WO2012122649A1 - Virus orf recombinant - Google Patents

Virus orf recombinant Download PDF

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Publication number
WO2012122649A1
WO2012122649A1 PCT/CA2012/050153 CA2012050153W WO2012122649A1 WO 2012122649 A1 WO2012122649 A1 WO 2012122649A1 CA 2012050153 W CA2012050153 W CA 2012050153W WO 2012122649 A1 WO2012122649 A1 WO 2012122649A1
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Prior art keywords
virus
orf virus
cells
recombinant
host range
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PCT/CA2012/050153
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English (en)
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Julia RINTOUL
Aimée Nicole LAPORTE
Monica KOMAR
John Bell
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Ottawa Hospital Research Institute
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Publication of WO2012122649A1 publication Critical patent/WO2012122649A1/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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • C12N2710/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2710/24211—Parapoxvirus, e.g. Orf virus
    • C12N2710/24232—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
    • C12N2710/24211—Parapoxvirus, e.g. Orf virus
    • C12N2710/24241—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24243—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present application relates to a recombinant orf virus for the treatment of hyperproliferative conditions.
  • Cancer is a complex disease affecting millions of people every year. The majority of current therapies often result in mild to severe side effects, poor efficacy, and a high mortality rate. New options for targeted, well-tolerated therapeutics with a high therapeutic index are needed.
  • Oncolytic viruses are viruses that preferentially infect and lyse cancer cells.
  • Oncolytic viruses may function by direct destruction of tumour cells, or, if modified, as vectors enabling genes expressing anticancer proteins to be delivered specifically to the tumor site.
  • Oncolytic viruses have also been shown to elicit both innate, and adaptive anti-tumour immune responses. Oncolytic viruses offer many benefits to traditional chemotherapeutic or immunotherapeutic drugs: they are targeted to cancer cells by nature, with little to no adverse affects, they offer a self-amplifying dose, some can target metastatic sites, they are immune-stimulating, they are easily manipulated, and they can be genetically modified to carry other therapeutic genes.
  • WO97/26904 and WO96/03997 disclose a mutant herpes simplex virus (HSV-1761) that inhibits tumour cell growth.
  • HSV-1716 comprising a 759 base pair deletion in each copy of ⁇ 34.5 of the long repeat region (R L ) to tumour cells kills these cells.
  • this virus is specific for neuronal cells, as HSV is known to selectively infect the neuronal system.
  • the use of common human pathogens as oncolytic viruses is limited, as it is likely that the general population has been infected and acquired an immune response to such viruses.
  • a pre-existing immune response to a viral strain similar to the one used as a therapeutic agent in the treatment of a cancer may attenuate the effectiveness of the virus as therapeutic agent.
  • ONYX-015 human adenovirus (produced by ONYX pharmaceuticals) is believed to replicate preferentially in p53 negative tumour cells. This virus shows promise in clinical trials with head and neck cancer patients (Kim, D., T. et al, Nat Med, 1998. 4: 1341-1342).
  • Reovirus type 3 is being developed by Oncolytics Biotech as a cancer therapeutic, which preferentially grows in PKR-/- cells (Yin, H. S., J. Virol Methods, 1997. 67:93-101 ; Strong, J. E. and P. W. Lee., J Virol, 1996. 70:612-616; Strong, J. E., et al, Virology, 1993.
  • Reovirus, type III exhibited enhanced replication properties in cells which expressed the mutant ras oncogene (Coffey, M. C, et al, Science, 1998. 282: 1332-1334; Strong, J. E., et al, Embo J, 1998. 17:3351-1362).
  • Mundschau and Faller Mundschau, L. J. and D. V. Faller, J Biol Chem, 1992.
  • WO 99/18799 reports the cytotoxic activity of Newcastle Disease Virus (NDV) and Sindbis virus towards several human cancer cells. Both viruses demonstrated selectivity in their cytotoxic activity towards tumor cells. WO 99/18799 also discloses the cytotoxic activity of VSV cells against KB cells (head and neck carcinoma) and HT 1080 (Fibrosarcoma), and alleviation of cytotoxicity in normal and tumor cells, by VSV, in the presence of interferon. No other cell types were tested for VSV cytotoxic activity.
  • NDV Newcastle Disease Virus
  • Sindbis virus Sindbis virus
  • the present application provides a parapoxvirus, methods for the treatment of hyperproliferative disorders, and uses of such parapoxviruses in the treatment of hyperproliferative disorders.
  • the parapoxvirus is an orf virus.
  • the present invention provides a recombinant orf virus
  • ORFV containing one or more heterologous host range genes or homologues thereof, wherein said heterologous host range genes enable replication of the recombinant orf in human cells.
  • the recombinant ORFV is an oncolytic virus.
  • the recombinant ORFV replicates in human cancer cells.
  • said host range genes are any heterologous host range genes that aid in or enable replication of the recombinant orf virus in human cells.
  • the heterologous host range genes are from Poxviridae, and include, but not limited to, one or more of: SPI-1, SPI-2, KIL, C7L, p28/NlR, B5R, E3L, K3L, M-T2, M-T4, M-T5, M11L, M13L, M063, and F11L.
  • the host range genes may further include homologues thereof.
  • the present invention provides an engineered orf virus containing one more host range genes from a poxvirus, such as from vaccinia virus.
  • such engineering allows the parapoxvirus to better replicate in human cells, while still maintaining the parapoxvirus' unique immune stimulatory profile.
