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WO2013038066A1 - Virus de la vaccine oncolytique modifié - Google Patents

Virus de la vaccine oncolytique modifié Download PDF

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Publication number
WO2013038066A1
WO2013038066A1 PCT/FI2012/050895 FI2012050895W WO2013038066A1 WO 2013038066 A1 WO2013038066 A1 WO 2013038066A1 FI 2012050895 W FI2012050895 W FI 2012050895W WO 2013038066 A1 WO2013038066 A1 WO 2013038066A1
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WIPO (PCT)
Prior art keywords
vaccinia virus
virus
cells
modified vaccinia
vaccinia
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PCT/FI2012/050895
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English (en)
Inventor
Akseli Hemminki
Sari Pesonen
Marko Ahonen
Markus VÄHÄ-KOSKELA
Suvi Parviainen
Vincenzo Cerullo
Julia DIACONU
Eerika Karli
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Oncos Therapeutics Ltd.
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Publication of WO2013038066A1 publication Critical patent/WO2013038066A1/fr

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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
    • 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
    • C12N15/863—Poxviral vectors, e.g. entomopoxvirus
    • C12N15/8636—Vaccina virus vectors
    • 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
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • 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
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • 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
    • 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
    • A61K48/0091—Purification or manufacturing processes for gene therapy compositions
    • 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
    • C12N2710/24171—Demonstrated in vivo effect

Definitions

  • the present invention relates to virus vectors for cancer therapy. Further, the present invention relates to a modified vaccinia virus vector, cells containing said vectors, modified vaccinia virus particles containing said vectors, methods for producing said vectors, pharmaceutical compositions and kits containing said vectors and methods for producing said vectors, methods for inhibiting malignant cell proliferation in a subject, methods for detecting the presence of a modified vaccinia virus vector in a subject, and use of said vectors for medicaments and cancer therapy.
  • Normal tissue homeostasis is a highly regulated process of cell proliferation and cell death. An imbalance in either of these processes may develop into a cancerous state. Lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphomas, pre-neoplastic lesions, pre-neoplastic lesions in the lung, colon cancer, melanoma, bladder cancer and any other cancer or tumor that may be treated, including metastatic or systemically distributed cancers are examples of cancers that can result. In fact, the occurrence of cancer is so high that over 500,000 deaths per year are attributed to cancer in the United States alone.
  • oncolytic viruses are able to enhance the induction of cell-mediated antitumoral immunity within the host. These viruses can also be engineered to express therapeutic transgenes within the tumor to enhance antitumoral efficacy.
  • Adenovirus therapy has been used previously for the treatment of various tumor types with excellent safety record and local responses have also been reported.
  • none of the existing clinical approaches have been able to cure metastatic advanced disease.
  • Vaccinia virus is a genetically complex DNA virus encoding a large number of genes, some of which have immune evading properties allowing the virus to establish local pockets of infection within an infected host.
  • Immunotherapy of cancer has resulted in recent clinical successes validating the potential of the approach.
  • a key realization has been that in addition to induction of an anti-tumor immune response, reduction of tumor immune suppressiveness is also required.
  • Oncolytic vaccinia virus seems a promising platform for immunotherapy.
  • an "arming" strategy with immunostimulatory molecules is useful to maximize the immunotherapeutic effect.
  • Antigen-presenting cells such as a dendritic cells (DCs) present antigens to T cells and have the ability to determine between immune response and tolerance.
  • peptides derived from endogenously expressed proteins are presented by APC in the context of MHC class I (MHC I) to CD8+ T cells, whereas peptides obtained from exogenously derived proteins are normally loaded onto MHC class II (MHC II) for presentation to CD4+ T cells.
  • MHC I MHC class I
  • MHC II MHC class II
  • exogenous antigens can be also loaded onto MHC I for "cross-presentation" to CD8+ T cells [6] .
  • CD154 also known as CD40L
  • CD40L is one such molecule. Normally it binds to CD40 on APC, which can trigger various signaling cascades on the target cell.
  • CD40L functions as a co-stimulatory molecule and induces activation in APC in association with T cell receptor stimulation by MHC molecules[8].
  • CD40L also promotes direct apoptosis of CD40+ cells[9-l l] .
  • Recombinant CD40L has been used in trials, with some efficacy, but systemic adverse events limited the dose that could be achieved locally, resulting in suboptimal efficacy[ 12] .
  • Monoclonal antibodies against CD40 have also provided exciting proof-of-concept data[ 12] .
  • CD40L as an arming device has been explored in the context of other viral platforms such as adenoviruses[ 13-17], or other gene therapy approaches[ 18-20] the combination of the oncolytic efficacy of vvdd and the immunological effects of CD40L have not been fully studied and the technology is not commonly used in cancer therapy.
  • Previous preclinical studies with non-replicative adenoviruses armed with CD40 have not provided oncolytic viruses that could have been adopted into wide clinical use.
  • Recent observations have also underlined the importance of the type of death tumor cells undergo. The immunogenicity of cell death can significantly influence subsequent anti-tumor immune response and the overall efficacy of a drug[21-23] .
  • An object of the present invention is to provide novel modified replicative oncolytic vaccinia virus vectors and methods for producing such vectors. Another object of the present invention is to provide novel uses of the inventive vaccinia virus vectors for cancer therapy, including gene therapy. Another object of the invention is to provide novel cancer therapies for treating mammalian subjects, in particular human, cat or dog subjects. Another object of the invention is to provide methods for tracking a vaccinia virus in subjects receiving therapy with, or being exposed to, oncolytic vaccinia virus according to the invention. Summary of the invention
  • the present invention provides modified oncolytic vaccinia virus vectors for cancer gene therapies for humans, dogs, and cats. More specifically, the invention provides construction of oncolytic vaccinia virus recombinants and cells and pharmaceutical compositions comprising said vectors which preferentially replicate in tumor cells and express at least one transgene to facilitate antitumor efficacy and apoptosis induction and to modulate host immune responses in a human, dog, or cat.
  • the present invention also provides said viruses for treating cancer in a subject and a method of treating cancer in humans, dogs, or cats.
  • the invention provides increased safety of oncolytic vaccinia virus due to a new arming device and incorporation of a fluorescent marker allowing tracking of the virus in subjects and the environment.
  • Oncolytic vaccinia virus is an attractive platform for immunotherapy. Oncolysis releases tumor antigens and provides costimulatory danger signals. However, arming the virus can improve efficacy further.
  • CD40 ligand CD40L, CD154
  • Thl T-helper type 1
  • the inventors have constructed a vaccinia oncolytic virus expressing human CD40L (vvdd-hCD40L-tdTomato), which in addition features a cDNA for the tdTomato fluorochrome for detection of virus, useful for biosafety evaluation and for detecting cells infected by the virus in living subjects and in samples derived from subjects.
  • vvdd-hCD40L-tdTomato human CD40L
  • tdTomato fluorochrome for detection of virus
  • the invention provides a modified vaccinia virus vector, a virus particle, a host cell, a pharmaceutical composition and a kit comprising vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein.
  • the modified vaccinia virus vector, the virus particle, the pharmaceutical composition or the kit above for use in cancer therapy, for eliciting immune response in a subject, for use in a method of inhibiting malignant cell proliferation in a mammal, for use in a therapy or prophylaxis of cancer, for detecting the presence of the modified vaccinia virus in a subject, and as an in situ cancer vaccine, optionally in combination with adenovirus.
  • a method of producing a modified vaccinia virus comprising vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein, comprising the steps of providing producer cells capable of sustaining production of vaccinia virus particles and carrying the modified vaccinia vector; culturing the producer cells in conditions suitable for virus replication and production; and harvesting the virus particles.
  • the inventors show effective expression of functional CD40L both in vitro and in vivo.
  • growth of tumors was significantly inhibited by oncolysis and apoptosis following both intravenous and intratumoral administration.
  • CD40-negative model CD40L expression did not add potency to vaccinia oncolysis.
  • vvdd-hCD40L-tdTomato oncolysis resulted in signs of immunogenic cell death in the presence and absence of human lymphocytes.
  • the inventive modified oncolytic vaccinia virus coding for CD40L mediates multiple anti-tumor effects including oncolysis, apoptosis and induction of T-cell responses through upregulation of Th l cytokines.
  • Figure 1 depicts schematically the cloning plasmids used in the present invention.
  • Figure 2 2.1a-c: The expression of the hCD40L transgene does not abrogate oncolytic efficacy.
  • Human lung adenocarcinoma cells (A549, CD40-) (a), CD40+ bladder cancer cells (EJ) (b), and breast cancer cells (M4A4-LM3, CD40-) (c) were infected with different concentrations of replication-competent vaccinia viruses either coding for CD40L or not. Three days later, cell viability was measured using an MTS assay.
  • 2.2a-c vvdd-hCD40L-tdTomato promotes immunogenic cell death.
  • A549, PC3MM2 and EJ cells were infected 1 pfu/cell of virus and cultured for 12 hours. After incubation markers for immunogenic cell death were assessed, (a) HMGB1 (b) ATP release (c) Calreticulin exposure.
