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WO2005111200A1 - Souches de reovirus et procedes d'utilisation de celles-ci - Google Patents

Souches de reovirus et procedes d'utilisation de celles-ci Download PDF

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WO2005111200A1
WO2005111200A1 PCT/CA2005/000749 CA2005000749W WO2005111200A1 WO 2005111200 A1 WO2005111200 A1 WO 2005111200A1 CA 2005000749 W CA2005000749 W CA 2005000749W WO 2005111200 A1 WO2005111200 A1 WO 2005111200A1
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reovirus
isolate
cells
interferon
seq
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Guy Lemay
Carole Danis
Penny Rudd
Sapha Barkati
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Universite de Montreal
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Universite de Montreal
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • 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
    • A61K35/765—Reovirus; Rotavirus
    • 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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12022—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
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12032—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • the present invention relates to novel strains of reoviruses and methods of uses thereof. More specifically, the present invention relates to a reovirus strain that is hypersensitive to interferon and more dependent of the transformed status of the host cells for its replication. This strain represents a promising alternative to wild type reoviruses for application as oncolytic agents in a clinical setting.
  • viruses are not only foes to fight but can become allies in our fight against diseases.
  • One field where viruses can be used is in the treatment of cancer as "so-called” oncolytic viruses.
  • reoviruses Classical mammalian Orthoreoviruses, referred to as “reoviruses”, are among candidates for such an application in oncolytic therapy against cancer and some of them are presently under clinical studies. These viruses present the advantage of being naturally oncolytic, while most other viruses require the selective inactivation of viral genes in order for them to acquire an adequate therapeutic index (i.e. an ability to replicate in and destroy specifically cancer cells and not (or only to an acceptable lower extent) their normal counterparts). Mammalian reoviruses exhibit low, or are devoid of pathogenic potential in human adults an advantage in any future therapeutic use.
  • Any oncolytic virus must be able to efficiently destroy cancer cells without causing pathology by indiscriminately also destroying normal untransformed cells counterparts of the targeted cancer cells.
  • the discrimination exhibited by any virus, or viral isolate may depend on the nature of the alterations found in the cancer cells, the nature of the virus itself and its sensitivity to resistance mechanisms present in untransformed cells.
  • PKR biological active PKR
  • normal untransformed cells such as "flat" mouse NIH-3T3 fibroblasts exhibiting highly developed contact inhibition of the cellular monolayer in cultured cells.
  • PKR inhibits the multiplication of certain reovirus and this inhibition is generally believed to occur via an increased phosphorylation level of the cellular elF-2 translation initiation factor.
  • PKR inhibition was shown to rely mostly on the synthesis of the ⁇ 3 viral protein that competes with PKR for the dsRNA activator (3,4,15,17,18,24,33).
  • the resistance conferred by the protein is certainly incomplete since reoviruses are not protected against high levels of PKR activity.
  • the ⁇ 3 protein forms a heterohexameric structure with the ⁇ 1 protein to make the outer capsid of the virion.
  • Interferon and PKR sensitivity of a given virus isolate could thus depend on the relative affinity of ⁇ 3 to bind either double-stranded RNA or ⁇ 1 , and could also varies depending on the amount and nature of ⁇ 1 (4,24).
  • the present invention concerns the novel finding that an increased interferon sensitivity of viruses correlates with their increased ability to discriminate between normal and Ras-transformed cells (23).
  • the present invention concerns isolates of mammalian reovirus
  • the present invention concerns a reovirus strain having a high sensitivity to interferon, the P4L-12 virus strain, which constitutes an attractive candidate for cancer treatment and for easy elimination by interferon post treatment (23).
  • the present application is the first to present a reovirus isolate having better ability than wild-type reovirus to discriminate between parental and Ras-transformed cells.
  • the present application is the first to present a reovirus isolate with apparently better replicative ability in Ras-transformed cells than wild-type reoviruses.
  • the P4L-12 virus could be easier to eliminate from inoculated individual using interferon treatment, an additional safety measure.
  • transformed cells and “cells expressing an activated Ras pathway”, refers to cells expressing characteristics traditionally associated with tumor cells via expression of oncogenic Ras or any other indirect activation of the Ras pathway leading to cellular transformation.
  • untransformed cells As used herein the terminologies "untransformed cells”, “normal cells” and “parental cells” refer to cells that do not express an activated Ras pathway nor characteristics traditionally associated with tumor cells.
  • isolate and “strain” are used interchangeably to refer to biologically-cloned viruses obtained by any known method including but not limited to consecutive limiting dilution and plaque purification .
