WO2018091680A1 - Vecteurs oncolytiques à base de la variole bovine - Google Patents

Vecteurs oncolytiques à base de la variole bovine Download PDF

Info

Publication number
WO2018091680A1
WO2018091680A1 PCT/EP2017/079656 EP2017079656W WO2018091680A1 WO 2018091680 A1 WO2018091680 A1 WO 2018091680A1 EP 2017079656 W EP2017079656 W EP 2017079656W WO 2018091680 A1 WO2018091680 A1 WO 2018091680A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
cowpox virus
cowpox
cancer
cells
Prior art date
Application number
PCT/EP2017/079656
Other languages
English (en)
Inventor
Marine RICORDEL
Philippe Erbs
Johann Foloppe
Original Assignee
Transgene Sa
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Transgene Sa filed Critical Transgene Sa
Publication of WO2018091680A1 publication Critical patent/WO2018091680A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24161—Methods of inactivation or attenuation
    • C12N2710/24162—Methods of inactivation or attenuation by genetic engineering

Definitions

  • the present invention is in the field of oncolytic viruses.
  • the invention provides new oncolytic viruses which are cowpox viruses. More precisely, the invention provides an alternative to the existing oncolytic virus vectors which are largely used for the vaccination. These alternative oncolytic vectors are used for prophylaxis or treatment of proliferative diseases, like cancers, tumors or restenosis, and for prophylaxis or treatment of diseases associated to an increased osteoclast activity, like rheumatoid arthritis or osteoporosis.
  • proliferative diseases like cancers, tumors or restenosis
  • prophylaxis or treatment of diseases associated to an increased osteoclast activity like rheumatoid arthritis or osteoporosis.
  • Oncolytic viruses are a class of theraplastic agents that have the unique property of tumor-dependent self-perpetuation (Hermiston et al., 2006, Curr. Opin. Mol. Ther., 8:322- 30). The benefit of using these viruses is that as they replicate, they lyse their host cells. Oncolytic viruses are capable of selective replication in dividing cells (e.g. cancer cells) while leaving non- dividing cells (e.g. normal cells) unharmed. As the infected dividing cells are destroyed by lysis, they release new infectious particles to infect the surrounding dividing cells. Therefore, oncolytic viruses offer new area for treating cancer, optionally in association with conventional treatments for cancer (Fisher et al., 2006, Curr. Opin. Mol.
  • Cancer cells are ideal hosts for many viruses because they have the antiviral interferon pathway inactivated or have mutated tumour suppressor genes that enable vira l replication to proceed unhindered (Chernajovsky et al., 2006, British Med. J., 332:170-2).
  • viruses including adenovirus, reovirus, measles, herpes simplex, Newcastle disease virus and vaccinia virus have now been clinically tested as oncolytic agents.
  • vaccinia virus VACV
  • VACV vaccinia virus
  • variola virus the causative agent of smallpox
  • Some viruses are naturally oncolytic and have an innate ability to selectively infect and kill tumor cells.
  • oncolytic viruses can also be engineered by modifying naturally occurring viruses.
  • the main strategies used currently to modify the viruses include: functional deletions in viral genes, the use of tumor- or tissue-specific promoters to control the expression of these viral genes, tropism modification to redirect virus to the cancer cell surface, among many other possibilities.
  • Viral modifications of the virus can be practiced in order to enhance the ability of viruses to infect and lyse 100% of the tumor cells which is difficult to achieve in in vivo context. Therefore, oncolytic viruses are often "armed" with enzyme-prodrug system which enhance the oncolytic efficacy of the virus therapy by exerting a strong bystander effect and thus permitting elimination of neighboring uninfected tumor cells.
  • armament with the so-called FCUl suicide gene encoding a bifunctional chimeric protein that combines the enzymatic activities of FCY1 and FUR1, efficiently catalyzed the direct conversion of 5- fluorocytosine (5-FC), a nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil (5-FU) and 5-fluorouridine-5'monophosphate (5-FUMP), thus bypassing the natural resistance of certain human tumor cells to 5-fluorouracil (Erbs et al., 2000, Cancer Res., 60(14):3813-22).
  • TK thymidine kinase
  • VACV oncolytic vaccinia viruses
  • Cowpox virus has oncolytic properties which make it particularly appropriate for anti-cancer oncolytic virotherapy considering its limited pathogenicity in humans: CPXV replicates very poorly in human normal tissues as illustrated herein. Moreover, the inventors discovered that CPXV can be modified, and that modifications aimed to inactivate CPXV105 CDS gene lead to an increased efficacy and safety compared to wild type CPXV. Moreover, a recombinant and CPXV105 CDS- defective cowpox virus engineered to express the suicide FCU-1 gene was shown particularly effective to replicate and lyse human tumor cells.
  • CPXV may be successfully used as an alternative oncolytic to VACV therapy virus for treating or preventing proliferative diseases such as cancer in smallpox vaccinated people as well as in non-vaccinated ones.
  • CPXV can also be exploited in combination with additional anticancer therapy/ies.
  • One aspect of the invention relates to a cowpox virus comprising a defective CPXV105
  • CDS gene as well as to a cowpox virus for use as an oncolytic virus for the prophylaxis or the treatment of a disease such as a proliferative disease or a disease associated with an increased osteoclast activity.
  • cowpox virus comprising defective CPXV083 CDS and/or CPXV051 CDS gene(s).
  • the cowpox virus is further defective for CPXV105 CDS gene.
  • the cowpox virus is further defective for CPXV049 CDS gene.
  • the CPXV of the present invention further com prises a truncated CPXV032 CDS gene.
  • said CPXV further comprises at least a nucleic acid of interest, in particular a suicide gene, a gene coding for an immunostimulatory polypeptide, a gene coding for an antigen, a gene coding for a permease, or a gene coding for other molecules of interest.
  • the present invention further provides a composition comprising the CPXV as described herein.
  • the CPXV is preferably formulated for intra-venous or intra-tumoral administration.
  • the present invention also concerns a process for preparing the CPXV, which comprises at least the steps of introducing said CPXV into a producer cell, culturing the producer cell under conditions that are appropriate for enabling said CPXV to be produced and recovering the produced CPXV from the cell culture.
  • the recovered CPXV can be purified at least partially.
  • the present invention provides a CPXV (e.g. wild type, or modified derivative CPXV, or recombinant CPXV), or a composition thereof, for use for the prophylaxis and/or the treatment of a disease.
  • said disease is a proliferative disease such as cancers, tumors and restenosis.
  • Said cancer is preferably selected from the group consisting of renal cancer, bladder cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, hepatic cancer, gastric cancer, pancreatic cancer, melanoma, ovarian cancer and glioblastoma, and especially metastatic ones.
  • said disease is a disease associated with an increased osteoclast activity, like rheumatoid arthritis and osteoporosis.
  • a method of treatment of a disease which comprises the administration into a host organism in need thereof of a therapeutically effective amount of a CPXV (e.g. a wild type, or modified derivative CPXV, or a recombinant CPXV) or a composition thereof.
  • Said method of treatment may be used in conjunction with one or more additional therapies such as ones selected from the group consisting of surgery, radiotherapy, chemotherapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy or cytokine therapy.
  • said CPXV is engineered to express a suicide gene
  • said recombinant virus may be used in conjunction with a pharmaceutically acceptable amount of the corresponding prodrug.
  • Figure 1 Generation of Cowpox virus (CPXV) expressing the GFP::FCU1 fusion gene and evaluation of the GFP-FCUl protein expression.
  • CPXV Cowpox virus
  • Figure 2 Specific CDase and UPRTase activities in LoVo cell lines.
  • CDase cytosine deaminase
  • ND not detectable
  • UPRTase uracil phosphoribosyltransferase
  • CDase and UPRTase activities are expressed as the number of nmoles of 5-FC deaminated per min per mg of protein and the number of nmoles 5-FU phosphorylated per min per mg of protein, respectively.
  • the indicated enzymatic activities were measured as described in Materials and Methods. Each value represents the average of three independent experiments ⁇ standard deviation.
  • LoVo cells were infected with the indicated vectors at a multiplicity of infection (MOI) of 0.001 and then incubated with 0.1 mM 5-FC from day 2 to day 5 post infection, the relative concentration of 5-FU in the media was measured by HPLC. The data are expressed as the percentage of 5-FU in media relative to the total amount of 5-FC + 5-FU.
  • MOI multiplicity of infection
  • a) Transduction efficiency a panel of human tumoral cells were infected with CPXVi/ - /gfp::fcul at MOI 0.01 and 0.1. Sixteen hours after infection, cells were harvested and washed with PBS and GPF fluorescence was evaluated by flow cytometry, b) Fold amplification between input viral titers and viral titers produced at 72 hpi was calculated in a panel of human tumoral cells, c) Viability by trypan blue exclusion: 3.10 5 cells/well were infected with CPXVtk-/gfp::fcul at various MOI (MOI 0.00001 to 0.1). Five days after, cells were counted by ViCell automate based on trypan blue exclusion method.
  • Figure 5 Combination oncolytic and prodrug activation cytotoxicity.
  • Figure 8 Organ distribution of CPXV in mice.
  • mice were injected i.v. with 10 6 pfu CPXVtk-/gfp::fcul. At day 2 and day 7, mice were euthanized and the indicated organs were collected and homogenized, and the viral titer (pfu/mg) contained in the lysate was measured on Vero cells.
  • Figure 9 In vivo CPXV activity in glioblastoma and colorectal tumor.
  • Figure 10 Replication of CPXwt and CPXtk-/qfp::fcul virus on 3D skin model.
  • Figure 11 Infection and replication of CPXwt and CPXtk-/qfp::fcul on hPBMC.
  • Fresh human PBMC were infected by CPXtk-/gfp::fcul at different MOI. Sixteen hours post infection, eGFP level was measured on flow cytometry. Four days post infection, cells and supernatants were harvested and sonicated. Viral titers were determined by plaque assay on Vero cells. Results are expressed as viral fold increased (corresponding to output/input ratio).
  • Figure 12 In vivo CPXV activity in pancreatic tumor.
  • Figure 13 In vitro evaluation of CPXV toxicity on human pancreatic islets.
  • Fresh pancreatic islets (InSphero ® ) (human primary cells) were infected with 100 pfu of CPXwt and CPXtk-/gfp::fcul. Seven days post infection, islets were collected and sonicated, viral titers were determined by plaque assay. Results are expressed as viral fold increased (corresponding to output/input ratio).
  • a and “an” are used in the sense that they mean “at least one”, “at least a first”, “one or more” or “a plurality” of the referenced components or steps, unless the context clearly dictates otherwise.
  • a cell includes a plurality of cells, including mixtures thereof.
  • one or more refers to either one or a number above one (e.g. 2, 3, 4, etc.).
  • the terms “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are open-ended and do not exclude additional, un-recited elements or method steps.
  • the expression “consisting essentially of” means excluding other components or steps of any essential significance. Thus, a composition consisting essentially of the recited components would not exclude traces, contaminants and pharmaceutically acceptable carriers. "Consisting of” shall mean excluding more than trace elements of other components or steps.
  • polypeptide refers to polymers of amino acid residues which comprise at least nine or more amino acids bonded via peptide bonds.
  • the polymer can be linear, branched or cyclic and may comprise naturally occurring and/or amino acid analogues and it may be interrupted by non-amino acids.
  • amino acid polymer is more than 50 amino acid residues, it is preferably referred to as a polypeptide or a protein whereas if it is 50 amino acids long or less, it is referred to as a "peptide”.
  • nucleic acid refers to any length of either polydeoxyribonucleotides (DNA) (e.g. cDNA, genomic DNA, plasmids, vectors, viral genomes, isolated DNA, probes, primers and any mixture thereof) or polyribonucleotides (RNA) (e.g. mRNA, antisense RNA, SiRNA) or mixed polyribo- polydeoxyribonucleotides.
  • DNA polydeoxyribonucleotides
  • RNA e.g. mRNA, antisense RNA, SiRNA
  • mixed polyribo- polydeoxyribonucleotides encompass single or double-stranded, linear or circular, natural or synthetic, modified or unmodified polynucleotides.
  • a polynucleotide may comprise non-naturally occurring nucleotides and may be interrupted by non- nucleotide components.
  • analogue refers to a molecule (polypeptide or nucleic acid) exhibiting one or more modification(s) with respect to the native counterpart. Any modification(s) can be envisaged, including substitution, insertion and/or deletion of one or more nucleotide/amino acid residue(s). Preferred are analogues that retain a degree of sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 98% identity with the sequence of the native counterpart.
  • identity refers to an amino acid to amino acid or nucleotide to nucleotide correspondence between two polypeptides or nucleic acid sequences.
  • identity refers to an amino acid to amino acid or nucleotide to nucleotide correspondence between two polypeptides or nucleic acid sequences. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps which need to be introduced for optimal global alignment and the length of each gap.
  • the term "host cell” should be understood broadly without any limitation concerning particular organization in tissue, organ, or isolated cells. Such cells may be of a unique type of cells or a group of different types of cells such as cultured cell lines, primary cells and dividing cells.
  • the term "host cells” include prokaryotic cells, lower eukaryotic cells such as yeast, and other eukaryotic cells such as insect cells, pla nt and ma mmalian (e.g. human or non-huma n) cells as well as cells ca pa ble of producing the cowpox virus of the invention. This term also includes cells which can be or has been the recipient of the virus described herein as well as progeny of such cells.
  • virus 'viral particle
  • viral vector viral vector
