signal peptides, etc.).

[0120] Optionally, recombinant polypeptide may advantageously contain additional regulatory elements to facilitate its expression, transport and biological activity. For example, a signal peptide may be included to facilitate secretion from infected cells. Signal peptide is typically inserted at the N-terminus of protein, immediately after Met initiator. The choice of signal peptide is wide and accessible to those skilled in the art. It may also be envisaged to add a transmembrane domain to facilitate the anchoring of encoded polypeptide in the appropriate membrane (e.g., plasma membrane) of infected cell. Transmembrane domain is typically inserted at the C-terminus of protein, immediately before or close to the stop codon. A wide variety of transmembrane domains are available in the art (see, for example, WO99 / 03885).

[0121] As a further example, peptide tags (typically short peptide sequences that can be recognized by available antisera or chemical compounds) may also be added to track expression, trafficking, or purification of the encoded gene product. A wide variety of tag peptides can be used in the context of the present invention, including but not limited to PK tags, FLAG octapeptides, MYC tags, HIS tags (usually a region of 4-10 histidine residues), and e-tags (US 6,686,152). Tag peptides may be independently placed at the N-terminus, or C-terminus, or internally, of the protein, and may be placed at any of these locations when several tags are used. Tag peptides can be detected by immunodetection assays using anti-tag antibodies.

[0122] As another example, glycosylation can be altered to enhance the biological activity of the encoded gene product. Such modifications can be accomplished, for example, by mutating one or more residues within the glycosylation site. Altered glycosylation patterns can enhance the ADCC ability and / or affinity of the antibody for the target.

[0123] Another approach that may be pursued in the context of the present invention is the coupling of the recombinant gene products encoded by the modified poxviruses described herein with external agents, such as cytotoxic and / or labeling agents. As used herein, the term "cytotoxic agent" refers to a compound that is directly toxic to cells (e.g., inhibits replication or growth), e.g., a toxin (e.g., an enzymatically active bacterial, fungal, plant, or animal toxin, or fragment thereof). As used herein, a "labeling agent" refers to a detectable compound. The labeling agent may be detectable itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze the chemical modification of a substrate compound that is detectable. The coupling may be performed by genetic fusion of the therapeutic polypeptide with the external agent.

[0124] Insertion of the recombinant nucleic acid (with appropriate regulatory elements) into the poxvirus genome is carried out by conventional means using appropriate restriction enzymes or, preferably, by homologous recombination.

[0125] In a further aspect, the invention provides a method for generating modified poxviruses, in particular recombinant oncolytic poxviruses, as described herein, by homologous recombination between a transfer plasmid containing the recombinant nucleic acid (and its regulatory elements) flanked 5' and 3' by viral sequences present upstream and downstream of the insertion site, respectively, and the viral genome. In one embodiment, the method comprises the steps of generating said transfer plasmid (e.g. by conventional molecular biology methods) and introducing said transfer plasmid into a suitable host cell, in particular together with a poxvirus genome (e.g. an M2L-inactivated virus) containing the flanking sequences present in the transfer plasmid. Preferably, the transfer plasmid is introduced into the host cell by transfection and into the virus by infection.

[0126] The size of each flanking viral sequence may vary, it is usually at least 100 bp and at most 1500 bp, preferably about 150-800 bp on each side of the recombinant nucleic acid, advantageously 180-600 bp, preferably 200-550 bp, more preferably 250-500 bp.

[0127] The recombinant nucleic acid molecule can be independently inserted anywhere in the poxvirus genome, and insertion can be performed by routine molecular biology well known in the art. Various insertion sites can be considered, for example, in unnecessary viral genes, intergenic regions, or non-coding parts of the poxvirus genome. In the context of the present invention, the J2R locus is particularly suitable. As mentioned above, when a foreign nucleic acid is inserted into the poxvirus genome, the viral locus at the insertion site is at least partially deleted, which can, for example, suppress the expression of viral gene products encoded by the fully or partially deleted locus, resulting in a virus with a defect in said viral function.

[0128] In certain embodiments, the identification of modified poxviruses may be facilitated by using a selection and / or detectable gene. In a preferred embodiment, the transfer plasmid further comprises a selection marker, particularly preferably the GPT gene (encoding guanine phosphoribosyltransferase) that allows growth in a selection medium (e.g. in the presence of mycophenolic acid, xanthine, and hypoxanthine), or a detectable gene encoding a detectable gene product, such as GFP, e-GFP, or mCherry. Furthermore, the use of an endonuclease capable of performing a double-strand break in said selection or detectable gene may also be considered. Said endonuclease may be in the form of a protein and may be expressed by an expression vector.

[0129] The homologous recombination allowing the generation of a modified poxvirus is preferably carried out in a suitable host cell (eg HeLa or CEF cells).

[0130] Poxvirus production Typically, the modified poxviruses of the invention are produced in suitable host cell lines using conventional techniques that involve culturing transfected or infected host cells under appropriate conditions such that infectious poxvirus particles are produced and harvested.

[0131] Thus, in another aspect, the present invention relates to a method for producing the modified poxvirus described herein, preferably 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 to allow the production of virus (e.g. infectious poxvirus particles); (d) recovering the produced virus from the culture of the producer cell line; and optionally (e) purifying the recovered virus.

[0132] In one embodiment, the producer cell is a HeLa cell (e.g., ATCC-CRM-CCL-2 商標 or ATCC-CCL-2.2 商標 ), HER96, PER-C6 (Fallaux et al., 1998, Human Gene Ther. 9: 1909-17), and a hamster cell line, e.g., BHK-21 (ATCC CCL-10), or an avian cell, e.g., one of those described in WO2005 / 042728, WO2006 / 108846, WO2008 / 129058, WO2010 / 130756, WO2012 / 001075, etc.), as well as primary chicken embryo fibroblasts (CEF) prepared from chicken embryos obtained from fertilized eggs.

[0133] The producer cells are preferably cultured in a suitable medium, which may or may not be supplemented with serum and / or suitable growth factors if necessary (e.g. synthetic medium, preferably synthetic medium without animal or human products). The suitable medium may be easily selected by the skilled artisan depending on the producer cells. Such media are commercially available. The producer cells are preferably cultured at a temperature comprised between +30°C and +38°C (more preferably about 37°C) for 1-8 days prior to infection. If necessary, several passages of 1-8 days may be performed to increase the total cell number.

[0134] In step (b), the producer cells are infected with the modified poxvirus under suitable conditions and with a suitable multiplicity of infection (MOI) that allows productive infection of the producer cells. For illustrative purposes, a suitable MOI is 10 -3 Preferably, the MOI is from 0.01 to 5, more preferably from 0.03 to 1. The infection step is carried out in a medium which may be the same as or different from the medium used for culturing the producer cells.

[0135] In step (c), the infected producer cells are then cultured under suitable conditions well known to those skilled in the art until progeny poxvirus (e.g. infectious viral particles) are produced. The culture of the infected producer cells is also preferably carried out for 1 to 5 days at +32°C to +37°C in a medium which may be the same or different from the medium used for culturing the producer cells and / or used for the infection step.

[0136] In step (d), the poxvirus produced in step (c) is collected from the culture supernatant and / or the producer cells. Recovery from the producer cells may require a step of disrupting the producer cell membrane to release the virus. Disruption of the producer cell membrane can be induced by a variety of techniques well known to those skilled in the art, including but not limited to freeze / thaw, hypotonic lysis, sonication, microfluidization, high shear (also called high speed) homogenization, or high pressure homogenization.

[0137] The poxvirus collected can then be distributed in dose units and at least partially purified before being used according to the present invention.Many purification steps and methods are available in the art, including, for example, clarification, enzymatic treatment (e.g., endonuclease, protease, etc.), chromatography steps, and filtration steps.Suitable methods are described in the art (see, for example, WO2007 / 147528; WO2008 / 138533, WO2009 / 100521, WO2010 / 130753, WO2013 / 022764).

[0138] In one aspect, the present invention also provides a cell infected with a modified poxvirus described herein.

[0139] composition The present invention also provides compositions comprising a therapeutically effective amount of the modified poxviruses described herein (active agents) and a pharma- ceutically acceptable vehicle. Such compositions may be administered once or several times, via the same or different routes.

[0140] A "therapeutically effective amount" corresponds to an amount of modified poxvirus sufficient to produce one or more beneficial results. Such a therapeutically effective amount may vary as a function of various parameters, in particular the method of administration; the disease state; the age and weight of the subject; the ability of the subject to respond to treatment; the type of concurrent treatment; the frequency of treatment; and / or the need for prophylaxis or therapy. When prophylactic use is concerned, the composition of the present invention is administered in a dose sufficient to prevent or delay the onset and / or establishment and / or recurrence of a proliferative disease (e.g., cancer), especially in a subject at risk. In the case of "therapeutic" use, the composition is administered to a subject diagnosed with a proliferative disease (e.g., cancer) with the goal of treating the disease, ultimately in conjunction with one or more conventional therapeutic modalities. In particular, a therapeutically effective amount may be the amount necessary to cause an observable improvement in the clinical condition compared to a baseline condition or compared to the expected condition if not treated, such as a reduction in tumor number, a reduction in tumor size, a reduction in the number or extent of metastases, an increase in the length of remission, a stabilization of the disease state (i.e., no worsening), a delay or slowing of disease progression or severity, a remission or palliation of the disease state, an increase in survival, an improvement in response to standard treatments, an improvement in quality of life, a decrease in mortality, etc. For example, techniques routinely used in laboratories (e.g., flow cytometry, histology, medical imaging) can be used to monitor tumors.