  • the recombinant orf virus is a live orf virus.
  • the present invention provides a method for the treatment of hyperproliferative disorders, comprising administering to a patient a therapeutically effective amount of a parapoxvirus in accordance with the invention.
  • the invention provides a parapoxvirus in accordance with the invention for use in the treatment of a hyperproliferative disorder or for use in the manufacture of a medicament for use in the treatment of a hyperproliferative disorder.
  • the invention provides a method for manufacturing a parapoxvirus, comprising producing the parapoxvirus in human cancer cells.
  • the invention provides a method for preventing or reducing surgery induced cancer metastasis, comprising administering to a patient, a therapeutically effective amount of an oncolytic parapoxvirus in accordance with any one of claims 1 to 7, in combination with cancer surgery.
  • the invention provides an orf virus expressing one or more heterologous host genes, such as those from vaccinia.
  • heterologous host range genes may include one or more of the following:
  • An Orf virus oncolytic virus is safer than other oncolytic viruses. Even wild type orf virus backbone has minimal pathogenicity in humans, which is different from other oncolytic viruses (herpes virus, vaccinia virus, Adeno-virus, measles, Polio virus, rhabdoviruses etc.).
  • Orf virus is one of the few viruses that has the ability to repeatedly infect its host, despite an apparent normal immune response. This characteristic is important, as it means the virus may be able to escape clearance by the immune system, allowing for multiple treatments of the virus in the clinic.
  • Orf virus infection leads to potent innate immune cell activation with minimal side effect: the immune stimulation is described in the literature as being 'self- regulated'. Meaning, the virus induces a very robust pro-inflammatory response, which the virus follows up with the induction of an anti-inflammatory response.
  • Orf virus may be better suited for intra-venous delivery than other similar oncolytic viruses since the virus expresses a homologue of Vascular Endothelial Growth Factor.
  • the lesions induced by orf virus infection have an extensive vascular accumulation, and dilation. This may allow for better delivery and spread of the virus within tumours beds.
  • ORFV growth curves were performed on B 16F10-LacZ and CT26-LacZ cell lines at an MOI of 0.3. Cells were infected at time 0, and cell lysates collected and processed by plaque assay at time-points post infection.
  • N 2, mean + SEM.
  • Figure 2 ORFV efficacy compared to other oncolytic viruses. (*P ⁇ 0.0001 using an unpaired, T test with Welch's correction). PBS, phosphate-buffered saline;
  • VAVC oncolytic vaccinia virus
  • MYXV oncolytic myxoma virus
  • RCNV oncolytic raccoonpoxvirus.
  • Figure 3 Flow cytometry analysis of Balb/c mouse splenocytes, 6 days post treatment (l e7 Live Orf virus, or PBS intravenously).
  • Figure 4 The quantitation of number of lung metastases in C57B1/6 mice in the presence and absence of NK cells.
  • Figure 5 Ex vivo cytotoxicity assays were performed on NK cells isolated from ORFV or PBS treated C57B1/6 mice.
  • FIG. 6 Figure 6 ORFV treatment of human xenograft tumours, (a) ORFV growth curves on A549 and nHDF cells were performed on a panel of human cancer cell lines at an MOI of 1. Cells were infected at time 0, and cell lysates collected and processed for virus by OA3.Ts plaque assay at time-points post infection. Virus titer was compared to input titers at 72 hours post infection to calculate the fold increase in ORFV.
  • FIG. 7 Sequence of amplified VV-E3L. Start site (ATG) and stop site
  • FIG. Sequence of C7L. Restriction sites used are in bold and underlined are the start (ATG) and stop (TAA) (SEQ ID NO:2).
  • FIG. 3 Sequence of K1L. Restriction sites used in bold and underlined are the start (ATG) and stop (TAA) (SEQ ID NO:3).
  • Figure 10 Characterization of recombinant OrfVmE3L by PCR analysis to confirm presence of inserted Vaccinia Virus (VV) E3L gene.
  • Figure 11 Characterization of recombinant OrfVmE3L by western blots analysis to confirm presence of inserted Vaccinia virus (VV) E3L gene.
  • Figure 12 Plaque phenotype assay, a) Visual representation of variability in plaque size at each time point, b) Plaque area of Wt OrfV and OrfVmE3L.
  • Figure 13 Infection Assay to compare the ability of OrfV and OrfVmE3L to productively infect a panel of cell lines.
  • Figure 16 In vivo lung metastasis model demonstrating tumour clearing capacity of OrfVmE3L.
  • parapoxvirus genus belongs to the Poxviridae family. Like all members of Poxviridae, parapoxviruses are oval, relatively large, double-stranded DNA viruses. Parapoxviruses have a unique spiral coat that distinguishes them from other poxviruses. Notable zoonotic hosts of Parapoxvirus include sheep, goats, and cattle. Parapoxviruses include orf virus, pseudocowpox, bovine papular stomatitis virus, parapoxvirus of red deer in New Zealand, and squirrel parapoxvirus. The term "parapoxvirus" refers to any parapoxvirus, such as orf virus, pseudocowpox, and bovine papular stomatitis virus.
  • Orf virus is a species of the Parapoxvirus genus of the Poxviridae family.
  • NZ-2 New Zealand-2
  • NZ-7 New Zealand-7
  • OV NZ2 OV NZ7
  • OV-SA00 OV-SA00
  • Orf virus has a worldwide distribution and causes dermal skin lesions in its natural host: goat and sheep, and occasionally humans. Typically, orf virus infections are initiated and maintained in the damaged skin, with no evidence of systemic spread. There have been no human deaths associated with the disease, even among immunocompromised patients (New England Joumal of Medicine 363(27):2621-7 and Clin Infect. Dis. 2007 Jun 1, 44(l l):el00-3 Epub 2007 Apr 19).