  • virion a virus comprising at least one element of a wild-type virus genome and may be packaged into a viral particle or to a viral particle.
  • viral particles may or may not contain nucleic acid (i.e. the viral genome) it is preferred that a virus comprises a DNA or RNA viral genome packaged into a viral particle (or virion) and is infectious (i.e. capable of infecting and entering into a host cell or subject).
  • the virus according to this invention is associated with a DNA genome, and most preferably a double-stranded DNA genome. I n the context of the present disclosure, a "virus” includes wild-type and engineered viruses.
  • Naturally occurring or wild-type or “native” is used to describe a biological molecule or organism that can be found in nature as distinct from being artificially produced by man.
  • a virus which can be isolated from a source in nature is wild- type.
  • the present invention a lso encom passes wild-type viruses that can be obtained from specific collections (e.g. ECCAC, ATCC, CNCM, etc.).
  • a biological molecule or an organism which has been intentionally modified by man in the laboratory is not naturally occurring.
  • non-naturally occurring viruses include, among many others, recombinant viruses engineered by insertion of one or more gene(s) of interest in the viral genome and mutated viruses engineered by total or partial deletion of a viral gene to make the modified virus defective for the encoded gene product (e.g. TK- virus) as well as chimeric viruses containing genomic fragments obtained from different virus origins.
  • recombinant viruses engineered by insertion of one or more gene(s) of interest in the viral genome and mutated viruses engineered by total or partial deletion of a viral gene to make the modified virus defective for the encoded gene product (e.g. TK- virus) as well as chimeric viruses containing genomic fragments obtained from different virus origins.
  • originating or “originate” is used to identify the original source of a component (e.g. polypeptide, nucleic acid molecule, virus, etc.) but is not meant to limit the method by which the component is made which can be, for example, by chemical synthesis or recombinant means.
  • a component e.g. polypeptide, nucleic acid molecule, virus, etc.
  • the term "oncolytic virus” refers to a virus capable of selectively replicating in dividing cells (e.g. a proliferative cell such as a cancer cell) with the aim of slowing the growth and/or lysing said dividing cell, either in vitro or in vivo, while showing no or minimal replication in non-dividing cells (e.g. primary cells).
  • replication and “propagation” are used interchangeably and refer to the ability of a virus to reproduce and proliferate.
  • Virus replication can be quantified at the level of nucleic acid or at the level of infectious viral particle using assays standard in the art and described herein such as a virus titer assay, plaque assay, absorbance, fluorescence detection, mass spectrometry, etc.
  • treatment encompasses prophylaxis (e.g. preventive measure in a subject at risk of having the pathological condition to be treated) and/or therapy (e.g. in a subject diagnosed as having the pathological condition), optionally in association with conventional therapeutic modalities.
  • the result of the treatment is to slow down, cure, ameliorate or control the progression of the targeted pathological condition.
  • a subject is successfully treated for a cancer if after administration of a cowpox virus as described herein, alone or in combination with other therapy/ies, the subject shows an observable improvement of its clinical status.
  • administering refers to the delivery to a subject of a therapeutic agent such as the cowpox virus described herein.
  • proliferative disease encompasses any disease or condition resulting from uncontrolled cell growth and spread including cancers, tumors and some cardiovascular diseases (restenosis that results from the proliferation of the smooth muscle cells of the blood vessel wall, etc.).
  • cancer may be used interchangeably with any of the terms “tumor”, “malignancy”, “neoplasm”, etc. These terms are meant to include any type of tissue, organ or cell, any stage of malignancy (e.g. from a pre-lesion to stage IV).
  • disease associated with an increased osteoclast activity encompasses any disease or condition resulting in bone resorption or destruction (e.g. rheumatoid arthritis, osteoporosis, etc.).
  • subject generally refers to an organism for whom any product and method of the invention is needed or may be beneficial.
  • the organism is a mammal, particularly a mammal selected from the group consisting of domestic animals, farm animals, sport animals, and primates.
  • the subject is a human who has been diagnosed as having or at risk of having a proliferative disease such as a cancer.
  • subject and patients may be used interchangeably when referring to a human organism and encompasses male and female.
  • the subject to be treated may be a new-born, an infant, a young adult, an adult or an elderly.
  • combination treatment may be used interchangeably and refer to a treatment of a subject with at least two different therapeutic agents.
  • one of therapeutic agent is a cowpox virus as described herein.
  • the second therapeutic agent may be any clinically established therapeutic agent, in particular one selected from the group consisting of surgery, radiotherapy, chemotherapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy, cytokine therapy, targeted cancer therapy, gene therapy, photodynamic therapy, transplantation etc.
  • a combinatorial treatment may include a third or even further therapeutic agent(s).
  • cowpox virus or "CPXV” refers to a group of viruses belonging to the Poxviridae family, more precisely to the Chordopoxvirinae subfamily, and even more precisely to the Orthopoxvirus genus.
  • Cowpox virus has a broad host range and is believed to persist in a reservoir comprising various rodents indigenous to parts of Europe and adjoining Asia (Chantrey et al., 1999, Epidemiol. Infect., 122:455-60.). Very often domestic cats cause intermediates CPXV infected hosts, which may transmit the virus to humans (Essbauer et al., 2002, Revue Med. Vet., 153, 10:635-42). However, in vitro and in vivo studies performed with laboratory strains of cowpox virus strongly support that compounds like cidofovir and ST-246 will be active against genetically diverse CPXV isolates.
  • Poxviruses are DNA viruses that replicate in the cytoplasm of infected cells. Recent genotypic data pointed to a much higher genomic diversity among CPXV as compared to isolates from other Orthopoxvirus species. In particular, they have the largest genome (more than 220 kbp), about 30kbp larger than the VACV genome (Carroll et al., 2011, PLoS One 6:e23086). Because the infected cell must deliver large amounts of DNA precursors to cytoplasmic replication sites, the virus encodes and expresses many enzymatic activities required for DNA metabolism and synthesis, including ribonucleotide reductase and deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase).
  • dUTPase deoxyuridine 5'-triphosphate nucleotidohydrolase
  • CPXV can be divided into two major monophyletic clades (Cowpox-like and Vaccinialike).
  • the cowpox-like clade can be further grouped into four distinct monophyletic clusters, clusters 1, 2, 3 and 4, respectively and Vaccinia-like clade comprises cluster 5.
  • the cowpox virus for use herein is any virus of the Cowpox phylogeny belonging to any clade, cluster and strain described in the art encompassing clinical, laboratory and vaccine isolates (see e.g. Carroll et al., 2011, PLoS One 6:e23086; Duraffour et al., 2013, PLoS One 8(2):l-8). Moreover, it may be isolated from any host organism including domestic and zoological garden animals (e.g. elephants, cats, pet rodents, etc.) as well as primate (e.g. humans), and may be pathogenic or have a reduced virulence or be avirulent at least with respect to the subject to be treated.
  • domestic and zoological garden animals e.g. elephants, cats, pet rodents, etc.
  • primate e.g. humans
  • the CPXV of the invention belongs to (wild-type virus) or is obtained from (e.g. engineered or recombinant virus) the Cowpox-like clade and more particularly to any of clusters 1, 2, 3 and 4.
  • Exemplary CPXV of Cluster 1 comprises CPXV_GER1980_EP4 (Genbank H 420895) and CPXV_GER2002_MKY (Genbank H 420898).
  • Exemplary CPXV of cluster 2 comprises CPXV_GER1991_3 (Genbank DQ 437593).
  • Exempla ry CPXV of cluster 3 comprises CPXV_FRA2001_NANCY (Genbank HQ420894), CPXV_GER1990_2 (Genbank HQ420896), CPXV_UK2000_K2984 (Genbank HQ420900), CPXV_BR (Genbank AF482758.2 or NC 003663) and CPXV_NOR1994-MAN (Genbank HQ420899).
  • Exemplary CPXV of cluster 4 comprises CPXV_GER1998_2 (Genbank HQ420897).
  • CPXV of vaccinia-like clade and cluster 5 of this clade or any derivative thereof are suita ble as well in the context of this invention, and in particular CPXV_gri (Genbank X94355), CPXV_FIN2000_MAN (Genbank HQ420893) and CPXV_AUS1999_867 (Genbank HQ407377).
  • a preferred embodiment is directed to a CPXV of clade 3 and especially to the Brighton red strain (CPXV_BR).
  • cowpox virus of the invention may be altered and may comprise one or more mutation(s) in its genome, i.e. deletion, substitution or addition of one or more nucleotides or any combination of these possibilities.
  • mutation(s) in its genome, i.e. deletion, substitution or addition of one or more nucleotides or any combination of these possibilities.
  • modifications may be consecutive or not. Desirably, said modifications lead to the inability for the virus to produce a protein having the activity of the protein produced by the unmodified gene, resulting in a defective virus for this particular activity.
  • modification(s) may occur in the promoter region or in the coding sequence or both
  • preferred modification(s) comprises deletion of a whole gene sequence (i.e.
  • Methods for modifying the genome of a poxvirus can be used to modify the genome of cowpox virus of the invention, for example, the methods disclosed in McCart et al., 2001, Cancer Res., 61:8751-57, Kim et al., 2006, Mol. Ther., 14:361-70, WO 2004/014314 in view of the information and sequence data given in the present application and those available in Genbank.
  • the Example section also illustrates appropriate methods to produce cowpox viruses according to the invention.
  • the cowpox virus of the invention comprises a defective CPXV105 CDS gene (UniprotKB Q8QMX0), resulting in a defective thymidine kinase (TK) activity.
  • TK thymidine kinase
  • the TK enzyme is involved in the synthesis of deoxyribonucleotides. TK is needed for viral replication in normal cells as these cells have generally low concentration of nucleotides whereas it is dispensable in dividing cells which contain high nucleotide concentration.
  • TK The reaction catalyzed by TK involves the transfer of a ⁇ -phosphoryl moiety from ATP to 2'deoxy-thymidine (dThd) to produce thymidine 5'- monophosphate (dTMP).
  • Cowpox viruses' TK is of type 2.
  • Type 2 TKs have a smaller polypeptide chain compared to type 1, being of ⁇ 25 KDa but form homotetramers. They are sensitive to the feedback inhibitors dTDP or dTTP, which are generated at the end of the metabolic pathway.
  • Type 2 TKs have a much narrower substrate specificity compared to type 1 TKs and only phosphorylate 2'deoxyuridine (dU) and/or dThd (El Omari et al., 2006, BMC Struct. Biol., 6:22). It is within the reach of the skilled in the art to generate a cowpox defective for CPXV105 CDS gene, based on the information given herein a nd the available CPXV genome sequence, using conventional molecular biology techniques (PCR, gene targeting, use of restriction enzymes, ligations, molecular cloning, CRISPR/Cas9, etc.).
  • the CPXV of the present invention is modified by altering at least one gene or both genes encoding ribonucleotide reductase (RR).
  • RR ribonucleotide reductase
  • this enzyme catalyzes the reduction of ribonucleotides to deoxyribonucleotides that represents a crucial step in DNA biosynthesis.
  • the viral enzyme is similar in subunit structure to the mammalia n enzyme, being composed of two heterologous subunits, designed Rl and R2 encoded respectively by the CPXV083 CDS (corresponding to Vaccinia virus I4L) and CPXV051 CDS (corresponding to vaccinia virus F4L locus).
  • the CPXV083 CDS gene encoding the Rl large subunit
  • CPXV051 CDS gene encoding the R2 small subunit
  • both may be inactivated.
  • the CPXV of the present invention comprises a defective CPXV049 CDS gene, corresponding to Vaccinia Virus F2L, and encoding deoxyuridine triphosphatase (dUTPase).
  • this enzyme catalyzes the conversion of dUTP to dU M P and pyrophosphate in the presence of Mg(2+) ions.
  • dUTPase in removing dUTP from the dNTP pool and generating dUMP, is involved in both maintaining the fidelity of DNA replication and in providing the precursor to produce TM P by thymidylate synthase.
  • the CPXV of the present invention comprises a truncated CPXV032 CDS gene, the homologue of C5L of Vaccinia Virus.
  • a truncated gene means may be defined as lacking ⁇ 80% of the amino acid length as compared to the wild-type gene. Truncation may be at the N or the C terminus or internally.
  • the CPXV of the present invention is modified by altering one or more of the following genes (named using VACV nomenclature) : genes encoding interferon-modulating polypeptide (including, but not limited to, B8R, B18R, B19R and/or vC12L) that results in the virus lacking at least an interferon-modulating function; genes encoding a complement control polypeptide (e.g.
  • VCP vaccinia virus com plement control protein
  • genes encoding a TNF-modulating polypeptide including, but not limited to, A53R and B28R
  • genes encoding a serine protease inhibitor including, but not limited to B13R, B22R, a nd/or K2L
  • genes encoding an I L- ⁇ modulator polypeptide e.g.
  • B15R, B16R that results in the virus lacking at least one I L- ⁇ modulator function
  • genes encoding inhibitor of I L-1 and TLR signal e.g. A46R and A52R
  • genes encoding NF- ⁇ inhibitor e.g. NIL, K7L, M2L
  • genes encoding IRF3/7 inhibitor e.g. C6L, N2L
  • genes encoding chemokine binding protein e.g. C23L, A41L, vCKBP, vCCI
  • genes encoding antiapoptotic proteins e.g. F1L
  • genes encoding proteins involved in nucleotide metabolism e.g. A48R, A57R
  • other examples are A26L, A56R, C4L, D4R, OIL, B7R, and A44L.
  • the CPXV of the present invention may also be modified by altering CPXV021 CDS gene (also called CPXV VGFgene, or C11R in vaccinia virus) which encodes proteins expressed early after cell infection and which function seems important for virus spread in normal cells; gene encoding ubiquitine ligase (e.g. CPXV023 CDS); gene encoding soluble I L-18 binding proteins (e.g. CPXV024 CDS) and A-type inclusion body ATI gene (e.g. CPXV158 CDS).