[0141] Therapeutically effective amount may also be the amount necessary to cause the generation of effective non-specific (innate) immune response and / or specific (adaptive) immune response. Typically, the generation of immune response, especially T cell response, may be evaluated in vitro, in suitable animal models, or using biological samples collected from subjects (ELISA, flow cytometry, histology, etc.). Various available antibodies may also be used to identify the different immune cell populations involved in anti-tumor response present in the treated subject, such as cytotoxic T cells, activated cytotoxic T cells, natural killer cells, and activated natural killer cells. Improvement of clinical condition can be easily evaluated by any relevant clinical measurement method that is usually used by physicians or other skilled medical staff.

[0142] The term "pharmaceutically acceptable vehicle" is intended to include any and all carriers, solvents, diluents, excipients, adjuvants, dispersion media, coatings, antibacterial and antifungal agents, absorption agents, and the like, that are compatible with administration in mammals, particularly human subjects. Non-limiting examples of pharmaceutically acceptable vehicles include water, NaCl, normal saline solution, lactated Ringer's solution, sugar solutions (e.g., glucose, trehalose, saccharose, dextrose, and the like), alcohol, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, and the like, and other physiologically balanced saline solutions may also be used (see, for example, the latest edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams&Wilkins).

[0143] In one embodiment, the compositions are suitably formulated to ensure stability of the modified poxvirus active agent under conditions of manufacture and long-term storage (i.e., at least 6 months, preferably at least 2 years) at freezer temperatures (e.g., −70° C. to −10° C.), refrigerated temperatures (e.g., 4° C.), or ambient temperatures (e.g., 20-25° C.). Such formulations generally include a liquid carrier, such as an aqueous solution.

[0144] Advantageously, the composition is suitably buffered for human use, preferably at physiological pH, or at a slightly basic pH (e.g., from about pH 7 to about pH 9, with a pH of 7 to 8 being particularly preferred, particularly close to 7.5). Suitable buffers include TRIS (tris(hydroxymethyl)methylamine), TRIS-HCl (tris(hydroxymethyl)methylamine-HCl), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), phosphate buffers (e.g., PBS), ACES (N-(2-acetamido)-aminoethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), MOPSO (3-(N-morpholino)-2-hydroxypropanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), DIPSO (3-[bis(2-hydroxyethyl)amino]-2-hydro

[0033] Examples of suitable hydroxypropane-1-sulfonic acid include, but are not limited to, N-(2-hydroxypropyl)-N'-pyrazine-N'-3-propanesulfonic acid ... Preferably, said buffer is selected from TRIS-HCl, TRIS, Tricine, HEPES and phosphate buffers, including a mixture of Na2HPO4 and KH2PO4 or a mixture of Na2HPO4 and NaH2PO4. Said buffers (especially those mentioned above, especially TRIS-HCl) are preferably present in a concentration between 10 and 50 mM.

[0145] It may also be beneficial to include a monovalent salt in the formulation to ensure appropriate osmolality, said monovalent salt may in particular be selected from NaCl and KCl, preferably said monovalent salt is NaCl, preferably at a concentration of 10-500 mM NaCl.

[0146] The composition may also be formulated to include a cryoprotectant to protect the modified poxvirus at low storage temperatures. Suitable cryoprotectants include, but are not limited to, sucrose (or saccharose), trehalose, maltose, lactose, mannitol, sorbitol, and glycerol, preferably at a concentration of 0.5-20% (weight in grams per volume in liters, referred to as w / v). For example, sucrose is preferably present at a concentration of 5-15% (w / v).

[0147] The modified poxvirus composition, in particular the liquid composition, may further comprise a pharma- ceutically acceptable chelating agent to improve stability. The pharma- ceutically acceptable chelating agent may in particular be selected from ethylenediaminetetraacetic acid (EDTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ethylene glycol tetraacetic acid (EGTA), dimercaptosuccinic acid (DMSA), diethylenetriaminepentaacetic acid (DTPA) and 2,3-dimercapto-1-propanesulfonic acid (DMPS). The pharma- ceutically acceptable chelating agent is present in a concentration of at least 50 μM, particularly preferably in a concentration of 50 to 1000 μM. Preferably, the pharma- ceutically acceptable chelating agent is EDTA, present in a concentration close to 150 μM.

[0148] Additional compounds may be present to enhance the stability of the modified poxvirus composition. Such additional compounds include, but are not limited to, C2-C3 alcohols (preferably at a concentration of 0.05-5% (volume / volume or v / v)), sodium glutamate (preferably at a concentration of less than 10 mM), non-ionic surfactants (US7,456,009, US2007-0161085), such as Tween 80 (also known as polysorbate 80) at low concentrations of less than 0.1%. Divalent salts such as MgCl2 or CaCl2 have been found to induce stabilization of various biological products in liquid state (see Evans et al. 2004, J Pharm Sci. 93:2458-75 and US7,456,009). Amino acids, particularly histidine, arginine, and / or methionine, have been found to induce stabilization of various viruses in liquid state (see WO2016 / 087457).

[0149] The presence of high molecular weight polymers such as dextran or polyvinylpyrrolidone (PVP) is particularly suitable for freeze-dried compositions obtained by processes involving vacuum drying and freeze-drying, as the presence of these polymers aids in the formation of a cake during freeze-drying (see, e.g., WO03 / 053463; WO2006 / 085082; WO2007 / 056847; WO2008 / 114021 and WO2014 / 053571).

[0150] According to the present invention, the formulation of the composition can also be adapted to the method of administration to ensure correct distribution or delayed release in vivo.Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polyethylene glycol can be used (see, for example, "Sustained and Controlled Release Drug Delivery Systems", edited by JR Robinson, Marcel Dekker, Inc., New York, 1978; WO01 / 23001; WO2006 / 93924; WO2009 / 53937).

[0151] By way of example, a Tris-buffered formulation (Tris-HCl pH 8) containing sucrose 5% (w / v), sodium glutamate 10 mM, and NaCl 50 mM is suitable for storage of the compositions described herein at -20°C to 5°C.

[0152] Dosage In a preferred embodiment, the composition is formulated in individual doses, each dose containing about 10 3 ~10 12 It contains vp (viral particles), iu (infectious units) or pfu (plaque forming units) of modified poxvirus. The amount of virus present in a sample can be determined by routine titration, for example by counting the number of plaques after infection of permissive cells (e.g. HeLa cells) to obtain a plaque forming unit (pfu) titer, by measuring the A260 absorbance (vp titer) or by quantitative immunofluorescence, for example using an antiviral antibody (iu titer). Further refinements of the calculations required to adapt the appropriate dosage to a subject or group of subjects can be routinely made by the expert, taking into account the relevant circumstances. As a general guide, a suitable individual dose for a poxvirus composition is about 10 3 ~about 10 12 pfu, advantageously about 10 4 pfu~about 1011 pfu, preferably about 10 5 pfu~about 10 10 pfu, more preferably about 10 6 pfu~about 10 9 pfu, in particular about 10 6 , 5x10 6 , 10 7 , 5x10 7 , 10 8 , or 5x10 8 Individual doses of pfu are particularly preferred.

[0153] Administration In the context of the present invention, any conventional route of administration can be applied, including parenteral, local, or mucosal routes. Parenteral routes are for administration as injections or infusions and include systemic as well as local routes. Parenteral injection types that can be used to administer the poxvirus composition include intravenous (into a vein), intravascular (into a blood vessel), intraarterial (into an artery such as the hepatic artery), intradermal (into the dermis), subcutaneous (under the skin), intramuscular (into a muscle), intraperitoneal (into the peritoneum), and intratumoral (into or in the immediate vicinity of a tumor), as well as scarification. Administration may take the form of a single bolus dose, for example, by a continuous perfusion pump. Mucosal administration includes, but is not limited to, oral / feeding, intranasal, intratracheal, intrapulmonary, intravaginal, or intrarectal routes. Local administration can also be performed using transdermal means (e.g., patches, etc.). Preferably, the modified poxvirus composition is formulated for intravenous administration, or for intratumoral administration at or near a tumor.

[0154] Administration may use a conventional syringe and needle (e.g., Quadrafuse needle), or any compound or device available in the art that can facilitate or improve the delivery of modified poxvirus in a subject (e.g., electroporation to facilitate intramuscular administration). An alternative is the use of a needleless injection device (e.g., Biojector™ device). Transdermal patches may also be envisioned.