  • a “mutation” is a change in the genetic material of a biological entity which can be transmitted to its descendent.
  • a mutation can be, for example, a change in one or more nucleotides.
  • nucleotides can be added, deleted or substituted for another nucleotide.
  • Spontaneous mutations and experimentally induced mutations exist.
  • a mutant polypeptide can be encoded from this mutant nucleic acid molecule.
  • a "mutant" of the novel strains of reoviruses of the present invention namely the P4L-12 isolate deposited in the International Depository Authority of Health Canada in Winnipeg under access no: IDAC 059 on May 20, 2004; the P4SC1-31 isolate, and the P4SC1-42 isolate may or may not have the same identifying biological characteristics of these isolates, as long as the mutant possesses the desirable oncolytic abilities of their parent strains.
  • Suitable methods for preparing mutants and variants of the inventive strain include, but are not limited to: mutagenesis by irradiation with ultraviolet light or X-rays, or by treatment with a chemical mutagen such as nitrosoguanidine (N-methyl-N'-nitro-N- nitrosoguanidine), methylmethanesulfonate, nitrogen mustard and the like. These methods are well known in the art and are described, for example in the case of reoviruses (9,14,22).
  • mutants of reoviruses encompassed within the present invention may comprise a mutation at position 221 of ⁇ 3 according to the numbering presented in SEQ ID NO: 1 and at position 315 of ⁇ l according to the numbering presented in SEQ ID NO: 4. They may further comprise mutations in ⁇ 3 responsible for resistance to E-64 which further enhance their ability to discriminate.
  • Mutated strains derived from the P4L-12 strain using known methods are then preferably selected or screened for improved ability to discriminate between Ras transformed cells and their untransformed counterparts, for their improved virulence towards cancer cells, or for other desirable properties related to their oncolytic potential towards mammal cancer cells and more particularly human cancer cells.
  • mutagenized viruses are selected on the basis of their improved ability to discriminate between transformed cells and their untransformed counterparts, their improved virulence towards cancer cells and/or their larger spectrum of action on various cancer cells.
  • a reovirus exhibiting hypersensitivity to interferon and/or improved ability to discriminate between cells expressing an activated Ras pathway and their untransformed counterparts which comprises at least one of a S4 gene which hybridizes under highly stringent conditions to a hybridization probe, the nucleotide sequence of which consists of SEQ ID NO: 7 and a M2 gene which hybridizes under highly stringent conditions to a hybridization probe, the nucleotide sequence of which consists of SEQ ID NO: 8.
  • the reovirus comprises a M2 gene which hybridizes under highly stringent conditions to a hybridization probe, the nucleotide sequence of which consists of SEQ ID NO: 8 and a S4 gene which hybridizes under highly stringent conditions to a hybridization probe, the nucleotide sequence of which consists of SEQ ID NO: 7.
  • the reovirus comprises a nucleotide sequence encoding a protein selected from the group consisting of 1) a ⁇ 3 protein comprising the polypeptide as set forth in SEQ ID NO: 3 or 2) a ⁇ 1 sequence protein comprising the polypeptide as set forth in SEQ ID NO: 6.
  • the reovirus comprises a nucleotide sequence encoding a ⁇ 3 protein comprising the polypeptide as set forth in SEQ ID NO: 3 and a ⁇ 1 sequence protein comprising the polypeptide as set forth in SEQ ID NO: 6.
  • a biologically pure isolate of a virus strain comprising all of the identifying characteristics of the P4L-12 strain deposited at the International Depository Authority of Health Canada in Winnipeg under accession number IDAC059, or a mutant thereof derived from said isolate.
  • a biologically pure isolate of a virus strain comprising all of the identifying characteristics of the P4SC1-31 strain or a mutant thereof derived from said isolate.
  • a biologically pure isolate of a virus strain comprising all of the identifying characteristics of the P4SC1 -42 strain.
  • a pharmaceutical composition comprising a reovirus isolate of the present invention, and a pharmaceutically acceptable carrier or excipient.
  • a method for lysing mammalian cancer cells in vitro comprising applying a cytotoxic amount of an isolate of the present invention on mammalian cells expressing an activated Ras pathway.
  • the mammalian cells expressing an activated Ras pathway are human cells expressing an activated Ras pathway.
  • a method for lysing mammalian cells expressing an activated Ras pathway in vivo comprising applying a cytotoxic amount of an isolate of the present invention to a mammal suspected of having cells expressing an activated Ras pathway.