  • CPXV021 CDS gene also called CPXV VGFgene, or C11R in vaccinia virus
  • gene encoding ubiquitine ligase e.g. CPXV023 CDS
  • gene encoding soluble I L-18 binding proteins e.g. CPXV024 CDS
  • A-type inclusion body ATI gene e.g. CPXV158 CDS
  • the cowpox virus of the invention is recombinant (i.e. engineered to express a nucleic acid of interest) and comprises inserted in its genome at least one nucleic acid of interest.
  • the nucleic acid of interest can be homologous or heterologous to the host organism into which it is introduced. More specifically, it can be of human origin or not (e.g. of bacterial, yeast or viral origin).
  • said nucleic acid of interest encodes a therapeutic molecule and, notably, all or part of a polypeptide.
  • a polypeptide is understood to be any translational product of a polynucleotide regardless of size, and whether glycosylated or not, and includes peptides and proteins.
  • the nucleic acid of interest encodes a therapeutic molecule of therapeutic or prophylactic interest which is capable of providing a biological activity when administered appropriately to a subject, which is expected to cause a beneficial effect on the course or a symptom of the pathological condition to be treated.
  • a therapeutic gene may be envisaged in the context of the invention such as those encoding therapeutic molecules that can compensate for defective or deficient proteins in the subject, or those that act through toxic effects to limit or remove harmful cells from the body or those that encode immunity conferring polypeptides. They may be native genes or genes obtained from the latter by mutation, deletion, substitution and/or addition of one or more nucleotides.
  • suitable molecule of therapeutic interest include, without limitation, polypeptides encoded by suicide genes which are capable of reinforcing the oncolytic nature of the cowpox virus of the present invention, as well as polypeptides capable of potentiating anti-tumor efficacy such as immunostimulatory polypeptides and antigens (for inducing or activating an immune humoral and/or cellular response).
  • Suicide gene product encoded by suicide genes which are capable of reinforcing the oncolytic nature of the cowpox virus of the present invention, as well as polypeptides capable of potentiating anti-tumor efficacy such as immunostimulatory polypeptides and antigens (for inducing or activating an immune humoral and/or cellular response).
  • suicide gene refers to a gene coding for a polypeptide able to convert a precursor of a drug, also named “prodrug”, into a cytotoxic compound.
  • Examples of suicide genes and corresponding prodrugs comprising one nucleobase moiety are disclosed in the following table:
  • Thymidine Kinase Ganciclovir Thymidine Kinase Ganciclovir
  • Ganciclovir elaidic acid ester Thymidine Kinase Ganciclovir
  • bromovinyl)-2'-deoxyuridine zidovudine
  • the cowpox of the invention carries in its genome a suicide gene encoding a polypeptide having at least cytosine deaminase (CDase) activity.
  • CDase cytosine deaminase
  • the prokaryotes and lower eukaryotes it is not present in mammals
  • CDase is involved in the pyrimidine 5 metabolic pathway by which exogenous cytosine is transformed into uracil by means of a hydrolytic deamination.
  • CDase also deaminates an analogue of cytosine, i.e.
  • 5-fluorocytosine thereby forming 5-fluorouracil (5-FU), a compound which is cytotoxic by itself but even more when it is converted into 5-fluoro-UMP (5-FUMP).
  • CDase encoding nucleic acid molecule can be obtained from any prokaryotes and lower eukaryotes such as
  • FCYl gene Saccharomyces cerevisiae
  • FCAl gene Candida Albicans
  • FCAl gene Escherichia coli
  • CDase proteins have been published and are available in specialized data banks (SWISSPROT EMBL, Genbank, Medline and the like).
  • Functional analogues of these genes may also be used.
  • Such analogues preferably have a degree of identity of at least 80%, preferably of at least 90%, and most preferably of at least
  • the cowpox virus of the invention carries in its viral genome a suicide gene encoding a polypeptide having uracil phosphoribosyl transferase (UPRTase) activity.
  • UPRTase uracil phosphoribosyl transferase
  • prokaryotes and lower eukaryotes uracil is transformed into UMP by the action of UPRTase.
  • This enzyme converts 5-FU into 5-FUMP.
  • UPRTase analogues may also be used such as the N-terminally truncated FUR1 mutant described in EP998568 (with a deletion of the 35 first residues up to the second Met residue present at position 36 in the native protein) which exhibits a higher UPRTase activity than that of the native enzyme.
  • the suicide gene inserted in the viral genome of the cowpox virus of the present invention encodes a polypeptide having CDase and UPRTase activities.
  • a polypeptide can be engineered by fusion of two enzymatic domains, one having the CDase activity and the second having the UPRTase activity.
  • Exemplary polypeptides include without limitation fusion polypeptides codA::upp, FCY1::FUR1 and FCYl::FURl[Delta] 105 (FCUl) and FCUl-8 described in W096/16183, EP998568 and WO2005/07857.
  • FCUl suicide gene (or FCYl::FURl[Delta] 105 fusion) encoding a polypeptide comprising the amino acid sequence represented in the sequence identifier SEQ. ID NO: 1 of WO2009/065546.
  • the present invention encompasses analogues of such polypeptides providing they retain the CDase and/or UPRTase activities.
  • the cowpox virus of the invention encodes suicide gene product(s)
  • a "permease” is a trans-membranous protein involved in the transfer of a drug comprising one nucleobase moiety, or a precursor thereof through the cell membrane.
  • Permeases comprise purine permeases, cytosine permeases and nucleoside transporters.
  • the permease is a purine or a cytosine permease of S. Cerevisiae with a specific preference for the purine-cytosine permease, known as FCY2, and the uracil permease, known as FU R4 or any analogue thereof (i.e. at least 80% identity with the wild-type genes).
  • FCY2 and Fur4 are preferably associated with 5-Fluorocytosine (5-FC).
  • FCY2 mediates symport of protons and adenine, guanine, hypoxanthine and cytosine across the yeast plasma membrane.
  • FCY2 protein mediates also the transport of 5-fluorocytosine, an analogue of cytosine (Grenson et al., 1970, J. Bacteriol., 103(3):770-7). Uracil uptake into 5. cerevisiae is mediated by the uracil permease, FUR4 which is an uracil-proton symporter.
  • FUR4 protein ca n also mediates the transport of 5-fluorouracil, an analogue of uracil (Jund and Lacroute, 1970, J.
  • FCY2 and Fur4 are available in the swissprot database (accession number P17064 and P05316 respectively).
  • Preferred permeases appropriate for expression in the cowpox virus of the invention, especially a CDase and/or UPRTase -encoding CPXV (e.g. FCUl) are disclosed in WO 2006/048768 (see e.g. the amino acid sequence SEQ. I D NO: 1 and SEO I D NO : 2).
  • immunostimulatory polypeptide refers to a polypeptide or protein, which has the ability to stimulate the immune system, in a specific or non-specific way.
  • suitable immunostimulatory polypeptides include, without limitation, agents such as, e.g. alpha, beta or gamma interferon, interleukin (in particular I L-2, I L-6, I L-10 or I L-12) and tumor necrosis factor (TNF); agents that affect the regulation of cell surface receptors such as, e.g.
  • inhibitors of Epidermal Growth Factor Receptor in particular cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab, gefitinib, eriotinib or lapatinib
  • agents that affect angiogenesis such as, e.g. inhibitor of Vascular Endothelial Growth Factor (in particular bevacizumab or ranibizumab)
  • GM-CSF granulocyte macrophage - colony stimulating factor
  • antigen generally refers to a substance that is recognized and selectively bound by an antibody or by a T cell antigen receptor, in order to trigger an immune response. It is contemplated that the term antigen encompasses native antigen as well as fragment (e.g. epitopes, immunogenic domains, etc.) and derivative thereof, provided that such fragment or derivative is capable of being the target of an immune response. Suitable antigens in the context of the invention are preferably polypeptides (e.g.
  • peptides, polypeptides, post translationally modified polypeptides, etc. including one or more B cell epitope(s) or one or more T cell epitope(s) or both B and T cell epitope(s) and capable of raising an immune response, preferably, a humoral or cell response that can be specific for that antigen.
  • the one or more antigen(s) is selected in connection with the disease to treat.
  • Preferred antigens for use herein are cancer antigens and antigens of pathogens.
  • the antigen(s) contained in or encoded by the cowpox virus is/are cancer antigen(s) (also called tumor-associated antigens) that is associated with and/or serve as markers for cancers.
  • Cancer antigens encompass various categories of polypeptides, e.g. those which are normally silent (i.e. not expressed) in normal cells, those that are expressed only at low levels or at certain stages of differentiation and those that are temporally expressed such as embryonic and foetal antigens as well as those resulting from mutation of cellular genes, such as oncogenes (e.g. activated ras oncogene), proto- oncogenes (e.g. ErbB family), or proteins resulting from chromosomal translocations.
  • oncogenes e.g. activated ras oncogene
  • proto- oncogenes e.g. ErbB family
  • proteins resulting from chromosomal translocations e.g. activated ras on
  • the cancer antigens also encompass antigens encoded by pathogenic organisms (bacteria, viruses, parasites, fungi, viroids or prions) that are capable of inducing a malignant condition in a subject (especially chronically infected subject) such as RNA and DNA tumor viruses (e.g. HPV, HCV, EBV, etc.) and bacteria (e.g. Helicobacter pilori).
  • pathogenic organisms bacteria, viruses, parasites, fungi, viroids or prions
  • cancer antigens include, without limitation, MART- 1/Melan-A, gplOO, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, Colorectal associated antigen (CRC)-C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, amll, Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-family of tumor anti
  • MUC1, MUC16, etc. see e.g. US6,054,438; WO98/04727; or WO98/37095), HER2/neu, p21ras, RCASl, alpha-fetoprotein, E-cadherin, alpha-catenin, beta-catenin and gamma-catenin, pl20ctn, gpl00.sup.Pmelll7, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, pl5, gp75, GM2 and GD2 gangliosides, Smad family of cancer antigens brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2 and viral antigens such as the HPV-16 and HPV
  • antigens suitable for use in this invention are marker antigens (beta- galactosidase, luciferase, green fluorescent proteins, etc.).
  • marker antigens beta- galactosidase, luciferase, green fluorescent proteins, etc.
  • the present invention also encompasses cowpox virus expressing two or more polypeptides of interest as described herein, e.g. at least two antigens, at least one antigen and one cytokine, at least two antigens and one cytokine, etc.
  • the cowpox virus of the invention further comprises at least one nucleic acid of interest including, but not limited to:
  • apoptotic genes including pro-apoptotic genes (e.g. Bax, Bak, Bok, Bad, Bid et Bim), inhibitors of pro-apoptotic genes (e.g. Bax inhibitor, Bak inhibitor, Bok inhibitor, Bad inhibitor, Bid inhibitor, Bim inhibitor), anti-apoptotic genes (e.g. Bcl-2, Bcl-xL, Bcl-w, Nrl3) and inhibitors of anti-apoptotic genes (e.g. Bcl2 inhibitor, Bcl-xL-inhibitor, Bcl- w inhibitor, Nrl3 inhibitor),
  • pro-apoptotic genes e.g. Bax, Bak, Bok, Bad, Bid et Bim
  • inhibitors of pro-apoptotic genes e.g. Bax inhibitor, Bak inhibitor, Bok inhibitor, Bad inhibitor, Bid inhibitor, Bim inhibitor
  • anti-apoptotic genes e.g. Bcl-2, Bcl-xL, Bcl-w
  • nucleic acid coding for endonuclease like restriction enzymes (e.g. restriction enzymes of type I, II, III, IV or V, artificial restriction enzymes like Transcription activator-like effector nucleases (TALEN) or zinc finger nuclease), CRISPR/Cas9 nucleic acid coding for immune checkpoint inhibitors, including, but not limited to anti-PDl, anti-PDLl, anti-PDL-2, anti-CTLA4, anti-Tim3, anti-LAG3, anti-BTLA, - RNA, including but not limited to miRNA, targets of miRNA, shRNA, siRNA.
  • restriction enzymes e.g. restriction enzymes of type I, II, III, IV or V, artificial restriction enzymes like Transcription activator-like effector nucleases (TALEN) or zinc finger nuclease
  • CRISPR/Cas9 nucleic acid coding for immune checkpoint inhibitors including, but not limited to anti-PDl, anti-PDLl, anti-PD
  • nucleic acid(s) of interest may be easily obtained by cloning, by PCR or by chemical synthesis according to the conventional techniques. They may be native genes or genes derived from the latter by mutation, deletion, substitution and/or addition of one or more nucleotides. Moreover, their sequences are widely described in the literature which can be consulted by persons skilled in the art.
  • nucleic acid(s) of interest can be optimized for providing high level expression in a particular host cell or subject. It has been indeed observed that, the codon usage patterns of organisms are highly non-random and the use of codons may be markedly different between different hosts.
  • nucleic acid(s) of interest might be from bacterial or lower eukaryote origin (e.g. the suicide gene), it may have an inappropriate codon usage pattern for efficient expression in higher eukaryotic cells (e.g. human).
  • codon optimization is performed by replacing one or more "native” (e.g. bacterial or yeast) codon corresponding to a codon infrequently used in the host organism of interest by one or more codon encoding the same amino acid which is more frequently used. It is not necessary to replace all native codons corresponding to infrequently used codons since increased expression can be achieved even with partial replacement.
  • expression in the host cell or subject can further be improved through additional modifications of the gene sequence.
  • the sequence of the nucleic acid of interest can be modified so-as to prevent clustering of rare, non-optimal codons being present in concentrated areas and/or to suppress or modify "negative" sequence elements which are expected to negatively influence expression levels.
  • negative sequence elements include without limitation the regions having very high (>80%) or very low ( ⁇ 30%) GC content; AT-rich or GC-rich sequence stretches; unstable direct or inverted repeat sequences; R A secondary structures; and/or internal cryptic regulatory elements such as internal TATA-boxes, chi-sites, ribosome entry sites, and/or splicing donor/acceptor sites.