[0155] The compositions described herein are suitable for single administration or a series of administrations. It is also possible to proceed through successive administration cycles repeated after a rest period. The interval between each administration may be from 3 days to about 6 months (e.g., 24 hours, 48 ​​hours, 72 hours, weekly, biweekly, monthly, or quarterly, etc.). The intervals may also be irregular. The dose may vary from administration to administration within the ranges mentioned above. A preferred treatment regime involves 2-10 weekly administrations of the poxvirus composition, possibly followed by 2-15 administrations at longer intervals (e.g., 3 weeks).

[0156] Methods of using the m2 defective poxviruses and compositions of the invention In another aspect, the compositions described herein are for use in treating or preventing proliferative diseases according to the modalities described herein.Thus, the present invention also provides a method of treatment comprising administering the composition to a subject in need of treatment (preferably a subject suffering from cancer) in an amount sufficient to treat or prevent such disease, as well as a method of inhibiting tumor cell proliferation comprising administering the composition to a subject.In the context of the present invention, the methods and uses described herein are aimed at slowing down, curing, ameliorating, or managing the onset or progression of proliferative diseases.

[0157] The term "proliferative disease" as used herein encompasses a broad group of various diseases resulting from uncontrolled cell proliferation and expansion, including cancer, and diseases associated with increased osteoclast activity (e.g., rheumatoid arthritis, osteoporosis, etc.), and cardiovascular diseases (e.g., restenosis due to proliferation of smooth muscle cells in the walls of blood vessels). Unregulated cell division and growth can lead to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. The term "cancer" can be used interchangeably with any of the terms "tumor," "malignant tumor," "neoplasm," etc., and is intended to include any type of tissue, organ, or cell, any stage of malignant tumor (e.g., prelesion to stage IV), including solid tumors as well as blood-borne tumors and primary and metastatic cancers.

[0158] Representative examples of cancers that may be treated using the compositions and methods of the present invention include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia, and more specifically, bone cancer, gastrointestinal cancer, liver cancer, pancreatic cancer, stomach cancer, colorectal cancer, esophageal cancer, oropharyngeal cancer, laryngeal cancer, salivary gland cancer, thyroid cancer, lung cancer, head and neck cancer, skin cancer, squamous cell carcinoma, melanoma, uterine cancer, cervical cancer, endometrial cancer, vulvar cancer, ovarian cancer, breast cancer, prostate cancer, endocrine system cancer, soft tissue sarcoma, bladder cancer, kidney cancer, glioblastoma, and various types of central nervous system (CNS) cancer. In one embodiment, the methods and uses according to the present invention are for treating a cancer selected from the group consisting of renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), breast cancer (e.g., metastatic breast cancer), colorectal cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer (e.g., hepatocellular carcinoma), gastric cancer, bile duct cancer, endometrial cancer, pancreatic cancer, and ovarian cancer.

[0159] Typically, administration of the compositions described herein results in a therapeutic benefit to the treated subject, which can be evidenced by an observable improvement in clinical condition compared to baseline condition or compared to expected condition if not treated. The improvement in clinical condition can be easily evaluated by any relevant clinical measurement method normally used by a physician or other skilled medical staff. In the context of the present invention, the therapeutic benefit can be transient (for one or several months after cessation of administration) or persistent (for several months or years). As the natural history of the clinical condition can vary widely from subject to subject, the therapeutic benefit does not have to be observed in every single subject treated, but in a significant number of subjects (e.g., a statistically significant difference between two groups can be determined by any statistical test known in the art, such as Tukey's parametric test, Kruskal-Wallis test, Mann and Whitney U test, Student's t test, Wilcoxon test, etc.).

[0160] For example, therapeutic benefit in a subject afflicted with cancer may be evidenced by, for example, a reduction in tumor number, a reduction in tumor size, a reduction in the number or extent of metastases, an increase in the length of remission, a stabilization of the disease state (i.e., no worsening), a decrease in the rate or severity of disease progression, an increase in survival, an improved response to standard treatment, remission of surrogate markers of disease, an improvement in quality of life, a reduction in mortality, and / or prevention of disease recurrence.

[0161] Clinical benefit can be assessed using appropriate measurements, such as blood tests, analysis of biological fluids and biopsies, and medical imaging methods. These can be performed before administration (baseline), at various times during treatment, and after treatment has been discontinued. Such measurements are routinely evaluated in medical laboratories and hospitals, and many kits are commercially available (e.g., immunoassays, quantitative PCR assays).

[0162] Preferred embodiments are directed to compositions comprising a modified poxvirus, desirably an oncolytic modified poxvirus, preferably an oncolytic vaccinia virus (e.g., the Copenhagen strain), particularly preferably an oncolytic vaccinia virus encoding an anti-CTLA-4 antibody as described herein, for use in treating subjects with cancer, preferably renal cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer), melanoma, and ovarian cancer.

[0163] In another embodiment, the modified poxvirus or composition described herein is for use to enhance an anti-tumor adaptive immune response or to enhance or prolong an anti-tumor response.

[0164] In another aspect, the modified poxvirus or composition thereof is used or administered to stimulate or improve an immune response in a subject to be treated. Thus, the present invention also encompasses a method for stimulating or improving an immune response, comprising administering to a subject in need of a stimulated or improved immune response, a composition in an amount sufficient to stimulate or improve the immunity of the subject according to the modalities described herein. The stimulated or improved immune response can be specific (i.e., epitope / antigen-directed) and / or non-specific (innate), humoral and / or cellular, in particular a CD4+ or CD8+-mediated T cell response. The ability of the compositions described herein to stimulate or improve an immune response can be evaluated in vitro (e.g., using a biological sample collected from the subject) or in vivo using a variety of direct or indirect assays standard in the art (see, for example, Coligan et al., 1992 and 1994, Current Protocols in Immunology; ed J Wiley & Sons Inc, National Institute of Health or later editions). Those cited above with regard to the antigenicity of the polypeptides are also suitable.

[0165] In particular, and in comparison to conventional (m2 positive) poxviruses, the modified poxviruses or compositions described herein also have the following objectives: Stimulating or improving lymphocyte-mediated immune responses (especially to antigenic polypeptides); Stimulate or improve APC activity; Stimulating or improving anti-tumor responses; Stimulating or improving the CD28 signaling pathway; improve the therapeutic efficacy provided by the modified poxviruses described herein in a treated subject or group of treated subjects; and / or Reduce the toxicity provided by the modified poxviruses described herein in a treated subject or group of treated subjects. or any combination thereof are also useful.

[0166] Combination therapy In one embodiment, the modified poxvirus, composition or method of the present invention is used as a single therapy.In another embodiment, they can be used or performed together with one or more additional therapies, particularly with standard treatment therapy suitable for the type of cancer that afflicts the subject to be treated.Standard treatment therapies for various types of cancer are well known to those skilled in the art and are usually disclosed in Cancer Network and clinical practice guidelines.Such one or more additional therapies are 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, and transplantation, etc.

[0167] Such additional anti-cancer therapy is administered to the subject according to standard practice before, after, simultaneously with, or in an intervening manner with the modified poxvirus or composition described herein. Co-administration of two or more therapies does not require that the agents be administered at the same time or by the same route, so long as there is an overlap in the period during which the composition and the additional anti-cancer therapy exert their therapeutic effect. Co-administration includes administering the modified poxvirus composition within the same day (e.g., 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours) as the other therapeutic agent. Although any order is contemplated by the present invention, it is preferred that the modified poxvirus composition is administered to the subject before the other therapeutic agent.

[0168] In certain embodiments, the modified poxvirus or composition described herein may be used in conjunction with surgery. For example, the composition may be administered after partial or total surgical resection of a tumor (e.g., by local application, e.g., by local application within the resected compartment).

[0169] In other embodiments, modified poxviruses or compositions described herein can be used concomitantly with radiation therapy. Those skilled in the art can easily assemble appropriate radiation therapy protocols and parameters (see, e.g., Perez and Brady, 1992, Principles and Practice of Radiation Oncology, 2nd Ed. JB Lippincott Co; with appropriate modifications and adaptations as will be readily apparent to those skilled in the art). Types of radiation that can be used, particularly in cancer treatment, are well known in the art and include electron beams, linear accelerators or high energy photons from radioactive sources such as cobalt or cesium, protons, and neutrons. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by tumor cells. Regular X-ray doses or high single doses for extended periods (3-6 weeks) are contemplated by the present invention.

[0170] In certain embodiments, modified poxvirus or composition described herein may be used together with chemotherapy.Representative examples of suitable chemotherapeutic agents currently available for the treatment of cancer include, but are not limited to, alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, platinum derivatives, tyrosine kinase receptor inhibitors, cyclophosphamide, metabolic antagonists, DNA damaging agents, and mitotic inhibitors.Representative examples of suitable chemotherapeutic agents currently available for the treatment of infectious diseases include, in particular, antibiotics, metabolic antagonists, antimitotic agents, and antiviral agents (e.g., interferon alpha).

[0171] In further embodiments, the modified poxviruses or compositions described herein may be used in conjunction with anti-neoplastic antibodies and immunotherapies such as siRNA and antisense polynucleotides.