  • orf virus as a vaccine vector to protect animals from infections of other, more severe viruses has been the subject of study. However, prior to work by the present inventors, it is believed that no live, replicating orf virus had ever been shown to be useful in treating hyperproliferative conditions.
  • the present invention relates to use of an orf virus in the treatment of hyperproliferative conditions.
  • wild type orf virus strain NZ2
  • wild type orf virus strain NZ2
  • Orf virus may be as effective or may, in some circumstances, outperform other known replicating, established oncolytic viruses.
  • the in vivo anti cancer properties of Orf virus is at least partially replication independent, since the virus is able to effectively reduce tumours even when the virus is UV-inactivated (unable to replicate).
  • live replicating Orf virus has superior anti-cancer properties to its inactivated counterpart.
  • NK cells natural killer cells
  • the Applicants have shown for the first time that orf virus can dramatically activate a cytotoxic and cytokine-producing phenotype of NK cells, which in turn are at least partially responsible for clearing tumour cells from the animals.
  • This immune stimulation profile is unique to orf virus, making it an attractive immunotherapy cancer therapeutic.
  • the present invention relates to use of an orf parapoxvirus in the treatment of hyperproliferative conditions.
  • ORFV ORF virus
  • ORF virus ORF virus
  • the range of host cells that a virus can infect is called its "host range”. This can be narrow or, as when a virus is capable of infecting many species, broad.
  • host range gene refers to a gene encoding a gene product (i.e. protein), which is necessary such that a given virus having said host range gene is able to replicate on cells of species on which the virus does not replicate in the absence of the functional host range. If the respective host range gene is deleted the viral replication may be restricted to cells from fewer or only one animal species.
  • poxvirus refers to any poxvirus, such as avipoxvirus (including fowlpox virus), capripoxvirus (including sheeppox virus), leporipoxvirus (including myxoma virus), molluscipoxvirus (including molluscum contagiosum), orthopoxvirus (including vaccinia virus), parapoxvirus (including orf virus), suipoxvirus (including swinepox virus), yatapoxvirus (including yaba monkey tumour virus), entomopoxvirus A (including Melolontha melolontha entomopoxvirus), entomopoxvirus B (including Amsacta moorei entomopoxvirus) and entomopoxvirus C (including Chironomus luridus entomopo
  • poxvirus tropism (a process of tropism that determines which cells can become infected by a poxvirus) is dependent not upon specific cell surface receptors, but rather upon: (1) the ability of the cell to provide intracellular complementing factors needed for productive virus replication, and (2) the ability of the specific virus to successfully manipulate intracellular signaling networks that regulate cellular antiviral processes downstream of virus entry.
  • the large genomic coding capacity of poxviruses enables the virus to express a unique collection of viral proteins that function as host range factors, which specifically target and manipulate host signaling pathways to establish optimal cellular conditions for viral replication.
  • the known host range factors from poxviruses have been associated with manipulation of a diverse array of cellular targets, which includes cellular kinases and phosphatases, apoptosis, and various antiviral pathways.
  • Poxviruses As a family of viruses, poxviruses collectively exhibit a broad host range and most of the individual members are capable of replicating in a wide array of cell types from various host species, at least in vitro.
  • Poxviruses can, as a family, infect both vertebrate and invertebrate animals.
  • Orthopox includes smallpox virus (variola), vaccinia virus, cowpox virus, and monkeypox virus.
  • Yatapox includes tanapox virus and yaba monkey tumor virus.
  • Molluscipox includes molluscum contagiosum virus (MCV).
  • the host range genes may be from the Orthopoxvirus genus.
  • the vaccinia virus genes K1L, C7L and E3L have been shown that the expression of either K1L or C7L allows vaccinia virus replication in human MRC-5 cells; the E3L to gene was shown to be required for vaccinia virus replication in monkey Vero and human HeLa cells (Wyatt et al, Virology 1998, 251 : 334-342).
  • Fl 1L a host range gene from the Orthopoxvirus genus.
  • Fl 1L is a gene that was deleted from MVA (a vaccine version of vaccina virus that is very attenuated for its ability to replicate). MVA is considered a very safe virus, with limited pathogenicity - which is because at least most if not all of its 'host range genes' have been deleted from the virus. These include C7L and K1L, among many others. Fl 1L therefore may not technically be considered a host range gene as defined in the prior art, but certainly is broadly associated with this title since it is one of the genes that was deleted from MVA to make the virus more attenuated. The actual function of the gene is a motility protein (helps the virus move around from cell to cell). However, for the purpose of the present application, Fl 1L shall be considered to be a host range gene and falls within the scope of the definition provided above.
  • Examples of vaccinia virus host range genes also include the genes CI 8L,
  • orthopoxvirus host range genes include SPI-1, SPI-2, p28/NlR, B5R, and K3L.
  • host range genes from the Leporipox virus genus are contemplated. These include M-T2, M-T4, M-T5, M11L, M13L, M063, found in the Myxoma virus species.
  • the host range genes may be from the Orthopoxvirus genus and/or from the Leporipoxvirus genus.
  • Preferred heterologous host range genes are host range genes, heterologous to orf virus, that confer replication competency on orf virus, such that when inserted into the orf genome, they provide or improve the ability of orf virus to replicate in human cells.