  • the cowpox virus comprises the elements necessary for the expression of the nucleic acid(s) of interest. More precisely, the nucleic acid(s) of interest inserted in the genome of the cowpox virus of the invention is/are operably linked to suitable regulatory elements for its/their expression in a host cell or subject.
  • regulatory elements or “regulatory sequence” refers to any element that allows, contributes or modulates the expression of the nucleic acid(s) of interest in a given host cell or subject, including replication, duplication, transcription, splicing, translation, stability and/or transport of the nucleic acid(s) or its derivative (i.e. mRNA).
  • operably linked means that the elements being linked are arranged so that they function in concert for their intended purposes.
  • a promoter is operably linked to a nucleic acid molecule if the promoter effects transcription from the transcription initiation to the terminator of said nucleic acid molecule in a permissive host cell.
  • the choice of the regulatory sequences can depend on such factors as the gene itself, the virus into which it is inserted, the host cell or subject, the level of expression desired, etc.
  • the promoter is of special importance. In the context of the invention, it can be constitutive directing expression of the nucleic acid(s) of interest in many types of host cells or specific to certain host cells (e.g. liver- specific regulatory sequences) or regulated in response to specific events or exogenous factors (e.g. by temperature, nutrient additive, hormone, etc.) or according to the phase of a viral cycle (e.g. late or early).
  • CMV cytomegalovirus
  • RSV cytomegalovirus
  • PGK phosphoglycero kinase
  • TK thymidine kinase promoter of herpes simplex virus
  • T7 polymerase promoter WO98/10088
  • Representative examples include without limitation the vaccinia 7.5K, H5R, 11K7.5 (Erbs et al., 2008, Cancer Gene Ther. 15(1): 18-28), TK, p28, pllK, Prl3.5 (WO2014/063832), pB8R, pFUL, pA44L, pCUR (WO2011/128704) and K1L promoter, as well as synthetic promoters such as those described in Chakrabarti et al. (1997, Biotechniques, 23: 1094-7; Hammond et al., 1997, J. Virol.
  • the regulatory elements controlling the expression of the nucleic acid(s) of interest may further comprise additional elements for proper initiation, regulation and/or termination of transcription (e.g. a transcription termination sequences), mRNA transport (e.g. nuclear localization signal sequences), processing (e.g. splicing signals), and stability (e.g. introns and non-coding 5' and 3' sequences), translation (e.g. an initiator Met, tripartite leader sequences, I RES ribosome binding sites, signal peptides), targeting sequences, transport sequences, secretion signa l, and sequences involved in replication or integration. Said sequences have been reported in the literature and can be readily obtained by those skilled in the art.
  • the nucleic acid(s) of interest can be inserted at any location of the viral genome, with a specific preference for a non-essential locus.
  • CPXV105 CDS gene, CPXV083 CDS gene, CPXV051 CDS gene or intergenic zones are particularly appropriated for insertion of the nucleic acid sequence of interest and appropriate regulatory sequences in cowpox virus.
  • the cowpox virus is defective for the CPXV105 CDS gene and comprises inserted in place of the CPXV105 CDS gene a nucleic acid of interest (e.g. the FCU- 1 gene) under the transcriptional control of a synthetic promoter (e.g. the pllK7.5 promoter).
  • a nucleic acid of interest e.g. the FCU- 1 gene
  • a synthetic promoter e.g. the pllK7.5 promoter
  • the invention also relates to a process for preparing a cowpox virus according to the invention, in which process:
  • a cowpox virus of the invention is introduced into a cell
  • said cell is cultured under conditions which are appropriate for enabling said cowpox virus to be produced
  • the cowpox virus of the present invention is produced into a suitable host cell line using conventional techniques including culturing the transfected or infected host cell under suitable conditions so-as to allow the production of infectious viral particles and recovering the produced infectious viral particles from the culture of said cell and optionally purifying said recovered infectious viral particles.
  • suitable host cells for production of the oncolytic virus include without limitation human cell lines such as HeLa (ATCC), Monkey cells such as Vero (ATCC CCL-081), 293 cells (Graham et al., 1997, J. Gen. Virol.
  • HER96 PER-C6 (Fallaux et al., 1998, Human Gene Ther., 9: 1909-17)
  • CV1 ATCC CCL-70
  • BSC1 ATCC CCL-26
  • avian cells such as those described in WO2005/042728, WO2006/108846, WO2008/129058, WO2010/130756, WO2012/001075, etc.
  • hamster cell lines such as BHK-21 (ATCC CCL-10) as well as primary chicken embryo fibroblasts (CEF) prepared from chicken embryos obtained from fertilized eggs.
  • Host cells are preferably cultivated in a chemically defined medium with no product of animal or human origin.
  • Culturing is carried out at a temperature, pH and oxygen content appropriate for the producer cell. Such culturing conditions are within the expertise of one of ordinary skill in the art. If growth factors are present, they are preferably recombinantly produced and not purified from animal material. Suitable animal-free media are commercially available, for example VP-SFM medium (Invitrogen) for culturing CEF producer cells. Producer cells are preferably cultivated at a temperature comprised between +30°C and +38°C (more preferably at about +37°C) for between 1 and 8 days (preferably for 1 to 5 days for CEF and 2 to 7 days for immortalized cells) before infection. If needed, several passages of 1 to 8 days may be made in order to increase the total number of cells. Producer host cells are infected by the CPXV with an appropriate multiplicity of infection (MOI) to permit productive infection, which can be as low as 0.001 (more preferably between 0.05 and 5).
  • MOI multiplicity of infection
  • infected producer cells are cultured under appropriate conditions well known to those skilled in the art until progeny viral vector (e.g. infectious CPXV particles) is produced.
  • progeny viral vector e.g. infectious CPXV particles
  • Culture of infected producer cells is also preferably performed in a chemically defined medium (which may be the same as or different from the medium used for culture of producer cells and/or for infection step) free of animal- or human-derived products at a temperature between +30°C and +37°C, for 1 to 5 days.
  • the viral CPXV particles may be collected from the culture supernatant and/or the producer cells.
  • Recovery from producer cells (and optionally also from culture supernatant) may require a step allowing the disruption of the producer cell membrane to allow the liberation of the virus from producer cells.
  • the disruption of the producer cell membrane can be induced by various techniques well known to those skilled in the art, including but not limited to, freeze/thaw, hypotonic lysis, sonication, microfluidization, or high speed homogenization.
  • the recovered Cowpox virus can be at least partially purified before being used according to the present invention.
  • Various purification steps can be envisaged, including clarification, enzymatic treatment (e.g. endonuclease such as benzonase, protease), ultracentrifugation (e.g. sucrose gradient or cesium chloride gradient), chromatographic and filtration steps.
  • enzymatic treatment e.g. endonuclease such as benzonase, protease
  • ultracentrifugation e.g. sucrose gradient or cesium chloride gradient
  • chromatographic and filtration steps e.g. WO2007/147528; WO2008/138533, WO2009/100521, WO2010/130753, WO2013/022764.
  • the invention also relates to a composition which comprises a therapeutically effective amount of a cowpox virus as described herein (e.g. wild type, modified derivative thereof such as a CPXV105 CDS-defective cowpox, or recombinant cowpox), or prepared according to the process described herein.
  • the composition further comprises a pharmaceutically acceptable vehicle.
  • the composition of the present invention is more specifically intended for the preventive or curative treatment of proliferative diseases (cancers, tumors, restenosis, etc.) or diseases associated to an increased osteoclast activity (e.g. rheumatoid arthritis, osteoporosis).
  • a preferred composition comprises a therapeutically effective amount of a CPXV105 CDS-defective cowpox (e.g. a BR cowpox) and notably a recombinant CPXV105 CDS- defective cowpox encoding a suicide gene product such as FCU-1.
  • a CPXV105 CDS-defective cowpox e.g. a BR cowpox
  • a recombinant CPXV105 CDS- defective cowpox encoding a suicide gene product such as FCU-1.
  • a “therapeutically effective amount” corresponds to the amount of cowpox virus that is sufficient for producing one or more beneficial results. Such a therapeutically effective amount may vary as a function of various parameters, in particular the mode of administration; the disease state; the age and weight of the subject; the ability of the subject to respond to the treatment; kind of concurrent treatment; the frequency of treatment; and/or the need for prevention or therapy.
  • the composition of the invention is administered at a dose sufficient to prevent or to delay the onset and/or establishment and/or relapse of a pathologic condition (e.g. a proliferative disease such as cancer), especially in a subject at risk.
  • a pathologic condition e.g. a proliferative disease such as cancer
  • the composition of the invention is administered to a subject diagnosed as having a pathological condition (e.g. a proliferative disease such as cancer) with the goal of treating the disease, optionally in association with one or more conventional therapeutic modalities.
  • a therapeutically effective amount could be that amount necessary to cause an observable improvement of the clinical status over the baseline status or over the expected status if not treated as described hereinafter.
  • An improvement of the clinical status can be easily assessed by any relevant clinical measurement typically used by physicians and skilled healthcare staff. For example, techniques routinely used in laboratories (e.g. flow cytometry, histology) may be used to perform tumor surveillance.
  • a therapeutically effective amount could also be the amount necessary to cause the development of an effective non-specific (innate) and/or specific anti-tumor response.
  • development of an immune response in particular T cell response can be evaluated in vitro, in suitable animal models or using biological samples collected from the subject.
  • pharmaceutically acceptable vehicle is intended to include any and all carriers, solvents, diluents, excipients, adjuvants, dispersion media, coatings, antibacterial and antifungal agents, absorption agents and the like compatible with administration in mammals and in particular human subjects.
  • the cowpox virus of the invention can independently be placed in a solvent or diluent appropriate for human or animal use.
  • the solvent or diluent is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength.
  • Representative examples include sterile water, physiological saline (e.g. sodium chloride), Ringer's solution, glucose, trehalose or saccharose solutions, Hank's solution, and other aqueous physiologically balanced salt solutions (see for example the most current edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams&Wilkins).
  • the cowpox virus is suitably buffered for human use.
  • Suitable buffers include without limitation phosphate buffer (e.g. PBS), bicarbonate buffer and/or Tris buffer capable of maintaining a physiological or slightly basic pH (e.g. from approximately pH 7 to approximately pH 9).
  • composition of the invention may also contain other pharmaceutically acceptable excipients for providing desirable pharmaceutical or pharmacodynamic properties, including for example osmolarity, viscosity, clarity, colour, sterility, stability, rate of dissolution of the formulation, modifying or maintaining release or absorption into a human or animal subject, promoting transport across the blood barrier or penetration in a particular organ.
  • excipients for providing desirable pharmaceutical or pharmacodynamic properties, including for example osmolarity, viscosity, clarity, colour, sterility, stability, rate of dissolution of the formulation, modifying or maintaining release or absorption into a human or animal subject, promoting transport across the blood barrier or penetration in a particular organ.
  • composition of the invention may be adjuvanted to further enhance immunity (especially a T cell-mediated immunity) or facilitate infection of tumor cells upon administration.
  • suitable adjuvants include, without limitation, alum, mineral oil emulsion such as, Freunds complete and incomplete (IFA), lipopolysaccharide or a derivative thereof (Ribi et al., 1986, Plenum Publ. Corp. ,407- 419), saponins such as QS21 (Sumino et al., 1998, J.Virol. 72: 4931; W098/56415), imidazo- quinoline compounds such as Imiquimod (Suader, 2000, J. Am Acad Dermatol.
  • the composition of the invention may be formulated with the goal of improving its stability in particular under the conditions of manufacture and long- term storage (i.e. for at least 6 months, with a preference for at least two years) at freezing (e.g. -70°C, -20°C), refrigerated (e.g. 4°C) or ambient temperatures.
  • freezing e.g. -70°C, -20°C
  • refrigerated e.g. 4°C
  • ambient temperatures e.g. Various virus0 formulations are available in the art either in frozen, liquid form or lyophilized form (e.g. WO98/02522, WO01/66137, WO03/053463, WO2007/056847 and WO2008/114021, etc.).
  • Solid e.g.
  • dry powdered or lyophilized compositions can be obtained by a process involving vacuum drying and freeze-drying.
  • buffered formulations including NaCI and/or sugar are particularly adapted to the preservation of viruses (e.g. Tris 10 mM pH5 8 with saccharose 5 % (W/V), sodium glutamate 10 mM, and NaCI 50 mM or phosphate- buffered saline with glycerol (10%) and NaCI).
  • the cowpox virus composition is preferably formulated in a way adapted to the mode of administration to ensure proper distribution and release in vivo.
  • gastro- resistant capsules and granules are particularly appropriate for oral administration,0 suppositories for rectal or vaginal administration, optionally in combination with absorption enhancers useful to increase the pore size of the mucosal membranes.
  • absorption enhancers are typically substances having structural similarities to the phospholipid domains of the mucosal membranes (such as sodium deoxycholate, sodium glycocholate, dimethyl- beta-cyclodextrin, lauryl-l-lysophosphatidylcholine).