  • Figure 3 In vitro and in vivo expression of hCD40L.
  • A549 cells were infected with different pfu/cell of vvdd-hCD40L. Supernatant was collected at different time points and analyzed by FACSarray for expression of hCD40L.
  • Nude mice bearing M4A4-LM3 breast tumors were intratumorally injected with vvdd-tdTomato or vvdd- hCD40L-tdTomato.
  • Mice were imaged by IVIS.
  • Blood was collected on days 3 and 13 and tumors were harvested on day 13, both were analyzed for hCD40l concentration with FACSarray.
  • FIG. 4 vvdd-hCD40L-tdTomato showed increased anti-tumor efficacy in CD40-positive tumors following intratumoral administration.
  • Nude mice bearing human tumors were treated with PBS, vvdd-tdTomato and vvdd-hCD40L- tdTomato and tumor growth was measured over time. Administration of the virus was performed intratumorally.
  • Figure 5 Efficacy of vvdd-hCD40L-tdTomato in CD40-negative tumors following intravenous administration. Nude mice bearing human tumors were treated with PBS, vvdd-tdTomato and vvdd-hCD40L-tdTomato and tumor growth was measured over time. Administration of the virus was performed intravenously, (a) hCD40L-sensitive bladder tumors (EJ cell line), (b) hCD40 negative tumors (A549 cell line).
  • vvdd-hCD40L-tdTomato activates human lymphocytes and boosts their cytokine production
  • Human derived lymphocytes are activated by the vaccinia virus expressed hCD40L. Filtered supernatant from A549 cells infected with different concentrations of vvdd-tdTomato or vvdd-hCD40L-tdTomato were used to stimulate Ramos-blue cells. After 48h immunological activation, as measured by NFkB activation, was determined using QUANTI-Blue.
  • Human PBMCs were stimulated with oncolysate from vvdd-hCD40L-tdTomato infected A549 cells. Media was collected and cytokines were assessed by FACSARRAY.
  • Figure 7 depicts that systemically injected vvdd-tdtomato (SEQ ID NO: 7) enters into mammary fat pad tumors and expresses transgene in tumors.
  • SEQ ID NO: 7 systemically injected vvdd-tdtomato
  • Figure 8 depicts that Vaccinia virus infected cancer cells produce GMCSF (A, ELISA assay) and the virus produced GMCSF is fully functionally active (B, TF1 cell assay).
  • A ELISA assay
  • B TF1 cell assay
  • Figure 9 depicts that Vaccinia viruses are able to kill hamster cancer cell lines in vitro.
  • Figure 10 depicts that Vaccinia viruses are able to eradicate HapTl tumors in immunocompetent Syrian Hamsters.
  • Figure 11 depicts that splenocytes collected from virus treated HapTl tumor bearing hamsters are able to kill HapTl cells ex vivo.
  • Figure 12 depicts that SCCF1 cells produce dominantly EEV form of vvdd-luc while A549 cells produce both EEV and IMV forms of viral particles.
  • FIG 13 depicts that 100% confluent feline squamous cell carcinoma SCCF1 cells are resistant to vaccinia virus oncolysis but are able to continuously produce EEV particles (A). EEV particles produced by SCCF1 cells and IMV produced by A549 cells. EEV are useful stealth vehicles for intravenous delivery as they are not neutralized by antibodies.
  • Figure 14 depicts the concept that Vaccinia virus shows increased tropism towards the producer cell line in comparison to non-parental cell types (A). Also, there might be tropism towards cells of the same animal. Virus produced on human cancer cells transduces human cancer cells better than monkey cells (B). Figure 15 depicts enhancing the release of EEV particles by silencing A34R gene.
  • Figure 16 depicts a summary of advantages from vaccinia virus - adenovirus combination therapy.
  • Figure 17 depicts that Vaccinia virus and human adenovirus type 5 combine to kill cancer cells.
  • Figure 18 depicts that Vaccinia virus is able to replicate and spread in cells already infected with human adenovirus.
  • Figure 19 depicts molecular mechanisms for autocrine and paracrine synergy mechanisms between adenovirus and vaccinia virus.
  • Figure 20 depicts that Adenovirus + vaccinia virus combination enhances therapeutic efficacy in an in vivo model of induced resistance towards oncolytic virotherapy
  • Figure 21 depicts Tumor destruction in immunocompetent mouse melanoma model and demonstrates the oncolytic potency of the vaccinia virus + adenovirus combination. Vaccinia followed by adenovirus was the optimal schedule.
  • Figure 22 depicts that splenocytes from adenovirus (lower line) or vaccinia virus (upper line) treated B16.0VA-tumor-bearing mice are able to destroy B16.0VA cells grown in culture.
  • Figure 23 depicts that combination immunovirotherapy with vaccinia virus and adenovirus generates antigen-specific cytotoxic T cells.
  • Figure 24 depicts that after Vaccinia virus + adenovirus combination treatment antitumor immune response dominates over antiviral responses.
  • Figure 25 depicts that feline (SCCF1) and canine (others) cancer cell lines can be transduced by vaccinia virus vvdd-luc.
  • Figure 26 depicts that subconfluent feline and canine cancer cell lines can be killed by vaccinia virus vvdd-tdtomato (SEQ ID NO: 7).
  • Figure 27 depicts that Vaccinia virus vvdd-luc (SEQ ID NO : 6) reduces the growth of canine ACE1 prostate cancer tumors in mice.
  • Figure 28 depicts that Vaccinia virus is able to infect feline fibrosarcoma tumor tissue ex vivo.
  • Figure 29 depicts that VV and Ad can transduce cells in canine osteosarcoma tissue even when combined.
  • Figure 30 depicts that hCD40L is active in canine PBMCs.
  • Figure 31 shows that both adenovirus and vaccinia virus replicate productively in co- infected human SKOV3 ovarian cancer cells.
  • Figure 32 shows the proportion of NK cells in tumors, as analysed by FACS. Vaccinia followed by adenovirus resulted in highest NK cell percentages.
  • Figure 33 shows virus load and levels of neutralizing antibodies with different virus dosings.
  • Figure 34 shows tumor volumes following Ad+VV dosing. At the end of the experiment, tumor sizes were smallest in the vaccinia followed by adenovirus group.
  • Figure 35 shows the results from Adenovirus (Ad5/3-D24-TK/GFP, 10 TU/cell) and VV (VV-tdTomato, 0.1 PFU/cell) infections. Antiviral effects of interferon gamma and beta were attenuated in Ad+VV combination treated cells.
  • Figure 36 shows the quantitation of virus titers with Vaccinia and Adenovirus in A549 cells. Even in the presence of antiviral cytokines TN Fa I pha and interferons gamma and beta, high titer of virus could be produced following co-infection.
  • Figure 37 shows that oncolytic vaccinia virus and adenovirus are able to co-infect primary surgical human ovarian cancer tissue.
  • Figure 38 shows the levels of infectious virus in primary ovarian cancer tumor tissue following infection with single viruses or their combination.
  • Figure 39 shows the results in vaccinia virus production on SCCF1 cells which preferentially produce EEV (the most appealing form of vaccinia for intravenous injection). Sucrose gradient ultracentrifugation of cell pellets results in the IMV form of vaccinia while centrifugation of the supernatant reveals the EEV band.
  • Figure 40 Vvdd-hCD40L-tdTomato targeting and replication in vivo following intratumoral and intravenous administration.
  • heterologous nucleic acid sequence refers to a nucleic acid sequence that originates from a source other than the specified virus.
  • heterologous nucleic acid sequence refers to a nucleic acid sequence that originates from a source other than the specified host cell.
  • mutation refers to a deletion, an insertion of heterologous nucleic acid, an inversion, or a substitution, including an open reading frame ablating mutations as commonly understood in the art.
  • gene refers to a segment of nucleic acid that encodes an individual protein or RNA (also referred to as a "coding sequence” or “coding region”), optionally together with associated regulatory regions such as promoters, operators, terminators and the like, that may be located upstream or downstream of the coding sequence.
  • mutant virus refers to a virus comprising one or more mutations in its genome, including but not limited to deletions, insertions of heterologous nucleic acids, inversions, substitutions or combinations thereof.
  • wild-type virus refers to the most frequent genotype of a virus found in nature and against which mutants are defined.
  • anti-viral response refers to a cell's response to viral infection and includes, for example, production of interferons, cytokine release, production of chemokines, production of lymphokines or a combination thereof.
  • normal host cell refers to a non-cancerous, non-infected cell with an intact anti-viral response.
  • oncolytic agent refers to an agent capable of inhibiting the growth of and/or killing tumour cells.
  • adjuvant refers to a substance which, when added to a vaccine, is capable of enhancing the immune response stimulated by the vaccine in a subject.
  • marker refers to a marker that would confer an identifiable change to the cell permitting identification of cells containing the vector.
  • subject refers to any living organism, including an animal, animal tissue, animal cell, human, human tissue, and human cell.