  • Heterologous means derived from a different organism, in this case a different virus.
  • heterologous host range genes confer replication competency for orf virus on human cells, and are for example E3L, K1L and C7L.
  • human cells includes human cancer cells.
  • parapoxviruses also contain some host range genes (e.g. OV20.0L (a)
  • E3L homologue E3L homologue
  • these genes are adapted for replication in the natural hosts of these species (i.e. ungulates for orf virus infection).
  • parapoxvirus host range genes are not adapted to allow the viruses to replicate well in human cancer cells.
  • the recombinant ORFV may contain its natural host range genes. In an alternative embodiment, one or more the natural host range genes are not expressed.
  • Some of the host range genes prevent the activation of protein kinase R (PKR), both directly and indirectly. Inhibition of PKR serves to inhibit the cellular anti-viral response. In one aspect, inhibition of PKR, i.e. preventing its activation so that PKR cannot signal, is desirable for the rORF virus of the invention.
  • PPKR protein kinase R
  • the term "homologue of a host range gene” refers to a gene having a homology of at least 50%, preferably at least 70%, more preferably of at least 80%, 90%, 95%, 97%, 98%, or 99% in the coding part of the nucleotide sequence with a host range gene, wherein the "homologue of the host range gene” has the same, similar or identical biological function of a host range gene.
  • the biological function and definition of a host range gene is defined above. Specific tests how to determine whether a gene has the biological function of a host range gene are known to the person skilled in the art.
  • the Applicant have shown for the first time that the oncolytic potency of orf virus can be increased by transferring heterologous host range genes into the parapoxvirus genome.
  • Orf virus, and the parapoxvirus genus generally, lack a number of the vaccinia virus host range genes, which may be one of the reasons why orf virus does not replicate well on human cancer cells. Furthermore, the host range genes that are present in orf are not particularly effective. [0066] Thus, in one aspect the present invention provides a recombinant orf virus containing one or more host range genes or homologues thereof.
  • the host range genes are not orf host range genes.
  • the host range genes enable replication of the recombinant orf in humans. Examples include host range genes from poxviridae, such as from the
  • orthopoxvirus genus including vaccinia and other members of the orthopoxvirus genus, and from the leporipoxvirus genus, including myxoma virus.
  • such engineering allows the orf virus to better replicate in human cancer cells, while still maintaining the orf virus' unique immune stimulatory profile.
  • the recombinant orf virus may be prepared using techniques commonly known in the art.
  • the one or more host range genes can be introduced at any location within the orf virus genome, so long as such introduction(s) does not serve to disrupt necessary functions of the orf virus, and in particular does not disrupt the immune stimulatory effect of the orf virus.
  • the recombinant orf virus is an oncolytic virus.
  • Oncolytic viruses are viruses that preferentially infect and lyse cancer cells.
  • oncolytic viruses productively infect (i.e. replicate) in cancer cells. They may function by direct destruction of tumour cells, or, by indirect destruction using an immune-mediated attack. If modified, they can also function as vectors enabling genes expressing anti-cancer proteins to be delivered specifically to the tumor site.
  • the recombinant oncolytic virus of this disclosure may be administered in a convenient manner such as by the oral, intravenous, intra-arterial, intra-tumoral, intramuscular, subcutaneous, intranasal, intradermal, or suppository routes or by implantation (e.g., using slow release molecules).
  • the agents may be coated by, or administered with, a material to prevent inactivation.
  • the recombinant oncolytic virus of the present invention may also be administered parenterally or intraperitoneally.
  • Dispersions of the recombinant oncolytic virus component can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms, such as an antibiotic like gentamycin.
  • pharmaceutically acceptable carrier and/or diluent includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for biologically active substances is well known in the art. Supplementary active ingredients, such as antimicrobials, can also be incorporated into the compositions.
  • the carrier can be a solvent or dispersion medium containing, for example, water, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be effected by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the recombinant oncolytic viruses of the present disclosure in the required amount of the appropriate solvent with various other ingredients enumerated herein, as required, followed by suitable sterilization means.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the recombinant oncolytic virus plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically or veterinary acceptable carrier.
  • compositions comprising the recombinant oncolytic virus of this disclosure may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical viral compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries that facilitate formulating active recombinant oncolytic virus into preparations that can be used biologically or pharmaceutically.
  • the recombinant oncolytic virus compositions can be combined with one or more biologically active agents and may be formulated with a pharmaceutically acceptable carrier, diluent or excipient to generate pharmaceutical or veterinary compositions of the instant disclosure.
  • recombinant oncolytic virus compositions may be formulated with a pharmaceutically or veterinary-acceptable carrier, diluent or excipient is aqueous, such as water or a mannitol solution (e.g., about 1% to about 20%), hydrophobic solution (e.g., oil or lipid), or a combination thereof (e.g., oil and water emulsions).
  • a pharmaceutically or veterinary-acceptable carrier is aqueous, such as water or a mannitol solution (e.g., about 1% to about 20%), hydrophobic solution (e.g., oil or lipid), or a combination thereof (e.g., oil and water emulsions).
  • any of the biological or pharmaceutical compositions described herein have a preservative or stabilizer (e.g., an antibiotic) or are sterile.
  • the biologic or pharmaceutical compositions of the present disclosure can be formulated to allow the recombinant oncolytic virus contained therein to be bioavailable upon administration of the composition to a subject.