[0172] In still further embodiments, the modified poxvirus or compositions described herein may be used in conjunction with an adjuvant. Representative examples of suitable adjuvants include, but are not limited to, TLR3 ligands (Claudepierre et al., 2014, J. Virol. 88(10): 5242-55), TLR9 ligands (e.g., Fend et al., 2014, Cancer Immunol. Res. 2, 1163-74; Carpentier et al., 2003, Frontiers in Bioscience 8, e115-127; Carpentier et al., 2006, Neuro-Oncology 8(1): 60-6; ​​EP1162982; US7,700,569 and US7,108,844), and PDE5 inhibitors, such as sildenafil (US5,250,534, US6,469,012, and EP463756).

[0173] In further embodiments, the modified poxvirus or compositions described herein may be used according to a prime boost regimen comprising sequential administration of a priming composition and a boosting composition. Typically, the priming composition and the boosting composition use different vectors encoding at least an antigenic domain that has in common at least one of the modified poxviruses described herein. Furthermore, the priming composition and the boosting composition can be administered at the same site or at different sites by the same route of administration or by different routes of administration.

[0174] Other features, objects, and advantages of the present invention will be apparent from the description and drawings, and from the claims. The following examples are included to illustrate preferred embodiments of the present invention. However, those skilled in the art, in light of this disclosure, will appreciate that changes can be made in the specific embodiments disclosed without departing from the spirit and scope of the present invention. EXAMPLES

[0175] material and method Proteins and viruses Recombinant Fc fusion proteins (human and mouse) with or without a C-terminal His tag were ordered from R&D Systems. Human CD80-Fc and CD86-Fc were biotinylated in-house using biotinamide hexanoyl-6-aminohexanoic acid N-hydroxysuccinimide ester (Sigma).

[0176] Various vaccinia viruses were used: · Wild-type vaccinia (Copenhagen, Wyeth, and Western Reserve strains); · a double-deleted vaccinia virus (strain Copenhagen) defective in both thymidine kinase and ribonucleotide reductase activities (tk-;rr-; described in WO2009 / 065546); Triple-deleted vaccinia virus (Copenhagen strain) defective in tk, rr-, and m2 activities. The triple-deleted virus was generated from the double-deleted tk-rr- by specific homologous recombination into the open reading frame of the M2L locus, as described below.

[0177] In addition to vaccinia virus, all poxviruses tested were wild-type strains unless otherwise specified.

[0178] M2L deletion in rr- and tk- vaccinia viruses For in vivo studies, double (tk-rr-) and triple (tk-rr-m2-) deletion vaccinia viruses were engineered to encode firefly luciferase under the p11K7.5 promoter at the J2R locus.

[0179] The M2L gene deletion introduced into the VV genome includes 64 nucleotides upstream of the m2 ORF and the first 169 codons of the m2 ORF. The deletion was performed by homologous recombination with a transfer plasmid derived from PUC18. This transfer plasmid contained a left (nt 26980-27479 of VV genome accession M35027) arm and a right (nt 28051-28550) arm separated by an expression cassette encoding the selection marker enhanced green fluorescent protein / xanthine-guanine phosphoribosyltransferase (EGFP / GPT) under the control of the vaccinia pH5R promoter. The resulting plasmid was transferred by electroporation using Amaxa Nucleofactor into chicken embryo fibroblasts (CEFs) infected with luciferase-encoding vaccinia virus (rr-; tk- / luciferase). Recombinant viruses were isolated by EGFP / GPT selection. Deletion of M2L and insertion of the EGFP / GPT cassette were confirmed by PCR analysis. The EGFP / GPT selection cassette was removed by passaging the recombinant virus in CEFs without selection. Primary research stocks were produced in CEFs. Deletion of the M2L gene was confirmed by PCR and sequencing.

[0180] Virus was produced in CEF after infection at MOI 0.05 and 3 days of incubation. Three days after infection, crude harvest containing infected cells and culture supernatant was harvested and stored at -20°C until use. Prior to purification, the suspension was homogenized to release virus particles. Large cell debris was then removed by depth filtration. The clarified virus suspension was then concentrated and diafiltered with formulation buffer by using tangential flow filtration and size hollow fiber microfiltration filters. Finally, the purified virus was further concentrated using the same tangential flow filtration system, aliquoted, and stored at -80°C until use.

[0181] ELISA assay for B7 binding 96-well plates (Nunc immune plate Medisorp) were coated overnight at 4°C with 100 μL of 0.5 μg / mL B7, CTLA4, or CD28 protein dissolved in coating buffer (50 mM Na-carbonate pH 9.6). Microplates were washed with PBS / 0.05% Tween 20 and saturated with 200 μL of blocking solution (PBS; 0.05% Tween 20; 5% Non-Fat Dry Milk (Biorad)). All antibody preparations and dilutions were made in blocking solution. In triplicate, 100 μL of sample was added to each well, with two-fold serial dilutions for some experiments (binding curves). Microplates were then incubated with 100 μL of anti-Flag-HRP (Sigma) diluted 1:10,000. The microplate was then incubated with 100 μL / well of 3,3',5,5'-tetramethylbenzidine (TMB, Sigma) and the reaction was stopped with 100 μL of 2M H2SO4. The absorbance was measured at 450 nm using a plate reader (TECAN Infinite M200PRO). The absorbance values ​​were transferred to the software GraphPadPrism for analysis and graph display.

[0182] Competitive ELISA Experimental conditions and solutions were the same as above unless otherwise specified. For CTLA4 / CD80, CD28 / CD80, and PDL1 / CD80 competition assays, 100 μL of CTLA4, CD28, and PDL1 were coated at 0.25 (CTLA4) or 1 μg / mL (CD28 and PD-L1). Samples were added and diluted (2-fold serial dilutions) in blocking solution containing a fixed concentration of CD80 (50 ng / mL, 250 ng / mL, or 500 ng / mL for CTLA4, CD28, and PD-L1, respectively). For CD86 / CTLA4 and CD28 / CD86 competition assays, 100 μL of CD86 or CD28 were coated at 0.25 (CD86) or 2 μg / mL (CD28). Samples were added and diluted (2-fold serial dilutions) in blocking solution containing a fixed concentration of CTLA4 (100 ng / mL) or CD86 (500 ng / mL), respectively. Anti-His tag-HRP (Qiagen) at 1 / 2000 or streptavidin-HRP (Southern Biotech) at 1 / 1000 were used as conjugation reagents. Plates were further processed and results were analyzed as described above.

[0183] Western blot Twenty-five microliter samples were prepared in Laemmli buffer with (reducing conditions) or without (non-reducing conditions) 5% β-mercaptoethanol (BME). After electrophoresis on Criterion TGX 4-15% stain free gels (Biorad), proteins were transferred to PVDF membranes (Transblot Turbo System). The IBind Flex Western system (Invitrogen) was used for protein / antibody incubation and washing. Blots were probed with 2.5 μg / mL CD80-Fc, CD86-Fc, CTLA4-Fc, or anti-Flag-HRP at 1 / 1000. For CD80-Fc, CD86-Fc, and CTLA4-Fc, HRP anti-human Fc (Bethyl) at 1 / 3000 was used as the conjugated antibody. 1X iBind Flex solution was used for blocking, diluting antibodies, washing, and wetting the iBind Flex Card. Immunocomplexes were detected using Amersham ECL Prime Western Blotting reagent. Chemiluminescence was recorded using a Molecular Imager ChemiDOC XRS (Biorad).

[0184] Affinity chromatography Supernatants of CEFs infected (MOI 0.05) with either MVA or Vaccinia Virus Copenhagen were collected 72 hours post-infection. Supernatants were centrifuged and filtered through 0.2 μm filters to remove most of the cell debris and vaccinia virus. Treated supernatants supplemented with 0.05% Tween 20 were then concentrated approximately 20-fold using vivaspin20 30000 MWCO cut-off concentrators (Sartorius). Streptavidin magnetic beads (GE healthcare) were coated with an irrelevant monoclonal biotinylated antibody (chCXIIG6), CTLA4-Fc-Biot, or CD86-Fc-Biot. To remove nonspecific binding, 4 mL of concentrated supernatant (MVA and Vaccinia Virus Copenhagen) was incubated with 24 μL of chCXIIG6-streptavidin beads. The flow-through from this first incubation was split into two equal parts and incubated with CTLA4-Fc-Biot-streptavidin beads or CD86-Fc-Biot-streptavidin beads to obtain the following four arms: MVA supernatant + CTLA4 beads (MVA A4); MVA supernatant + CD86 beads (MVA CD); Vaccinia virus + CTLA4 beads supernatant (VV A4) and Vaccinia virus + CD86 beads (VV CD86). After extensive washing of the beads with PBS, 0.05% Tween 20, followed by PBS, bound proteins were eluted twice with 50 μL of 0.1 M acetic acid and immediately neutralized by adding 4 μL of 2 M Tris Base. The two eluates were then pooled before MS analysis.