  • the method further comprises a step of eliminating residual isolate from said mammal comprising administration of interferon to said mammal.
  • the mammal is a human.
  • the isolate is P4L- 12.
  • a method of identifying an isolate of a mammalian reovirus mutant having an increased ability to discriminate between cells expressing an activated Ras pathway and their normal counterparts comprising comparing the sensitivity to interferon of a candidate reovirus isolate to that of a wild-type reovirus isolate, wherein the candidate reovirus isolate is selected if it is more sensitive to interferon than the wild-type reovirus isolate.
  • the step of comparing the sensitivity to interferon of a candidate reovirus isolate to that of a wild-type reovirus isolate is performed in L929 cells.
  • a method of identifying an isolate of a mammalian reovirus mutant having an increased ability to discriminate between cells expressing an activated Ras pathway and their normal counterparts comprising identifying a reovirus which hybridizes to at least one hybridization probe selected from the group consisting of a first probe, the nucleotide sequence of which consists of SEQ ID NO: 7 and a second probe, the nucleotide sequence of which consists of SEQ ID NO: 8.
  • a method of selecting a reovirus strain for treating a tumor in a patient in need thereof comprising determining whether said tumor comprises cells expressing an activated Ras pathway wherein if said tumor comprises cells expressing an activated Ras pathway, a treatment comprising the administration of a reovirus of the present invention is selected.
  • a use of a reovirus isolate of the present invention for lysing cells expressing an activated Ras pathway is provided.
  • a reovirus isolate of the present invention in the manufacture of a medicament for lysing cells expressing an activated Ras pathway.
  • a method for lysing mammalian cancer cells comprising applying a cytotoxic amount of an isolate according to the present invention on mammalian cancer cells.
  • the isolate is P4L-12 and the mammalian cancer cells expresses Ras.
  • the method further comprises a step of eliminating residual P4L-12 isolate from the treated individual or animal comprising administration of interferon following treatment or during treatment.
  • a method of identifying an isolate of a mammalian reovirus mutant having an increased ability to discriminate between cancer cells and their normal counterparts comprising comparing the sensitivity to interferon of a candidate reovirus isolate to that of a wild-type reovirus isolate, wherein the candidate reovirus isolate is selected if it is more sensitive to interferon than the wild-type reovirus isolate.
  • a method of identifying an isolate of a mammalian reovirus mutant that preferentially replicates in Ras-transformed cells comprising comparing the sensitivity to interferon of a candidate reovirus isolate to that of a wild-type reovirus isolate, wherein the candidate reovirus isolate is selected if it is more sensitive to interferon than the wild-type reovirus isolate.
  • a method of identifying a factor influencing the ability of a reovirus isolate to discriminate between normal and Ras-transformed cells comprising identifying genetic alterations in the nucleotide sequence or alteration in the protein expression of a candidate reovirus isolate having a sensitivity to interferon that differs from that of wild-type reovirus isolate by comparing the nucleotide sequence or the protein expression of the candidate reovirus isolate with that of wild-type reovirus isolate.
  • the present invention therefore also relates to the use of the reovirus isolates of the present invention for determining the nature of the genetic alterations responsible for reovirus sensitivity to interferon and preferential ability to multiply in Ras-transformed cells and its increased virulence as compared to wild-type reoviruses towards these cells.
  • the present invention is especially concerned with the use of P4L- 12 strain for this purpose.
  • the present invention relates to methods of optimizing reovirus sensitivity to interferon and discrimination between normal and cancer cells by combining different mutations in ⁇ 3 encoding and ⁇ 1 encoding genes.
  • the present invention concerns combining mutations that affect virus uncoating with those mutations in P4L-12 that affect the discrimination between parental and Ras-transformed cells based on the different sensitivity to interferon.
  • the recombinant reovirus could be generated by co-infection of mammalian cells with different subtypes of reoviruses or by screening mutants resistant to E64 from P4L-12.
  • the present invention also concerns the use of the different viral isolates described herein, especially the interferon hypersensitive P4L-12 virus strain to determine their replicative potential in different transformed cells, especially of human origin.
  • P4L-12 appears to be the most interesting virus for future clinical applications, it cannot be excluded that viruses with intermediate sensitivity could be appropriate in specific cancer cell types.
  • the present invention therefore also concerns the use of the intermediate sensitivity novel viral isolates to determine their potency in various animals tumor cells, especially urine, models of tumors produced by inoculation of murine or human tumor cells.
  • reovirus refers to any mammalian or avian reovirus classified in the Orthoreovirus genus, whether naturally occurring, modified or recombinant. In more specific embodiments, mammalian reoviruses are used.