  • Vaccinia virus is a member of the Orthopoxvirus genus of the Poxviridae. It has large double-stranded DNA genome ( ⁇ 200 kb, ⁇ 200 genes) and a complex morphogenic pathway produces distinct forms of infectious virions from each infected cell. Viral particles contain lipid membranes(s) around a core. Virus core contains viral structural proteins, tightly compacted viral DNA genome, and transcriptional enzymes. Dimensions of vaccinia virus are ⁇ 360 x 270 x 250 nm, and weight of ⁇ 5-10 fg . Genes are tightly packed with little non-coding DNA and open-reading frames (ORFs) lack introns. Three classes of genes (early, intermediate, late) exists.
  • Early genes code for proteins mainly related to immune modulation and virus DNA replication.
  • Intermediate genes code for regulatory proteins which are required for the expression of late genes (e.g. transcription factors) and late genes code for proteins required to make virus particles and enzymes that are packaged within new virions to initiate the next round of infection.
  • Vaccinia virus replicates in the cell cytoplasm.
  • the genome of WR vaccinia has been sequenced (Accession number AY243312).
  • EEV particles have an extra membrane derived from the trans-Golgi network. This outer membrane has two important roles: a) it protects the internal IMV from immune aggression and, b) it mediates the binding of the virus onto the cell surface.
  • CEVs and EEVs help virus to evade host antibody and complement by being wrapped in a host-derived membrane.
  • IMV and EEV particles have several differences in their biological properties and they play different roles in the virus life cycle. EEV and IMV bind to different (unknown) receptors (1) and they enter cells by different mechanisms (2). EEV particles enter the cell via endocytosis and the process is pH sensitive. After internalization, the outer membrane of EEV is ruptured within an acidified endosome and the exposed IMV is fused with the endosomal membrane and the virus core is released into the cytoplasm. IMV, on the other hand, enters the cell by fusion of cell membrane and virus membrane and this process is pH-independent. In addition to this, CEV induces the formation of actin tails from the cell surface that drive virions towards uninfected neighboring cells.
  • EEV is resistant to neutralization by antibodies (NAb) (3, 4) and complement toxicity (5), while IMV is not. Therefore, EEV mediates long range dissemination in vitro and in vivo (6).
  • Comet-inhibition test has become one way of measuring EEV-specific antibodies since even if free EEV cannot be neutralized by EEV NAb, the release of EEV from infected cells is blocked by EEV NAb and comet shaped plaques cannot be seen (4, 7).
  • EEV has higher specific infectivity in comparison to IMV particles (lower particle/pfu ratio) (1) which makes EEV an interesting candidate for therapeutic use.
  • EEV outer membrane of EEV is an extremely fragile structure and EEV particles need to be handled with caution which makes it difficult to obtain EEV particles in quantities required for therapeutic applications.
  • EEV outer membrane is ruptured in low pH (pH ⁇ 6) (2). Once EEV outer membrane is ruptured, the virus particles inside the envelope retain full infectivity as an IMV.
  • Some host-cell derived proteins co-localize with EEV preparations, but not with IMV, and the amount of cell-derived proteins is dependent on the host cell line (8) and the virus strain. For instance, WR EEV contains more cell-derived proteins in comparison to VV IHD-J strain (9).
  • Host cell derived proteins can modify biological effects of EEV particles. As an example, incorporation of the host membrane protein CD55 in the surface of EEV makes it resistance to complement toxicity (5).
  • human A549 cell derived proteins in the surface of EEV particles may target virus towards human cancer cells. Similar phenomenon has been demonstrated in the study with human immunodeficiency virus type 1, where host- derived ICAM-1 glycoproteins increased viral infectivity (10).
  • IEV membrane contains at least 9 proteins, two of those not existing in CEV/EEV. F12L and A36R proteins are involved in IEV transport to the cell surface where they are left behind and are not part of CEV/EEV (9, 11). 7 proteins are common in (IEV)/CEV/EEV: F13L, A33R, A34R, A56R, B5R, E2, (K2L).
  • IEV International Health Department
  • J International Health Department
  • the IHD-W phenotype was attributed largely to a point mutation within the A34R EEV lectin-like protein (12, 13). Also, deletion of A34R increases the number of EEVs released (13, 14). EEV particles can be first detected on cell surface 6 hours post-infection (as CEV) and 5 hours later in the supernatant (IHD-J strain). Infection with a low multiplicity of infection (MOI) results in higher rate of EEV in comparison to high viral dose. The balance between CEV and EEV is influenced by the host cell (15) and strain of virus.
  • adenoviruses activate innate responses (characterized by IL-6 and IL-12 secretion) through both MyD88 and TLR9 dependent and independent mechanisms (16).
  • MyD88/TLR9 knockout DCs showed reduced secretion of these cytokines upon challenge with Ad5 virus, there was no difference in cytokine induction in peritoneal macrophages from MyD88/TLR9-negative mice compared to those from WT mice.
  • adenovirus In vivo in mice, adenovirus induces strong type I interferon mainly in splenic mDCs (IFN-beta) via a RIG-I/MDA5-independent and TLR- independent mechanisms associated with endosomal release and subsequent IRF-7 activation, and in pDCs (IFN-alpha) via endosomal TLR-dependent recognition (17). Besides IFN induction by DCs, adenovirus elicits IL-6 by non-DC cells in the spleen.
  • adenovirus infection triggers a biphasic response including tumor necrosis factor alpha, macrophage inflammatory protein 2 (MIP-2), and interferon gamma-inducible protein 10 (IP-10) at 6 hr and 5 days post i.v. administration.
  • Adenovirus dsDNA may be recognized by TLR9 and NLRP3 inflammasome, leading to robust inflammatory cytokine secretion by infected macrophages (18), but also components of the virion activate macrophages independently of these sensors (19).
  • Adenoviruses do not efficiently activate macrophages in vitro unless cocultured with epithelial cells (20). Adenoviruses do not directly activate NK cells but instead do so via macrophages or DCs, via NKG2D upregulation (21). At the same time, adenovirus encoded E1A upregulates NKG2D also on tumor cells, rendering them targets for NK mediated destruction (22). Adenovirally transduced DCs are susceptible to Treg- mediated immunosuppression, regardless of maturing conditions (23). Immune responses elicited by oncolytic vaccinia virus
  • Systemic VV infection elicits a rapid chemokine response involving Mig and Crg-2 in several organs (24).
  • serum levels of TNF-alpha, IL-6, IFN- gamma or MCP-1 do not increase notably over background at 24, 48 or 72 hours post infection (25).
  • Murine plasmacytoid dendritic cells also do not produce type I IFN or TNF-alpha upon vaccinia virus infection, unlike infection with myxoma virus (26). This is in contrast to MVA, which in addition to these chemo/cytokines induces noticeable blood serum levels of MIP-lalpha, IP-10 and IL-lbeta, also in humans (27).
  • VV gene product H I blocks phosphorylation of STAT-1, whereas E3L prevents induction of IFN-beta by blocking detection of VV dsDNA or RNA intermediates (26).
  • VV gene product A46 protein on the other hand, interferes with TLR signaling by binding to both MyD88 and TRIF (28, 29). While type I IFN is important for clearance of VV from mice, PKR-independent mechanisms are likely at play since E3L is a potent inhibitor of PKR (30). These and several other VV components may contribute to enhancement of otherwise sensitive viruses (31). Clinical use of vaccinia viruses
  • Vaccinia has been used for eradication of smallpox and later, as an expression vector for foreign genes and as a live recombinant vaccine for infectious diseases and cancer.
  • Vaccinia virus is the most widely used pox virus in humans and therefore safety data for human use is extensive.
  • Those are generalized vaccinia (systemic spread of vaccinia in the body), erythema multiforme (toxic/allergic reaction), eczema vaccinatum (widespread infection of the skin), progressive vaccinia (tissue destruction), and postvaccinia! encephalitis.
  • CD40L is a type II transmembrane protein belonging to the tumor necrosis factor family.
  • CD40L is also known as CD154 or gp39 and is predominately expressed on CD4+ T-cells and binds to the CD40 receptor on the membrane of antigen-presenting cells (APCs) [ 1, 2] .
  • APCs antigen-presenting cells
  • CD40 is expressed on macrophages and dendritic cells (DCs) where its activation by CD40L leads to antigen presentation and cytokine production followed by T-cell priming and a strong innate immune response [3] .
  • Granulocyte-macrophage colony stimulating factor is among the most potent inducers of anti-tumor immunity (Dranoff, G. GM-CSF-based cancer vaccines. Immunol Rev 188, 147-154 (2002)). It acts through several mechanisms, including direct recruitment of Natural Killers (NK) and APCs such as dendritic cells (DC) (Degli- Esposti, M.A. & Smyth, MJ. Close encounters of different kinds: dendritic cells and NK cells take centre stage. Nat Rev Immunol 5, 112-124 (2005); Andrews, D. M., et al. Cross-talk between dendritic cells and natural killer cells in viral infection. Mol Immunol 42, 547-555 (2005)). GMCSF can also specifically activate DCs at the tumor site to increase their expression of co-stimulatory molecules to enhance cross-priming and T cell activation rather than cross-tolerance.