[0185] Protein preparation for digestion 10 μL or 20 μL samples were evaporated and reduced by solubilization in 10 μL of 10 mM DTT in 25 mM NH4HCO3 (1 h at 57 °C). Reduced cysteine ​​residues were alkylated with 10 μL of 55 mM iodoacetamide in 25 mM NH4HCO3 for 30 min at room temperature in the dark. Trypsin (12.5 ng / μL; Promega V5111), freshly diluted in 25 mM NH4HCO3, was added to the samples in a 1:100 (enzyme / protein) ratio to a final volume of 30 μl and incubated for 5 h at 37 °C. Trypsin activity was inhibited by acidification with 5 μL of H2O / TFA 5%.

[0186] MS / MS analysis Samples were analyzed by a nanoUPLC system (nanoAcquity, Waters) coupled to a quadrupole-Orbitrap hybrid mass spectrometer (Q-Exactive plus, Thermo Scientific, San Jose, CA). The UPLC system was equipped with a Symmetry C18 precolumn (20х0.18mm, 5μm particle size, Waters, Milford, USA) and an ACQUITY UPLC® BEH130 C18 separation column (75μm×200mm, 1.7μm particle size, Waters). The solvent system consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). 2μL of each sample was injected. Peptides were captured at 5μL / min with 99%A and 1%B during 3 min. Elution was performed using a linear gradient of 1 to 35% B in 79 min at 60 °C with a flow rate of 400 nL / min. To minimize carryover, a column wash (50% ACN for 20 min) was included between each sample in addition to a solvent blank injection performed after each sample.

[0187] The Q-Exactive Plus was operated in positive ion mode with a source temperature of 250 °C and a spray voltage of 1.8 kV. Full scan MS spectra (300–1800 m / z) were acquired with a resolution of 140,000 at m / z 200, a maximum injection time of 50 ms, and an AGC target of 3x106 charges, with the lock-mass option enabled (445.12002 m / z). Using a 2 m / z window, up to 10 of the most intense precursors were isolated per full scan and fragmented using higher energy collision dissociation (HCD, standardized collision energy of 27 eV), with dynamic exclusion of already fragmented precursors set to 60 sec. MS / MS spectra were acquired with a resolution of 17,500 at m / z 200, a maximum injection time of 100 ms, and an AGC target of 1x105. The system was fully controlled by XCalibur software (v3.0.63; Thermo Fisher Scientific).

[0188] MS / MS Data Interpretation Using Mascot (version 2.5.1, Matrix science, London, England), MS / MS data were searched against a combined target-decoy database (01-04-2018, containing 33939 target sequences and the same number of reverse decoy sequences) derived from the chicken (Gallus gallus) and vaccinia virus Uniprot databases. The target proteins hCTLA4, hCD86, and hCXIIG6, as well as the target-decoys, were added manually to the database. The database included common contaminants (human keratin and porcine trypsin) and was created using an in-house database creation toolbox (http: / / msda.u-strasbg.fr). The following parameters were applied: one missed cleavage by trypsin and variable modifications (oxidation of methionine (+16 Da), carbamidomethylation of cysteine ​​(+57 Da) were considered. The search window was set at 25 ppm for precursor ions and 0.07 Da for fragment ions. Mascot result files (.dat) were imported into Proline software (http: / / proline.profiproteomics.fr / ) and proteins were validated with pretty rank equal to 1, 1% FDR on peptide-spectrum matches based on adjusted e-value, at least one unique peptide per protein, 1% FDR on protein sets, and Mascot Modified Mudpit scoring.

[0189] Mixed lymphocyte reaction (MLR) The ability of m2-viruses to activate lymphocytes was assessed in an MLR assay. CEF cells were infected with COPTG19289, VVTG18058, or MVAN33 (MOI 0.05), and culture supernatants were collected 48 hours postinfection and concentrated approximately 20-fold using vivaspin 20 30000 MWCO cutoff concentrators (Sartorius). Concentrated supernatants were added undiluted or diluted 10- or 100-fold to a final "supernatant concentration" of 2-, 0.2-, and 0.02-fold, respectively (20 μL into 200 μL).

[0190] Blood from different healthy donors was purchased at Etablissement Francais du sang (EFS Grand Est, 67065 Strasbourg). PBMCs were purified by the Ficoll-Paque method (Ficoll-Paque PLUS, GE Healthcare) and dissolved in RPMI medium supplemented with 20% FBS (fetal bovine serum) and 10% DMSO at approximately 1 × 10 7 The PBMCs were thawed at 37°C, resuspended in RMPI medium with 10% FBS, and centrifuged at 300g for 5 min. The collected cells were resuspended in RMPI medium + 10% FBS to a cell concentration of 3x10 6 The cells / mL were adjusted. 100 μL of PBMCs from two different donors were mixed in triplicate into wells of a 96-well microplate. 20 μL of the infected cell supernatant described above was added to each well, and the microplate was incubated at 37°C in a 5% CO2 atmosphere for 72 hours.

[0191] The culture supernatants of the MLRs were then collected and human interleukin-2 (IL-2) was assayed by ELISA. * IL-2 was measured using the -2 ELISA MAX™ deluxe Set kit (BioLegend). Measurements were normalized by dividing the mean of the IL-2 concentrations of three replicates of a given sample by the mean of the IL-2 concentrations of three replicates of PBMCs incubated with medium.

[0192] In vivo experiments in humanized NCG-34+ mice Mouse humanization NOD / Shi-scid / IL-2Rγ null immunodeficient mouse strain (NCG) was provided by Taconic. Four-week-old animals were treated intraperitoneally with busulfan (chemoablation) and the following day, CD34+ human stem cells (50,000 cells / mouse) were injected intravenously (IV). Engraftment levels were monitored 14 weeks after cell injection by flow cytometric analysis of human CD45+ cells among whole blood leukocytes. Humanization rate was defined as the ratio of circulating hCD45 / total CD45 (mCD45+hCD45).

[0193] T lymphocyte immunophenotype Blood (100 μL) was collected from the retro-orbital sinus 2 days before tumor implantation. Human CD45+, CD3+, CD3+CD4+, and CD3+CD8+ lymphocyte populations were assessed by flow cytometry (Attune NxT, Lifetechnologies) using antibodies directed against hCD45 (Ref 563879; BD), CD4 (Ref 130-092-373; Miltenyi), CD3 (Ref 130-109-462; Miltenyi) and CD8 (Ref 130-096-561; Miltenyi) and the live / dead yellow marker (Ref L34968; Thermofisher). Briefly, blood samples were incubated with the various antibodies for 30 min at 4°C. Red blood cells were then lysed using High Yield Lysis buffer (HYL250; Thermo Fischer Scientific) for 15 min at room temperature (RT) immediately followed by flow cytometry analysis (Attune NxT, Life technologies).

[0194] Treatment with oncolytic viruses Human colorectal cancer cells HCT-116 were purchased from ATCC (CCL-247™), grown in McCoy's 5A medium supplemented with 10% FBS + penicillin / streptomycin, and detached with trypsin for 10 min at 37° C. After washing, cells were resuspended in sterile PBS at 5×10 7 Resuspend at 5x10 cells / ml and add 100 μl of cell suspension (5x10 6 Mice were injected subcutaneously into one flank with 100 cells each. The average tumor volume was approximately 70 mm 3 Upon reaching this age, mice were randomized into 5 groups (5 mice / group) based on humanization rate and tumor size. Group 1 received the vehicle. Group 2 has 10 5 The mice were given VVTG18058 with pfu. Group 3 has 10 6 The mice were given VVTG18058 with pfu. Group 4 has 10 5 The pfu strain COPTG19289 was given. Group 5 has 10 6 The pfu strain COPTG19289 was given.

[0195] Each group received a single intravenous (IV) injection of 100 ul of the virus preparation on the randomization day, defined as D0. Mice were monitored daily for any signs of unexpected distress. Body weight and tumor volume were monitored three times a week. Tumor diameters were measured using calipers. Tumor volume (mm 3 ) using the following formula: Volume = 1 / 2(Length x Width) 2 ) was calculated. 3 Animals were sacrificed when the body weight loss exceeded 25% or when the body weight loss exceeded 25%.

[0196] Example 1: Identification of the ability of vaccinia virus m2 protein to interfere with the B7-mediated costimulatory pathway and characterization of its binding properties Supernatants from vaccinia virus-infected cells inhibit the interaction of CTLA4 with CD80 or CD86. Two assays were set up to quantitatively monitor the CD80 / CTLA4 and CD86 / CTLA4 blocking activities offered by the different virus candidates. In these assays, human CTLA4 (hCTLA4) was immobilized on an ELISA plate and soluble tagged hCD80 or hCD86 was added. In this setup, any competing molecule that binds to either the immobilized or soluble partner induces a signal reduction (competitive assay). The anti-hCTLA4 antibody ipilimumab (Yervoy) and the supernatant of uninfected DF1 (a commercially available chicken cell line; e.g., available at ATCC® CRL-12208™) were used as positive and negative controls, respectively. Surprisingly, we found that all the supernatants from cells infected with vaccinia virus (Copenhagen, Wyeth, and Western Reserve strains) competed in a dose-response manner in both the CD80 / CTLA4 and CD86 / CTLA4 assays, similar to Yervoy interacting with coated hCTLA-4 (Figures 1A and 1B). In contrast, the supernatants from uninfected DF1 cells had no effect. Interestingly, the supernatants from DF1 infected with modified vaccinia virus Ankara (MVA) did not inhibit the hCTLA4 / hCD80 and hCTLA4 / hCD86 interactions at all. This indicates that this interference ability is not preserved in this virus, which lost six genome fragments (deletions I-VI) during the attenuation process (data not shown). These results suggest that something in the VV supernatants interfered with the binding of CTLA-4 to CD80 and CD86.