  • the level of recombinant oncolytic virus in serum, tumors, and other tissues after administration can be monitored by various well-established techniques, such as antibody-based assays (e.g., ELISA).
  • recombinant oncolytic virus compositions are formulated for parenteral administration to a subject in need thereof (e.g., a subject having a tumor), such as a non-human animal or a human.
  • Preferred routes of administration include
  • systemic formulations are an embodiment that includes those designed for administration by injection, e.g. subcutaneous, intra- arterial, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for intratumoral, transdermal, transmucosal, oral, intranasal, or pulmonary administration.
  • the systemic or intratumoral formulation is sterile.
  • the recombinant oncolytic virus compositions of the instant disclosure may be formulated in aqueous solutions, or in physiologically compatible solutions or buffers such as Hanks's solution, Ringer's solution, mannitol solutions or physiological saline buffer.
  • any of the recombinant oncolytic virus compositions described herein may contain formulator agents, such as suspending, stabilizing or dispersing agents.
  • penetrants, solubilizers or emollients appropriate to the harrier to be permeated may be used in the formulation.
  • l-dodecylhexahydro-2H-azepin-2-one (Azon®), oleic acid, propylene glycol, menthol, diethyleneglycol ethoxyglycol monoethyl ether
  • Administration can be achieved using a combination of routes, e.g., first administration using an intra-arterial route and subsequent administration via an intravenous or intratumoral route, or any combination thereof.
  • the present invention provides methods and uses involving the orf viruses of the invention in order to inhibit the growth of tumors, cancers, neoplastic tissue and other premalignant and non-neoplastic hyperproliferative disorders, all of which are together referred to as hyperproliferative disorders herein. Also included are methods of preventing metastasis and/or recurrence.
  • tumors, cancers and neoplastic tissue that can be treated by the present invention include but are not limited to malignant disorders such as lymphomas; ovarian cancers; breast cancers; osteosarcomas; angiosarcomas; fibrosarcomas and other sarcomas; leukemias; sinus tumors; uretal, bladder, prostate and other genitourinary cancers; colon esophageal and stomach cancers and other gastrointestinal cancers; lung cancers; myelomas; pancreatic cancers; liver cancers; kidney cancers; endocrine cancers; skin cancers; and brain or central and peripheral nervous (CNS) system tumors, malignant or benign, including gliomas and neuroblastomas.
  • malignant disorders such as lymphomas; ovarian cancers; breast cancers; osteosarcomas; angiosarcomas; fibrosarcomas and other sarcomas; leukemias; sinus tumors; uretal, bladder,
  • premalignant and nonneoplastic hyperproliferative disorders include but are not limited to myelodysplastic disorders; cervical carcinoma-in-situ;
  • familial intestinal polyposes such as Gardner syndrome; oral leukoplakias; histiocytoses; keloids; hemangiomas; hyperproliferative arterial stenosis; EBV-induced
  • lymphoproliferative disease hyperkeratoses and papulosquamous eruptions including arthritis, autoimmune disorders such as lupus, inflammatory arthritis, graft-vs-host disease.
  • the methods of treatment disclosed herein may be employed with any subject known or suspected of carrying or at risk of developing a hyperproliferative disorder as defined herein.
  • treatment refers to methods of killing, inhibiting or slowing the growth or increase in size of a body or population of hyperproliferative cells or tumor or cancerous growth, reducing
  • hyperproliferative cell numbers or preventing spread to other anatomic sites, as well as reducing the size of a hyperproliferative growth or numbers of hyperproliferative cells.
  • treatment is not necessarily meant to imply cure or complete abolition of hyperproliferative growths.
  • a treatment effective amount is an amount effective to result in the killing, the slowing of the rate of growth of hyperproliferative cells, the decrease in size of a body of hyperproliferative cells, and/or the reduction in number of hyperproliferative cells.
  • Subjects to be treated by the methods of the present invention include both human subjects and animal subjects for veterinary purposes.
  • Animal subjects are preferably mammalian subjects including horses, cows, dogs, cats, rabbits, sheep, and the like.
  • the present disclosure provides methods of inhibiting the growth or promoting the killing of a tumor cell or treating cancer, by administering a recombinant oncolytic virus according to the instant disclosure at a multiplicity of infection sufficient to inhibit the growth of a tumor cell or to kill a tumor cell.
  • the recombinant oncolytic virus is administered more than once, preferably twice, three times, or up to 10 times.
  • the tumor cell can be treated in vivo, ex vivo, or in vitro.
  • tumor cells or cancers examples include breast cancer (e.g., breast cell carcinoma), ovarian cancer (e.g., ovarian cell carcinoma), renal cell carcinoma (RCC), melanoma (e.g., metastatic malignant melanoma), prostate cancer, colon cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), bone cancer, osteosarcoma, rhabdomyosarcoma, leiomyosarcoma, chondrosarcoma, pancreatic cancer, skin cancer, fibrosarcoma, chronic or acute leukemias including acute lymphocytic leukemia (ALL), adult T-cell leukemia (T-ALL), acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphangiosarcoma, lymphomas (e.g., Hodgkin's and non-Hodgkin's lymphom
  • leukemia/lymphomas ATLL
  • entroblastic/centrocytic (eb/cc) follicular lymphomas cancers diffuse large cell lymphomas of B lineage, angioimmunoblastic lymphadenopathy (AILD)-like T cell lymphoma and HIV associated body cavity based lymphomas
  • AILD angioimmunoblastic lymphadenopathy
  • Castleman's disease Kaposi's Sarcoma, hemangiosarcoma, multiple myeloma,
  • the methods involve parenteral administration of a recombinant oncolytic virus, preferably via an artery or via an in-dwelling medical device.