  • NK Natural Killers
  • APCs such as dendritic cells (DC) (De
  • Vaccinia virus is appealing for cancer gene therapy due to several characteristics. It has natural tropism towards cancer cells [ 16] and the selectivity can be significantly enhanced by deleting some of the viral genes.
  • the present invention relates to the use of double deleted vaccinia virus (vvdd) in which two viral genes, viral thymidine kinase (TK) and vaccinia growth factor (VGF), are at least partially deleted. TK and VGF genes are needed for virus to replicate in normal but not in cancer cells [ 17] .
  • the partial TK deletion may be engineered in the TK region conferring activity.
  • TK deleted vaccinia viruses are dependent on cellular nucleotide pool present in dividing cells for DNA synthesis and replication. Therefore TK deletion limits virus replication significantly in resting cells allowing efficient virus replication to occur only in actively dividing cells (e.g. cancer cells).
  • VGF is secreted from infected cells and has a paracrine priming effect on surrounding cells by acting as a mitogen [ 18] .
  • Replication of VGF deleted vaccinia viruses is highly attenuated in resting (non- cancer) cells [ 19] .
  • the effects of TK and VGF deletions have been shown to be synergistic.
  • the oncolytic vaccinia virus vector according to the present invention may comprise further transgenes depending on the intended application of the virus.
  • transgenes suitable for including in the inventive vaccinia vectors alone or in combination with others are TNF-alpha, hNIS, interferon alpha, interferon beta and interferon gamma, and monoclonal antibodies directed against various targets such as CTLA-4 or TGF-beta
  • vaccinia constructs have been usually made by inserting transgenes into the middle of vaccinia virus thymidine kinase (TK) region without actually deleting any part of TK region, only disrupting it. This facilitates back-recombination for an intact TK region, and since such a construct would have a replicative selection advantage over TK deleted virus, it would quickly out-grow the original strain.
  • TK thymidine kinase
  • the plasmid has been modified so that after transgene insertion into the insetion site in the TK region, there is no possible way for virus to gain intact TK gene (Example 3). Ensuring that the transgene is present makes the virus safer since an immunogenic virus is more rapidly cleared from normal tissues.
  • Tdtomato (Accession code: AY678269) is used as a transgene in several constructs of the present invention. It is an ideal fluorescent protein for live imaging studies due to its excellent brightness and photostability (Example 4). It can be detected as deep as 1 cm below the surface and extremely small lesions can be visualized. This feature gives a possibility to follow virus spread in the animal (normal vs. malignant tissue) and helps to optimize the treatment schedule (new injection when no virus is left in the body). No adverse effects are seen in a safety evaluation of vaccinia viruses coding for tdtomato and luciferase. Also, tdTomato allows the virus to be detected in organs, tissues, secretions, excretions and environment. These aspects are relevant from the point of view of understanding possible adverse events (their association with treatment) and biosafety. No other oncolytic viruses with tdTomato have been reported previously.
  • the VV recombinants of the present invention carry in their VGF region an enzymatically inactive form of beta galactosidase, which nevertheless is recognized by antibodies. Because beta galactosidase is foreign to humans, it may be recognized by the immune system and function as an adjuvant in therapeutic settings (Example 5). Therefore, this novel aspect increases the immunogenicity of the virus making it safer (faster clearance from normal tissues) and more effective (enhanced anti-tumor immunity). However, to avoid metabolic issues in treated patients, in the present invention enzymatically inactive variant is used while retaining the immunogenicity and capacity for detection with antibodies.
  • the vaccinia virus strain Western Reserve (WR) A34R gene encodes a lectin-like glycoprotein, which is expressed in the outer membrane of extracellular enveloped virus (EEV). The glycoprotein binds EEV-particles to the host cell membrane and inhibits the release of the particles. It has been shown that WR A34R deletion mutant virus is capable of releasing up to 24-fold more EEVs from infected cells than normal WR virus. Also vaccinia virus strain International Health Department-J (IHD-J) is able to produce large amounts of EEV particles because of a mutation within the A34R protein.
  • IHD-J International Health Department-J
  • feline squamous cell carcinoma cell line (SCCF1) produces almost exclusively EEV form of vaccinia virus particles if cells are 100% confluent at the time of infection. EEV particles are released from the host cells via exocytosis. Host cells are not lysed in this process which allows continuous production of EEV from infected cells. EEV particles can be concentrated from cell culture medium by using Optiprep gradient purification (see below).
  • EEV particles can be purified from the cell culture medium with Optiprep (Sigma) system, which is based on the use of continuous gradient of iodixanol solution (47). Both IMV and EEV particles can be collected separately from the cell lysate by using the same continuous gradient (48).
  • Iodixanol purification method offers many advantages in comparison to CsCI or sucrose purification methods. Virus infectivity is retained well during iodixanol purification (49) and also particle: infectivity ratio is lower in comparison to CsCI purification methods (50). Viscosity of iodixanol is lower if compared to sucrose solution of the same density and this may help retaining glycoprotein structures on the viral surface (51).
  • iodixanol purified virus prep can be used directly without any further processing (such as dialysis). Additional processing steps often decrease the infectivity of the virus and potentially damage the outer membrane of EEV particles. Optiprep is available as a sterile solution which is critically important for clinical applications.
  • Human lung adenocarcinoma A549 cell line as a host cell for enhanced tumor cell targeting Oncolytic vaccinia virus has previously been produced in non-tumor monkey cells.
  • virus produced in foreign organisms causes several problems, including regulatory issues related to injecting non-human material in humans.
  • a virus produced in non-human system is also rapidly cleared from the subject receiving therapy due to recognition of foreign surface structures of the virus by the subject's immune system. This may result in poor therapeutic effect when a foreign host is used as a production host.
  • human lung adenocarcinoma A549 cells to produce human cancer targeted viral preparations is used in the present invention.
  • host cell proteins exist in the EEV outer membrane, these proteins may help virus to be targeted for the same cell type.
  • increased tumor targeting for viral preparations produced in human cancer cells is observed, as is readily seen in Example 18.
  • the profile of proteins associated with EEV varies with cell type, indicating involvement of host factors (6, 15).
  • the association of cell-derived antigens is also influenced by the virus strain.
  • Several host membrane proteins that are present in the trans-Golgi network (TGN), early endosomes or plasma membrane fractions have been found in EEV preparations e.g. CD46, CD55, CD59, MHC class I and others (5). In electron microscopy studies, low levels of these proteins have been found in EEV particles as well.
  • Host cell proteins from the ER, intermediate compartment (IC), and early Golgi membranes are not found in EEV or IMV preparations, suggesting these membranes are not utilized for EEV formation.
  • Vaccinia virus infects a single type of vertebrate host cell during its life cycle (ICTVdB Virus Descriptions 00.058.1.01.001. Vaccinia virus).
  • the methods of the present invention administer an oncolytic vaccinia virus, optionally followed or preceded by administration of a composition comprising an agent used in cancer therapy, including oncolytic adenovirus.
  • routes of administration vary, naturally, with the location and nature of the tumor, and include, e.g., intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, regional (e.g., in the proximity of a tumor, particularly with the vasculature or adjacent vasculature of a tumor), percutaneous, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, and oral administration.
  • Compositions are formulated relative to the particular administration route.
  • the methods of the present invention comprise administering an oncolytic vaccinia virus, followed or preceded by one or more traditional cancer therapy, such as chemotherapy, radiotherapy, surgery or immunotherapy.
  • Suitable treatments in therapeutic applications may include various "unit doses."
  • Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered and the particular administration route and formulation are within the skill of those in the clinical arts.
  • a unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time.
  • Unit dose of the present invention may conveniently be described in terms of TCID50 units (median tissue culture infective dose, AdEasy Vector System) for a viral construct.
  • suitable unit doses in therapy may range from 10 3 to 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 TCID50 units.
  • the lower limit of the therapeutic unit dose range may be 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 TCID50 units and the upper limit of the therapeutic unit dose range may be 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , or 10 13 TCID50 units.
  • variation in dosage will necessarily occur depending for example on the condition of the subject being treated, route of administration and the subject's response to the therapy. The person responsible for administration will in any event determine the appropriate dose for the individual subject.
  • both adenovirus and VV have an extensive history of use in humans, and they both have excellent safety profiles.
  • both viruses are potent vaccine vectors and have been used successfully in vaccination, particularly in heterologous prime-boost settings (52). Whereas prime-boost with the same virus biases the immune response against the vector rather than the immunogen, vaccination using immunologically different viruses has been shown to circumvent this problem and to provide superior vaccination efficacy against the target antigen (53).
  • oncolytic Ad appears to be restricted by type I IFN-inducible mechanisms in vivo (55).
  • VV is able to facilitate replication of Ad in a manner similar to VSV (56).
  • Ad is also sensitive to antiviral effects mediated by IFN-gamma (57), a cytokine to which VV carries countermeasures (soluble B8R protein).
  • vaccinia virus expressing adenovirus 14.7K protein has been reported to exhibit reduced sensitivity to TNF-alpha (58) and thus vaccinia could benefit from the presence of adenovirus (Example 23).