  • Another and particularly appropriate5 example is a formulation adapted to the administration through microneedle means (e.g. transcutaneous or intradermal patches).
  • a formulation may comprise resuspension of the immunotherapeutic product in endotoxin-free phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • cowpox virus ca n be adapted as a function of various parameters and may be routinely determined by a practitioner in the light of the relevant0 circumstances.
  • individual doses for the cowpox virus may vary within a range extending from approximately 10 3 to approximately 10 12 vp (viral particles), iu (infectious unit) or pfu (plaque-forming units) depending on the virus and the quantitative technique used.
  • Cowpox virus suitable doses are selected preferably between approximately 10 4 pfu to approximately 10 11 pfu, more preferably between 10 5 pfu to approximately 10 10 pfu; doses of approximately 10 6 pfu to approximately 5xl0 9 pfu being particularly adapted (e.g.
  • the quantity of virus present in a sample can be determined by routine titration techniques, e.g. by counting the number of plaques following infection of permissive cells (e.g. BHK-21 or CEF), immunostaining (e.g. using antivirus antibodies; Caroll et al., 1997, Virology 238: 198-211), by measuring the A260 absorbance (vp titers), or still by quantitative immunofluorescence (iu titers).
  • permissive cells e.g. BHK-21 or CEF
  • immunostaining e.g. using antivirus antibodies; Caroll et al., 1997, Virology 238: 198-211
  • A260 absorbance vp titers
  • iu titers quantitative immunofluorescence
  • the cowpox virus or the composition of the invention may be administered in a single dose or multiple doses. If multiples doses are contemplated, administrations may be performed by the same or different routes and may take place at the same site or at alternative sites. Intervals between each administration can be from several hours to 8 weeks (e.g. 24h, 48h, 72h, weekly, every two or three weeks, monthly, etc.). Intervals can also be irregular. It is also possible to proceed via sequential cycles of administrations that are repeated after a rest period (e.g. cycles of 3 to 6 weekly administrations followed by a rest period of 3 to 6 weeks). The dose can vary for each administration within the range described above.
  • Parenteral routes are intended for administration as an injection or infusion and encompass systemic as well as locoregional routes.
  • Locoregional administrations are restricted to a localized region of the body (e.g. intraperitoneal or intrapleural administration).
  • Common parenteral injection types are intravenous (into a vein), intra-arterial (into an artery), intradermal (into the dermis), subcutaneous (under the skin), intramuscular (into muscle) and intratumoral (into a tumor or at its proximity). Infusions typically are given by intravenous route.
  • Topical administration can be performed using transdermal means (e.g. patch and the like).
  • Mucosal administrations include without limitation oral/alimentary, intranasal, intratracheal, intrapulmonary, intravaginal or intra-rectal route.
  • Preferred routes of administration for the oncolytic CPXV of the invention include intravenous and intratumoral routes.
  • Administrations may use conventional syringes and needles (e.g. Quadrafuse injection needles) or any compound or device available in the art capable of facilitating or improving delivery in the subject.
  • Transdermal systems are also appropriate, e.g. using solid, hollow, coated or dissolvable microneedles (see e.g., Van der Maaden et al., 2012, J. Control release 161: 645-55) and preferred are silicon and sucrose microneedle patches (see, e.g., Carrey et al., 2014, Sci Rep 4: 6154 doi 10.1038; and Carrey et al., 2011, PLoS ONE, 6(7) e22442).
  • the cowpox virus or composition is administered to the subject in combination with a pharmaceutically acceptable quantity of prodrug(s).
  • the cowpox virus and the prodrug can be administered concurrently (within the same time period), sequentially (e.g., the cowpox virus being administered first and the prodrug given second, or vice-versa), in an interspersed manner or in any combination of these types of administration. It is possible to administer a single dose of prodrug or doses which are repeated for a time which is sufficiently long to enable the toxic metabolite to be produced within the host organism or cell.
  • prodrug comprised between 50 and 500 mg/kg/day, preferably between 50 mg/kg/day and 200 mg/kg/day, and more preferably of 100 mg/kg/day.
  • the prodrug is administered in accordance with standard practice.
  • the oral or intravenous route is preferred.
  • a preferred embodiment relates to intravenous or intratumoral administration(s) of the cowpox virus (e.g.
  • a FCU-1 expressing and CPXV105 CDS-defective cowpox advantageously combined with oral or intravenous administration of the corresponding prodrug in a sequential schedule of administration with a specific preference for the prodrug therapy starting after the cowpox virus therapy, preferably at least 3 days, more preferably at least 4 days and even more preferably at least 7 days after the first administration of the virus.
  • the prodrug is advantageously an analogue of cytosine, in particular 5-FC or 5-FU.
  • a particularly preferred composition comprises 10 6 pfu to 5xl0 9 pfu of a CPXV105
  • CDS-defective cowpox e.g. a BR cowpox
  • CPXV105 CDS- defective cowpox encoding a suicide gene product such as FCU-1 formulated for intravenous or intratumoral administration, optionally in association with 5-FC or 5-FU prodrug.
  • the present invention provides a cowpox virus or a composition thereof for use as an oncolytic virus to treat or prevent a disease or a pathologic condition in a subject in need thereof.
  • the present invention also relates to a method of treatment comprising administering such a cowpox virus or composition thereof in a n amount sufficient for treating or preventing a disease or a pathologic condition in a subject in need thereof.
  • the cowpox virus is as described herein (e.g. wild type, modified derivative thereof such as a CPXV105 CDS-defective cowpox, or recombinant cowpox), or prepared according to the process described herein, or comprised in a composition as described herein.
  • the cowpox virus is a CPXV105 CDS-defective cowpox (e.g. CPXV_BR) and, notably, a recombinant CPXV105 CDS-defective cowpox encoding a suicide gene product such as FCU-1.
  • the cowpox virus is wild type.
  • a “disease” (and any form of disease such as “disorder” or “pathological condition”) is typically characterized by identifiable symptoms.
  • a preferred use consists in treating or preventing a proliferative disease and a disease associated to an increased osteoclast activity.
  • proliferative diseases that may be prevented or treated using the CPXV of the invention or composition thereof include cancers, tumors or restenosis; examples of diseases associated to an increased osteoclast activity that may be prevented or treated using the combination and methods of the invention include rheumatoid arthritis and osteoporosis.
  • the present invention is particularly suited for treating or preventing cancers and particularly Adrenocortical Carcinoma, Adrenal Cortex Cancer, Anal Cancer, Gastrointestinal Carcinoid Tumors (for example Appendix Cancer and Carcinoid Tumor), Bile Duct Cancer (for example Cholangiocarcinoma), Bladder Cancer, Bone Cancer (for example Ewing Sarcoma, Malignant Fibrous Histiocytoma of Bone and Osteosarcoma), Brain Tumors (for example Astrocytomas, Embryonal Tumors, Germ Cell Tumors, Central Nervous System Atypical Teratoid/Rhabdoid Tumor, Craniopharyngioma, Ependymoma, Gliomas and Glioblastoma), Breast Cancer (for example Ductal Carcinoma I n Situ), Bronchial Tumors, Carcinoma of Unknown Primary, Cardiac (Heart) Tumors, Cervical Cancer, Chordoma,
  • the cowpox virus or a composition according to the invention is used for treating glioblastoma, lung cancer, liver cancer, colorectal cancer, pancreatic cancer and cervical cancer, and it is preferably administered either intratumorally or intravenously.
  • a particularly preferred method comprises 1 to 6 intravenous or intratumoral administrations of the cowpox virus of the invention or composition thereof given at weekly to monthly intervals with a specific preference for 3 bi-weekly administrations of a composition comprising 10 6 -5xl0 9 pfu of a CPXV105 CDS-defective cowpox (e.g.
  • the beneficial effects provided by the methods of the present invention can be evidenced by an observable improvement of the clinical status over the baseline status or over the expected status if not treated according to the modalities described herein.
  • An improvement of the clinical status can be easily assessed by any relevant clinical measurement typically used by physicians and skilled healthcare staff.
  • the therapeutic benefit can be transient (for one or a couple of months after cessation of administration) or sustained (for several months or years).
  • the natural course of clinical status which may vary considerably from a subject to another, it is not required that the therapeutic benefit be observed in each subject treated but in a significant number of subjects (e.g.
  • a re particularly appropriate for treating cancer such methods ca n be correlated with one or more of the followings: inhibiting or slowing tumor growth, proliferation and metastasis, preventing or delaying tumor invasion (spread of tumor cells in neighbouring tissues), reducing the tumor number; reducing the tumor size, reducing the number or extent of metastases, providing a prolonged overall survival rate (OS), increasing progression free survival (PFS), increasing the length of remission, stabilizing (i.e. not worsening) the state of disease, providing a better response to the standard treatment, improving quality of life and/or inducing an anti-tumor response (e.g. non-specific (innate) and/or specific such as a cytotoxic T cell response) in the subject treated in accordance with the present invention.
  • an anti-tumor response e.g. non-specific (innate) and/or specific such as a cytotoxic T cell response
  • the present invention also relates to a method for treating a disease or a pathologic condition in a subject in need thereof comprising administering the cowpox virus described herein (wild type, or modified derivative CPXV, or recombinant), or prepared according to the process described herein, or comprised in the composition described herein, or a cowpox for use described herein. More precisely, the present invention related to a method for inhibiting tumor cell growth in vivo comprising administering a cowpox virus or a composition thereof in a subject in need thereof so-as to inhibit the growth of a tumor. For general guidance, inhibition of tumor cell growth can be evaluated routinely, for example by radiography means.
  • the administration(s) of the cowpox virus or a composition thereof desirably result(s) in at least a 10% decrease of the tumor mass.
  • the present invention also relates to a method of decreasing lytic activity of a cowpox virus or a composition thereof in a non-dividing cell (e.g. as compared to a method relying on the use of a vaccinia virus such as a Copenhagen VV).
  • the administration(s) of the cowpox virus or a composition thereof desirably result(s) in at least a 10% decrease of lytic activity in a primary cell (e.g. administration of the cowpox virus of the invention is at least 15%, at least 20%, et least 25%, at least 30%, at least 40%, or at least 50% less cytotoxic in primary cells than administration of a vaccinia virus under the same experimental conditions).
  • the cowpox virus of the present invention or the cowpox virus for use according to this invention does not replicate in a primary cell, meaning that the output to input ratio is 2 or less.
  • said primary cell is hepatocyte.
  • any of the methods of the present invention may be implemented with a suicide gene expressing cowpox virus or composition thereof in association with a pharmaceutically acceptable quantity of a prodrug corresponding to the encoded suicide gene product.
  • a preferred association is directed to a CDase-encoding cowpox virus or a UPRTase-encoding cowpox virus (or both CDase and UPRTase) and 5-FC or 5-FU.
  • the CPXV, composition thereof or method according to the invention can be combined with one or more substances which potentiate the cytotoxic effect of the compounds obtained after conversion of the prodrug (e.g. 5-FU or 5-FUMP).
  • substances which potentiate the cytotoxic effect of the compounds obtained after conversion of the prodrug e.g. 5-FU or 5-FUMP.
  • Mention may in particular be made of drugs which inhibit the enzymes of the pathway for the de novo biosynthesis of the pyrimidines (for example those mentioned below), drugs such as Leucovorin (Waxman et al., 1982, Eur. J. Cancer Clin.
  • the drugs which inhibit the enzymes of the pathway for the de novo biosynthesis of the pyrimidines are preferably selected from the group consisting of PALA (N-(phosphonoacetyl)-L-aspartate; Moore et al., 1982, Biochem. Pharmacol. 31, 3317-21), Leflunomide, A771726 (active metabolite of Leflunomide; Davis et al., 1996, Biochem. 35, 1270-1273) and Brequinar (Chen et al., 1992, Cancer Res. 52, 3251- 7).
  • PALA N-(phosphonoacetyl)-L-aspartate
  • Leflunomide A771726
  • Brequinar Choen et al., 1992, Cancer Res. 52, 3251- 7.
  • the cowpox virus of the present invention can be administered in association with any conventional therapeutic modalities which are available for treating or preventing the targeted disease or pathological condition.
  • conventional therapy include, without limitation, surgery, radiotherapy, chemotherapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy, cytokine therapy, transplantation (e.g. stem cell), hyperthermia, photodynamic therapy.
  • the CPXV, composition or method according to the invention can also be used in association with radiotherapy.
  • radiotherapy Those skilled in the art can readily formulate appropriate radiation therapy protocols and parameters (see for example Perez and Brady, 1992, Principles and Practice of Radiation Oncology, 2nd Ed. JB Lippincott Co; using appropriate adaptations and modifications as will be readily apparent to those skilled in the field).
  • the types of radiation that may be used in cancer treatment are well known in the art and include electron beams, high-energy photons from a linear accelerator or from radioactive sources such as cobalt or cesium, protons, and neutrons.
  • Radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. Regular X-rays doses for prolonged periods of time (3 to 6 weeks), or high single doses are contemplated by the present invention.