[0197] To exclude artifacts involving cells or media components, different cell lines from different origins (primary avian cell lines and human tumor cell lines) were tested, also assayed with the FACS competition method.

[0198] Competitive FACS analysis was performed using a human cell line (i.e., KM-H2, Hodgkin's lymphoma) that naturally displays hCD80 and hCD86 on its surface. Binding of soluble recombinant CTLA4-Fc to KM-H2 cells was demonstrated using a fluorochrome-conjugated anti-Fc antibody. When co-incubated with CTLA4-Fc, supernatants of vaccinia virus-infected cells competed with KM-H2 cells for CTLA4-Fc binding, in striking contrast to MVA-infected cells, which behaved as a negative control (data not shown).

[0199] To evaluate their ability to interfere with the binding of CTLA4 to CD80 or CD86, a competitive ELISA was performed using the supernatants of HeLa cells infected with different poxviruses (instead of DF1). Various vaccinia virus strains (Wyeth, WR, and Copenhagen) as well as other orthopox (e.g., raccoonpox, rabbitpox, cowpox, MVA), avipox (fowlpox), and parapoxviruses (pseudocowpox virus) were tested. Uninfected HeLa cells were used as negative control. In this screening experiment, HeLa cells were infected with the different poxviruses at a high MOI (MOI 1) to ensure optimal infection. The resulting supernatants were collected and tested by evaluating their ability to inhibit the binding of CTLA4-Fc to CD80 (expressed at OD450nm). As illustrated in Figure 2, the supernatants of cells infected with three vaccinia virus strains or raccoonpox (RCN), rabbitpox (RPX), and cowpox (CPX) were all able to interfere with the binding of hCTLA4 to hCD80. These results indicate that factors secreted during infection with these poxviruses interfered with the CTLA4-B7 pathway. The new unknown factors responsible for this inhibitory activity were termed "interference factors" (IFs). Again, the supernatants of cells infected with MVA and several other poxviruses, such as pseudopox virus (PCPV) and fowlpox virus (FPV), as well as uninfected HeLa cells (HeLa), did not show any inhibition of the CTLA4 / CD80-CD86 interaction.

[0200] "Interfering factors" are present in vaccinia virus supernatants but not in MVA supernatants To find out with which molecules present in VV-infected supernatants IFs interact, Western blots of supernatants of CEFs (also called CEPs) uninfected or infected with MVA or vaccinia virus were probed with the three components of the ELISA assay described above (i.e., hCD80, hCD86, and hCTLA4). CEFs were chosen because they tolerate both vaccinia virus, which produces IFs, and MVA, which does not produce IFs. Each of the proteins used to probe the Western blots was specifically fused with an Fc moiety that allowed dimerization and detection with the same anti-Fc binding antibody. Each supernatant was used either as is or concentrated 20-fold (x20). The blots shown in Figure 3 clearly demonstrated that a large molecule of approximately 200 kDa was present only in vaccinia virus-infected supernatants, and was highlighted (at least under these immunoblot conditions) by both hCD80 and hCD86, but not by hCTLA4. This band was also easily detected in non-concentrated supernatants. Reactivity with both hCD80-Fc and hCD86-Fc was lost under reducing conditions (no bands were detected), indicating that intra- and / or inter-disulfide bonds are required to maintain IF structure and interaction with CD80 and CD86 (data not shown). In sharp contrast, no bands were highlighted in MVA supernatants.

[0201] Characterization of binding properties: "Interfering factors" present in vaccinia virus supernatants inhibit the binding of CD80 and CD86 to CTLA4 and CD28, but potentialize the binding of CD80 to PD-L1 As discussed above, CD80 and CD86 are important costimulatory antigens involved in the regulation of adaptive T cell responses. As CD80 and CD86 are involved in several molecular interactions encompassing negative (CTLA4 in both cases, PD-L1 in CD80 alone) and positive (CD28) outcomes in terms of immune responses, different ELISAs were set up to decipher the IF effect on each of these five specific interactions. Undiluted supernatants from CEFs infected with non-recombinant vaccinia virus (VV) were tested in these different assays and compared with supernatants from MVA-infected CEFs and the anti-hCTLA4 antibody Yervoy (10 μg / ml). Supernatants from uninfected CEF cells were used as negative controls. As illustrated in FIG. 4, VV supernatants inhibited the interaction of CD80 and CD86 with CTLA4 (as evidenced by the striking decrease in OD450 nm absorbance) similar to Yervoy (as expected by the binding of Yervoy and its CTLA-4 target, which prevents access to CD80 and thus CTLA4 / CD80 ligation). In striking contrast, MVA-infected cell supernatants had no effect (same absorbance as the negative CEF control). Furthermore, VV supernatants were also able to abolish the positive interaction of CD80 or CD86 with CD28 (significant decrease in OD450 nm absorbance compared to the absorbance measured with the supernatant of uninfected CEF cells). In contrast, MVA-infected cell supernatants and Yervoy had no effect (same absorbance as the negative control) (as expected for an antibody that targets only the CTLA4 receptor). These results confirm the presence of "IFs" in the supernatants of VV-infected cells, whereas the MVA genome does not produce such factors.

[0202] Surprisingly, the presence of vaccinia virus supernatant increased the PD-L1 / CD80 interaction (significantly increased OD450nm absorbance compared to negative controls), thus enhancing PDL1-mediated immunosuppressive signaling. In contrast, Yervoy and MVA-infected CEF supernatants had no effect on PDL1 / CD80 (same absorbance as uninfected controls). As expected, recombinant hCD80, hCTLA4, and hPD1 abolished this interaction. This result indicates that IF and the CTLA4 binding site on CD80 do not completely overlap. It should be noted that CD80 / PD-L1 interaction has recently been linked to Treg survival.

[0203] These results highlight the improved immunosuppression exhibited by the poxvirus m2 polypeptide: indeed, m2 promotes immunosuppressive pathways by blocking CD80 / CD28, CD86 / CD28, and by potentiating the PDL1-CD80 pathway, whereas CTLA4-Fc inhibits these three pathways, including the immunosuppressive PDL1-CD80 interaction.

[0204] Identifying the m2 poxvirus protein as an interference factor Based on the apparent molecular weight of approximately 200 kDa and the fact that IFs are not present in MVA-infected supernatants, we examined 37 genes that differ between the vaccinia Copenhagen strain and MVA for potential candidates, without finding any obvious candidates. No proteins of approximately 200 kDa could be identified. Of these 37 gene candidates, the largest protein encoded is the DNA-dependent RNA polymerase subunit rpo147(J6R), which has a theoretical mass of 147 kDa. Thus, nothing smaller than 200 kDa was observed. Based on primary structure, there were no obvious viral protein candidates that could be linked to IFs.

[0205] Therefore, an experimental approach to identify IFs was attempted using affinity chromatography (see schematic in Figure 5A) to capture IFs. The affinity chromatography was loaded with 20-fold concentrated supernatants of vaccinia virus-infected CEFs (VV-infected). In parallel, 20-fold concentrated supernatants of MVA-infected cells (MVA-infected) were processed. VV and MVA supernatants were applied to immobilized CTLA4 (negative control) or immobilized CD86-Fc fusions, followed by elution by acid. The different eluates of the affinity chromatography arms were analyzed by MS / MS (mass spectrometry) after trypsin digestion. The m / z data obtained were used to probe chicken (Gallus gallus) and vaccinia virus data banks. Only one hit was obtained from the supernatant of vaccinia-infected CEFs incubated with CD86-coated beads. This covers 75% (including peptide signal) or 82% (excluding peptide signal) of the vaccinia virus protein m2 protein encoded by the M2L locus (Figure 5B; sequences covered by the detected peptides are shown in bold). This result is entirely consistent with the absence of an M2L locus in the MVA genome and the fact that m2 has a predicted signal peptide and is therefore a putative secreted protein.

[0206] However, m2 protein has been reported to migrate under reducing conditions on SDS-PAGE as a 35 kDa protein, with a calculated molecular mass of only 25 kDa, far from the 200 kDa mass of IF observed on SDS-PAGE (Hinthong et al. 2008). Nevertheless, to our knowledge, the behavior of m2 protein on SDS-PAGE under non-reducing conditions has not been documented. Therefore, we hypothesize that IF may be a homo- or heteromultimeric complex involving VV m2 protein with intersubunit disulfide bonds, resulting in an apparent mass of approximately 200 kDa on SDS-PAGE.