  • each virus genome occupies its own subcellular compartment (adenovirus in the nucleus, vaccinia in the cytoplasm) there is minimal risk of recombination.
  • VV activated mouse mDCs and conferred antigen (OVA)-specific therapeutic potential with similar efficacy to Ad (59).
  • OVA antigen
  • plasmacytoid dendritic cells were infected by both viruses, but whereas Ad infection resulted in pDC activation, it also reduced CTL activation compared to VV or saline (60).
  • Vaccinia on the other hand, induced CTL activation but did so without activating and maturing the pDCs.
  • pDCs from IFNAR KO mice also fail to undergo maturation upon Ad challenge, yet are able to instigate specific CTL responses.
  • adenovirus type 5 and modified vaccinia Ankara (MVA) vectors displayed reduced vaccine efficacy compared to either vector alone (63).
  • MVA was found to suppress adenovirus gene transcription partly through soluble factors. Since MVA was obtained by serial passage of an ancestral vaccinia virus strain, resulting in multiple gene deletions including the type I and type II interferon (IFN) scavengers B18R and B8R, respectively, it is likely the combined interferon and/or other cytokines released into the culture supernatant by MVA infection could potentially interfere with adenovirus replication.
  • IFN type II interferon
  • adenovirus may be partially sensitive to these antiviral mechanisms (55).
  • oncolytic vaccinia virus deleted for the vaccinia virus growth factor (VGF) as well as thymidine kinase (TK) do not hamper the IFN-neutralizing activity of VV, and VV has been demonstrated to enhance replication and spread of IFN-sensitive VSV by antagonizing antiviral responses (56). Therefore the VV vector of the present invention does not inhibit oncolytic Ad - and in fact enhances Ad instead -, through soluble components. Indeed, in contrast to MVA and Ad which did not co-infect A549 cells in culture, we constantly detected double-infected cells at many different ratios (Examples 21-22).
  • Ad5 alone in comparison to all other vectors was able to generate antigen-specific protective responses in mice against lethal VV challenge (64), underscoring the oft superior immunogenic property of Ad compared to most other viruses used in gene therapy today.
  • Ad5 was superior to VV in inducing hCMV- specific immune responses in mice (65).
  • MVA after Ad (63), and MVA interferes with Ad replication via induction of type I IFN.
  • oncolytic vaccinia virus instead suppresses type I IFN, for the duration of its own replication it should enhance replication of Ad, which once VV has been cleared would elicit the immunotherapeutically critical IFN responses enhancing heterologous prime-boost.
  • viral TK activity may also be controlled effectively in the construct by an open reading frame ablating mutation outside the active region, e.g. to the 5' direction from the region conferring TK activity.
  • the precise size of the TK deletion is not crucial as long as it provides a partially deleted TK which is not able reconstitute an active TK.
  • the partial deletion of TK comprises deletion of as few as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides, but may also comprise deletion of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350 or 400 nucleotides as long as enough wild type TK remains for recombination and inserting the transgene.
  • the length of the deletion may depend on the method used to insert the transgene and it may be longer or shorter than what is practical when inserting the transgene with homologous recombination. This completely avoids any possibility of reconstitution of the wild type TK gene in the event of expulsion of the transgene, which has been reported to occur and shown also in this application. It is logical that vaccinia without a transgene that disrupts TK gene would have an advantage over the recombinant virus and therefore there is selective pressure for back-recombinants.
  • the imaging device we developed for imaging mice can be used in a human trial featuring a tdTomato coding virus, to evaluate presence, persistence and amplification in tumor versus normal tissues. While formal toxicity studies are needed, tdTomato is not expected to be toxic[30, 31, 35] .
  • Vaccinia virus has legendary immunogenicity due to its use in eradication of small pox. However, this was achieved mainly through antibody induction and in fact vaccinia per se is not very potent in inducing cellular immunity[ l, 36] . It has a complex genome that encodes for several immunomodulatory proteins, including B18R which naturally antagonizes innate cellular and antiviral responses initiated by type I interferons[ l] . Its immunomodulatory properties make this virus an intriguing platform to express immune stimulatory molecules by rational design.
  • a modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein.
  • the modified vaccinia virus vector comprises a thymidine kinase gene which comprises at least one insertion site for inserting the heterologous protein and thymidine kinase inactivation is carried out by a deletion in the thymidine kinase region conferring activity.
  • the first heterologous protein is an enzymatically inactive beta-galactosidase, capable of producing an immune response, inserted in the place of the deleted vaccinia growth factor gene and another heterologous protein is at least one of CD40L, a fluorescent marker protein, and GMCSF inserted in the insertion site of the partially deleted thymidine kinase gene.
  • the modified vaccinia virus is Western Reserve (WR) strain vaccinia virus and the modified vaccinia virus vector comprises the genes for expressing human CD40L (hCD40L) and tdTomato as heterologous proteins.
  • human CD40L (hCD40L) gene and tdTomato gene are inserted in the in the partially deleted thymidine kinase gene of the modified vaccinia virus vector.
  • a host cell carrying a modified vaccinia virus vector in another aspect is provided a host cell carrying a modified vaccinia virus vector.
  • the host cell in which the host cell is a cancer cell originating from feline squamous cell carcinoma cell line SCCF1 or from human lung adenocarcinoma A549 cell line.
  • a modified vaccinia virus particle containing a modified vaccinia virus vector
  • the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted
  • the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein and in which the virus particle is of the type intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV), or extracellular enveloped virus (EEV), more preferably the virus particle is of the type EEV or IMV, and most preferably the virus particle is of the type EEV.
  • IMV intracellular mature virus
  • IEV intracellular enveloped virus
  • CEV cell-associated enveloped virus
  • EEV extracellular enveloped virus
  • modified vaccinia virus particle in which the modified vaccinia virus particle comprises inactivation of the viral A34R protein by gene silencing, by post-translational gene silencing, by RNA interference, or by small interfering RNA (si RNA).
  • modified vaccinia virus particle comprises inactivation of the viral A34R protein by gene silencing, by post-translational gene silencing, by RNA interference, or by small interfering RNA (si RNA).
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more of the following : the modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein; the host cell comprising the modified vaccinia vector above; and the virus particle comprising the modified vaccinia vector above.
  • the pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more of the following : the modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein; the host cell comprising the modified vaccinia vector above; and the virus particle comprising the modified vaccinia vector above; and a therapeutically active amount of an oncolytic adenovirus.
  • the modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the
  • a kit comprising one or more containers and one or more of the following : the modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein; the host cell comprising the modified vaccinia vector above; the virus particle comprising the modified vaccinia virus vector above; the pharmaceutical composition according to the invention; and the pharmaceutical composition according to the invention wherein the oncolytic adenovirus is provided in the same or a separate container.
  • an in vitro method for producing a modified vaccinia virus wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein and comprising the steps of providing producer cells capable of sustaining production of vaccinia virus particles and carrying a modified vaccinia vector; culturing the producer cells in conditions suitable for virus replication and production; and harvesting the virus particles.
  • an in vitro method for producing a modified vaccinia virus wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein and comprising the steps of providing SCCF1 or A549 producer cells capable of sustaining production of vaccinia virus particles and carrying a modified vaccinia vector; culturing the producer cells in conditions suitable for virus replication and production; and harvesting the EEV virus particles.
  • a modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein; a host cell comprising the modified virus vector above; a virus particle comprising the modified virus vector above, a pharmaceutical composition comprising the modified virus vector above, or the kit comprising the modified virus vector above for use in cancer therapy.
  • a modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein; a host cell comprising the modified virus vector above; a virus particle comprising the modified virus vector above, a pharmaceutical composition comprising the modified virus vector above, or the kit comprising the modified virus vector above for use as a medicament to elicit immune response in a subject.
  • an in situ cancer vaccine comprising the modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein; a host cell comprising the modified virus vector above; a virus particle comprising the modified virus vector above, a pharmaceutical composition comprising the modified virus vector above, or the kit comprising the modified virus vector above.
  • a modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein; a pharmaceutical composition comprising the modified virus vector above, or the kit comprising the modified virus vector above for use in a method of inhibiting malignant cell proliferation in a mammal wherein the method comprises administering to the mammal the modified vaccinia virus vector, the composition or the kit an amount sufficient to inhibit malignant cell proliferation compared to the malignant cell proliferation that would occur in the absence of the said modified vaccinia virus vector or the composition.
  • a modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein; a host cell comprising the modified virus vector above; a virus particle comprising the modified virus vector above, a pharmaceutical composition comprising the modified virus vector above, or the kit comprising the modified virus vector above for use in combination therapy or prophylaxis of cancer, wherein in the combination therapy or prophylaxis comprises administering the modified vaccinia virus vector and adenovirus.
  • an in vitro or in vivo method for detecting the presence of a modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein; a host cell comprising the modified virus vector above; or a virus particle comprising the modified virus vector above wherein the method comprises the step of detecting the presence of a marker present in the virus.
