WO2009095167A1 - Attenuated oncolytic paramyxoviruses encoding avian cytokines - Google Patents

Attenuated oncolytic paramyxoviruses encoding avian cytokines Download PDF

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Publication number
WO2009095167A1
WO2009095167A1 PCT/EP2009/000308 EP2009000308W WO2009095167A1 WO 2009095167 A1 WO2009095167 A1 WO 2009095167A1 EP 2009000308 W EP2009000308 W EP 2009000308W WO 2009095167 A1 WO2009095167 A1 WO 2009095167A1
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virus
recombinant
ndv
transgene
oncolytic
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PCT/EP2009/000308
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English (en)
French (fr)
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Rudolf Beier
Florian PÜHLER
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Bayer Schering Pharma Aktiengesellschaft
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Priority to MX2010008370A priority Critical patent/MX2010008370A/es
Priority to EP09704993A priority patent/EP2237790A1/en
Priority to EA201001202A priority patent/EA201001202A1/ru
Priority to CA2711571A priority patent/CA2711571A1/en
Priority to JP2010544616A priority patent/JP2011510638A/ja
Priority to AU2009210301A priority patent/AU2009210301A1/en
Priority to BRPI0906354A priority patent/BRPI0906354A2/pt
Priority to CN2009801035310A priority patent/CN102099045A/zh
Publication of WO2009095167A1 publication Critical patent/WO2009095167A1/en
Priority to IL206382A priority patent/IL206382A0/en
Priority to ZA2010/06151A priority patent/ZA201006151B/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/565—IFN-beta
    • 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
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18132—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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18141—Use of virus, viral particle or viral elements as a vector
    • C12N2760/18143—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
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18161—Methods of inactivation or attenuation

Definitions

  • the present invention refers to a recombinant oncolytic RNA Newcastle Disease Virus comprising at least one transgene coding for an avian cytokine, wherein the recombinant oncolytic RNA Newcastle Disease Virus is obtainable from a velogenic or mesogenic oncolytic RNA Newcastle Disease Virus.
  • Virus-mediated expression of the cytokine in the natural host cells leads to a reduced pathogenicity of the virus for avian species.
  • the virus in the present invention is suitable for the treatment of diseases, especially for oncolytic tumor treatment.
  • Recombinant viruses are produced that encode an avian cytokine, wherein the pathogenicity of the virus is reduced for an avian species leading to a diminished environmental toxicity of the virus.
  • the oncolytic activity of the virus is not impaired by the described method of pathogenicity reduction.
  • the virus genome may encode additional therapeutic transgenes, preferably binding proteins (antibodies, ankyrin repeat molecules, peptides etc.), prodrug-converting enzymes or/and proteases.
  • binding proteins antibodies, ankyrin repeat molecules, peptides etc.
  • prodrug-converting enzymes or/and proteases The activity of these binding proteins, prodrug-converting enzymes and/or proteases increases the anti-tumor effect of the virus.
  • the invention describes manufacture and the use of such modified viruses for treatment of cancer.
  • Newcastle Disease Virus has been used as an experimental therapeutic agent for more than 40 years and is reviewed by Sinkovics and Horvath (2000). The Newcastle Disease Virus in general is described in the book by Alexander (1988).
  • NDV strain PV701 is being developed as an anticancer treatment for glioblastoma (Lorence et al., 2003).
  • the NDV strain MTH68 has been used as an experimental cancer treatment and has been administered to humans for more than 30 years (Csatary et al., 2004).
  • VSV Vesicular Stomatitis Virus
  • Paramyxoviruses contain single-stranded RNA genomes of negative polarity having genomes of 15-19 kb in length (wild-type) and the genomes contain 6-10 genes.
  • the viral envelope is formed by the surface glycoproteins and a membrane part derived from the host cell.
  • the surface glycoproteins F and HN or H or G) mediate entry and exit of the virus from the host cell.
  • the nucleocapsid is inside the envelope and contains the RNA genome and the nucleocapsid protein (NP), phospho- (P) and large (L) proteins responsible for intercellular virus transcription and replication.
  • the matrix (M) protein connects the viral envelope and the nucleocapsid.
  • Paramyxoviridae may contain "accessory" genes which may be additional transcriptional units interspersed with the genes mentioned above.
  • the accessory genes are mostly ORFs that overlap with the P gene transcriptional unit. A comprehensive description of paramyxoviridae can be found in (Lamb, 2001 ).
  • NDV is the prototypic member of the genus Avulavirus in the family Paramyxoviridae belonging to the order Mononegavirales.
  • the viral genome is a single-stranded negative-sense RNA coding for six major proteins: the nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin protein (HN) 1 and the polymerase protein (L).
  • NP nucleocapsid protein
  • P phosphoprotein
  • M matrix protein
  • F fusion protein
  • HN hemagglutinin protein
  • L polymerase protein
  • NDV is in detail characterized in Alexander (1988) and Lamb (2001 ).
  • Newcastle Disease virus as an avian pathogen
  • NDV strains are classified on their pathogenicity for chicken as velogenic strains (highly virulent) leading to acute lethal infection of chicken of all ages, mesogenic isolates (intermediate virulence) that are only lethal in young chicks, and lentogenic strains (nonvirulent) manifested in a mild or unapparent form of the disease.
  • Classification of NDV isolates in velogen, mesogen or lentogen is determined by the mean death time (MDT) of the chicken embryo in 9 day-old embryonated eggs after inocculation with the minimum lethal dose to kill the embryo.
  • MDT mean death time
  • NDV is primarily transmitted by aerosols or large droplets that are inhaled by susceptible birds. During the course of infection new infectious virus particles will be shed from the infected respiratory tract or excreted in the feces. By ingestion of this virus-containing material by healthy birds, new infections can be established and virus-spreading from one bird to another can be maintained.
  • NDV as a threat for poultry farming and the need for vaccination has been already recognised at the beginning of the last century. Iyer and Dobson have performed studies in the 1930s on the attenuation of virulent NDV strains, leading to the development of some mesogenic NDV vaccine strains.
  • NDV Newcastle disease virus
  • US 2004/0043035 relates to a recombinant NDV mutant that is not able to express an immunodominant epitope of the nucleoprotein (NP) and is suited as a marker vaccine strain.
  • NP nucleoprotein
  • US 2003/0224017 describes a reverse genetic system for NDV for the production of a recombinant NDV vaccine.
  • This system allows the expresssion of transgenes, e.g. avian cytokines (chicken IL-2, chicken IL-4) from the NDV genome to generate NDV vaccine strains.
  • transgenes e.g. avian cytokines (chicken IL-2, chicken IL-4) from the NDV genome to generate NDV vaccine strains.
  • EP 1 300 157 relates to an attenuated mutant Newcastle disease virus strain suitable for in ovo vaccination of avian species comprising a mutation in the gene sequences encoding the HN and/or F glycoproteins.
  • US 6,719,979 relates to a process for generating infectious Newcastle disease virus (NDV) entirely from cloned full-length cDNA and to the use of vaccines and diagnostic assays generated with and derived from the process.
  • NDV Newcastle disease virus
  • WO 2007/025431 describes a method for producing a recombinant attenuated Newcastle Disease La Sota strain and its use in the preparation of vaccine for the prevention of the diseases caused by Newcastle Disease virus (NDV).
  • NDV Newcastle Disease virus
  • WO 2000/067786 concerns cDNAs for making attenuated, infectious Newcastle disease virus (NDV).
  • NDV Newcastle disease virus
  • vaccines comprising attenuated, infectious NDV.
  • EP-A-O 702 085 relates to genetically manipulated infectious replicating non- segmented negative-stranded RNA virus mutants, comprising an insertion and/or deletion in an open reading frame, a pseudogen region or an intergenic region of the virus genome.