  • the recombinant oncolytic virus treatment may be combined with surgery (e.g., tumor excision), radiation therapy, chemotherapy, or immunotherapy, and can be administered before, during or after a complementary treatment.
  • surgery e.g., tumor excision
  • radiation therapy e.g., chemotherapy, or immunotherapy
  • the oncolytic virus may be administered as a method for preventing or reducing surgery induced cancer metastasis, comprising administering to a patient, a therapeutically effective amount of the oncolytic parapoxvirus in combination with cancer surgery (e.g. tumour excision).
  • cancer surgery e.g. tumour excision
  • the oncolytic parapoxvirus can be administered before, during, or after a surgical cancer treatment, such that a metastasis is reduced or inhibited.
  • Wild type orf virus has been demonstrated to replicate in and kill approximately 50% of the 30 human cancer cell lines tested from the NCI-60 cell panel (see Table 1). The cell lines were screened by infecting at a multiplicity of infection
  • the concentration of orf virus was determined at each of the time points so as to conclude which cell lines were permissive to infection. A cut-off of more than 10-fold increase in orf virus particles was deemed significant. Interestingly, even in those cell lines deemed non-permissive to orf virus infection, the virus was still able to cause cell death in a percentage of those tested (data not shown). [0092] The concentration of Orf virus particles was determined at each time point, and the maximal fold increase in Orf virus production determined by comparing the highest Orf virus output to the input concentration determined from the concentration of Orf virus particles at the day 0 time-point. Each cell line was deemed permissive (+) or non-permissive (-) based on a greater than 10-fold increase in Orf virus concentration.
  • Table 1 In vitro screen of the NCI-60 cell panel with Orf virus.
  • OVCAR-8 Ovarian carcinoma - 8 2
  • Orf virus has the ability to replicate in and kill mouse cancer cell lines CT26-LacZ and BlFlO-LacZ in vitro.
  • live replicating virus performs better than inactivated virus is important.
  • Others have reported the use of chemically inactivated ORFV as an anti cancer agent.
  • the Applicant shows the benefit of using live replicating Orf virus as an oncolytic agent; the present invention has the added benefit of viral induced oncolysis.
  • Applicant demonstrates that live virus is also better at stimulating the immune system for added anti cancer benefit: refer to figures 4 and 5.
  • UV inactivated Orf virus does not reduce lung mestastases in the absence of NK cells, suggesting that the NK cell immune stimulation is likely attributed to a component of the virus particle, and is independent of productive viral infection.
  • NK cells NK cytotoxicity towards B16LacZ tumour cells was analyzed using an ex vivo killing assay (Figure 5).
  • Tumour naive C57B1/6 animals were treated intravenously with le7 UV inactivated orf virus, or Live orf virus or PBS control. 24 hours post treatment, animals were sacrificed, and spleens were harvested, and cell sorted for NK cells by DX5 + cell sorting.
  • NK cells were mixed with chromium labeled B16LacZ target cells in triplicate, at different effectortarget ratios, and the amount of cancer cell death was measured by the release of chromium into the media after a 4 hour incubation. Shown is the percent killing of target B16F10-LacZ cells at 24 hours post infection.
  • N 3, mean + SEM.
  • NK cells from Live and UV inactivated orf virus treated animals induce robust cytotoxicity towards B16LacZ targets at 24 hours post virus injection.
  • live Orf virus performs better than UV inactivated Orf virus.
  • Orf virus can specifically kill human tumour cells
  • Applicant also demonstrates that Orf virus has in vivo efficacy in an A549 xenograft model of human cancer in CD-I nude mice.
  • 2xl0 6 A549 cells were injected subcutaneously into the right flank of mice.
  • mice were treated intra-tumorally with 5 doses of 10 7 pfu Orf virus or PBS control.
  • Subcutaneous tumours were measured 2-3 times per week using digital calipers. Animals were sacrificed when tumour burden reached a volume of 1500 mm 3
  • VACV vaccinia virus
  • Wildtype parental NZ2 orf virus was obtained from Dr. Mercer at Otago in New Zealand.
  • pV41 cloning vector from Dr. Mercer was used as the backbone into which vaccinia Virus (VV) host range genes were introduced.
  • VV vaccinia Virus
  • the Applicant introduced vaccinia host range cDNA into the ankyrin-like region of the orf virus genome following the 11-10 locus.
  • Parental pV41 was digested with Xbal
  • E3L cDNA was amplified from vaccinia Western Reserve strain (purchased from ATCC) using primers:
  • FWD CCTCTAGAGAAACGACGAACCACCAGAG (SEQ ID NO:4)
  • REV GGCCAGATCTTCAGAATCTAATGATGAC (SEQ ID NO:5) allowing for the insertion of specific restriction sites for cloning into the pV41 vector.
  • Vaccinia virus host range genes were subcloned into the pT7-Blue-3 (Novagen) expression vector.
  • the pT7-Blue-3-mE3L vector was transformed into Nova Blue competent cells (Novagen), amplified and extracted. The insertion was sequenced to demonstrate proper amplification.
  • pT7-Blue-3-mE3L clone #9 was sequenced effectively (see Figure 7, SEQ ID NO: 1 ).