  • Cancer cell lines used in this study included human breast cancer cell line M4A4- LM,[39], EJ cells, lung adenocarcinoma cells A549 and CV-1 cells (African green monkey kidney fibroblasts) (ATCC, Manassas, VA USA). All cell lines were maintained in the recommended conditions. Viruses
  • vaccinia viruses used in this study are of the Western Reserve strain with disrupted TK and VGF genes for enhanced cancer cell specificity.
  • the tdTomato gene[35] was cloned into pSC65 (a kind gift from Bernie Moss, National Institutes of Health, Bethesda, MD) under the control of the P7.5 promoter to create pSC65-tdTomato.
  • hCD40L cDNA was inserted under the control of the pE/L promoter to create pSC65-tdTomato-hCD40L.
  • These shuttle plasmids were co-transfected with vvdd-luc in CV-1 cells.
  • vvdd-tdTomato and vvdd-tdTomato-hCD40L were selected by picking plaques positive for red fluorescence and negative for luciferase.
  • Viruses were amplified on A549 cells and purified over a sucrose cushion, and titers were determined with a standard plaque assay on Vero cells as described previously[4] .
  • PFU virus titers (PFU/ml) were determined by plaque assay. The presence of the inserted genes was verified by PCR, with fluorescence microscope and with FACSarray. UV light inactivation of viruses.
  • viruses were suspended in lOug/ml psoralen in Hanks balanced salt solution containing 0.1% bovine serum albumin. The suspension was incubated for 10 min at room temperature and then irradiated in a CL-1000 UV cross-linker (UVP, Cambridge, United Kingdom) with UV-A light (365 nm) for 3 min. A 5-day plaque assay was used to confirm lack of replication competent virus.
  • Cells on 96-well plates were infected with different concentrations of virus suspended in growth medium containing 2% FCS. One hour later, cells were washed and incubated in growth medium containing 5% FCS for 72 h. Cell viability was then analyzed using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium] (Cell Titer 96 AQueous One Solution proliferation assay; Promega).
  • mice Animal experiments were approved by the Experimental Animal Committee of the University of Helsinki, Finland. Mice were purchased from Taconic (Ejby, Denmark, and Hudson, NY) at the age of 4 to 5 weeks and housed under standard conditions with food and water ad libitum. M4A4-LM3 cells were injected subcutaneously into flanks of nude Naval Medical Research Institute (NMRI) mice. When tumors reached the size of approximately 5 by 5 mm, virus was injected either intratumorally or intravenously. Bioluminescence images were captured using the IVIS imaging series 100 system (Xenogen, Alameda, CA). hCD40L concentration in mouse serum and from tumor lysate was determined with FACSarray.
  • NMRI nude Newcastle Medical Research Institute
  • mice were injected intravenously with virus, and bioluminescent imaging and blood samples were taken 3 and 13 days post injection. On day 13 tumors were collected and lysed with ultrasonification. Samples were analysed with FACSarray for hCD40L quantification. Human derived lymphocyte stimulation
  • Ramos-Blue cells are B lymphocyte cell lines that stably expresses an NFkB/AP-l-inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene. When stimulated, they produce SEAP in the supernatant.
  • SEAP secreted embryonic alkaline phosphatase
  • the levels of SEAP can be readily monitored using QUANTI-BlueTM, a medium that turns purple/blue in the presence of SEAP. Levels of activation were read with microplate reader at the wave length of 450nm.
  • Example 1 Generation of human CD40L-expressing vaccinia virus CD40 ligand can induce apoptosis of tumor cells and it also able to trigger several immune mechanisms.
  • One of these is a T-helper type 1 (Th l) response that leads to activation of cytotoxic T-cells and reduction of immune suppression.
  • Th l T-helper type 1
  • the virus construct also includes a cDNA expressing the tdTomato fluorochrome in order to facilitate identification of the virus in vitro and in vivo[30] .
  • tdTomato Compared to prior commonly used fluorophores, such as green fluorescent protein, tdTomato possesses greater tissue penetration [31] and is superior to the more frequently used EGFP.
  • tdTomato In contrast to previous Western Reserve strain based vaccinia designs which have only featured a transgene insertion into TK, we engineered a deletion of the TK region (in addition to the transgene insert) to completely avoid the possibility of back- recombination which could result in a wild type TK gene.
  • Example 2 (depicted in Figure 1) : Schematic presentation of cloning plasmids.
  • TK disruption was changed to TK deletion by PCR with primers F (SEQ ID NO: 1) : TAATC TAGAG CCGTG GGTCA TTG and R (SEQ ID NO : 2) : TAAGG TACCC ATGCG TCCAT AGTCC on shuttle plasmid pSC65 to create insert which was inserted back to pSC65 creating pMA.
  • Tdtomato gene from pTdTomato was cut with Asel and EcoRI and was inserted to pMA (cut with Ndel and EcoRI) to create pMA-tdtomato.
  • pMA-tdtomato-hCD40L (SEQ ID NO: 3) was cloned from pShuttle/CMV-hCD40l (cut with accl and hinpll) and inserted to pMA-tdtomato (cut with Accl).
  • pMA-tdtomato-hGMCSF (SEQ ID NO: 4) was cloned from pSC65- hGMCSF-hNIS (SEQ ID NO: 5) (cut with Taql) and inserted to pMA-tdtomato (cut with Accl).
  • 117 bases are missing in the active region of the partially deleted TK in the construct.
  • Example 3 Partial deletion in TK region prevents formation of functional TK in recombinant viruses resulting in increased safety.
  • Partial deletion in the vaccinia virus thymidine kinase (TK) region prevents back-recombination for an intact TK increasing the safety of the virus
  • vvdd-luc virus with a disrupted TK region SEQ ID NO: 6 (without a partial deletion) was serially passaged in A549 cells and luc- negative plaques were collected for PCR analysis. Back-recombination for an intact TK region was seen in virus extracted from plaque 5 (p5) (TK region identical in size with wild type WR; wt).
  • Example 4 Described vaccinia virus expresses enzymatically inactive but antigenic beta galactosidase.
  • Human A549 lung cancer cells cultured in 12-well plates were infected with indicated vaccinia recombinants at MOI 1 and 24 hours later fixed with 3.7% paraformaldehyde for 30 minutes at room temperature.
  • Parallel wells were stained using X-gal solution, which is converted into a blue dye by enzymatically active beta galactosidase encoded in the VGF region of the vaccinia virus genome, and with polyclonal anti-beta galactosidase antibody, which does not discriminate between enzymatically active or inactive forms of beta galactosidase.
  • VV recombinant VV-tdTomato (SEQ ID NO: 7) carries in its VGF region an enzymatically inactive form of beta galactosidase, which nevertheless is recognized by antibodies. Because beta galactosidase is foreign to humans, it can be recognized by the immune system and function as an adjuvant in therapeutic settings. Lack of enzymatic activity is important from a safety point-of-view as there is no risk of adverse events related to enzymatic activity, while the benefits of enhanced immunity are retained, and immunostaining can also be used for detection of infected cells.
  • Example 5 (depicted in Figure 2) : Expression of the hCD40L transgene does not compromise the oncolytic activity of vvdd-hCD40L-tdTomato.
  • vvdd-hCD40L-tdTomato promotes a more immunogenic form of cell death compared with mock infection or vvdd-tdTomato Calreticulin exposure as well as ATP and HMGB1 release have been recently proposed as in vitro measurable indicators of immunogenic cell death[33] .
  • Example 7 (depicted in Figure 3) : vvdd-hCD40L-tdTomato displays tumor- restricted expression of the transgene
  • Example 8 (depicted in Figure 4) : vvdd-hCD40L-tdTomato results in increased anti-tumor activity in CD40-positive tumors following intratumoral and intravenous administration
  • Example 11 (depicted in Figure 7) : Systemically injected vvdd-tdtomato (SEQ ID NO: 7) enters into mammary fat pad tumors and expresses transgene in tumors. 5xl0e6 pfu of vvdd-tdtomato-hCD40L (SEQ ID NO: 8) virus was injected systemically into nude mice carrying GFP-positive M4A4-LM3 mammary fat pad tumors. Noninvasive imaging was done on indicated days to assess the expression of transgene (tdtomato) in animals (upper panel). Lower panel shows tumor cells that stably express GFP.
  • SEQ ID NO: 7 Systemically injected vvdd-tdtomato enters into mammary fat pad tumors and expresses transgene in tumors. 5xl0e6 pfu of vvdd-tdtomato-hCD40L (SEQ ID NO: 8) virus was injected systemically into nude mice carrying GFP-positive M4
  • Example 12 (depicted in Figure 8) : Vaccinia virus infected cancer cells produce active form of hGMCSF.
  • A549 cells were infected with vvdd-tdtomato-hGMCSF (SEQ ID NO: 9) at given viral doses and hGMCSF concentration in the supernatant was assessed at different time points with FACSArray (A).
  • Human erythroleukemic TF1 cells which are dependent on fully functional human GMCSF for viability, were incubated with supernatant from virus (vvdd-tdtomato-hGMCSF (SEQ ID NO: 9) or vvdd-tdtomato (SEQ ID NO : 7) ) infected A549 cells.