  • WO 99/66045 relates to genetically modified NDV viruses obtained from full- length cDNA molecules of the virus genome.
  • WO 00/62735 relates to a method of tumor treatment comprising administering an interferon-sensitive, replication-competent clonal RNA virus, e.g. NDV.
  • an interferon-sensitive, replication-competent clonal RNA virus e.g. NDV.
  • WO 01/20989 (PCT/USOO/26116) a method for treating patients having tumor with recombinant oncolytic paramyxoviruses is described.
  • the tumor is reduced by administering a replication-competent Paramyxoviridae virus.
  • Various methods are described that can be used to engineer the virus genome in order to improve the oncolytic properties.
  • WO 03/005964 relates to recombinant VSV comprising a nucleic acid encoding a cytokine.
  • US 2004/0170607 relates to the treatment of melanoma by administering a virus which is not a common human pathogen.
  • NDV can be genetically manipulated using the reverse genetics technology as described e.g. in EP-A-O 702 085.
  • NDV constructs comprising additional nucleic acids coding for secreted alkaline phosphatase (Zhao and Peeters, 2003), green fluorescent protein (Engel-Herbert et al., 2003), VP2 protein of infectious bursal disease virus (Huang et al., 2004), influenza virus hemagglutinin (Nakaya et al., 2001 ) and chloramphenicol acetyl transferase (Huang et al., 2001 ) (Krishnamurthy et al., 2000).
  • RNA viruses as virotherapy agents are reviewed in Russell (2002). The content of any of these documents is herein incorporated by reference.
  • Oncolytic NDV strains have been studied since the early 1960s as tumor therapeutics. Most virus strains used for oncolytic tumor therapy are belonging to the class of mesogenic viruses, that are described as pathogens for poultry. To develop an oncolytic NDV as an antitumoral biological drug, potential existing environmental toxicity, especially the pathogenicity for poultry should be reduced. However NDVs attenuated for poultry must have the continuing ability to lyse tumor cells and keep its oncolytic potential.
  • Existing strategies for the development of vaccines can not be applied, because they are focussing on the stimulation of the bird immune systeme either by application of completely inactivated virus particles or apathogenic lentogenic virus strains. Both vaccine types are no more able to replicate in cancer cells and subsequently have lost the potential to lyse tumor cells.
  • the attenuation of NDV for poultry without diminishing the oncolytic activity of the virus can be reached with the help of the reverse genetic technology.
  • Recombinant attenuated NDVs are created by the insertion of transgenes coding for cytokines, in particular antiviral or immune-stimulating cytokines.
  • NDV vaccine is not suitable for such an approach because the objective of a vaccine is to induce a strong immune response with a long lasting immunity to protect the animal against a secondary infection.
  • the described attenuation of NDV has the main goal to allow an animal to control the (undesired) primary infection and thus to decrease environmental toxicity and to increase safety of the virus when applied in a therapy of a proliferative disease.
  • a subject of the present invention is the lysis of tumor cells by an oncolytic virus.
  • An indication for such an oncolytic virus approach is cancer therapy in humans.
  • a NDV vaccine for poultry as disclosed in US 2003/0224017 is applied to animals and therefore located in the field of animal health care.
  • an object of the present invention is a recombinant oncolytic RNA Newcastle Disease Virus comprising at least one transgene coding for an avian cytokine, wherein the recombinant oncolytic RNA Newcastle Disease Virus is obtainable from a velogenic or mesogenic oncolytic RNA Newcastle Disease Virus.
  • the virus of the present invention may comprise at least one further transgene having therapeutic activity when expressed by a virus-infected tumor cell.
  • the at least one further transgene is partially allogene or syngene for the host.
  • the virus of the present invention may be an attenuated virus.
  • Attenuation includes reduction of the pathogenicity of the virus for an avian species and reduction of pathogenicity in a biological assay system that is predictive for avian species (see Example 5).
  • a further aspect of the present invention is a recombinant oncolytic RNA virus attenuated for poultry comprising a nucleic acid comprising at least one transgene coding for a cytokine.
  • the virus is attenuated for chicken.
  • the "transgene coding for a cytokine” is in particular the "a transgene coding for an avian cytokine” as described herein.
  • the invention relates to the nucleocapsid of the recombinant virus of the present invention comprising viral RNA complexed with capsid proteins.
  • the invention relates to an RNA which is RNA of the virus of the present invention.
  • the invention also relates to an RNA complementary to the RNA of the virus of the present invention.
  • the invention relates to a DNA, e.g. a cDNA encoding the RNA of the present invention and/or a DNA complementary to the RNA of the present invention. Furthermore, the invention relates to the prevention or treatment of tumor diseases, cancer or/and proliferative diseases.
  • RNA or/and the DNA of the present invention may be provided in an isolated form.
  • the cytokine encoded by the transgene as described herein may be secreted from a bird cell infected by the virus of the present invention and, after binding to its appropriate interferon-receptor on the neighbouring cell, may induce an antiviral state in the receptor-carrying cell. Therefore replication of the virus of the present invention in the interferon stimulated bird cell can be inhibited at least partially.
  • the similarity between chicken type I interferons and mammalian type I interferons is very low ( ⁇ 25% identity at amino acid level) (Staeheli et al. 2001 ).
  • a cytokine exhibiting such antiviral effect in a bird cell does not show essentially biological activity in a human cell and may have essentially no influence on virus replication in human tumor cells. Furthermore, essentially no adverse effects by these cytokines are expected in the human organism.
  • the expression of the transgene encoding a cytokine as described herein leads to a reduced pathogenicity of the virus for an avian species, especially poultry, especially chicken and thereby to a diminished environmental toxicity.
  • the activity of the transgene encoding a cytokine as described herein has essentially no detrimental effect on the therapeutic effect of the virus.
  • the invention generally relates to RNA viruses, preferably negative strand RNA viruses, more preferably such viruses that have both oncolytic properties and can be genetically engineered.
  • viruses are: - paramyxoviruses, preferably Newcastle Disease Virus (NDV), measles virus, mumps virus, Sendai virus;
  • orthomyxoviruses preferably influenza virus
  • - rhabdoviruses preferably vesicular stomatitis virus.
  • the virus of the present invention is an avian pathogen, in particular a pathogen of poultry, more particular a pathogen of chicken.
  • the virus of the present invention is a negative strand RNA virus.
  • the recombinant RNA virus of the present invention may be a paramyxovirus, preferably a Newcastle Disease Virus (NDV).
  • NDV Newcastle Disease Virus
  • the NDV may be a mesogenic or velogenic strain. It is preferred that the NDV is a mesogenic NDV.
  • the virus of the present inventon is obtainable from a velogenic or mesogenic oncolytic RNA Newcastle Disease Virus, in particular from strain MTH68.
  • the virus of the present invention is preferably replication competent.
  • the virus of the present invention may have a pathogenicity reduced for an avian species, in particular with respect to the virus from which the recombinant virus is obtainable. It is preferred that in the virus of the present invention, the pathogenicity reduction is a capability of the virus to reduce bird cell lysis about 48h after infection with MOI 0,01 measured by increasing cell viability, whereby cell viability is increased to at least about 25% (such as at least about 25% up to about 50%) surviving cells, more preferably to at least about 50% (such as at least about 50 % up to about 75%) surviving cells and most preferably to at least about 75% (such as at least about 75% up to 100%) surviving cells with respect to the virus from which the recombinant virus is obtainable.