  • a "mammal suspected of having a proliferative disorder” is meant to refer to a mammal that may have a proliferative disorder or tumor or has been diagnosed with a proliferative disorder or tumor or has been previously diagnosed with a proliferative disorder or tumor, the tumor or substantially all of the tumor has been surgically removed and the mammal is suspected of harboring some residual tumor cells.
  • hyposensitivity to interferon is defined as a sensitivity to interferon that is higher than that of wild-type H8-2 and than that of original stock of reovirus serotype 3 Dearing obtained from the American Type Culture Collection.
  • the term "improved ability to discriminate between Ras transformed cells and their untransformed counterparts” is defined as an ability to discriminate such cells that is higher than that of H8-2 or than that of original stock of reovirus serotype 3 Dearing obtained from the American Type Culture Collection (ATCC VR824).
  • compositions which contain, as the active ingredient, one or more of the reoviruses associated with a "pharmaceutically acceptable carrier or excipient".
  • the active ingredient reovirus is usually mixed with an excipient, diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule, sachet, paper or other container.
  • the pharmaceutically acceptable excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient.
  • compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
  • excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose.
  • the formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy- benzoates; sweetening agents; and flavoring agents.
  • the compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
  • compositions of the present invention preferably contain about 10 2 pfus to about 10 13 pfus of the reovirus.
  • the predetermined quantity of reovirus is calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • compositions of the present invention can be administered orally, intravenously, intraperitoneally, intracranially intranasally, intramuscularly, subcutaneously, sublingually, intrathecally, or intradermally.
  • the route of administration can depend on a variety of factors, such as the environment and therapeutic goals.
  • mammals generally refer to humans, but also can include domesticated mammals (e.g., dogs, cats, and livestock such as cows, horses, pigs, and sheep) in which prevention, delay or treatment of proliferative diseases such as cancer is desirable.
  • composition of the present invention may be administered in conjunction with removal of the tumor. It may also be administered in combination with any other known anticancer therapy including radiotherapy, chemotherapy, interferon treatment, interleukin treatment or any other immunotherapeutic approach.
  • high stringency conditions are meant to refer to conditions enabling only sequences with a high homology to bind. Without being so limited, examples of such conditions are listed In the handbook "Molecular cloning, a laboratory manual, second edition of 1989 from Sambrook et al.: 6XSSC or 6XSSPE, Denhardt's reagent or not, 0.5% SDS and the temperature used for obtaining high stringency conditions is most often in around 68°C (see pages 9.47 to 9.57 of Sambrook and particularly pages 9.56 and 9.57) oligonucleotides.
  • the optimal temperature to be used for a specific nucleic acid probe may be empirically calculated, and although there is room for alternatives in the buffer conditions selected, within these very well known condition ranges, the nucleic acid captured will not vary significantly. Indeed, Sambrook clearly indicates that the "choice depends to a large extent on personal preference" (see page 9.47).
  • the present invention seeks to meet these needs and other needs.
  • Figure 1 graphically illustrates the effect of interferon on reovirus isolates replication in L929 cells through reduction in virus titer
  • Figure 2 shows a comparison of the multiplication of reovirus in Ras- transformed and parental NIH-3T3 cells as detected by immunoblotting of proteins recovered from infected cells. Position of ⁇ 1C, the major viral outer capsid protein, is indicated;
  • FIG. 3 Panel A shows infectious virus production in parental and Ras- transformed NIH-3T3 cells as determined by viral titration; Panel 3B shows viral release in the supernatant of the same cells as detected by immunoblotting;
  • Figure 4 shows the effect of interferon on H8-2 and P4L-12 in parental and Ras-transformed cells as detected by metabolic radiolabeling of proteins.
  • the positions of major viral proteins ( ⁇ class, ⁇ 1C and ⁇ 3) are indicated;
  • Figure 5 compares the replicative ability of wild type virus isolate H8-2 and of the P4L-12 isolate on primary human peripheral blood mononuclear cells (PBMCs) using immunoblotting in panel A and metabolic radiolabeling in panel B. The position of the ⁇ 3 viral protein is indicated;
  • PBMCs peripheral blood mononuclear cells
  • Figure 6 shows the effect of the cathepsin inhibitor E-64 on viral replication in L929 cells using metabolic radiolabeling. The position of the ⁇ 3 viral protein is indicated;
  • Figure 7 compares the amino acid sequence of the ⁇ 3 protein of the P4L-12 (SEQ ID NO: 1) isolate with that of the H8-2 (SEQ ID NO: 2) and of a consensus sequence (SEQ ID NO: 3) derived from publicly available ⁇ 3 sequences;
  • Figure 8 compares the amino acid sequence of the ⁇ l protein of the P4L-12 (SEQ ID NO: 4) isolate with that of the H8-2 (SEQ ID NO: 5) and of a consensus sequence (SEQ ID NO: 6) derived from publicly available ⁇ l sequences.