  • Negative and positive controls were incubated with growth medium and commercial hGMCSF (2 ng/ml; Invitrogen), respectively. Growth medium was changed every other day and cell viability was assessed 7 days later (B).
  • Example 13 (depicted in Figure 9) : Vaccinia viruses are able to kill hamster cancer cell lines HapTl and Hak in vitro. Hamster cancer cell lines were infected with vvdd-tdtomato (SEQ ID NO : 7) or vvdd-tdtomato-hGMCSF (SEQ ID NO : 9) at viral doses of 0.01, 0.1, 1, or 1 pfu/cell. Cell viability was assessed 48 hours later.
  • Example 14 (depicted in Figure 10) : Vaccinia viruses are able to eradicate HapTl tumors in immunocompetent Syrian Hamsters.
  • HapTl cells (7* 106 cells/tumor) were injected subcutaneously into flanks of Syrian hamsters (8 animals; 2 tumors/animal). Tumors were allowed to grow until average diameter of 5mm. Tumors were injected i.t. with 1* 10 ⁇ 6 pfu / tumor of vvdd-tdtomato (SEQ ID NO : 7) or vvdd-tdtomato- hGMCSF (SEQ ID NO : 9). Mock animals received only growth media. Tumor size was followed.
  • Example 15 Splenocytes collected from virus treated HapTl tumor bearing hamsters are able to kill HapTl cells ex vivo. Spleens were collected from Hamsters previously cured from HapTl tumors with vvdd-tdtomato (SEQ ID NO : 7) or vvdd-tdtomato-hGMCSF (SEQ ID NO: 9) viruses (data presented in Example 14). Splenocytes were isolated and cultured in 10% RMPI growth media for two days.
  • HapTl and Hak cells were seeded 50000 cells/well on 96-well-plates and splenocytes were added next day (in ratios splenocytes to cancer cells were 1 : 1, 10: 1, or 20: 1).
  • the cell viability for HapTl and Hak cells was assessed 24 hours later with MTS assay (490nm). Data show specific cell killing of HapTl cells by splenocytes collected from virus treated HapTl tumor bearing hamsters. As a control, HaK cells (foreign tumor cell type) are spared.
  • Example 16 (depicted in Figure 12) : SCCF1 cells produce dominantly the EEV form of vvdd-luc while A549 cells produce both EEV and IMV forms of viral particles. Immunofluorescence visualization of EEV and IMV particles in SCCFland A549 cells 48h after infection with vv-tdtomato at 0.1 MOI.
  • Example 17 100% confluent feline squamous cell carcinoma SCCF1 cells are resistant to vaccinia virus oncolysis but are able to continuously produce EEV particles. SCCF1 cells were infected with vvdd-luc at 0.1 pfu/cell and cell viability was assessed 3, 4, and 11 days later with MTS-assay. A549 cells were used as a control cell line (A). To visualize EEV particles, 100% confluent SCCF1 cells were infected with vvdd-luc at a viral dose of 0.1 pfu/cell and supernatant was collected 10 days later.
  • Example 18 (depicted in Figure 14) : Vaccinia virus shows increased tropism towards producer cell line in comparison to non-parental cell types.
  • Vaccinia virus (vvdd-luc) was produced in 11 different cancer cell lines from 5 different species (A).
  • Producer cell line and other cell lines were infected with vvdd-luc at 1 pfu/cell and luciferase expression was assessed 6 hours later.
  • Purified viral preparations were used for viral transduction assay in parental and other cancer cell lines. Transduction efficiency of viral preparations was enhanced in parental cell line in comparison to other cancer cell lines (B). The data shows that production in A549 human tumor cells results in higher transduction of the same cells in comparison to monkey cells.
  • Example 19 Enhancing the release of EEV particles by silencing A34R gene. Schematic presentation of silencing viral gene A34R by using designed siRNA constructs against the gene to enhance the release of EEV particles.
  • Example 20 Summary of advantages from vaccinia virus - adenovirus combination therapy.
  • Example 21 (depicted in Figure 17) : Vaccinia virus and human adenovirus type 5 combine to kill cancer cells.
  • vaccinia virus and adenovirus were mixed at varying ratios and used to infect 786-0 human renal cell carcinoma cells in culture (where the immune system is not present) at indicated MOIs, additive cell killing was seen.
  • a complex pattern of cell killing was observed 72 hours post infection : at some MOIs, VV and Ad are antagonistic (where the graph bulges outward compared to single infection as shown on the far left and right walls), at others synergistic (where graph bends inward compared to singly infected cells).
  • Example 22 (depicted in Figure 18) : Vaccinia virus is able to replicate and spread in cells already infected with human adenovirus.
  • Human A549 cells in culture were infected with Ad5/3-D24-TK/GFP at MOI 10 and incubated for 12 hours. Subsequently, VV-tdTomato was added at varying concentrations to parallel wells and washed out 1 hour later. Agarose overlay was added and plaques (VV in red) were visualized under fluorescence microscope 72 hours later [left panel] . Singly VV infected cells were used as comparison [right panel] . Results show VV is able to form plaques on cells expressing late adenovirus genes indicative of full adenovirus replication cycle. Plaque size is similar to singly VV infected cells, further suggesting that the presence of adenovirus does not adversely affect VV replication cycle and that both viruses can therefore be combined to target the same cancer.
  • Example 23 (depicted in Figure 19) : Molecular mechanisms for autocrine and paracrine synergy mechanisms between adenovirus and vaccinia virus.
  • Example 24 (depicted in Figure 20) : Adenovirus + vaccinia virus combination enhances therapeutic efficacy in an in vivo model of induced resistance towards oncolytic virotherapy.
  • 5-to-7-week old SCID mice (groups of five mice each) were injected intraperitoneally with 3e5 SKOV3Luc cells in 100 ul PBS. Three days later, mice received an i.p. injection of either PBS (Vehicle) or le9 VP adenovirus (Ad5/3- D24) [triangles] . Two days after that, mice received i.
  • Example 25 Tumor destruction in immunocompetent mouse melanoma model demonstrates oncolytic potency of the vaccinia virus + adenovirus combination.
  • Mouse B16.0VA cells were implanted subcutaneously in C57/BL6 mice (3e5 cells per mouse) and allowed to form palpable tumors ( ⁇ 5mm in diameter). Groups of mice received an intratumoral injection of either PBS or virus (for adenovirus, we used Ad5/3-D24 at 2el0 VPs and for vaccinia, le8 PFUs).
  • mice Six days later, a separate set of mice (4 each, see Figure 2) were sacrificed for immunological analysis and another set of mice received another intratumoral injection, forming the indicated treatment groups (5-6 mice each) in the left panel), and six days after that the mice were sacrificed and organs and tumor extracted for analysis.
  • Upper panel shows the size of the extracted tumors from each treatment group at study endpoint.
  • Lower panel shows the quantitated data of tumor sizes, demonstrating that vaccinia virus followed by adenovirus provides greatest tumor control.
  • Example 26 Splenocytes from adenovirus or vaccinia virus-treated B16.0VA-tumor-bearing mice are able to destroy B16.0VA cells grown in culture. Singly treated mice from the experiment described in Figure 6, having received a single intratumoral injection of either adenovirus (2el0 VP) or vaccinia virus (le8 PFU), were sacrificed 6 days after virus injection for analysis. Spleens were harvested and pooled (from 4 mice) and single-cell suspensions of splenocytes generated by gentle needle aspiration.
  • adenovirus 2el0 VP
  • vaccinia virus le8 PFU
  • splenocytes effectors
  • trypsinized B16.0VA cells targets
  • MTS assay 72 hours later shows that splenocytes from adenovirus-treated animals carry a larger tumor-destroying capacity compared to splenocytes from vaccinia-treated animals.
  • Example 27 Combination immunovirotherapy with vaccinia virus and adenovirus generates antigen-specific cytotoxic T cells.
  • Tumors from B16.0VA tumor bearing mice treated with adenovirus or vaccinia virus were extracted and single cell suspensions generated by trituration and passing through a 40 um nylon mesh. Cells were stained with fluorescent antibodies against CD8+ T cells as well as with pentamer against mouse MHC loaded with the ovalbumin immunodominant peptide epitope SIINFEKL and analysed by flow cytometry (A).
  • B FACS data plotted into a bar graph.
  • Example 28 (depicted in Figure 24) : Vaccinia virus - adenovirus combination treatment: anti-tumor immune response dominates over antiviral responses. The best therapy effect is achieved when vaccinia virus is followed by adenovirus by a novel mechanism : On one hand, vaccinia virus reduces antiviral effects in tumors and increases adenovirus replication. On the other hand, vaccinia virus induces central memory T cells against the tumor, which receive a strong inflammatory signal to proliferate and attack the tumor when adenovirus is injected. No other combination yields this effect; vaccinia followed by vaccinia lacks robust inflammation and the immune response recognizes the virus rather than the tumor.
  • Adenovirus-adenovirus regimen also gears the immune response against the virus rather than the tumor, and while there is inflammation, adenovirus has not stimulated central memory T cells.
  • inflammation after vaccinia infection is poor, and there are no central memory T cells to stimulate.