  • the pathogenicity reduction is a capability of the virus to reduce bird cell lysis about 48h after infection with MOI 0,01 measured by increasing cell viability, whereby cell viability is increased to at least about 25% (such as at least about 25% up to about 50%) surviving cells, more preferably to at
  • the pathogenicity reduction is a survival time prolongation of virus infected chicken embryos in 11 day old embryonated eggs measured by mean death time (MDT) determination, whereby the MDT is prolonged by at least about 15h (such as at least 15 h up to about 2Oh), more preferably at least about 2Oh (such as at least 20 h up to about 3Oh and most preferably by more than 3Oh compared to the virus from which the recombinant virus is obtainable.
  • MDT mean death time
  • the oncolytic activity of the virus of the present invention for human tumor cells is essentially not reduced.
  • the oncolytic activity of the virus for human tumor cells measured by cell viability after 48h after infection is not reduced to more than 50% compared to the virus from which the recombinant virus is obtainable and more preferably is essentially not reduced with respect to the virus from which the recombinant virus is obtainable.
  • the cytokine encoded by the at least one transgene as indicated herein may be any cytokine.
  • the cytokine may be a cytokine capable of inhibiting at least partially virus replication in a bird cell, in particular in poultry cell, more particular in chicken cell. It is preferred that virus replication is essentially completely inhibited.
  • the cytokine may be a cytokine having essentially no biological activity in a mammal, in particular in a human being.
  • biological activity refers in particular to the ability of a cytokine to inhibit at least partially virus replication, in particular replication of a virus of the present invention.
  • Biological activity also refers to any other activity a cytokine may exhibit in a non-mammalian cell or/and in a non-mammal.
  • the cytokine may be a cytokine capable of inhibiting at least partially virus replication in a bird, in particular in poultry, more particular in chicken, and may have essentially no biological activity in a mammal, in particular in a human being.
  • the cytokine may be selected from avian cytokines, in particular from poultry cytokines, more particular from chicken cytokines.
  • the cytokine may be selected from interferons, in particular from avian interferons, more particular from chicken interferons, more particular from chicken type I interferons.
  • the interferon may be interferon-beta or a member of the interferon-alpha family.
  • the recombinant oncolytic RNA virus is an NDV comprising a nucleic acid comprising a transgene encoding a chicken interferon-alpha or/and chicken interferon-beta.
  • a transgene is defined in the context of the NDV genome as additional nucleic acids that are introduced in the viral genome.
  • the nucleic acids can be selected from different genomic sources (e.g. NDV genome, different virus class, prokaryotic or eukaryotic sources, mammalian or non- mammalian species). Also fused transgenes from two or more different genomic sources are possible. Also synthetic transgenes based on de novo synthesis of nucleic acid sequences can be constructed.
  • the nucleic acids must be located within at least one transcriptional cassette.
  • the transgene is translated into a protein in the infected cell. For this reason the transgenic sequence should include a translational start and stop-codon.
  • the recombinant oncolytic virus of the present invention may comprise a nucleic acid encoding at least one further transgene independently selected from transgenes coding for binding proteins, prodrug-converting enzymes, and proteases.
  • the at least one further transgene codes preferably for a binding protein that has a therapeutic activity when expressed by the virus-infected tumor cell.
  • the at least one further transgene codes for a prodrug-converting enzyme that has a therapeutic activity when expressed by the virus-infected tumor cell.
  • the at least one further transgene codes for a protease that has a therapeutic activity when expressed by the virus-infected tumor cell.
  • transgene or “at least one transgene” as used herein refers to a transgene encoding an avian cytokine, or the at least one further transgene having therapeutic activity when expressed by a virus- infected tumor cell, or a combination thereof. This definition of “transgene” applies in particular in the position and number of transgenes within the virus genome, as described herein, if not indicated otherwise.
  • RNA virus, the genome, antigenome, nucleocapsid and/or DNA molecule of the present invention may comprise the transgene as described herein which may be located within the transcriptional cassette as described herein.
  • the recombinant oncolytic virus of the present invention may comprise at least two, at least three, at least four, or at least five nucleic acids each comprising a transgene as described herein.
  • the recombinant oncolytic virus of the present invention may comprise at the maximum five nucleic acids each comprising a transgene as described herein.
  • recombinant oncolytic virus of the present invention comprises one, two, three, four, or five nucleic acids each comprising a transgene as described herein. If the recombinant virus of the present invention comprises at least two transgenes, they may be identical or different.
  • the nucleic acid comprising the at least one transgene may be located at any position between the reading frames of the viral genes.
  • the nucleic acid comprising the at least one transgene may be located 5' of the N gene, between the N and the P gene, between the P and the M gene, between the M and the F gene, between the F and the HN gene, between the HN and the L gene, or/and 3' of the L gene. It is preferred that the nucleic acid is located in a more 5 1 position, such as 5 1 of the N gene, between the N and the P gene, or/and between the P and the M gene, as such location leads to an improved expression compared with a more 3' location.
  • the virus of the present invention exhibits a tumor-selective infection that leads to a tumor-selective expression of the transgene as described herein.
  • the recombinant RNA virus of the present invention may comprise in total up to five transgenes, up to four transgenes, or up to three transgenes.
  • the transgene encoding an avian cytokine, or the at least one further transgene having therapeutic activity when expressed by a virus-infected tumor cell, or a combination thereof is preferably heterologous to the oncolytic RNA virus on which the recombinant RNA virus of the present invention is based.
  • heterologous refers to the complete gene or a part thereof, which may be the coding region of the gene or a part thereof.
  • the heterologous nucleic acid may be an artificial nucleic acid or may be obtained from natural sources or by recombination of at least two nucleic acids selected from nucleic acids obtained from natural sources and/or artificial nucleic acids.
  • Natural sources include animals such as mammals, plants, fungi, and microorganisms such as bacteria, protozoa and viruses, which may be different from oncolytic RNA viruses of the present invention.
  • the transgene may also encode for a fusion protein.
  • the transgene encoding an avian cytokine and the at least one further transgene having therapeutic activity when expressed by a virus-infected tumor cell may be located on at least two separated transcription units.
  • At least one transcription unit comprising the nucleic acid comprising the at least one transgene encoding an avian cytokine may also be transcribed in a tumor cell as described herein.
  • At least one transcription unit comprising the nucleic acid comprising the at least one second transgene having therapeutic activity when expressed by a virus-infected tumor may also be transcribed in a bird cell as described herein.
  • At least two separated transcription units each may be transcribed in a tumor cell as described herein and in a bird cell as described herein.
  • At least one transgene encoding an avian cytokine and the at least one further transgene having therapeutic activity when expressed by a virus-infected tumor cell are translated in a tumor cell as described herein and in a bird cell as described herein.
  • the at least one further transgene having therapeutic activity when expressed by a virus- infected tumor cell may code for a binding protein.
  • a pharmaceutical composition comprising a recombinant oncolytic virus of the present invention, a virus genome of the present invention, a virus antigenome of the present invention, and/or a DNA molecule of the present invention as an active ingredient optionally together with pharmaceutically acceptable carriers, diluents and/or adjuvants, which virus, virus genome, antigenome and/or DNA molecule comprises at least one one further transgene having therapeutic activity when expressed by a virus-infected tumor cell encoding for a binding protein.
  • Binding proteins are proteins, which, when expressed in a target cell, are capable of binding to a component of said cell and/or a neighbouring cell.
  • binding proteins are proteins which bind to intracellular components.
  • a binding protein is selected from the following group consisting of a natural ligand, a genetically modified ligand, a recombinant soluble domain of a natural receptor and a modified version thereof, a peptide ligand, a polypeptide ligand, an antibody molecule and fragments and derivatives thereof, and an antibody-like molecule like an ankyrin-repeat protein and fragments and derivatives thereof.
  • binding proteins as described herein might be of human, murine or closely related origin or a chimeric version, i.e. a protein which may be a fusion protein comprising sequences from different species, e.g. human and mouse.