  • MRV-3 mammalian reovirus serotype 3, strain Dearing, genus Orthoreovirus, family Reoviridae isolates differing in interferon sensitivity were obtained and their ability to replicate and destroy parental or Ras-transformed NIH-3T3 cells were tested.
  • reovirus isolates differing in their interferon sensitivity are presented that were obtained by chemical mutagenesis. Their replicative properties in parental and Ras-transformed mouse NIH-3T3 cells are also presented. It was observed that most isolates can bypass resistance mechanisms of parental cells at high multiplicity of infection and that there is at least a partial correlation between the ability to discriminate between transformed and parental cells and this isolates' interferon sensitivity. Most interestingly, an interferon-hypersensitive mutant virus was more dependent on Ras activation than any other viral isolate. Altogether, this suggests that optimal viral isolates could be selected to attack tumoral cells depending on these cells' level of PKR expression and/or activation as well as that of upstream or downstream factors affecting the ability of PKR to influence reovirus' replication.
  • Mouse fibrobiasts L929 cells were originally obtained from the American Type Culture Collection (ATCC) and were propagated in MEM medium supplemented with 5% fetal bovine serum.
  • SC1 feral mouse embryo fibrobiasts cells were also obtained from the American Type Culture Collection (Rockville, MD) and propagated in MEM medium supplemented with 1% non- essential amino acids and 5% fetal bovine serum.
  • Parental NIH-3T3 murine fibrobiasts cells and their Harvey-Ras G72, -transformed counterparts (herein referred to as Ras-transformed) were a generous gift from Dr. Robert Nabi (Departement de Pathologie et biologie cellulaire, Universite de Montreal).
  • Viruses [0073] Original stock of reovirus serotype 3 (Dearing) was originally obtained from the American type culture collection (ATCC). The PI3-1 virus exhibiting a Y354H amino acid substitution in the ⁇ 3 protein was obtained from Dr Terence S. Dermody (Vanderbilt University) and was described as exhibiting an increased kinetic of disassembly of its outer capsid protein and resistance to lysosomal proteases inhibitors such as E-64 (2,8,30). All virus stocks were propagated at a multiplicity of infection of one plaque forming unit (PFU)/cell in mouse L929 cells.
  • PFU plaque forming unit
  • EXAMPLE 1 Generation of viral isolates [0074] Chemical mutagenesis was used to obtain a panel of viral isolates that differ in their sensitivity to interferon. It was reasoned that these viruses should also differ in their sensitivity to PKR, and therefore in their ability to discriminate between normal and transformed (or cancerous) cells. The classical reovirus serotype 3 Dearing was chosen for mutagenic treatment.
  • EXAMPLE 2 Isolation of viral cloned isolates [0077] All viral isolates were cloned using two consecutive cloning steps. First, viral stocks were diluted and plated in 96-wells microplates seeded with L929 cells such as to obtain less than 50% of visibly infected wells after 7 days. Viruses were then propagated on L929 cells monolayers and recloned by plaque formation on L929 cells monolayers overlaid with agar. Isolated plaques were picked and virus propagated again in L929 cells. Final viral stocks were titered using the TCID 50 method on L929 cells (7).
  • L929 cells (5 X 10 5 cells seeded in a 60 mm diameter petri dish) were pretreated with a saturating concentration of 500 international units (IU)/ml of mouse ⁇ -interferon (Calbiochem, La Jolla CA) for 24 hours before being infected at an MOI of 10 PFU/cell. Infected cells were incubated in MEM containing 2% heat-decomplemented fetal bovine serum and interferon concentration was maintained at the same level.
  • Nuclei were pelleted by centrifugation (10 000 rpm for 20 minutes) and supernatant recovered for analysis by SDS-PAGE (8% acrylamide-bis-acrylamide gel) and autoradiography on a Kodak X-omatTM AR film (Amersham, Piscataway NJ).
  • Sensitivity to interferon was further examined by quantitation of the decrease in virus titer in interferon-treated L929 compared to that in non treated cells as graphically illustrated in Figure 1.