  • CPXV or composition thereof may be administered upon excision of the tumor (e.g. by local application within the excised zone for example).
  • the CPXV may be used in combination with one or more substances effective in anticancer therapy, like chemotherapeutic drugs or immunotherapeutic products.
  • the CPXV may be used in conjunction with chemotherapeutic drugs currently used for treating cancer.
  • any chemotherapy drug conventionally used in anti-cancer therapy may be used in combination with the cowpox virus of the present invention or the composition thereof, there may be mentioned more specifically alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, platinum derivatives, inhibitors of tyrosine kinase receptors, antimetabolites and antimitotic agents.
  • the CPXV may be used in conjunction with immunotherapy, and especially with anti-neoplastic antibodies as well as siRNA and antisense polynucleotides.
  • Representative examples include, among others, monoclonal antibodies blocking immune checkpoint (e.g.
  • the cowpox virus or composition thereof is administered in combination with an immunotherapeutic product, like for example a vaccine.
  • immunotherapeutic vaccines suitable for use in the present invention are plasmid DNA vector, or viral vectors such as vaccinia virus (e.g. Copenhagen, WR, Wyeth, MVA, etc.), adenovirus, lentivirus, herpes virus, recombinant polypeptides, among many others.
  • a preferred vaccine is different from the cowpox virus of the present invention, e.g. preferably an oncolytic virus based-vaccine, and more preferably a Vaccinia Virus based- vaccine (e.g. wild-type, attenuated e.g. by TK defectiveness and/or recombinant VACV).
  • the course of treatment may be routinely determined by a practitioner and various protocols are encompassed by the present invention. For example, 1 to 6 administrations of the CPXV (e.g. 3 bi-weekly injections) may be given during one cycle of chemotherapy.
  • CPXV e.g. 3 bi-weekly injections
  • Human colon cancer cell lines LoVo (ATCC ® CCL-229TM), HCT 116 (ATCC ® CCL-247TM), human lung cancer cell line A549 (ATCC ® CCL-185TM), hepatocarcinoma human cell line HepG2 (ATCC ® HB 8065TM), glioblastoma human cancer cell line U-87 MG (ATCC ® HTB-14), cervical human cancer cell line HeLa (ATCC ® CCL-2TM), pancreatic human cancer cell line MIA- Paca-2 (ATCC ® CRL-1420TM) and Vero cell line (ATCC ® CCL-81) were obtained from the American Type Culture Collection (ATCC, Rockville, MD).
  • Human esophagus cancer cell line OE-19 (ECACC n°96071721) was obtained from European Collection of Cell Culture (ECACC). All cell lines were grown in recommended media supplemented with 10% fetal calf serum (FCS). Fresh human hepatocytes were purchased from Kalycell (Plobsheim, France) and maintained in hepatocyte medium (Kalycell, France).
  • Viruses CPXV (CPXVwt) (ATCC ® VR-302TM) used in this study was obtained from ATCC.
  • Recombinant CPXV expressing the enhanced green fluorescent protein fused to FCUl were generated in Vero cells infected with CPXVwt at a MOI of 0.01. After being incubated at 37°C for 3 hours, the cells were then transfected with a shuttle plasmid containing the fusion gene GFPr.FCUl positioned under the control of the synthetic pllK7.5 promoter and surrounded by the flanking sequence of the TTC gene.
  • Virus was isolated from GFP-fluorescent plaques and submitted to additional plaque purification cycles in Vero cells. Virus structure was confirmed by multiple PCRs and DNA sequencing and the resulting virus is named CPXVtk-/gfp::fcul. CPXVw ⁇ and CPXVtk ⁇ /gfp::fcul were amplified in Hela cells and purified by sucrose gradient. Virus stocks were titrated on Vero cells by plaque assay. Western blotting
  • LoVo tumor cells were infected by CPXVwt and CPXVtk /gfp::fcul at an MOI of 0.1 and incubated for 24 h.
  • Cell lysate proteins (30 ⁇ g) (determined using a Bio-Rad protein assay) were run on a 10% SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions and transferred onto a nitrocellulose membrane.
  • the mem brane was incubated with mouse monoclonal antibody 3H 1 directed against FCUl (Foloppe et al., 2008, Gene Ther., 15 :1361-71), washed and incubated with secondary antibody coupled horseradish peroxidase (Amersham, Les Ulis, France). Blots were developed using enhanced chemiluminescence (Amersham).
  • Enzymatic assays were developed using enhanced chemiluminescence (Amersham).
  • CDase activity and UPRTase activity were determined in LoVo cells using 5-FC (Toronto Research Chemicals Inc., North York, Canada) and 5-FU (Sigma) as substrates.
  • Lovo human tumor cells (3 x 10 6 cells) were infected with each CPXV vector at a MOI of 0.001.
  • enzymatic assays were determined as previously described (Erbs et al., 2000, Cancer Res., 60(14):3813-22).
  • 5-FC, 5-FU and 5-FUM P were separated isocratically using HPLC (supelcosil LC-18-S column and UV detection at 260 nm and 280 nm).
  • the mobile phase was 50 mM phosphoric acid adjust to pH 2.1 with ammonium hydroxide.
  • the mobile phase was 20 mM KH 2 P0 4 , 5mM tetrabutylammoniumsulfate, 5% methanol adjusted to pH5 with potassium hydroxyde.
  • CDase activity was also measured indirectly by measuring 5-FU released in the culture media .
  • LoVo cells were infected with the different vectors at a MOI of 0.0001 and plated in 6-well culture dish (10 6 cells/well). After 48 h, 1 mM 5-FC was added to the cultures. Every day during 1 week, the concentrations of 5-FC a nd 5-FU in the media were measured using HPLC. Fifty ⁇ of media were quenched with 1 ml of ethyl acetate/2-propanol/0.5M acetic acid solution (84:15 :1). The samples were vortexed and centrifuged.
  • Human LoVo and A549 tumor cells were transduced in suspension at a MOI of 0.01 and 0.000001, respectively.
  • a total of 5 x 10 5 cells/well were plated in 6-well culture dishes in 2 ml of medium supplemented with 10 % FCS.
  • cells were exposed 5 to various concentrations of 5-FC for 3 days, before determination of cell viability by trypan blue exclusion using a Vi-Cell Cell Counter.
  • Human tumor cells were infected in suspension by CPXVwt and CPXVtk ⁇ /gfp::fcul at a MOI of 0.1, 0.01, 0.001 and 0.0001.
  • a total of 3 x 10 5 cells/well were plated in 6-well culture 10 dishes in 2 ml of medium supplemented with 10 % FCS. Cells were then cultured at 37°C for 5 days and the viable cells were counted by trypan blue exclusion using a Vi-Cell Cell Counter (Beckmann Coulter, California).
  • Human tumor cells were infected with CPX 'tk /gfp::fcul at MOI 0.0001 to 1. A total of 5 x 15 10 5 cells/well were plated in 6-well culture dishes in 2 ml of medium supplemented with 10 % FCS. At 16 hours post infection, cells were harvested, washed with PBS, and GFP signal was measured by flow cytometry using NaviosTM flow cytometer.
  • human hepatocarcinoma cells Hep G2 and human primary hepatocytes were infected in 6- well plates (1 x 10 6 cells/well) by CPXVwt and QPYSItk /gfpy.fcul at a MOI of 0.0001 (100 PFU/well). Cells were incubated in fresh growth medium supplemented with 10 % FCS until ha rvesting. At 72h post-infection, supernatant and cells were collected, freeze-thawed and sonicated and viral progeny were quantified on Vero cells by plaque assay. In vivo viral pathogenicity and biodistribution experiment
  • Viral pathogenicity was assessed by survival studies in immunocompetent BALB/c mice (female, 6 weeks old from Charles Rivers Laboratories). I ncreasing dose ranging from 1 x 10 4 PFU to 1 x 10 7 PFU of CPXVwt and CPXV 'tk/gfp::fcul were injected intravenously by tail vein injection. The animals were followed daily throughout the course of the experiment for sign of illness, examining weight loss, general appearance, lesion formation.
  • mice Female Swiss nude mice were obtained from Cha rles River Laboratories. Animals used in the studies were uniform in age (6 weeks) and body weight (20-23 g).
  • mice were randomized in a blinded manner and treated with the recombinant CPXV.
  • mice were perfused intracardially with an exsanguinating solution (0.9 % NaCI with heparin 50 Ul/ml) until all the blood was removed. Tumors and other organs were collected and weighted, homogenized in PBS, sonicated and titers were determined on Vero cells by plaque assay. Viral titers were standardized to milligram of tissue. In vivo antitumor activity of the recombinant CPXV
  • CPX 'tk ⁇ /gfp::fcul at 1 x 10 6 pfu (in 100 ⁇ PBS) was injected once intratumorally in established s.c U-87 MG or LoVo model.
  • a control group was injected in the same manner with PBS.
  • 5-FC was given by oral gavage for 3 weeks at 5 100 mg/kg (0.5 ml 5-FC 0.5% in water) twice a day.
  • Tumor size was measured twice a week using calipers.
  • Tumor volumes were calculated in mm 3 using the formula ( 7/6) (length x width 2 ).
  • CPXVtk-/gfp::fcul at 1 x 10 6 pfu (in 100 ⁇ PBS) was injected once intratumorally in0 nude mice bearing subcutaneous MIA-Paca-2 tumors.
  • a control group was injected in the same manner with PBS. In this experience, no 5-FC was administrated to the mice.
  • Tumor size was measured twice a week using calipers. Tumor volumes were calculated in mm 3 using the formula ( ⁇ /6) (length x width 2 ).
  • Virus engineering 0 The engineered virus is shown in Figure la.
  • the coding sequence of fusion green fluorescent protein-FCUl (GFP-FCUl) was introduced into the tk locus under transcriptional control of the synthetic vaccinia promoter pllk7.5, as described in the Materials and Methods.
  • the chimeric GFP::FCU1 gene was generated by directly fusing in frame the coding sequences of GFP and FCUl, followed by a precise deletion of the translation stop and start codons of GFP5 and FCUl, respectively.
  • GFP-FCUl fusion protein exhibits CDase and UPRTase activities similar to the FCUl protein and this chimeric protein displays a fluorescent signal intensity equivalent to the native GFP protein.
  • Virus structures were confirmed by PCR and sequencing.
  • a major strength of any prodrug activation model is the potential to extend the 20 cytotoxic therapeutic effect to untransfected cells.
  • an efficient bystander effect has been reported as 5-FU can reach neighboring cells by simple diffusion.
  • An analysis of supernatant by high performance liquid chromatography (HPLC) revealed a progressive amount of 5-FU in the extracellular medium of LoVo cells transduced with CPXVtk-/gfp::fcul at MOI 0.001 and incubated with O.lmM 5-FC ( Figure 3).
  • QPXMtk/gfpr.fcul reached a fold increase of, respectively, 100 000, 70 000, 62 000, 32 000 and 25 000, demonstrating the high capacity of CPX 'tk-/gfp fcul to kill tumor cell lines in vitro.
  • New experiments showed that the CPXVtk-/gfp::fcul virus could also infect and replicate in the U87MG cell line.
  • Oncolytic activity was evaluated by determining the percentage of viability of 8 cell lines upon QPX ⁇ ltk /gfp::fcul infection according to different MOI (from 0.1 to 0.0001). Cells were infected with the virus at different MOI and counted five days later. Trypan blue exclusion test demonstrates that CPXVtk ⁇ /gfp::fcul infection rapidly kills the majority of cells (Figure 4c). We observed an interesting and encouraging dose-response of the virus in all cell lines tested. Indeed, 75-80% of A549 (lung carcinoma) and U87MG (glioblastoma) cell lines were killed even at the lowest virus dose (MOI 0.0001).
  • CPXtk ⁇ /gfp::fcul and 5-FC were used to infect LoVo and A549 cancer cells at MOI of respectively 0.01 and 0.000001.
  • 5-FC was added to the cultures at a range of concentrations from 0,1 to ⁇ , and cell viability was determined 3 days later by trypan blue exclusion.
  • the oncolytic effect of CPXV in the absence of prodrug resulted in approximately 15% reduction in viable cell number.
  • CPXtk ⁇ /gfp::fcul in absence of prodrug showed no difference in cytotoxicity at these low MOI.
  • mice were infected i.v. with CPXwt or CPXtk ⁇ /gfp::fcul at a range of 1 x 10 4 to 1 x 10 7 pfu/mouse and formulation buffer was used as control (Figure 6). Weight loss and mortality were monitored for 83 days. All CPXtk ⁇ /gfp::fcul infected group mice were healthy and no clinical signs of illness were observed in mice. CPXwt at 1 xlO 7 pfu/dose killed 9/10 mice at day 1 and one died later ( Figure 6d).
  • Vaccinia virus strain Copenhagen and i/ -deleted vaccinia virus were also used in this study as control. As shown in Figure 7, Vaccinia viruses can strongly replicate in hepatocarcinoma but also in human primary hepatocytes although at a lesser extent. Conversely, neither CPXwt nor CPXtk ⁇ /gfp::fcul replicate in primary hepatocytes. Again, we demonstrate that CPXi/ " /gfp::fcul selectively replicate in tumoral cells.
  • a biodistribution assay was performed in U87-MG glioblastoma tumor-bearing immunodeficient mice.
  • a dose of 10 6 pfu CPXVtk ⁇ /gfp::fcul was injected by the i.v. route.
  • At 2 and 7 days p.i., 3 mice were euthanized by exsanguination and the organs were collected.
  • CPXVtk ⁇ /gfp::fcul virus titers were determined by a standard plaque assay on Vero cells.
  • CPXVtk ⁇ /qfp::fcul therapy reduces tumor in a glioblastoma and colorectal xenograft model of cancer
  • Tumor cells were implanted subcutaneously into the right flank of 7 weeks old Swiss Nude mice. 17 days later, when tumor size reached approximately 100mm 3 , one single dose of CPXwt or CPXtk ⁇ /gfp::fcul was injected into the tumor. Control groups were injected with buffer. After 5 days, to allow for the viruses to replicate, 5-FC treatment was started. Mice were treated twice a day with soluble 5-FC at a dose of 200mg/kg/day for 2 weeks. After 5 days of treatment, mice showed classical signs of illness due to 5-FU toxicity, mainly diarrhea and loss of weight. This discomfort was controlled by a recovery time of two days between two phases of 5-FC treatment.
  • CPXV infection resulted in an impressive inhibition of the U87- MG tumors growth of approximately 77%.
  • Treatment with 200 mg/kg/day 5-FC subsequently increases CPXV anti-tumor activity to more than 88% inhibition of tumor size progression.
  • PBMCs peripheral blood mononuclear cells
  • CPXtk-/gfp::fcul penetrate poorly into PBMCs, with less than 5% of cells infected at MOI 1.
  • no viral amplification was observed 4 days post infection.
  • the replication of the recombinant CPXV was totally abortive into these blood cells, showing that CPXtk-/gfp::fcul has a negligible impact on these cells.
  • CPXVtk ⁇ /qfp::fcul therapy reduces tumor in a pancreatic model of cancer
  • CPXtk-/gfp::fcul treatment resulted in the stabilization of the tumorgrowth with a reduction of more than 90% of tumoral mass as compared to the control group (p ⁇ 0.0005).
  • Pox lesions appeared 26 days after virus injection, with 3 to 10 pox/mouse allocated on tail, footpad, back and face.