  • Example 29 (depicted in Figure 25) : Feline and canine cancer cell lines can be transduced by vaccinia virus.
  • Feline (SCCF1) and canine (Abrams, D17, ACE1, MDCK) cell lines were infected with vvdd-luc at viral doses of 0.04, 0.2, 1, or 5 pfu/cell. Luciferase expression was measured 24 hours later.
  • ACE1 prostatic carcinoma
  • Abrams osteosarcoma
  • D17 osteosarcoma
  • MDCK kidney cell line
  • SCCF1 squamous cell carcinoma
  • Example 30 Feline and canine cancer cell lines can be killed by vaccinia virus vvdd-tdtomato (SEQ ID NO: 7). Subconfluent feline and canine cell lines were infected with vvdd-tdTOM at viral doses of 0.01, 0.1, 1, or 1 pfu/cell. Cell viability was assessed 4 days later.
  • ACE1 Canine prostatic carcinoma
  • Abrams Canine osteosarcoma
  • D17 Canine osteosarcoma
  • MDCK Canine kidney cell line
  • SCCF1 Feline squamous cell carcinoma
  • Example 31 (depicted in Figure 27) : Vaccinia virus vvdd-luc reduces the growth of canine ACE1 prostate cancer tumors, lx 107 ACE1 (canine prostatic carcinoma) cells were injected subcutaneously into nude mice. Two intratumor injections of vvdd-luc at a dose of 1x105 pfu were done at indicated time points (arrows) and tumor growth was followed.
  • Example 32 (depicted in Figure 28) : Vaccinia virus is able to infect feline fibrosarcoma tumor tissue ex vivo.
  • Cat fibrosarcoma tumor tissue was obtained from tumor surgery following owner informed consent and manually dissected into multiple, roughly equally sized fragments. These were split into 24-well plates containing 0.5 ml fresh growth media supplemented with antibiotics (P/S), serum (10%) and L-glutamine. 24 hours later, tissue pieces were either left untreteated or infected with le5 PFU VV-tdTomato virus. Fluorescence micrographs taken 24 hours later show evidence of VV replication [red] . Importantly, data shows that clinical tumor specimen can be infected with our vaccinia constructs.
  • Example 33 (depicted in Figure 29) : VV and Ad can transduce cells in canine osteosarcoma tissue even when combined.
  • Primary cancer tissue was obtained during surgery of a male dog with solid osteosarcoma.
  • Cultured slices were infected at le6 PFU VV-tdTomato and/or le9 TU Ad5/3-D24-TK/GFP virus and followed under microscope. Results show both VV and Ad can transduce cells in dog osteosarcoma tissue even when combined.
  • Example 34 (depicted in Figure 30) : hCD40L is active in canine PBMCs.
  • PBMC's were isolated from blood donor dogs.
  • PBMCs were cultured with the supernatant collected from virus infected A549 cells (control virus Ad5/3hTERTE3 or CD40L coding virus Ad5/3hTERT-CD40L) and supernatant was filtered through 2um filter to remove virus particles before adding to the PBMCs.
  • Example 35 ( Figure 31) : Both adenovirus and vaccinia virus replicate productively in co-infected human SKOV3 ovarian cancer cells (adenovirus Ad5/3-D24 100 TU/cell, vaccinia virus VV-tdTomato 1 PFU/cell) as seen by EM 72 hours post infection.
  • Example adenovirus particles are indicated by black arrows, vaccinia particles by white arrows. Magnification ⁇ 40 OOOx.
  • Example 37 Tumor tissue was analyzed by FACS : increased infiltration of NK cells was observed in tumors treated with vaccinia virus (either VV+Ad or VV+VV). Heterologous combination with vaccinia virus followed by adenovirus yielded the greatest responses, demonstrating that the effect is not dependent on either virus alone but likely is dependent on vaccinia virus.
  • Example 38 ( Figure 33): Virus load and levels of neutralizing antibodies.
  • B16.0VA tumor tissue was extracted at study endpoint and snap-frozen. After rotor homogenization in 1000 ul PBS, samples were freeze-thawed three times to release infectious virus. Then tumor homogenate was titered for adenovirus by TCID50 and vaccinia virus by plaque assay on A549 cells. Results show repeated dosing of adenovirus reduces virus load in the tumors at study end below detection level. Switching the latter dosing to vaccinia virus rescues adenovirus presence in the tumors. On the other hand, vaccinia virus is able to persist in mouse B16 tumors for longer than adenovirus.
  • Example 39 Evaluation of the role of NK cells in vaccinia/adeno combination therapy 6.67 ⁇ 10 ⁇ 9 VP of adenovirus was given intratumorally per injection. For vaccinia virus, the dose was 3.33 ⁇ 10 ⁇ 7 per injection.
  • mice were given intraperitoneal injections of 33 ul polyclonal rabbit anti-mouse Asialo GM 1 antibody (to deplete NK cells) on days indicated by yellow circles. Animals were sacrificed and sampled 14 days post first virus injection. The results show NK cells are not critical for the anti-tumor effect of vaccinia virus, despite induction of such cells (Example 37, Figure 32).
  • Example 41 ( Figure 35): Adenovirus (Ad5/3-D24-TK/GFP, 10 TU/cell) and VV (VV- tdTomato, 0.1 PFU/cell) were used to infect human SKOV3Luc ovarian cancer cells either pre-treated or not for 6 hours with putative antiviral recombinant human cytokines; interferon (IFN) beta, IFN gamma and/or tumor necrosis factor alpha. Fluorescence micrographs taken 72 hours post infection suggest a reduction in replication of both viruses in response to the antiviral cytokines.
  • IFN interferon
  • Figure 42 Quantitation of virus titers in the experiment described in the previous figure.
  • plaque assay on A549 cells was conducted using whole well freeze-thaw lysate (top panel), and for adenovirus, TCID50 assay in A549 cells was performed (bottom panel). Results show increased vaccinia virus titers in cells pretreated with IFN gamma and co-infected with vaccinia and adenovirus compared to vaccinia virus alone (*, p ⁇ 0.05, studen't t test on log-transformed titers, duplicate wells per virus/regimen).
  • adenovirus may be antagonizing the antiviral activity of IFN gamma against vaccinia virus.
  • IFN beta and IFN gamma appear to mediate a more potent antiviral effect against both adenovirus and vaccinia virus than TNF alpha.
  • Co-infection of cells does not markedly reduce (or increase) replication of either virus, despite pretreatement with antiviral cytokines, suggesting heterologous virus interference is minimal and does not involve antiviral signaling mechanisms.
  • Example 43 Oncolytic vaccinia virus and adenovirus are able to co- infect primary surgical human cancer tissue. Shown are fluorescence micrographs of double-infected human ovarian cancer tissue (adenovirus Ad5/3-D24-TK/GFP, 1 ⁇ 10 ⁇ 8 TU/well, vaccinia virus VV-tdTomato 1x107 PFU/well in a 24-well plate in 0.5ml standard growth medium) 72 hours post infection. Double-infected cells appear yellow in the overlay micrograph.
  • Example 44 ( Figure 38): Analysis of levels of infectious virus in primary ovarian cancer tumor tissue at experiment end (previous figure) shows productive replication of vaccinia virus over time, irrespective of whether adenovirus was co-infecting the tissue or not, and productive replication of adenovirus when combined with vaccinia virus, suggesting enhancement of adenovirus replication by vaccinia virus.
  • Example 45 ( Figure 39): Vaccinia virus production on SCCF1 cells which preferentially produce EEV (the most appealing form of vaccinia for intravenous injection). Sucrose gradient ultracentrifugation of cell pellets results in the IMV form of vaccinia while centrifugation of the supernatant reveals the EEV band.
  • Tube 1 Collected cell pellet (containing IMV)
  • Tube 2 harvested supernatant (containing mostly EEV and only a small amount of IMV)
  • Tube 3 uninfected cell supernatant (no virus) Both bands of the middle tube are infectious.
  • Example 46 Vvdd-hCD40L-tdTomato targeting and replication in vivo following intratumoral and intravenous administration. Mice bearing EJ and A549 tumors were injected with vvdd-tdTomato and vvdd-hCD40L-tdTomato (a) intratumorally and (b) intravenously. tdTomato expression was visualized by IVIS.
  • Boulter EA Appleyard G. Differences between extracellular and intracellular forms of poxvirus and their implications. Prog Med Virol 1973; 16: 86-108.
  • Mcintosh AA Smith GL.
  • Vaccinia virus glycoprotein A34R is required for infectivity of extracellular enveloped virus. J Virol 1996;70: 272-81.

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Abstract

L'invention concerne des vecteurs de virus de la vaccine oncolytique modifié et leurs utilisations thérapeutiques contre le cancer. Lesdits vecteurs de virus de la vaccine consistent à neutraliser des mutations dans le gène thymidine kinase et dans le gène du facteur de croissance de la vaccine outre les gènes codant des protéines marqueurs ou d'addition. L'invention concerne également un procédé de production de la vaccine. Les vecteurs de virus de la vaccine sont destinés à l'usage thérapeutique systémique et séquentiel contre le cancer.
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