  • the recombinant binding molecules based on the description above can be monomeric, dimeric, trimeric, tetrameric or multimeric.
  • the recombinant binding molecules based on the description above can be monospecific, bispecific or multispecific.
  • the preferred binding proteins are selected from binding proteins having a therapeutic activity.
  • a natural ligand as described herein can be a growth factor or a peptide.
  • a genetically modified ligand may be an analogue of a naturally occurring growth factor or peptide.
  • Recombinant soluble domains of a natural receptor or modified versions of it as described herein are recombinantly expressed soluble extracellular domains of a cell-surface receptor and/or fragments of it, a recombinantly expressed soluble extracellular domain of a cell adhesion molecule and/or fragments thereof.
  • Antibody molecules as mentioned above may be monoclonal immunoglobulin antibodies of any known specificity and isotype, fragments thereof and/or fragments thereof fused to effector proteins.
  • the antibody molecules may be chimeric, humanized or human antibodies.
  • Antibody fragments contain at least one antigen-binding domain of an antibody. Antibody fragments have been described extensively in the literature (reviewed eg. in Allen (2002), herein incorporated by reference).
  • Preferred examples are single-chain Fv fragments, Fab fragments, F(ab2'), domain- deleted versions called minibodies, and other immunoactive portions, fragments, segments and other smaller or larger partial antibody structures wherein the latter possess sufficient targeting properties or immunological stimulatory or inhibitory activity so as to be therapeutically useful within the methods of the present invention.
  • Such antibodies may be derived from hybridoma cloning experiments by use of transgenic mice or from phage display selections, ribosome display selections, or colony filter screening of antibody libraries containing human antibody sequences or related methodologies.
  • Binding proteins with antibody like properties as described herein may be genetically modified proteins or domains of it in which one or more peptide loops are randomized on the level of amino acids in such a way that high affinity binding molecules with high specificity can be enriched against any antigen from libraries of such molecules by phage display, ribosome display, colony filter screen or related methodologies.
  • the selected proteins usually have high thermal and thermodynamic stability and are well expressed in recombinant expression systems such as E. coli, yeast, insect and mammalian expression system. Examples for such binding proteins with antibody like properties are ankyrin repeat proteins as described in Binz et al.
  • Antibody-like molecules can be monomeric or repetitive molecules either constructed as single-chain molecules or as multichain molecules wherein the antibody-like molecule possesses sufficient targeting properties or immunological stimulatory or inhibitory activity so as to be therapeutically useful within the methods of the present invention.
  • Another subject of the present invention is a method for treatment of a proliferative disease, in particular a hyperproliferative disease, such as a tumor or cancer, comprising administering in a pharmaceutically effective amount to a subject in need thereof a recombinant oncolytic virus of the present invention, a virus genome of the present invention, a virus antigenome of the present invention, and/or a DNA molecule of the present invention comprising at least one further transgene having therapeutic activity when expressed by a virus-infected tumor cell encoding for a binding protein as described herein.
  • a proliferative disease in particular a hyperproliferative disease, such as a tumor or cancer
  • the binding protein may be a fusion protein comprising at least one binding domain, e.g. from an antibody, and at least one heterologous domain.
  • “Heterologous” has the meaning as discussed above in the context of heterologous genes.
  • the binding proteins described above are able to deliver a payload to a disease specific site (e.g. a tumor) as a so called intrabody or as extracellular available binding protein.
  • the delivered payload can be a heterologous domain, e.g. a toxin such as human RNAse (De Lorenzo et al., 2004) (Zewe et al., 1997) Pseudomonas exotoxin (Chaudhary et al., 1989) (Kreitman and
  • beta-glucosidase (Rowlinson-Busza, 1992), carboxypeptidase, (Antoniw et al., 1990), (Bagshawe et al., 1988), beta- lactamase with therapeutic efficacy, or an immune-stimulatory protein with cytokine activity such as IL-2, IL-12, TNF-alpha, IFN-beta or GM-CSF (see e. g. review by Allen (2002).
  • cytokine activity such as IL-2, IL-12, TNF-alpha, IFN-beta or GM-CSF
  • binding proteins described above have themselves antagonistic or agonistic efficacy which is therapeutically useful.
  • antagonistic/blocking binding molecules are the VEGF inhibitory antibody Avastin (Ferrara et al., 2004), the HER2/neu receptor blocking antibody Herceptin (Noonberg and Benz, 2000) or the EGF-receptor blocking antibody Erbitux (Herbst and Langer, 2002).
  • Agonistic binding proteins can be binding proteins which induce for example apoptosis (Georgakis et al., 2005) or have regulatory activity on DNA, RNA or proteins (e.g. induce transcription, stabilize proteins). The review by (Adams and Weiner, 2005) describes various therapeutic antibodies that could also be incorporated into an oncolytic virus
  • the at least one further transgene having therapeutic activity when expressed by a virus- infected tumor cell may code for a prodrug-converting enzyme.
  • a prodrug is a derivative or a precursor of a therapeutically active compound, which can be enzymatically converted into the active compound.
  • Prodrug- converting enzymes are enzymes capable of converting a prodrug into the therapeutically active drug.
  • a pharmaceutical composition comprising a recombinant oncolytic virus of the present invention, a virus genome of the present invention, a virus antigenome of the present invention, and/or a DNA molecule of the present invention as an active ingredient optionally together with pharmaceutically acceptable carriers, diluents and/or adjuvants, which virus, virus genome, antigenome and/or DNA molecule comprises at least one one further transgene having therapeutic activity when expressed by a virus-infected tumor cell encoding for a prodrug-converting enzyme.
  • the pharmaceutical composition may further comprise a prodrug which can be converted into a therapeutically active compound by the prodrug- converting enzyme encoded by the virus, virus genome, antigenome and/or DNA molecule.
  • the pharmaceutical composition may be suitable for treatment and/or alleviation of a proliferative disorder.
  • the prodrug may be formulated in a single composition with the recombinant oncolytic virus of the present invention, a virus genome of the present invention, a virus antigenome of the present invention, and/or a DNA molecule of the present invention as an active ingredient, or may be formulated in a composition distinct from the oncolytic virus formulation.
  • the oncolytic virus of the present invention encodes for a prodrug-converting enzyme
  • the oncolytic virus of the present invention causes selective expression of the prodrug-converting enzyme in a virus- infected target cell (in particular a tumor cell) which is usually not or not sufficiently expressing the prodrug converting enzyme.
  • a virus- infected target cell in particular a tumor cell
  • the prodrug is specifically converted into the pharmaceutical active compound in a target cell, in particular in a tumor cell, but may essentially not be converted into the therapeutically active compound in a non-target cell, in particular in a healthy cell of the subject to be treated.
  • undesired side-effect of the therapeutically active compound are reduced compared with treatment of the therapeutically active compound alone.
  • the prodrug may be a derivative or a precursor of a therapeutically active compound suitable for treatment and/or alleviation of a proliferative disorder, tumor or/and cancer, which prodrug can be converted by a prodrug converting enzyme.
  • the prodrug may be a compound known by a person skilled in the art. Derivatives and/or precursors are known by a person skilled in the art.
  • the prodrug is essentially pharmaceutically inactive and/or nontoxic.
  • prodrug-converting enzymes of the present invention are beta- glucuronidase, beta-galactosidase, beta-glucosidase, carboxypeptidase, beta-lactamase, D-amino acic oxidase. Further examples are known by a person skilled in the art. It is preferred that the prodrug-converting enzyme is essentially not expressed in non-tumor cells.
  • the prodrug-converting enzyme may be obtained from an organism selected from mammals, plants, fungi, and microorganisms such as bacteria, protozoa and viruses.