Abstract

La présente invention concerne un virus de la variole bovine comprenant au moins un gène CPXV105 CDS défectif, une composition comprenant celui-ci et un procédé de préparation d'un tel virus de la variole bovine. La présente invention concerne également un virus de la variole bovine et une composition associée destinés à être utilisés en tant que virus oncolytique à des fins prophylactiques ou thérapeutiques, et plus particulièrement pour le traitement du cancer.
PCT/EP2017/079656 2016-11-18 2017-11-17 Vecteurs oncolytiques à base de la variole bovine WO2018091680A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP16306513.9 2016-11-18
EP16306513 2016-11-18
EP17306016 2017-07-28
EP17306016.1 2017-07-28

Publications (1)

Publication Number Publication Date
WO2018091680A1 true WO2018091680A1 (fr) 2018-05-24

Family

ID=60331636

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2017/079656 WO2018091680A1 (fr) 2016-11-18 2017-11-17 Vecteurs oncolytiques à base de la variole bovine

Country Status (1)

Country Link
WO (1) WO2018091680A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020136235A1 (fr) * 2018-12-28 2020-07-02 Transgene Sa Poxvirus déficient en m2
CN113832115A (zh) * 2021-08-25 2021-12-24 居颂光 融合基因ril-7联合ccl19重组溶瘤牛痘病毒及其在制备抗肿瘤药物中的应用
WO2022232375A1 (fr) * 2021-04-30 2022-11-03 Kalivir Immunotherapeutics, Inc. Virus oncolytiques d'expression de cmh modifiée

Citations (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5168062A (en) 1985-01-30 1992-12-01 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter-regulatory DNA sequence
WO1996016183A1 (fr) 1994-11-17 1996-05-30 Cayla Genes suicide et associations d'analogues de nucleobases et nucleosides pyrimidiques avec des genes suicide en vue d'une therapie genique
WO1998002522A1 (fr) 1996-07-16 1998-01-22 Transgene S.A. Procede de conservation de virus recombinants infectieux, suspension aqueuse virale et utilisation comme medicament
WO1998004727A1 (fr) 1996-07-25 1998-02-05 Therion Biologics Corporation Poxvirus recombinant pour l'immunisation contre des antigenes associes aux tumeurs
WO1998010088A1 (fr) 1996-09-06 1998-03-12 Trustees Of The University Of Pennsylvania Procede inductible de production de virus adeno-associes recombines au moyen de la polymerase t7
WO1998037095A2 (fr) 1997-02-24 1998-08-27 Therion Biologics Corporation Poxvirus recombine destine a une immunisation contre l'antigene associe aux tumeurs muc1
WO1998056415A1 (fr) 1997-06-11 1998-12-17 Aquila Biopharmaceuticals, Inc. Saponines purifiees servant d'adjuvants oraux
US6054438A (en) 1987-01-07 2000-04-25 Imperial Cancer Research Technology Limited Nucleic acid fragments encoding portions of the core protein of the human mammary epithelial mucin
EP0998568A1 (fr) 1998-04-17 2000-05-10 Transgene S.A. Mutant ayant une activite uracile phosphoribosyl transferase
WO2001066137A1 (fr) 2000-03-07 2001-09-13 Merck & Co., Inc. Formulations d'adenovirus
WO2003053463A2 (fr) 2001-12-10 2003-07-03 Bavarian Nordic A/S Preparations contenant un poxvirus et procede de preparation de compositions stables contenant un poxvirus
WO2004014314A2 (fr) 2002-08-12 2004-02-19 David Kirn Procedes et compositions concernant les poxvirus et le cancer
WO2005007857A1 (fr) 2003-07-21 2005-01-27 Transgene S.A. Polypeptide a activite cytosine-desaminase amelioree
WO2005042728A2 (fr) 2003-11-03 2005-05-12 Probiogen Ag Lignees de cellules aviaires immortalisees pour la production de virus
WO2006048768A2 (fr) 2004-11-08 2006-05-11 Transgene S.A. Kit de parties conçues pour la mise en oeuvre d'un traitement antitumoral ou antiviral d'un mammifere
WO2006108846A1 (fr) 2005-04-11 2006-10-19 Vivalis Procede de fabrication de vaccins viraux dans des lignees de cellules souches embryonnaires aviaires derivees en suspension
WO2007056847A1 (fr) 2005-11-21 2007-05-24 Sanofi Pasteur Limited Formulations de stabilisation pour virus recombinants
WO2007147528A1 (fr) 2006-06-20 2007-12-27 Transgene S.A. Procédé de production de poxvirus et compositions de poxvirus
WO2007147529A2 (fr) 2006-06-20 2007-12-27 Transgene S.A. Vaccin viral recombinant
WO2008114021A1 (fr) 2007-03-19 2008-09-25 Stabilitech Ltd. Procédé de conservation de particules virales
WO2008129058A1 (fr) 2007-04-24 2008-10-30 Vivalis Lignées de cellules souches dérivées de l'embryon de canard pour la production de vaccins viraux
WO2008138533A1 (fr) 2007-05-14 2008-11-20 Bavarian Nordic A/S Purification de vaccins à base de virus vaccinia et de virus vaccinia recombinés
WO2009065546A1 (fr) 2007-11-19 2009-05-28 Transgene Sa Vecteurs oncolytiques dérivés de poxvirus
WO2009100521A1 (fr) 2008-02-12 2009-08-20 Sanofi Pasteur Limited Procédés d’utilisation de chromatographie d'échange d'ions et de chromatographie d'exclusion diffusion pour la purification du poxvirus
WO2010130756A1 (fr) 2009-05-12 2010-11-18 Transgene Sa Lignées cellulaires aviaires immortalisées et applications associées
WO2011128704A1 (fr) 2010-04-16 2011-10-20 Isis Innovation Limited Système d'expression de poxvirus
WO2012001075A2 (fr) 2010-07-02 2012-01-05 Transgene Lignees cellulaire aviaires immortalisées
WO2013022764A1 (fr) 2011-08-05 2013-02-14 David Kirn Procédés et compositions de production du virus de la vaccine
US8394385B2 (en) 2009-03-13 2013-03-12 Bavarian Nordic A/S Optimized early-late promoter combined with repeated vaccination favors cytotoxic T cell response against recombinant antigen in MVA vaccines
WO2014063832A1 (fr) 2012-10-28 2014-05-01 Bavarian Nordig A/S Promoteur pr13.5 pour lymphocytes t robustes et réponses d'anticorps
US8772023B2 (en) 2008-11-27 2014-07-08 Bavarian Nordic A/S Promoters for recombinant viral expression
WO2015155263A1 (fr) * 2014-04-10 2015-10-15 Transgene S.A. Vecteurs oncolytiques poxviraux

Patent Citations (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5168062A (en) 1985-01-30 1992-12-01 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter-regulatory DNA sequence
US6054438A (en) 1987-01-07 2000-04-25 Imperial Cancer Research Technology Limited Nucleic acid fragments encoding portions of the core protein of the human mammary epithelial mucin
WO1996016183A1 (fr) 1994-11-17 1996-05-30 Cayla Genes suicide et associations d'analogues de nucleobases et nucleosides pyrimidiques avec des genes suicide en vue d'une therapie genique
WO1998002522A1 (fr) 1996-07-16 1998-01-22 Transgene S.A. Procede de conservation de virus recombinants infectieux, suspension aqueuse virale et utilisation comme medicament
WO1998004727A1 (fr) 1996-07-25 1998-02-05 Therion Biologics Corporation Poxvirus recombinant pour l'immunisation contre des antigenes associes aux tumeurs
WO1998010088A1 (fr) 1996-09-06 1998-03-12 Trustees Of The University Of Pennsylvania Procede inductible de production de virus adeno-associes recombines au moyen de la polymerase t7
WO1998037095A2 (fr) 1997-02-24 1998-08-27 Therion Biologics Corporation Poxvirus recombine destine a une immunisation contre l'antigene associe aux tumeurs muc1
WO1998056415A1 (fr) 1997-06-11 1998-12-17 Aquila Biopharmaceuticals, Inc. Saponines purifiees servant d'adjuvants oraux
EP0998568A1 (fr) 1998-04-17 2000-05-10 Transgene S.A. Mutant ayant une activite uracile phosphoribosyl transferase
WO2001066137A1 (fr) 2000-03-07 2001-09-13 Merck & Co., Inc. Formulations d'adenovirus
WO2003053463A2 (fr) 2001-12-10 2003-07-03 Bavarian Nordic A/S Preparations contenant un poxvirus et procede de preparation de compositions stables contenant un poxvirus
WO2004014314A2 (fr) 2002-08-12 2004-02-19 David Kirn Procedes et compositions concernant les poxvirus et le cancer
WO2005007857A1 (fr) 2003-07-21 2005-01-27 Transgene S.A. Polypeptide a activite cytosine-desaminase amelioree
WO2005042728A2 (fr) 2003-11-03 2005-05-12 Probiogen Ag Lignees de cellules aviaires immortalisees pour la production de virus
WO2006048768A2 (fr) 2004-11-08 2006-05-11 Transgene S.A. Kit de parties conçues pour la mise en oeuvre d'un traitement antitumoral ou antiviral d'un mammifere
WO2006108846A1 (fr) 2005-04-11 2006-10-19 Vivalis Procede de fabrication de vaccins viraux dans des lignees de cellules souches embryonnaires aviaires derivees en suspension
WO2007056847A1 (fr) 2005-11-21 2007-05-24 Sanofi Pasteur Limited Formulations de stabilisation pour virus recombinants
WO2007147528A1 (fr) 2006-06-20 2007-12-27 Transgene S.A. Procédé de production de poxvirus et compositions de poxvirus
WO2007147529A2 (fr) 2006-06-20 2007-12-27 Transgene S.A. Vaccin viral recombinant
WO2008114021A1 (fr) 2007-03-19 2008-09-25 Stabilitech Ltd. Procédé de conservation de particules virales
WO2008129058A1 (fr) 2007-04-24 2008-10-30 Vivalis Lignées de cellules souches dérivées de l'embryon de canard pour la production de vaccins viraux
WO2008138533A1 (fr) 2007-05-14 2008-11-20 Bavarian Nordic A/S Purification de vaccins à base de virus vaccinia et de virus vaccinia recombinés
WO2009065546A1 (fr) 2007-11-19 2009-05-28 Transgene Sa Vecteurs oncolytiques dérivés de poxvirus
WO2009100521A1 (fr) 2008-02-12 2009-08-20 Sanofi Pasteur Limited Procédés d’utilisation de chromatographie d'échange d'ions et de chromatographie d'exclusion diffusion pour la purification du poxvirus
US8772023B2 (en) 2008-11-27 2014-07-08 Bavarian Nordic A/S Promoters for recombinant viral expression
US8394385B2 (en) 2009-03-13 2013-03-12 Bavarian Nordic A/S Optimized early-late promoter combined with repeated vaccination favors cytotoxic T cell response against recombinant antigen in MVA vaccines
WO2010130753A1 (fr) 2009-05-12 2010-11-18 Transgene Sa Procédé de préparation et de purification d'orthopoxvirus
WO2010130756A1 (fr) 2009-05-12 2010-11-18 Transgene Sa Lignées cellulaires aviaires immortalisées et applications associées
WO2011128704A1 (fr) 2010-04-16 2011-10-20 Isis Innovation Limited Système d'expression de poxvirus
WO2012001075A2 (fr) 2010-07-02 2012-01-05 Transgene Lignees cellulaire aviaires immortalisées
WO2013022764A1 (fr) 2011-08-05 2013-02-14 David Kirn Procédés et compositions de production du virus de la vaccine
WO2014063832A1 (fr) 2012-10-28 2014-05-01 Bavarian Nordig A/S Promoteur pr13.5 pour lymphocytes t robustes et réponses d'anticorps
WO2015155263A1 (fr) * 2014-04-10 2015-10-15 Transgene S.A. Vecteurs oncolytiques poxviraux