[0207] Example 2: m2 defective poxviruses no longer produce IFs Construction of an M2L-deleted poxvirus The involvement of m2 in IF was further investigated by deleting the M2L gene in the vaccinia virus genome. Specifically, a double-deleted (DD) vaccinia virus expressing luciferase (i.e., tk1, described in WO2009 / 065546 and designated VVTG18277) was used to express vaccinia luciferase. - ,rr- - ) to disrupt the M2L locus, thereby generating a recombinant triple deletion (TD) virus (i.e., tk - rr - , m2 - ) (COPTG19289). An M2L partial deletion extending from 64 nucleotides upstream of the m2 ORF to the first codon 169 suppressed m2 protein expression (m2-) and did not significantly affect virus replication in CEFs compared with the parent (data not shown).

[0208] M2L-deleted viruses no longer produce IFs Supernatants obtained upon infection of human HeLa and avian DF1 cells with DD and TD viruses were tested by competitive ELISA as described above. As shown in Figure 6, supernatants collected upon infection with the M2L deleted vaccinia virus COPTG19289 were no longer able to inhibit the CTLA4 / CD80 interaction (as evidenced by the same absorbance values ​​measured in uninfected HeLa or DF1 cells), unlike the parental DD virus (VVTG18277), which showed a strongly reduced absorbance reading compared to the negative control.

[0209] Furthermore, when subjected to Western blotting as described above, the large complex migrating at 200 kDa detected with CD80-Fc or CD86-Fc probes was no longer detectable when supernatants from M2L-deleted viruses were used (data not shown), confirming that m2 is at least part of the IFs.

[0210] Example 3: m2 defective recombinant poxvirus The construction of tk-rr-m2-oncolytic vaccinia virus expressing luciferase (the gene was inserted into the J2R locus) was described above.

[0211] Tumor-regressing activity LOVO (ATCC® CCL-229™) and HT116 (ATCC® CCL-247™) colon cancer cells were cultured in 96-well plates for 8.10 min. 5 Cells were seeded at a cell density of 1000 cells / well. Plates were incubated at 37°C, 5% CO2 for 4 hours before infection. Cells were infected with either the tk-rr-m2-COPTG19289 virus or the tk-rr-VVTG18277 virus, both of which express luciferase. -1 ~10 -4 Cells were infected at a range of MOIs of particles / cell. At 96 hours post-infection (D4), cell viability was determined by trypan blue exclusion using a cell counter (Vi-Cell, Beckman coulter). Quantification of % cell viability in LOVO (Figure 7A) and HCT116 (Figure 7B) demonstrated that the oncolytic potential provided by the m2-defective COPTG19289 virus was comparable to that obtained with m2-positive VVTG18277 in LOVO cells and HCT116. Specifically, VVTG18277 and COPTG19289 were infected at 10 -1 and 10 -2 When infected with an MOI of 10, LOVO cells were lysed. -3 At a low MOI of 10, 80% cell viability was observed; -4 In HCT116 cells, the MOI of 10 -1 , 10 -2 , and 10 -3 At an MOI of 10, no cell survival was detected (0%). -4At an MOI of 100, less than 50% of the cells were viable, so the viral oncolytic activity was even higher. Mock treatment had no effect on the cells and was used to determine 100% viability of LOVO and HCT116 cells.

[0212] This lack of difference between the double and triple deleted viruses was confirmed in other tumor cell lines, including melanoma B16F10 (ATCC® CCL-6475™), mouse colon carcinoma CT26WT (ATCC® CRL-2638™), and mouse colon adenocarcinoma MC38WT cells (available from Kerafast and Cellosaurus CVCL_B288). Both vaccinia viruses showed a 10 -1 At an MOI of 10 for B16F10 and MC38WT, -2 At an MOI of 100 mg / kg, CT26 was tumorigenic, whereas CT26WT was partially tumorigenic.

[0213] In conclusion, recombinant m2-defective viruses displayed oncolytic activity comparable to their m2-positive counterparts, confirming that disruption of the M2L locus did not adversely affect oncolytic activity in tumor cell lines.

[0214] Transgene expression in vivo Luciferase expression resulting from tk-rr-m2-oncolytic vaccinia virus (COPTG19289) was evaluated and compared with that obtained with tk-rr-VVTG18277 virus in B16F10 tumor-implanted C57BL / 6 mice after subcutaneous injection of each virus (10 7pfu) were injected intratumorally, and tumor sampling was collected on days 1, 2, 6, 9, 13, and 16 to assess luciferase activity per gram of tumor (RLU / g tumor). As illustrated in Figure 8, for both viruses, strong luciferase activity was detected on the first day (D1 and D2) after virus injection, which then decreased. However, luciferase expression reached background levels after 13 days of infection with VVTG18277. In contrast, weak but persistent expression levels were measured after infection with COPTG19289, which were maintained over a long period of time (D9, D13, and D16).

[0215] Antitumor activity The antitumor activity provided by tk-rr-m2-oncolytic vaccinia virus (COPTG19289) was assayed in three tumor models, B16F10, CT26, and HT116, respectively.

[0216] In the first set up, C57BL / 6 mice (10 mice / group) were implanted with B16F10 tumors subcutaneously. 3 When the tumors reached a volume of 10 μg / kg, the tumors of each animal were measured, the mice were randomized, and the mice were injected with 10 μg / kg / day of the tumor by intratumoral route on D0, D3, D6, D10, and D14. 7 pfu of COPTG19289, VVTG18277, or mock vehicle (negative control) were injected. Animal survival and tumor growth were followed twice weekly (tumor volumes >2000 mm 3 (Mice were killed when tumor volume reached ≥ 2000 mm ). There was no significant difference between the two VV-treated groups. Notably, some animals showed slow tumor growth in both groups. In contrast, tumor growth in mock-treated animals was very rapid, reaching 2000 mm in 24 days. 3, which resulted in the death of all mice on D24. Mice survival was improved by vaccinia virus treatment. Specifically, in this experiment, 50% survival time was obtained at D23 for mice treated with tk-rr-VVTG18277 and at D28 for mice treated with tk-rr-m2-COPTG19289. For clarity, survival curves were consistent between the two VV groups, except that 2 of 10 injected mice died after a few days (data not shown).

[0217] Antitumor activity was also assayed in Balb / c mice implanted with CT26 tumors by subcutaneous injection. 3 When tumors reached a volume of 1000 mg / kg, they were individually measured and mice were randomized (D0) before intratumoral injection of tk-rr-m2-oncolytic vaccinia virus (COPTG19289) or tk-rr-VVTG18277 virus or mock vehicle (10 mice / group). 7 Tumors were injected with 50 μl of pfu of each vaccinia virus preparation (or mock). Tumor growth was followed twice weekly and tumor volumes were monitored until tumor burden reached 2000 mm. 3 Mice were sacrificed when tumor volume reached 2000 mm3 or greater. As shown in Figure 9, the tumor volume in mock-treated animals increased rapidly and reached 2000 mm3 by D28. 3 In contrast, tumor growth was delayed in the VV-treated groups, and tumor volumes reached 1000 mm only with the tk-rr-m2-vaccinia virus. 3 It was less.

[0218] Antitumor activity was also assayed in Swiss nude mice implanted with HT116 tumors by subcutaneous injection (10 mice / group). Ten days after tumor implantation, two different doses, 10 5 and 10 7 pfu of vaccinia virus was injected intravenously. Tumor growth was monitored twice weekly, and tumor volumes were increased to 2000 mm 3 Mice were sacrificed when the tumor mass reached 2000 mm 45 days after tumor implantation. As expected, the tumor mass in mock-treated animals increased rapidly, reaching 2000 mm 45 days after tumor implantation. 3In contrast, 10 5 Tumor growth was delayed in the group treated with pfu VV. In particular, as shown in FIG. 7 Tumor growth was completely inhibited in the two groups injected with pfu vaccinia virus.

[0219] In conclusion, modification of the VV M2L locus, which renders vaccinia virus unable to produce the immunosuppressive M2 protein, does not affect the oncolytic activity, antitumor efficacy, or expression of the transgene.

[0220] Example 4: Mixed Lymphocyte Reaction (MLR) Assay Supernatants from CEF cells infected with COPTG19289 (tk-, rr-, and m2-), VVTG18058 (tk-rr-), or MVAN33 were assessed in the MLR for their ability to activate lymphocytes. After 48 h of infection (MOI 0.05), culture supernatants were collected and concentrated approximately 20-fold.