  • Viral stocks prepared from infected L929 cells were titered by TCID5 0 on L929 cells, as described in Example 3.
  • Treatment with interferon at 500IU/ml was performed 24 hours before titration in 96-wells microplate. Results are presented as the average of three independent experiments with errors bars representing standard deviation of the mean.
  • Infected cells were incubated in MEM containing 2% heat-decomplemented fetal bovine serum.
  • TBS-NP40 TBS-NP40 and analysis was achieved by using a rabbit antireovirus ahtiserum followed by a phosphatase-conjugated anti-rabbit immunoglobulin antibody (1/3000 Cedarianes Hornby ON). Antigen-antibody complexes were detected using NBT-BCIP substrate (InVitrogen life technologies, Burlington ON).
  • EXAMPLE 7 Infection of Ras-transformed cells by the viral isolates measured by viral assembly and release [0093] Virus release was also examined in infected cells by immunoblotting of cell supernatant of cells infected as in Example 6 (Fig. 3, panel B). A small aliquot of the tissue culture medium infected at either a MOI of 10 or 250 was collected and analyzed by immunoblotting using antireovirus antiserum followed by peroxydase conjugated secondary antibody and enhanced chemiluminescence detection.
  • NIH-3T3 cells as judged by phase contrast microscopy and trypan blue staining.
  • cytopathic effect was only apparent at high multiplicity of infection, trypan blue staining indicated between 30 and 50% of cell death in these conditions.
  • Virus release was seen in Ras-transformed cells for both viruses at high MOI while it was only observed for the P4L-12 virus at low MOI.
  • parental cells only the H8-2 virus was detected in the medium at high MOI.
  • Example and Example 6 confirm that the P4L-12 isolate is better suited to Ras-transformed cells not only in terms of protein synthesis level but also in terms of virus assembly and release.
  • EXAMPLE 8 Effect of interferon in parental and Ras-transformed NIH-3T3 cells [0096] The effect of interferon on the wild type H8-2 and the P4L-12 isolate in parental as compared to Ras-transformed cells was also assessed. ( Figure 4).
  • Panel B cells were treated 24 hours prior infection and during infection so as to maintain antiviral response with 250 lU/ml of interferon. Proteins were analyzed by metabolic radiolabeling at 48 hours post-infection followed by SDS-PAGE and autoradiography.
  • the P4L-12 virus isolate appears to be more strongly restricted than a wild type isolate to untransformed mouse NIH-3T3 cells.
  • P4L-12's ability to discriminate between parental and transformed mouse fibrobiasts cells may not be completely predictive of the situation occurring in human cells.
  • Systemic inoculation could be a very interesting route of administration to ensure that even sites distal to the tumor are reached by the virus (12). With that route, human blood cells will be the first putative target encountered by the virus. The ability of the virus to infect normal blood cells should thus be minimized in an effort to prevent side effects or uncontrolled viral replication.
  • Panel A cells were infected at a MOI of 40 and recovered 48 hours later for analysis by immunoblotting using a monoclonal anti- ⁇ 3 antibody (4F2, obtained from Dr. Kevin M. Coombs) followed by a phosphatase conjugated secondary antibody. Antigen-antibody complexes were revealed with NBT/BCIP substrate. The control was a small aliquot of purified reovirus serotype 3, showing the position of the ⁇ 3 protein. Cells were similarly infected at different multiplicities of infection or left uninfected (MOI of 0) (Panel B). Metabolic radiolabeling was performed 48 hours post-infection.
  • L929 cells were thus seeded at 2,5 X 10 5 cells per 60 mm- diameter petri dish and treated the next day for two hours with 100 ⁇ M of E-64 (Sigma-Aldrich, Oakville, Ontario) before being infected at a multiplicity of infection of 40 with either the H8-2 or P4L-12 viral isolates or the control PI3-1 virus known to exhibit relative resistance to E-64.
  • E-64 Sigma-Aldrich, Oakville, Ontario
  • cells were fed with medium containing E-64 and incubated for 22 hours at 37°C before being analyzed for virus infection. Synthesis of viral proteins was detected by metabolic radio labeling. Results are shown on Figure 6 where the position of the ⁇ 3 outer capsid protein is indicated. Non-infected cells were similarly treated as a control.