Non-Patent Citations (73)

* Cited by examiner, † Cited by third party
Title
ADRA ET AL., GENE, vol. 60, 1987, pages 65 - 74
ANDERSEN ET AL., EUROPEAN J. BIOCHEM., vol. 204, 1992, pages 51 - 56
ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, 1981, pages 482 - 9
BULLER ET AL., NATURE, vol. 317, no. 6040, 1985, pages 813 - 5
BULLER ET AL., NATURE, vol. 317, no. 6040, 1985, pages 813 - 815
CADMAN ET AL., SCIENCE, vol. 250, 1979, pages 1135 - 1137
CADMAN ET AL., SCIENCE, vol. 250, 1979, pages 1135 - 7
CAROLL ET AL., VIROLOGY, vol. 238, 1997, pages 198 - 211
CARREY ET AL., PLOS ONE, vol. 6, no. 7, 2011, pages e22442
CARREY ET AL., SCI REP, vol. 4, 2014, pages 6154
CARROLL ET AL., PLOS ONE, vol. 6, 2011, pages e23086
CARROLL ET AL., PLOS ONE, vol. 6, no. 8, 2011, pages e23086
CHAKRABARTI ET AL., BIOTECHNIQUES, vol. 23, 1997, pages 1094 - 7
CHANTREY ET AL., EPIDEMIOL. INFECT., vol. 122, 1999, pages 455 - 460
CHANTREY ET AL., EPIDEMIOL. INFECT., vol. 122, 1999, pages 455 - 60
CHEN ET AL., CANCER RES., vol. 52, 1992, pages 3251 - 3257
CHEN ET AL., CANCER RES., vol. 52, 1992, pages 3251 - 7
CHERNAJOVSKY ET AL., BRITISH MED. J., vol. 332, 2006, pages 170 - 2
CHU ET AL., J. EXP. MED., vol. 186, 1997, pages 1623
CODY J J ET AL: "Expression of osteoprotegerin from a replicating adenovirus inhibits the progression of prostate cancer bone metastases in a murine model", LABORATORY INVESTIGATION, NATURE PUBLISHING GROUP, THE UNITED STATES AND CANADIAN ACADEMY OF PATHOLOGY, INC, vol. 93, no. 3, 1 March 2013 (2013-03-01), pages 268 - 278, XP002742297, ISSN: 0023-6837, [retrieved on 20130128], DOI: 10.1038/LABINVEST.2012.179 *
DAVIS ET AL., BIOCHEM., vol. 35, 1996, pages 1270 - 1273
DAYHOFFED, vol. 3, 1981, pages 482 - 9
DURAFFOUR ET AL., PLOS ONE, vol. 8, no. 2, 2013, pages 1 - 8
EDWARD JENNER: "The Three Original Publications on Vaccination Against Smallpox, by Edward Jenner : I. An Inquiry Into the Causes and Effects of the Variolae Vaccinae, Or Cow-Pox. 1798", THE HARVARD CLASSICS VOL. XXXVIII, PART 4, 1909-14, 25 April 2001 (2001-04-25), New York, XP055431236, Retrieved from the Internet <URL:http://www.bartleby.com/38/4/1.html> [retrieved on 20171201] *
EL OMARI ET AL., BMC STRUCT. BIOL., vol. 6, 2006, pages 22
ERBS ET AL., CANCER GENE THER, vol. 15, no. 1, 2008, pages 18 - 28
ERBS ET AL., CANCER GENE THER., vol. 15, no. 1, 2008, pages 18 - 28
ERBS ET AL., CANCER RES., vol. 60, no. 14, 2000, pages 3813 - 22
ESSBAUER ET AL., REVUE MED. VET., vol. 153, no. 10, 2002, pages 635 - 42
ESSBAUER ET AL., REVUE MED. VET., vol. 153, no. 10, 2002, pages 635 - 642
FALLAUX ET AL., HUMAN GENE THER., vol. 9, 1998, pages 1909 - 17
FEND LAETITIA ET AL: "Oncolytic virotherapy with an armed vaccinia virus in an orthotopic model of renal carcinoma is associated with modification of the tumor microenvironment", ONCOIMMUNOLOGY, LANDES BIOSCIENCE, US, vol. 5, no. 2, 1 January 2016 (2016-01-01), pages e1080414 - 1, XP008180456, ISSN: 2162-402X *
FISHER ET AL., CURR. OPIN. MOL. THER., vol. 8, no. 4, 2006, pages 301 - 13
FOLOPPE ET AL., GENE THER., vol. 15, 2008, pages 1361 - 1371
FOLOPPE ET AL., GENE THER., vol. 15, 2008, pages 1361 - 71
GRAHAM ET AL., J. GEN. VIROL., vol. 36, 1997, pages 59 - 72
GRENSON ET AL., J. BACTERIOL., vol. 103, no. 3, 1970, pages 770 - 7
GRENSON ET AL., J. BACTERIOL., vol. 103, no. 3, 1970, pages 770 - 777
HAMMOND ET AL., J. VIROL. METHODS, vol. 66, 1997, pages 135 - 8
HERMISTON ET AL., CURR. OPIN. MOL. THER., vol. 8, 2006, pages 322 - 30
JUND; LACROUTE, J. BACTERIOL., vol. 102, no. 3, 1970, pages 607 - 15
JUND; LACROUTE, J. BACTERIOL., vol. 102, no. 3, 1970, pages 607 - 615
KERN ET AL., GENE, vol. 88, 1990, pages 149 - 57
KIM ET AL., BIOCHEM. MOL. BIOL. INTERNAT., vol. 41, 1997, pages 1117 - 24
KIM ET AL., MOL. THER., vol. 14, 2006, pages 361 - 70
KRITSCH ET AL., J. CHROMATOGR. ANAL. TECHNOL. BIOMED. LIFE SCI., vol. 822, 2005, pages 263 - 70
KUMAR; BOYLE, VIROLOGY, vol. 179, 1990, pages 151 - 8
MARINE RICORDEL ET AL: "Cowpox Virus: A New and Armed Oncolytic Poxvirus", MOLECULAR THERAPY - ONCOLYTICS, vol. 7, 24 August 2017 (2017-08-24), pages 1 - 11, XP055430885, ISSN: 2372-7705, DOI: 10.1016/j.omto.2017.08.003 *
MARTINUSSEN ET AL., J. BACTERIOL., vol. 176, 1994, pages 6457 - 63
MARTINUSSEN ET AL., J. BACTERIOL., vol. 177, 1995, pages 271 - 4
MCCART ET AL., CANCER RES., vol. 61, 2001, pages 8751 - 57
MCCART; 2001 ET AL., CANCER RES., vol. 61, pages 8751 - 57
MCCOLLUM ET AL., JID, 2012, pages 206
MCCOLLUM ET AL., JOURNAL OF INFECTIOUS DISEASE, 2012, pages 206
MOORE ET AL., BIOCHEM. PHARMACOL., vol. 31, 1982, pages 3317 - 21
MOORE ET AL., BIOCHEM. PHARMACOL., vol. 31, 1982, pages 3317 - 3321
NEEDLEMAN; WUNSCH, J.MOL. BIOL., vol. 48, 1970, pages 443 - 453
PEREZ; BRADY: "Principles and Practice of Radiation Oncology", 1992, JB LIPPINCOTT CO
REMINGTON; A. GENNARO: "The Science and Practice of Pharmacy", LIPPINCOTT, WILLIAMS&WILKINS
RIBI ET AL., PLENUM PUBL. CORP., 1986, pages 407 - 419
ROTH SWAANTJE J ET AL: "Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC)", VETERINARY RESEARCH, BIOMED CENTRAL LTD, LONDON, UK, vol. 42, no. 1, 11 January 2011 (2011-01-11), pages 3, XP021085706, ISSN: 1297-9716, DOI: 10.1186/1297-9716-42-3 *
SMORLESI, GENE THER, vol. 12, 2005, pages 1324
SMORLESI, GENE THER., vol. 12, 2005, pages 1324
SUADER, J. AM ACAD DERMATOL., vol. 43, 2000, pages S6
SUMINO ET AL., J.VIROL., vol. 72, 1998, pages 4931
TRITEL ET AL., J. IMMUNOL., vol. 171, 2003, pages 2358
VAN DER MAADEN ET AL., J. CONTROL RELEASE, vol. 161, 2012, pages 645 - 55
VERARDI ET AL., HUM. VACCIN. IMMUNOTHER., vol. 8, 2012, pages 961 - 70
VERARDI ET AL., HUM. VACCIN. IMMUNOTHER., vol. 8, 2012, pages 961 - 970
W XU ET AL: "The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer", GENE THERAPY, vol. 22, no. 3, 1 March 2015 (2015-03-01), GB, pages 31 - 40, XP055431016, ISSN: 0969-7128, DOI: 10.1038/gt.2014.110 *
WAXMAN ET AL., EUR. J. CANCER CLIN. ONCOL., vol. 18, 1982, pages 685 - 692
WAXMAN ET AL., EUR. J. CANCER CLIN. ONCOL., vol. 18, 1982, pages 685 - 92
Z. XU ET AL: "Generation of a Complete Single-Gene Knockout Bacterial Artificial Chromosome Library of Cowpox Virus and Identification of Its Essential Genes", JOURNAL OF VIROLOGY., vol. 88, no. 1, 1 January 2014 (2014-01-01), US, pages 490 - 502, XP055431033, ISSN: 0022-538X, DOI: 10.1128/JVI.02385-13 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020136235A1 (fr) * 2018-12-28 2020-07-02 Transgene Sa Poxvirus déficient en m2
CN113453712A (zh) * 2018-12-28 2021-09-28 特兰斯吉恩股份有限公司 M2缺陷型痘病毒
WO2022232375A1 (fr) * 2021-04-30 2022-11-03 Kalivir Immunotherapeutics, Inc. Virus oncolytiques d'expression de cmh modifiée
US11963990B2 (en) 2021-04-30 2024-04-23 Kalivir Immunotherapeutics, Inc. Oncolytic viruses for modified MHC expression
CN113832115A (zh) * 2021-08-25 2021-12-24 居颂光 融合基因ril-7联合ccl19重组溶瘤牛痘病毒及其在制备抗肿瘤药物中的应用

Similar Documents

Publication Publication Date Title
AU2017387549B2 (en) Oncolytic viruses and therapeutic molecules
US20240024437A1 (en) Immunotherapeutic vaccine and antibody combination therapy
JP5710262B2 (ja) ポックスウイルス腫瘍細胞崩壊性ベクター
JP5710261B2 (ja) ポックスウイルス腫瘍細胞崩壊性ベクター
Fu et al. Recent advances in oncolytic virus-based cancer therapy
US20200000862A1 (en) Oncolytic virus therapy
WO2018091680A1 (fr) Vecteurs oncolytiques à base de la variole bovine
JP2024050588A (ja) パラポックスウイルスベクター
WO2019020543A1 (fr) Virus oncolytiques exprimant des agents ciblant des modulateurs immunitaires métaboliques
US20190134190A1 (en) Combination therapy with cpg tlr9 ligand
WO2020011754A1 (fr) Virus de la vaccine chimériques
EP3129037B1 (fr) Vecteurs oncolytiques poxviraux
WO2016131945A1 (fr) Produit de combinaison modulateur de l&#39;autophagie
US20220033445A1 (en) Method for producing an antitumoral arenavirus as well as arenavirus mutants
WO2024038175A1 (fr) Poxvirus chimériques
TW202413636A (zh) 嵌合痘病毒

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17798246

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17798246

Country of ref document: EP

Kind code of ref document: A1