  • a most preferred combination of the prodrug-converting enzyme and a prodrug is E. coli beta-glucuronidase and a prodrug which can be converted by beta-glucuronidase into an active cytotoxic compound.
  • E. coli beta-glucuronidase and a prodrug which can be converted by beta-glucuronidase into an active cytotoxic compound.
  • An example is HMR1826 (doxorubicin-glucuronide) which can be converted into doxorubicin which is a known compound for treatment of cancer.
  • Another subject of the present invention is a method for treatment of a proliferative disease, in particular a hyperproliferative disease, such as a tumor or cancer, comprising administering in a pharmaceutically effective amount to a subject in need thereof a recombinant oncolytic virus of the present invention, a virus genome of the present invention, a virus antigenome of the present invention, and/or a DNA molecule of the present invention comprising at least one further transgene encoding for a prodrug converting enzyme as described herein.
  • a proliferative disease in particular a hyperproliferative disease, such as a tumor or cancer
  • subject of the present invention is a method for treatment of a proliferative disease, in particular a hyperproliferative disease, such a cancer, comprising administering in a pharmaceutically effective amount to a subject in need thereof (i) a recombinant oncolytic virus of the present invention, a virus genome of the present invention, a virus antigenome of the present invention, and/or a DNA molecule of the present invention comprising at least one transgene encoding for a prodrug-converting enzyme, and (ii) a prodrug suitable for treatment of the proliferative disease, which prodrug can be converted into a pharmaceutically active compound by the prodrug-converting enzyme of (i).
  • the method may comprise the administration of a single pharmaceutical composition comprising both components (i) and (ii), or may comprise the administration of two distinct pharmaceutical compositions, one of which comprises component (i) and the other comprises (ii).
  • At least one further transgene having therapeutic activity when expressed by a virus- infected tumor cell may code for a protease.
  • a pharmaceutical composition comprising a recombinant oncolytic virus of the present invention, a virus genome of the present invention, a virus antigenome of the present invention, and/or a DNA molecule of the present invention as an active ingredient optionally together with pharmaceutically acceptable carriers, diluents and/or adjuvants, which virus, virus genome, antigenome and/or DNA molecule comprises at least one further transgene having therapeutic activity when expressed by a virus-infected tumor cell encoding for a protease.
  • the pharmaceutical composition may be suitable for treatment and/or alleviation of a proliferative disorder.
  • the oncolytic virus of the present invention may cause selective expression of the protease in a virus-infected target cell (in particular a tumor cell) which is usually not or not sufficiently expressing the protease.
  • a virus-infected target cell in particular a tumor cell
  • the protease may irreversibly cleave a target polypeptide in a target cell, thereby inhibiting proliferation and/or growth of the target cell or killing the target cell, but may essentially not cleave the target molecule in a non-target cell, in particular in a healthy cell of the subject to be treated.
  • the protease is a sequence-specific protease. More preferred is a protease specifically cleaving a target polypeptide.
  • the protease may either be of natural origin and may be derived from any species or it may be engineered. Amino acid sequences suitable for a specific cleavage of a predetermined target polypeptide can be determined by a person skilled in the art, e.g. on the basis of publicly available sequence databases. US 2005-0175581 and US 2004-0072276 describe the generation of protein-engineered proteases with a predetermined substrate specifity. These two documents are herein included by reference.
  • the target molecule of the protease may be any target molecule as described below for targets of binding proteins.
  • Another subject of the present invention is a method for treatment of a proliferative disease, in particular a hyperproliferative disease, such as a tumor or cancer, comprising administering in a pharmaceutically effective amount to a subject in need thereof a recombinant oncolytic virus of the present invention, a virus genome of the present invention, a virus antigenome of the present invention, and/or a DNA molecule of the present invention comprising at least one further transgene having therapeutic activity when expressed by a virus-infected tumor cell encoding for a protease.
  • a proliferative disease in particular a hyperproliferative disease, such as a tumor or cancer
  • the transgene of the present invention may encode a fusion protein of a prodrug-converting enzyme as defined above, a binding molecule as defined above and/or a protease as defined above. Especially preferred is a fusion protein of a prodrug-converting enzyme and a binding molecule or a fusion protein of a protease and a binding molecule.
  • the present invention relates to a pharmaceutical composition which comprises as an active ingredient a virus as described herein, a nucleocapsid of the virus, a genome of the virus or a DNA molecule encoding the genome or/and an antigenome of the virus, optionally together with pharmaceutically acceptable carriers, diluents and/or adjuvants.
  • the pharmaceutical composition may be provided as a solution, suspension, a lyophilisate or in any other suitable form.
  • the composition may comprise carriers, buffers, surfactants and/or adjuvants as known in the art.
  • the composition may be administered e.g. orally, topically, nasally, pulmonally or by injection locally or intravenously.
  • the pharmaceutical composition is administered in a pharmaceutically effective amount depending on the type of disorder, the patient's condition and weight, the route of administration etc.
  • 10 9 to 10 12 virus particles, 10 8 to 10 11 , 10 7 to 10 10 , or 10 6 to 10 9 virus particles are administered per application.
  • the oncolytic therapy may be optionally combined with other tumor therapies such as surgery, radiation and/or chemotherapy such as cyclophosphamide treatment and/or hyperthermia treatment.
  • Yet another aspect is a method for treatment of a proliferative disease or/and cancer, comprising administering in a pharmaceutically effective amount to a subject in need thereof a recombinant oncolytic virus comprises as described herein, a nucleocapsid of the virus, a genome of the virus or a DNA molecule encoding the genome or/and an antigenome of the virus, optionally together with pharmaceutically acceptable carriers, diluents and/or adjuvants.
  • a recombinant oncolytic paramyxovirus can express a soluble binding protein, a prodrug-converting enzyme and/or a protease that may remain either in the infected cell or may be secreted, such as an antibody, an antibody fragment, an ankyrin repeat protein or another binding molecule as specified below.
  • the strain MTH68 was chosen in the present application because it has an inherent oncolytic property with promising data from experimental clinical treatments of patients (Sinkovics and Horvath, 2000). In principle, however, most NDV strains with multibasic fusion protein cleavage sites may be used as oncolytic agents for the treatment of tumors. The reverse genetics technology is applicable to all strains.
  • Binding proteins as described herein have been demonstrated to be of high therapeutic potential.
  • the combination of oncolytic NDV with therapeutic binding proteins, prodrug- converting enzymes and/or proteases of the above described properties will have additional or even synergistic efficacy of two therapeutical principles.
  • the oncolytic self-replicating virus targets the binding protein drug, the prodrug-converting enzyme and/or the protease to the preferred site of action where it is expressed in situ in high local concentrations.
  • Such protein expression is expected to be very selective and the binding protein, the prodrug-converting enzyme and/or the protease with its respective mode of action will add to the intrinsic therapeutic oncolytic activity of the NDV.
  • the amount of expressed transgene [binding protein, the prodrug-converting enzyme and/or protease] is expected to be roughly proportional to the mass of the tumor.
  • Antibody molecules or antibody like molecules or derivatives thereof are ideal binding proteins to be used with the NDV-system. Antibody molecules have been the subject of intensive research and technologies are now available to generate antibody molecules which are non-immunogenic, very selective and of high affinity. The local expression of antibody molecules at high concentrations lead to very significant agonistic or antagonistic efficacy or efficient targeting of effector molecules with reduced toxicity profile compared to standard therapy.
  • antibody-like molecules in the NDV system is expected to be even superior. These molecules are designed for selective high affinity binding with very high thermal stability and yield compared to normal antibodies.
  • the repetitive nature of the molecule can be finetuned according to the respective target for optimized targeting, binding, inhibition or activation.