  • L929 cells were infected at a multiplicity of infection of 10 for approximately 20 hours and recovered by scraping. Virions were extracted by Freon using standard procedures (27). Double-stranded genomic RNA was obtained by SDS-phenol-chloroform extraction of these semipurified virions and was recovered by ethanol precipitation. Oligonucleotides corresponding to untranslated 5' and 3' regions of the genomic RNA fragments of either S4 or M2 (encoding respectively ⁇ 3 and ⁇ 1) were used in a standard reverse transcriptase reaction with Moloney-MuLV reverse transcriptase. Overlapping fragments were then obtained by PCR reactions using oligonucleotides located at various positions along the sequence of each gene. Resulting PCR fragments were then sequenced at the sequencing center of Universite Laval.
  • a consensus sequence for ⁇ 3 amino acids sequence was obtained by aligning the 31 sequences from mammalian reoviral isolates of all 4 serotypes from different laboratories and published in the PubMedTM database (Accession numbers: AF332135; AF332137; AF368037; DQ004470 DQ004471 ; DQ004472; DQ004473; DQ004474; DQ004475; K02739; M13139 NC004265; U15072; U15073; U15074; U15075; U15076; U15077; U15078 U15079; U15080; U15081 ; U15082; U15083; U63303; U63304; U63305 U63306; U63307; X60066; X61586); the H8-2 wild type sequence as well as the previously reported sequence of the ts453 strain (6,25) are also included in the consensus.
  • the 100% consensus was prepared in such a way that, whenever there was variation at a particular position between the 31 sequences compared, a X appears in the consensus sequence (SEQ ID NO: 3).
  • the 31 reoviral isolates include 24 serotype 3, 5 serotype 1, one serotype 2 and one serotype 4.
  • This consensus sequence (SEQ ID NO: 3) was compared with that of the wild type H8-2 isolate (SEQ ID NO: 2) and of the P4L-12 virus mutant (SEQ ID NO: 1) ( Figure 7).
  • the P4L-12 isolate has a glutamic acid while H8-2 has a glycine.
  • Eighteen of the 31 sequences used to derive the consensus along with the original stock used to produce P4L-12 through chemical mutagenesis have a glutamic acid at this position and do not display the particular properties of P4L-12 discussed herein. This indicates that this position is not involved in P4L-12's advantageous phenotype.
  • a consensus sequence for ⁇ 1 amino acids sequence was obtained by aligning 8 sequences from reoviral isolates of different laboratories obtained from the PubMedTM database (Accession numbers: AF490617; AF368034; J02323; M19345; M19408; M20161 ; NC004270; NC004278) along with the H8-2 sequence.
  • the 8 reoviral isolates include 3 serotype 3, 3 serotype 1 , one serotype 2 and one serotype 4.
  • This consensus sequence (SEQ ID NO: 6) was compared with that of the wild type H8-2 isolate (SEQ ID NO: 5) and of the P4L-12 virus mutant (SEQ ID NO: 4). In Figure 8, the 11 variable amino acids position are boxed while the unique amino acid substitution at position 315 in P4L-12 is circled.
  • nucleotide sequence around mutations found in both S4 and M2 gene of P4L-12 is strictly conserved for at least 12 nucleotides on both side of the mutation in serotype 3 viruses.
  • a 25 nucleotide-long oligonucleotide with sequence ATGGGGTGTGATCGTTTATGATTAC (SEQ ID NO: 7), where bold-underlined C in P4L-12 replaces a G in the wild type sequence, is thus specific to the S4 gene of P4L-12 and could be developed as a specific probe for wild type vs P4L-12 mutant of serotype 3 viruses.
  • sequence GTGTTTGCCATAJCACCTAAACCAG (SEQ ID NO: 8) is specific to M2 gene where bold-underlined T replaces a C in the wild type sequence.
  • Adequate hybridization conditions using either wild type or mutant oligonucleotides could be used to probe for the presence of mutations at the same position in different viral stocks or isolates.
  • EXAMPLE 13 Selection of viral uncoating mutants [00132] As previously mentioned, uncoating of reovirus virions by lysosomal proteases is often a limiting factor that determines the ability of a given cell line, or cell type, to be efficiently infected by reovirus.
  • P4L-12's ⁇ 3 The amino acid substitution found in P4L-12's ⁇ 3 is distinct from those known to affect uncoating. It could thus be possible to use P4L-12 as an initial virus isolate to further select viruses that possess additional mutations that favor viral uncoating and thus virus infectivity in addition to P4L-12 advantageous replicative properties.
  • the P4L-12 virus will be serially passaged in L929 cells in the presence of E-64 protease inhibitor. This procedure is known to put a selective pressure to select viruses harboring mutations that favor viral uncoating (8).