  • the vector of clone #9 was digested with Xbal (Invitrogen) and Bglll (NEB) and the vaccinia mE3L was isolated by gel extraction. The mE3L was then cloned into the pV41 cloning vector at the multiple cloning site.
  • pV41-mE3L-09_01 was transformed into Nova Blue competent cells (Novagen), amplified and extracted The pV41-mE3L-09_01 vector was linearized with Seal (Invitrogen).
  • 293T cells were infected at an MOI of 0.05 with wildtype orf virus and subsequently transfected with the linearized pV41-mE3L vector using Lipofectamine 2000 reagent (Invitrogen).
  • the recombinant pV41 vector introduces the vaccinia host range cDNA into the ankyrin-like region of the orf virus genome following the 11-10 locus.
  • Orf-mE3L was selected for using the X-gluc (Cedarlane) GUS reporter system ( ⁇ -glucuronidase) and plaque purified 3X on OA3.Ts cells.
  • the recombinant was filtered through a 0.22 ⁇ pore to eliminate any wildtype- recombinant aggregates. The pure recombinant was then grown on OA3.Ts and harvested using a standard virus manufacturing protocol.
  • C7L orf virus (orf-mC7L): C7L cDNA was amplified from vaccinia Copenhagen strain (ATCC) using:
  • C7L gene was PCR amplified and subcloned into the pT7blue.3 expression vector (Novagen).
  • pT7blue.3-mC7L vector was transformed into Nova Blue competent cells (Novagen). Clones were amplified and DNA was extracted to allow for sequencing of clones, to confirm proper orientation using SeqMan (DNASTAR).
  • pT7blue.3-mC7L1.3 was sequenced effectively ( Figure 8, SEQ ID NO:2).
  • pT7blue.3-mC7L1.3 was digested with Xbal (Invitrogen) and Bglll (NEB) and VV mCL7 was isolated by gel extraction. The mC7L fragment was then cloned into pV41 cloning vector.
  • pV41-mC7L1.3 vector was transformed into Nova Blue competent cells (Novagen), amplified and extracted.
  • pV41-mC7L1.3 vector was linearized with Xhol (Invitrogen). 293T cells (le6 cells/wells) were infected at an MOI of 5, 0.5, 1 and 0.1 with wildtype orf virus and subsequently transfected with either 1.6 ⁇ g or 3 ⁇ g linearlized pV41-mC7L1.3 vector using
  • Lipofectamine 2000 reagent (Invitrogen). Orf-mC7L is selected for by using the X-Gluc (Cedarlane) GUS reporter system ( ⁇ -glucuronidase) and is plaque purified 3X on OA3.Ts cells. The recombinant is filtered through a 0.22 ⁇ pore to eliminate any wildtype- recombinant aggregates. The pure recombinant is grown on OA3.Ts, harvested and purified using a standard virus manufacturing protocol.
  • K1L orf virus (orf-mKIL).
  • K1L cDNA was amplified from vaccinia Copenhagen strain (ATCC) using: forward primer: GGTCTAGAATGTTAACAAAAATGTGGGAG (SEQ ID NO: 8), and
  • K1L gene was PCR amplified and subcloned into the pT7blue.3 expression vector (Novagen).
  • pT7blue.3-mKlL vector was transformed into Nova Blue competent cells (Novagen). Clones were amplified and DNA was extracted to allow for sequencing of clones, to confirm proper orientation using SeqMan (DNASTAR).
  • pT7blue.3-mKlL.9 was sequenced effectively ( Figure 9, SEQ ID NO:3).
  • pT7blue.3-mKlL.9 is digested with Sacl (Invitrogen) and Kpnl (Invitrogen) and VV mKlL is isolated by gel extraction. The mKIL fragment is then cloned into pV41 cloning vector.
  • pV41-mKlL.9 vector is transformed into Nova Blue competent cells (Novagen), amplified and extracted.
  • pV41- mKlL.9 vector is linearized with Xhol (Invitrogen).
  • 293T cells (le6 cells/wells) are infected at an MOI of 5, 0.5, 1 and 0.1 with wildtype orf virus and subsequently transfected with either 1.6 ⁇ g or 3 ⁇ g linearlized pV41-mKlL.9 vector using
  • Orf-mKIL Lipofectamine 2000 reagent (Invitrogen). Orf-mKIL is selected for by using the X-Gluc (Cedarlane) GUS reporter system ( ⁇ -glucuronidase) and is plaque purified 3X on OA3.Ts cells. The recombinant is filtered through a 0.22 ⁇ pore to eliminate any wildtype- recombinant aggregates. The pure recombinant is grown on OA3.Ts, harvested and purified using a standard virus manufacturing protocol.
  • Orf virus recombinants Other recombinant Orf viruses containing one or more heterologous host range genes can readily be manufactured by a person of the art using similar methods to those described above.
  • VV-E3L Characterization of recombinant OrfVmE3L by western blots analysis to confirm presence of inserted Vaccinia virus (VV) E3L gene.
  • the VV-E3L protein was demonstrated to be present in VV Western Reserve (positive control), as well as, OrfVmE3L, but not in the parental OrfV.
  • HeLa cells were seeded at le6 cells/well in 6-well dishes. The next day, the wells were infected at MOI 1 with either Vaccinia Virus Western Reserve strain (positive control), OrfV (negative control) or recombinant OrfVmE3L. 24 hours post infection, whole cell protein lysate was collected using NP-40 lysis buffer with complete protease inhibitor and complete phosphatase inhibitor.