  • different binding specificities can be combined within one ankyrin molecule, exploiting the possibility of joining in one ankyrin-repeat molecule several units with different binding specificities.
  • This modular structure allows the multivalent binding of greater protein surfaces than it is possible for antibodies, which can be extremely important in blocking protein-protein interactions.
  • the modular structure can also be exploited to block several effectors with only one single blocking ankyrin-repeat-protein.
  • ankyrin-repeat-molecules are extremely stable even under reducing condition these molecules can be designed to target proteins inside the cell ("Intrabody").
  • Possible targets for binding molecules or/and proteases can be all structures of a target cell or of the extracellular matrix surrounding the target cell which can be recognized by the described binding proteins or/and proteases and which are relevant to a certain type of pathological phenotype. These can be structural proteins, enzymes, growth factors, growth factor receptors, integrins, transcription factors etc.
  • binding proteins could interfere with are the ras, Wnt and Hedgehog pathway, where for example protein protein interactions can be blocked.
  • binding proteins intervening beneficially in the above described pathways in cancer cells are:
  • Ras effectors eg. GEFs
  • hypoxia induced proteins e.g. HIF1 ⁇
  • HIF1 ⁇ hypoxia induced proteins
  • Myc/Max DNA-binding or/and cofactor binding
  • VEGF-R vascular endothelial growth factor receptor
  • protease of the present invention, the prodrug-converting enzyme and/or the therapeutically active compounds derived from prodrugs of the present invention by the prodrug-converting enzyme may also beneficially intervene in the above described pathways of cancer cells.
  • Recombinant virus means a virus that has an engineered defined alteration in its genomic RNA sequence. This alteration may be one or more insertions, deletions, point mutations or combinations thereof.
  • a recombinant RNA virus of the present invention may comprise the full genomic sequence of a natural (unmodified) RNA virus or a sequence derived thereof and may additionally comprise at least one recombinant transcriptional cassette.
  • the at least one transcriptional cassette may be located in between two genes (transcriptional units) of the viral genome. In this case, the at least one transcriptional cassette is flanked by transcriptional start and stop sequences.
  • the at least one transcriptional cassette may also be located within a transcriptional unit of the viral genome. In this case, no additional transcriptional start and stop sequences are required.
  • the at least one transcriptional cassette may comprise restriction sites, such as Pad or/and Ascl, which may be unique. If two transcriptional cassettes are present, they may comprise different restriction sites.
  • RNA virus of the present invention comprises one or two recombinant transcriptional cassettes.
  • transgene located which may encode for a binding protein, a prodrug- converting enzyme and/or a protease as described herein.
  • any intergenic region between each of two genes (transcriptional units) of the viral genome is suitable for introducing the at least one recombinant transcriptional cassette. If more than one recombinant transcriptional cassette is present, they may be located in the same or different intergenic regions. It is preferred that at least one recombinant transcriptional cassette is located between the viral F and HN genes, in particular if the RNA virus of the present invention is a recombinant Newcastle Disease Virus.
  • transgene transgene encoding an avian cytokine, or the at least one further transgene having therapeutic activity when expressed by a virus-infected tumor cell, or a combination thereof, as described herein
  • the transgene independently have a size of up to about 10 kb, more preferred up to about 5 kb, most preferred up to about 2 kb.
  • expression control sequences such as transcriptional start and stop sequences and sequences controlling the translation are used.
  • the expression control sequences of an RNA virus may be used which may be the RNA virus on which the recombinant RNA virus of the present invention is based.
  • transcriptional start and stop sequences may be obtained from an RNA virus.
  • Expression control sequences may also be obtained from a target cell, in particular sequences controlling the translation and/or protein transport.
  • RNA virus a further subject of the present invention is a nucleocapsid of a recombinant oncolytic RNA virus of the present invention.
  • the nucleocapsid comprises the RNA molecule encoding the genome or/and the antigenome of the RNA virus and the nucleocapsid protein.
  • the nucleocapsid may also comprise the polymerase protein L or/and the phosphoprotein P.
  • Also subject of the present invention is the anti-genome of the genome of the present invention as described herein.
  • a further aspect of the present invention is a DNA molecule encoding the genome or/and the anti-genome of a recombinant oncolytic RNA virus of the present invention.
  • the DNA molecule may be a plasmid.
  • the DNA molecule of the present invention can be used for genetically engineering the RNA virus of the present invention. Further, the DNA molecule may be used for producing the RNA virus of the present invention. Therefore, the DNA molecule may be operatively linked to a transcriptional control sequence e.g. a prokaryotic or eukaryotic transcription control sequence.
  • Another aspect of the present invention is a method for producing a recombinant oncolytic RNA virus expressed from a DNA molecule encoding the genome and/or the anti-genome of a recombinant oncolytic virus of the present invention, in particular the recombinant oncolytic NDV of the present invention.
  • a further aspect of the present invention is a cell comprising the recombinant oncolytic virus of the present invention, a virus genome of the present invention, a virus anti-genome of the present invention and/or a DNA molecule of the present invention.
  • the cell may be a prokaryotic cell or a eukaryotic cell.
  • the cell may be a cell line, in particular a mammalian cell line, more particularly a human or murine cell line.
  • the cell may be used in the method of the present invention for producing the RNA virus of the present invention.
  • Suitable systems for transcribing a DNA molecule are known by a person skilled in the art, e.g. in prokaryotic systems such as E. coli or eukaryotic systems such as HeLa or CHO.
  • RNA virus a genome or anti-genome thereof or a DNA molecule comprising the full set of genes of Paramyxoviridae or a set of genes of Paramyxoviridae in which at least one gene or intergenic region is genetically modified, and further comprising at least one recombinant transcriptional cassette as described herein.
  • a virus, genome or antigenome or DNA molecule may be used for the manufacture of a medicament and/or treatment of cancer.
  • RNA virus, genome, anti-genome or DNA molecule is suitable for constructing a recombinant Paramyxoviridae virus, in particular a recombinant Newcastle Disease Virus by genetic engineering techniques in order to introduce a recombinant sequence into the transcription cassette.
  • the at least one transcription cassette may comprise a restriction site. If more than one transcription cassettes are present, the unique restriction sites of the transcriptional cassettes may be different.
  • An example is plasmid pflMTH68_Asc_Pac of Fig. 1 of WO 2006/050984, the disclosure of which is included herein by reference.
  • Another example is pflMTH68 murine IgG EDB as disclosed in Fig. 2 of WO 2006/050984, the disclosure of which is included herein by reference. Therapeutical relevance
  • Treatment of cancer or/and of a tumor includes inhibition of tumor growth, preferably the killing of the tumor cells or the blocking of proliferation in a time gap by infection.
  • NDV replicates selectively in tumor cells.
  • the virus of the present invention can be used to treat proliferative disorders, in particular hyperproliferative disorders.
  • neoplasms can be treated with the described virus, preferably cancers from the group consisting of lung, colon, prostate, breast and brain cancer can be treated.
  • a solid tumor can be treated.
  • tumors with low proliferation rate are prostate cancer or breast cancer.
  • a brain tumor can be treated.
  • glioblastoma More preferably a glioblastoma can be treated.
  • Mammals include human beings, mice, and rats.
  • RNA virus of the present invention in particular the recombinant NDV, can be constructed as described in Romer-Oberdorfer et al. (1999).
  • the construction of the new nucleic acid sequences is on the level of the cDNA which then is translated into RNA within a eucaryotic cell using the following starting plasmids: pCITE P, pCITE N, pCITE L, pX8 ⁇ T flNDV.
  • NDV can be any strain of Newcastle Disease Virus, more preferred a strain that is oncolytic in its wildtype form.