[0221] PBMCs were purified from blood collected from healthy donors using Ficoll-Paque PLUS (GE Healthcare). More specifically, 3 x 10 PBMCs derived from two different donors were 5 PBMCs were mixed in a 96-well microplate. Concentrated supernatants were added (20 μL into 200 μL) undiluted or diluted 10- or 100-fold to PBMC cultures for final "supernatant concentrations" of 2-fold, 0.2-fold, and 0.02-fold, respectively, and incubated for 72 h at 37°C in a 5% CO2 atmosphere. Addition of RPMI medium was used as a negative control. IL-2 secretion in the culture supernatants was measured as a lymphocyte activation marker by ELISA (IL-2 * IL-2 levels were quantified using the IL-2 MAX™ deluxe Set kit (-2 ELISA). Measurements were normalized by dividing the average of the 3 replicates of a given sample by the average of the IL-2 concentrations of the 3 replicates of PBMCs incubated with medium.

[0222] The negative control is the normalized lymphocyte activation state, which is 1. As illustrated in Figure 11, PBMCs incubated in the presence of supernatants from cells infected with MVA and COPTG19289 (tk-rr-m2-; TD) induced lymphocyte activation, reaching values ​​approaching 1 when diluted 10- or 100-fold and exceeding 1 when tested undiluted. In sharp contrast, VVTG18058 (tk-rr-; DD)-infected supernatants showed a pronounced inhibition of lymphocyte activation at all dilutions tested, confirming the immunosuppressive activity of the M2-encoding virus.

[0223] Example 5: Antitumor activity in humanized NCG-CD34+ mice The antitumor activity provided by the m2-COPTG19289 virus was evaluated in NOD / Shi-scid / IL-2Rγ null immunodeficient mouse strain (NCG) humanized with CD34+ human stem cells and implanted with human colorectal cancer cells HCT-116 (5x10 6 Mice were injected SC into one flank (corresponding to D0). Twelve days after transplantation (D12), mice were 5 pfu or 10 6 Mice were given a single IV injection of COPTG19289 (tk-rr-m2-; TD) or its m2+ counterpart VVTG18058 (tk-rr-; DD) at a dose of pfu. Vehicle-treated mice were used as negative controls. Tumor growth and mouse survival were monitored for at least 60 days after cell implantation.

[0224] As illustrated in Figure 12A and B, tumor burden rapidly increased in the vehicle-treated mice group. In sharp contrast, tumor growth was clearly inhibited in mice treated with m2-COPTG19289 (TD) or m2+VVTG18058 (DD) whatever dose was injected, although some animals experienced dose-dependent toxicity issues. Therefore, inhibition of tumor growth was monitored over a 60-day period. 10 6At this dose, both viruses delayed tumor growth with similar potency (Figure 12A), but lower toxicity was observed with the TD virus COPTG19289 compared to the DD VVTG18058 virus. Notably, one TD-treated animal was tumor-free 55 days after cell implantation and remained tumor-free beyond 85 days. 5 At this dose, the TD virus COPTG19289 showed improved antitumor efficacy compared to the DD VVTG18058 virus (Figure 12B). More specifically, tumor growth was significantly inhibited in 5 / 5 animals in the TD group, whereas it was inhibited in 2 / 5 animals in the DD group. Furthermore, reduced toxicity was observed in the TD group compared to the DD group.

[0225] Comparing mouse survival, 10 6 pfu (Figure 13A) or 10 5 It was confirmed that the antitumor effect provided by m2-COPTG19289(TD) was improved compared to m2+VVTG18058(DD) after a single IV injection of pfu (Figure 13B). More specifically, 100% of vehicle-treated animals died at day 52, whereas survival was clearly extended by VVTG18058(DD) treatment and even further by COPTG19289(TD) treatment. For example, the median survival time (Figure 13A) was 52 days for the negative control and 10 days for DD-10. 6 In the pfu-treated group, TD-10 6 In the pfu-treated group it was estimated to be 70 days.

[0226] In addition, 10 5 At the pfu dose, the median survival time was 52 days for DD virus and 80 days for TD virus.

[0227] These results demonstrate the improved therapeutic benefit offered by m2 defective poxviruses for treating conditions such as cancer.

[0228] References TIFF0007625521000002.tif232146TIFF0007625521000003.tif244134TIFF0007625521000004.tif158128

Claims

1. 1. A modified poxvirus, the genome of which contains an M2L locus that in its native (wild-type) context encodes a functional m2 poxvirus protein, and which has been modified to be defective in said m2 function, the functional m2 poxvirus protein is capable of binding to a CD80 costimulatory ligand or a CD86 costimulatory ligand, or is capable of binding to both a CD80 costimulatory ligand and a CD86 costimulatory ligand, and the defective m2 function is incapable of binding to the CD80 costimulatory ligand and a CD86 costimulatory ligand, and the modified poxvirus is oncolytic; and further modified at the J2R and I4L / F4L loci, resulting in a modified poxvirus defective in m2, tk, and rr activity; The modified poxvirus.

2. 2. The modified poxvirus of claim 1, which is produced or obtained from the subfamily Chordopoxvirinae.

3. 3. The modified poxvirus of claim 2, which is a member of the Orthopoxvirus or Leporipoxvirus genus.

4. 4. The modified poxvirus of claim 3, which is a vaccinia virus or a myxoma virus.

5. The modified poxvirus of claim 4, which is a vaccinia virus selected from the group consisting of Western Reserve (WR), Copenhagen (Cop), Lister, LIVP, Wyeth, Tashkent, Tian Tan, Brighton, Ankara, LC16M8, and LC16M0 strains.

6. The modified poxvirus of any one of claims 1 to 5, wherein the inability to bind to CD80 costimulatory ligand and CD86 costimulatory ligand results from a genetic lesion within the M2L locus or from an aberrant interaction that impairs m2 function either directly or indirectly.

7. The modified poxvirus of claim 6, wherein the genetic lesion comprises a partial or complete deletion and / or one or more non-silent mutations either within the m2 coding sequence or in a regulatory element controlling M2L expression.

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

9. The modified poxvirus of any one of claims 1 to 8, which is recombinant.

10. The modified poxvirus of claim 9, which has been engineered to express at least one polypeptide selected from the group consisting of an antigenic polypeptide, a polypeptide having a nucleoside / nucleotide pool regulating function, and an immunomodulatory polypeptide.

11. The modified poxvirus of claim 10, wherein the immunomodulatory polypeptide is selected from the group consisting of a cytokine, a chemokine, a ligand, and an antibody, or any combination thereof.

12. The modified poxvirus of claim 11, wherein the antibody specifically binds to an immune checkpoint protein.

13. The modified poxvirus of claim 12, wherein the immune checkpoint protein is selected from the group consisting of CD3, 4-1BB, GITR, OX40, CD27, CD40, PD1, PDL1, CTLA4, Tim-3, BTLA, Lag-3, and Tigit.

14. The modified poxvirus of claim 12 or 13, which expresses an antagonistic antibody that specifically binds to PD-L1 or CTLA4.

15. Defective in m2 activity, tk activity, and rr activity (a) an anti-CTLA-4 antibody; or (b) Anti-PD-L1 antibody The modified poxvirus of claim 14, which encodes

16. (a) the anti-CTLA-4 antibody is ipilimumab or tremelimumab; or (b) the anti-PD-L1 antibody is selected from atezolizumab, durvalumab, or avelumab; 16. The modified poxvirus of claim 15.

17. A method for producing a modified poxvirus according to any one of claims 1 to 16, comprising the steps of: (a) preparing a producer cell line; (b) transfecting or infecting the prepared producer cell line with a modified poxvirus; (c) culturing the transfected or infected producer cell line under appropriate conditions to allow for the production of virus; (d) recovering the produced virus from the culture of the producer cell line; The method comprising:

18. (e) purifying the recovered virus.

20. The method of claim 17, further comprising:

19. A pharmaceutical composition comprising a therapeutically effective amount of a modified poxvirus according to any one of claims 1 to 16 and a pharma- ceutically acceptable vehicle.

20. 10 3 ~10 12 p.f.u., 10 4 pfu~10 11 p.f.u., 10 5 pfu~10 10 pfu, or 10 6 pfu~10 9 20. The pharmaceutical composition of claim 19, comprising a modified poxvirus of pfu.

21. 21. The pharmaceutical composition of claim 19 or 20, formulated for intravenous or intratumoral administration.

22. 22. The pharmaceutical composition of any one of claims 19 to 21 for use in treating or preventing a proliferative disease selected from the group consisting of cancer, diseases associated with increased osteoclast activity, and cardiovascular diseases.

23. 23. The pharmaceutical composition of claim 22, wherein the cancer is selected from the group consisting of renal cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, gastric cancer, bile duct cancer, endometrial cancer, pancreatic cancer, and ovarian cancer.

24. - to stimulate or improve lymphocyte-mediated immune responses; - to stimulate or improve APC activity; - to stimulate or improve anti-tumor responses; - to stimulate or improve the CD28 signaling pathway; - to improve therapeutic efficacy in a treated subject or group of treated subjects compared to m2-positive poxviruses; and / or To reduce toxicity in a treated subject or group of treated subjects compared to m2 positive poxviruses 22. A pharmaceutical composition according to any one of claims 19 to 21 for use.

25. 25. The pharmaceutical composition of any one of claims 19 to 24, for use as the sole therapy or in combination with one or more further therapies.

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