  • Proteins were separated by SDS-PAGE (Bio-Rad), transferred to PVDF (Amersham Biosciences) and subsequently blocked with 5% (wt/vol) skim milk. The membrane was then probed with mouse monoclonal E3 antibody (1 :500; a gift from Stuart Isaacs, University of Pennsylvania). B-Tubulin was used as a loading control).
  • the Applicant compared the size of virus plaque on primary ovine testis cells (OA3.Ts) over time ( Figure 12).
  • OrfVmE3L The entire contents of each well was harvested 72 h.p.i. and subsequently titred to quantify viral output.
  • infection was not enhanced as a result of the E3L mutation, while on cancer cell lines HeLa and CT2A as well as goat cell line OA3t.s, increased viral replication of mE3L ORFV was demonstrated.
  • OrfVmE3L demonstrated an enhanced level of replication as well as a greater host cell range when compared to Wt ORFV in the cancer and sheep cell lines, but was not enhanced in the normal human cell line.
  • Figure 14 shows the results of a comparison of recombinant OrfV mE3L killing ability to parental OrfV.
  • HeLa cells and BHK21 cells were seeded at le4 cells/well in 96-well dishes. The next day, HeLa cells and BHK21 cells were infected at MOI 0.1 and 1, respectively, with either OrfV or recombinant OrfVmE3L. 72 hours post infection, cytotoxicity was assessed using alamar blue reagent., OrfVmE3L induces significantly more cell death in HeLa and BHK21 cells compared to WT Orf virus in vitro at 72 hours post infection.
  • OrfVmE3L virus a spreading assay was performed ( Figure 15). HeLa and nHDF cells were seeded in 12 well dishes to produce confluent monolayers. Cells were infected at an MOI of 0.01 and 0.005. Wells were stained with X-gluc 24 hpi and pictures were taken at 48 and 72 hpi. OrfVmE3L more readily infects and spreads in the cancer cells than in normal cells, as demonstrated by X-gluc staining (blue) of viral replication.
  • FIG. 16 shows an in vivo lung metastasis model demonstrating in vivo anti -tumour capacity of OrfVmE3L.
  • CT26LacZ and B16LacZ cells were injected IV (intra-venously) into Balb/c and C57B1/6 mice, respectively.
  • mice were either treated with le7 pfu OrfV or OrfVmE3L or PBS.
  • Mice were sacrificed on day 11, lungs harvested, stained with X-Gal and lung mets were enumerated.
  • OrfVmE3L significantly reduced tumor burden in both models compared to PBS controls.

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Abstract

La présente invention concerne un virus orf recombinant, et son utilisation et des méthodes impliquant un tel virus orf recombinant dans le traitement de troubles d'hyperprolifération. Le virus orf recombinant contient un ou plusieurs gènes hétérologues de la gamme des hôtes. De façon avantageuse, de telles modifications génétiques permettent au virus orf de mieux se répliquer dans des cellules humaines, tout en conservant le profil immunostimulateur unique du virus orf.
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JP2018516599A (ja) * 2015-05-29 2018-06-28 コーロン ライフ サイエンス インコーポレイテッドＫｏｌｏｎ Ｌｉｆｅ Ｓｃｉｅｎｃｅ， Ｉｎｃ． ポックスウイルス由来のプロモーターおよびそれを含むベクター
CN112522215A (zh) * 2019-09-18 2021-03-19 重庆市畜牧科学院 一种缺失008基因的重组羊口疮病毒及其构建方法
WO2022033573A1 (fr) 2020-08-13 2022-02-17 苏州般若生物科技有限公司 Virus de la dermatite pustulaire infectieuse d'ovis spp. mutant et son utilisation
US11433108B2 (en) * 2016-03-28 2022-09-06 Suzhou Prajna Biotech Co., Ltd. Anti-cancer oncolytic virus combination therapies and elite responder selection platforms

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Cited By (8)

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Publication number Priority date Publication date Assignee Title
JP2016501538A (ja) * 2012-12-21 2016-01-21 オタワ ホスピタル リサーチ インスティチュート 非複製型ウイルス由来粒子及びその使用
JP2020014467A (ja) * 2012-12-21 2020-01-30 セルベルム インコーポレイテッドＣｅｌｖｅｒｕｍ Ｉｎｃ． 非複製型ウイルス由来粒子及びその使用
US11110138B2 (en) 2012-12-21 2021-09-07 Celverum Inc. Non-replicating virus-derived particles and uses thereof
JP2018516599A (ja) * 2015-05-29 2018-06-28 コーロン ライフ サイエンス インコーポレイテッドＫｏｌｏｎ Ｌｉｆｅ Ｓｃｉｅｎｃｅ， Ｉｎｃ． ポックスウイルス由来のプロモーターおよびそれを含むベクター
US11433108B2 (en) * 2016-03-28 2022-09-06 Suzhou Prajna Biotech Co., Ltd. Anti-cancer oncolytic virus combination therapies and elite responder selection platforms
CN112522215A (zh) * 2019-09-18 2021-03-19 重庆市畜牧科学院 一种缺失008基因的重组羊口疮病毒及其构建方法
CN112522215B (zh) * 2019-09-18 2023-06-02 重庆市畜牧科学院 一种缺失008基因的重组羊口疮病毒及其构建方法
WO2022033573A1 (fr) 2020-08-13 2022-02-17 苏州般若生物科技有限公司 Virus de la dermatite pustulaire infectieuse d'ovis spp. mutant et son utilisation

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