  • the plasmid pX ⁇ T is described in EP 0 702 085 (Conzelmann KK).
  • the recombinant RNA virus of the present invention in particular the recombinant NDV, can be recovered initially from T7 polymerase expressing cells, eg. BHK T7 cells or transiently with T7 polymerase transfected CHO cells. It can be amplified in cells like 293, CEC32, HT29 or A431. It can also be amplified in the allantoic fluid of embryonated chicken eggs.
  • T7 polymerase expressing cells eg. BHK T7 cells or transiently with T7 polymerase transfected CHO cells. It can be amplified in cells like 293, CEC32, HT29 or A431. It can also be amplified in the allantoic fluid of embryonated chicken eggs.
  • the recombinant RNA virus in particular the recombinant NDV, is stored under the following conditions.
  • the recombinant RNA-virus, in particular NDV is stable in 5% D-mannitol / 1 % (w/v) L-lysine / pH 8,0 or standard cell culture medium. At -20 0 C for up to one month. At -80 0 C for up to 10 years.
  • the recombinant RNA virus of the present invention may be manufactured using a wild type-virus or a recombinant virus as starting material.
  • a nucleic acid such as DNA, including such wild-type or recombinant sequence, may be used.
  • the recombinant RNA virus or/and DNA molecule as described in WO 2006/050984 may be employed as starting material.
  • An example is plasmid pflMTH68_Asc_Pac of Fig. 1 of WO 2006/050984.
  • Another example is pflMTH68 murine IgG EDB of Fig. 2 of WO 2006/050984.
  • the diclosure of WO 2006/050984 is included herein by reference.
  • the disclosure of WO 2006/050984 concering recombinant oncolytic RNA viruses and the construction thereof is included herein by reference.
  • the recombinant RNA virus of the present invention in particular the purified recombinant NDV according to the invention can be used as a medicament, because it shows pharmacological effects.
  • the recombinant RNA virus, in particular the NDV of the invention, the virus genome, antigenome, nucleocapsid and/or DNA molecule of the present invention can be used for the manufacture of a medicament especially for prevention, alleviation or/and treatment of cancer, a tumor or/and a proliferative disease, especially for prevention, alleviation or/and treatment of cancer, such as lung cancer, prostate cancer, brain cancer, colon cancer, breast cancer.
  • the pharmaceutical composition of the present invention optionally comprises pharmaceutically acceptable carrier and diluents.
  • pharmaceutically acceptable carrier and diluents are described in Remington's Pharmaceutical Science, 15* n ed. Mack Publishing Company, Easton Pennsylvania (1980).
  • the virus titers used in the pharmaceutical composition or/and applied in the method of treatment of the present invention may be in the range of 10 9 to 10 12 pfu per dose, in a range of 10 8 to 10 11 pfu, in a range of 10 7 to 10 10 pfu or in a range of 10 6 to 10 9 pfu dependent on the indication of treatment.
  • the pharmaceutical composition of the present invention may be used for the prevention or/and treatment of a proliferative disorder, such as cancer.
  • the pharmaceutical composition of the present invention may comprise an emulsion of the recombinant oncolytic RNA virus of the present invention, in particular the NDV of the invention and may be administered by inhalation, intravenous infusion, subcutaneous injection, intraperitoneal injection or intratumoral injection.
  • a pharmaceutically effective amount is a titre of the oncolytic RNA virus of the present invention, in particular the NDV of the present invention, the virus genome of the present invention, or the DNA molecule of the present invention which prevents, alleviates or/and suppresses the disease.
  • the acceptable dosis may depend for example on the construct, the patient, the ways of administration and the type of cancer.
  • the subject is a mammal, more preferably a human patient.
  • Figure 1 describes plasmid pflMTH68 ChlFN-alpha comprising the full genome of NDV and one transcription cassette comprising the chicken interferon-alpha (ChlFN-alpha) transgene.
  • Figure 2 describes plasmid pflMTH68 ChlFN-beta comprising the full genome of NDV and one transcription cassette comprising the chicken interferon-beta (ChlFN-beta) transgene.
  • Figure 3a demonstrates that the avian CEC-32 cell line is partial protected from lysis 48h after infection with NDV-ChlFN-alpha and strong protected after infection with NDV-ChlFN-beta. Infection with a GFP expressing NDV completely destroys the CEC-32 monolayer. In contrast no difference in the lytic effect between the three viruses NDV-GFP, NDV-ChlFN-alpha and NDV-ChlFN-beta is seen after infection of the tumorigenic HeIa cell line, lndependend of the used virus the HeIa cell monolayer is 48h after infection completely destroyed.
  • Figure 3b shows a quantification of the cell survival of CEC-32 and HeIa cells 48h after infection with the viruses NDV-GFP, NDV-ChlFN-alpha and NDV-ChlFN-beta.
  • NDV-GFP viruses that kill CEC-32 cells
  • NDV-ChlFN-alpha keeps 26% of the infected cells alive.
  • the best protection is observed after infection with the NDV-ChIFN- beta virus, 96% of the CEC-32 cells are viable.
  • the viability of the tumorigenic HeIa cells is reduced under 10% independend of the used recombinant NDV.
  • Figure 4 shows that a large therapeutic window exists after the infection of tumor and fibroblast cells with NDV-ChlFN-alpha and NDV-ChlFN-beta.
  • the proliferation inhibition of the oncolytic viruses NDV-ChlFN-alpha and NDV- ChlFN-beta is very strong on tumor cells and in contrast nearly no growth inhibition is observed after infection of primary fibroblast cells, especially at low MOIs like 0.1 and MOI 0.01.
  • Figure 5a depicts the survival curves of NDV infected embryonated chicken eggs with the four viruses NDV-GFP, NDV-ChlFN-alpha, NDV-ChlFN-beta and the apathogenic strain LaSota.
  • Chick embryos infected with NDV- ChlFN-alpha or NDV-ChlFN-beta are surviving longer than the NDV-GFP infected embryos.
  • the curves are shifted clearly in the direction of the survival curve of the lentogenic NDV LaSota strain.
  • Figure 5b From the results of the survival curves a MDT (Mean Death Time) is calculated for each infection group. The MDT is increased for the viruses NDV-ChlFN-alpha and ChlFN-beta compared with NDV-GFP. The highest MDT was observed with the apathogenic strain LaSota.
  • MDT Mal Death Time
  • the oncolytic strain MTH68 of NDV was used to obtain viral RNA.
  • RT-PCR several fragments of cDNA were obtained and in a multi-step cloning procedure they were assembled into a full-genome cDNA that was cloned into the vector pX8 ⁇ T (Schnell et al., 1994) yielding the plasmid pflMTH68.
  • This vector can be used for transfection in order to rescue recombinant virus from a T7-polymerase expressing cell line.
  • ChlFN-alpha Two DNA transgenes coding for ChlFN-alpha (NM_205427) and ChlFN-beta ( N M_001024836) were amplified by PCR.
  • ChlFN-alpha fw 5 1 - ccttaattaagccaccatggctgtgcctgcaagccc-3 1 SEQ.
  • ChlFN-alpha- rev 5'-ccttaattaactaagtgcgcgtgttgcctgtg-3' SEQ. ID NO:4
  • Pad ChlFN-beta fw 5'-ccttaattaacgcaccatgactgcaaaccatcagtctccagg-3' SEQ. ID NO:5
  • ChlFN-beta-rev 5'-ccttaattaatcactgggtgttgagacgtttggatg-3' SEQ. ID NO:6 were used.
  • each of the two chlFN-transgenes were cloned into the Pad site of the plasmid pflMTH68Pac, respectively.
  • the total length of the genome was adjusted to be a multiple of 6 to follow the "rule of six" for the length of the viral genome.