  • viruses are selected by ammonium chloride selective pressure (that affects the endosomai pH and thus lysosomal proteases' activity) or by establishing persistently infected cells (that also put a pressure to favor viral uncoating), such as in PI3-1 (2,30).
  • Viruses are cloned, and their replication examined in L929 cells in the presence of interferon, E-64 or both. Their replication in untransformed and Ras-transformed cells are then be examined for those viruses presenting an interferon-hypersensitive/E-64-resistant phenotype.
  • P4L-12 may be considered a « second generation » of oncolytic reovirus
  • viruses combining P4L-12 mutations (and replicative phenotype) with easier uncoating (and thus potentially increased infectivity in transformed/cancer cells) would be considered « third generation
  • EXAMPLE 14 Viral infection and replication in human cancer cell lines [00140] In addition to murine Ras-transformed cells, the viral isolates are further characterized in human tumoral (cancer) cell lines.
  • NCI 60 collection (26) A classical collection of human cancer cell lines, the NCI 60 collection (26), is examined. Cell lines from different human tumor cell types are infected with H8-2, P4L-12, and future third-generation viral isolates.
  • infectious subviral particles » prepared by in vitro pre-treatment with proteolytic enzymes, are used to infect. Such particles are known to bypass restriction at the level of viral uncoating, as previously mentioned.
  • Viral replication are examined as in previous examples by looking at synthesis of viral proteins, viral release, and virus titer.
  • P4L-12 mutant virus presents a greater restriction to replication in untransformed mouse cells and human PBMCs, rendering this virus potentially safer.
  • maximum tolerated dose via different route of inoculation is established in murine models.
  • maximum tolerated dose is defined by the maximum dose that does not kill any animals in a group of at least 10 animals up to 4 weeks post-inoculation.
  • virus growth in various tissues is examined by virus titration in a few animals.
  • Viruses prepared for inoculation is recovered by extraction from infected L929 cells in suspension culture and concentrated by ultracentrifugation through a sterile cesium chloride cushion at a density of 1.32 g/ml.
  • mice Suckling mice are more susceptible to the reovirus-induced disease, although adult mice can support viral replication to a certain extent. Both models are thus studied.
  • Intravenous, intraperitoneal, intracranial and intranasal routes of inoculation are examined. These different routes represent a large spectrum of possibility for treatment of primary tumors (such as gliomas) and metastatic tumors and are also classical routes of inoculation to demonstrate reovirus pathogenic potential.
  • nude mice as a model of immunodeficient animal is inoculated solely via the intravenous route.
  • Wild type virus is used at the known maximum tolerated dose for each route and P4L-12 is used at the same dose, and at 10-fold increment up to 100-fold.
  • Groups of approximately 10-12 animals from at least two different litters are inoculated for each virus, inoculation route and virus dose. These inoculated animals are examined daily for obvious signs of disease and cumulative deaths (and development of disease) are measured.
  • human tumoral cells forming solid tumors are implanted as xenografts in the hind flank of nude mice (11). Again, maximum tolerated dose of the different viruses are inoculated via the tail vein in order to assess the ability of these viruses to kill tumor cells via systemic inoculation.
  • thermosensitive ts453 reovirus mutant increased dsRNA binding of sigma 3 protein correlates with interferon resistance.
  • Reovirus variants selected during persistent infections of L cells contain mutations in the viral S1 and S4 genes and are altered in viral disassembly. J Virol 71 :1362-1369.
  • RNA-dependent protein kinase (PKR) is regulated by reovirus structural proteins. Virology 234:364-371.

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Abstract

L'invention concerne un réovirus présentant une hypersensibilité aux interférons et/ou une capacité améliorée à distinguer des cellules transformées par Ras et des contreparties non transformées de celles-ci comprenant au moins un gène parmi un gène S4 s'hybridant, dans des conditions très exigeantes, à une sonde d'hybridation, la séquence nucléotidique comprenant SEQ ID NO: 7, et un gène M2 s'hybridant, dans des conditions très exigeantes, à une sonde d'hybridation, la séquence nucléotidique comprenant SEQ ID NO: 8. L'invention concerne également des procédés d'utilisation de celui-ci.
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US20110020288A1 (en) * 2007-03-12 2011-01-27 Oncolytics Biotech Inc. Reoviruses Having Modified Sequences
US8691241B2 (en) * 2007-03-12 2014-04-08 Oncolytics Biotech Inc. Reoviruses having modified sequences
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JP2010527593A (ja) * 2007-05-21 2010-08-19 オンコリティクス バイオテク，インコーポレーテッド 変異レオウイルスならびにその作製および使用方法
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