  • the sequence identity of the ChlFN-beta insert was confirmed by nucleotide sequencing.
  • In the ChlFN-alpha insert one G to A nucleotide exchange in position 89 in comparision with the sequence NM_205427 was detected.
  • Recombinant virus was rescued from T7-expressing cells transfected with the full-length viral genomic plasmid containing the genes for ChlFN-alpha (Fig. 1 ) or ChlFN-beta (Fig.
  • the resulting ChIFN-alpha-expressing virus was designated NDV- ChlFN-alpha and the ChIFN-beta expressing NDV was named NDV-ChIFN- beta.
  • the viruses were cultivated either in tissue culture or in the allantoic fluid of chicken eggs to produce high titres.
  • ChIFN-alpha and ChIFN-beta expressed from a recombinant NDV is biological active.
  • CEC-32 cells avian quail cell line
  • HeIa cells cervical cancer cell line
  • CEC-32 cells avian quail cell line
  • HeIa cells cervical cancer cell line
  • a chicken interferon-specific bioassay was used (Schwarz et al, JICR, 2005).
  • the assay is based on the stable transfected quail cell line CEC-511 carrying a luciferase-gene controlled by the IFN-responsive chicken Mx promotor.
  • the luciferase activity is induced when the CEC-511 indicator cells where incubated with ChIFN-alpha or ChIFN-beta. To perform this assay 15.000 CEC-511 cells were seeded in 96- well plate.
  • the supernatants of the NDV-ChlFN-alpha and NDV- ChlFN-beta infected CEC-32 and HeIa cells have a strong luciferase inducing activity on CEC-511 cells indicating virus-mediated expression of ChlFN-alpha and ChlFN-beta in the infected cells.
  • supernatant of MOCK-infected cells or infected with a control virus expressing GFP show no luciferase-inducing activity on the CEC-511 indicator cell line.
  • a 1 :1000 dilution of supernatant containing recombinant ChlFN-alpha or ChlFN-beta also demonstrate a ChlFN-dependent Mx promotor inducing activity.
  • CEC-32 cells avian quail cell line
  • HeIa cells cervical cancer cell line
  • CEC-32 cells avian quail cell line
  • HeIa cells cervical cancer cell line
  • CEC-32 cells avian quail cell line
  • HeIa cells cervical cancer cell line
  • CEC-32 cells avian quail cell line
  • HeIa cells cervical cancer cell line
  • the CellTiter-Glo ® Luciferase Assay Promega was performed to measure cell viability, following the protocol provided by the manufacturer. To determine the mean values, each datapoint was generated from six independend values.
  • NDV expressing ChlFN-alpha or ChlFN-beta.
  • NDV-GFP NDV-GFP
  • NDV-ChlFN-alpha NDV-ChlFN-alpha
  • NDV-ChlFN-beta NDV-ChlFN- beta
  • Fibroblast cells were infected with the indicated virus concentrations.
  • As a negative control fibroblasts cells were MOCK infected. After 1 h the inocculum was removed, cells were washed with PBS and incubated with 200 ⁇ l cell culture medium for 4 days in the cell culture incubator. Afterwards the remaining cells were fixed with 4% formaldehyde solution and stained with crystal violet. The stain was solubilized with 10% glacial acetic acid and the absorbence of each well was measured at 595 nm in an ELISA Reader.
  • This assay allows to compare the pathogenicity of diffferent NDV strains by comparing the survival time of the infected embryos.
  • the method is based on the "Mean Death Time of the Minimum Lethal Dose (MDT/MLD)" determination described in R. P. Hanson (1980).
  • MDT/MLD Minimum Lethal Dose
  • the protocol was modified in that way that not serial dilutions of the virus stock were inocculated in the eggs, instead of one defined virus dose injection with a concentration higher than the minimal lethal dose. For ethical reasons this assay modification reduces the number of embryos used for the test.
  • the MDT is determined by the following formula:
  • the survival of the NDV-ChlFN-alpha and NDV-ChlFN-beta infected chick embryos is improved in comparision with the NDV-GFP infected embryos.
  • the NDV-GFP infected embryonated eggs are losing signs of vitality between 24h and 62h, leading to a MDT (Mean Death Time) of 5Oh.
  • the NDV-ChlFN-beta infected embryos are surviving up to 96h with a calculated MDT of 69h.
  • a comparable survival time with a MDT of 74h is measured in the NDV-ChlFN-beta infected embryos.
  • Most of the NDV-ChlFN-beta infected embryos are dying between 48h and 96h.
  • the sensitivity of the assay is indicated by usage of the apathogenic or lentogenic strain LaSota. Even embryonated eggs inocculated with the NDV LaSota strain are dying between 62h to 115h with a calculated MDT of 85h. This experiments shows that the MDT of the mesogenic NDV MTH68 is shifted towards a lentogenic NDV strain.
  • a cytotoxic agent can be generated selectively at cancer sites. Br J Cancer, 58, 700- 703.
  • Binz H. K., Amstutz, P., Kohl, A., Stumpp, M.T., Briand, C, Forrer, P., Grutter, M.G. and Pluckthun, A. (2004) High-affinity binders selected from designed ankyrin repeat protein libraries. Nat Biotechnol, 22, 575-582.
  • NDV Newcastle disease virus
  • IBDV infectious bursal disease virus
  • Newcastle disease virus as a vaccine vector. J Virol, 75, 11868- 11873.
PCT/EP2009/000308 2008-01-29 2009-01-20 Attenuated oncolytic paramyxoviruses encoding avian cytokines WO2009095167A1 (en)

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US10308913B2 (en) 2005-12-02 2019-06-04 Icahn School Of Medicine At Mount Sinai Chimeric viruses presenting non-native surface proteins and uses thereof
US10035984B2 (en) 2009-02-05 2018-07-31 Icahn School Of Medicine At Mount Sinai Chimeric newcastle disease viruses and uses thereof
US10251922B2 (en) 2013-03-14 2019-04-09 Icahn School Of Medicine At Mount Sinai Newcastle disease viruses and uses thereof
US10519426B2 (en) 2013-09-03 2019-12-31 Medimmune Limited Compositions featuring an attenuated Newcastle disease virus and methods of use for treating neoplasia
KR20160047570A (ko) * 2013-09-03 2016-05-02 메디뮨 리미티드 약독화된 뉴캐슬병 바이러스를 특징으로 하는 조성물 및 신생물을 치료하기 위한 사용 방법
EP3508209A1 (en) * 2013-09-03 2019-07-10 MedImmune Limited Compositions featuring an attenuated newcastle disease virus and methods of use for treating neoplasia
WO2015032755A1 (en) * 2013-09-03 2015-03-12 Medimmune Limited Compositions featuring an attenuated newcastle disease virus and methods of use for treating neoplasia
KR20210048605A (ko) * 2013-09-03 2021-05-03 메디뮨 리미티드 약독화된 뉴캐슬병 바이러스를 특징으로 하는 조성물 및 신생물을 치료하기 위한 사용 방법
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KR102310692B1 (ko) 2013-09-03 2021-10-12 메디뮨 리미티드 약독화된 뉴캐슬병 바이러스를 특징으로 하는 조성물 및 신생물을 치료하기 위한 사용 방법
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US11471499B2 (en) 2013-09-03 2022-10-18 Medimmune Limited Compositions featuring an attenuated newcastle disease virus and methods of use for treating neoplasia
EP4101457A1 (en) * 2013-09-03 2022-12-14 MedImmune Limited Compositions featuring an attenuated newcastle disease virus and methods of use for treating neoplasia
US11389495B2 (en) 2014-02-27 2022-07-19 Merck Sharp & Dohme Llc Combination method for treatment of cancer

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