Improvements in extraction of viruses from cell cultures

Abstract

Provided herein are methods of producing a virus from a culture of a host cell. The method comprises providing a culture of a host cell that has been infected with the virus; contacting the host cell with a first buffer comprising a detergent and incubating the host cell in the presence of the first buffer for a first period of time, thereby producing a cell lysate; contacting the cell lysate with a second buffer comprising an endonuclease for a second period of time to degrade host cell nucleic acids; and collecting the virus.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 461,216, filed April 21, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] Reference to an electronic sequence listing

[0004] The contents of the electronic sequence listing (2024-013-02_SL.xml; size: 71,043 bytes; and creation date: April 16, 2024) are incorporated herein by reference in their entirety. Background Art

[0005] Triton X-100 was added to the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) list in December 2012, and the expiration date of REACH Annex XIV is January 2, 2021. After this date, Triton X-100 cannot be used in commercial production in the EU due to its environmental impact. Therefore, there is a need to identify alternative detergents to replace the use of Triton X-100 in the production of viral drug products. Summary of the Invention

[0006] Provided herein are methods for producing viruses from a culture of host cells. The methods include providing a culture of host cells infected with a virus; contacting the host cells with a first buffer comprising a detergent and incubating the host cells in the presence of the first buffer for a first period of time, thereby producing a cell lysate; contacting the cell lysate with a second buffer comprising an endonuclease for a second period of time to degrade host cell nucleic acids; and collecting the virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 is shown for use in virus-containing cell cultures with 1% The cell lysates obtained after incubation for 2 h with 20 and 20 mM Na2HPO4 (pH 8.0) were tested for increasing concentrations from 10 to 20 U / mL. Figure 3 is a graph of the results of residual host cell (HC) DNA after clarification at different concentrations and durations ranging from 60 to 240 minutes. Figure 1 Increasing the concentration of Benzonase was shown to reduce the residual HC DNA levels to those obtained with Triton X-100 cell lysis.

[0008] Figure 2 is shown with Figure 1Figure 2 is a graph showing the residual HCDNA results for the same test material after one overnight (O / N) storage at 2-8°C following clarification. Figure 2 It is shown that for the same extended storage at 2-8°C after clarification, increasing Benzonase concentrations reduced residual HC DNA levels to levels obtained with Benzonase treatment of Triton X-100 cell lysates for at least 90 minutes prior to clarification.

[0009] Figure 3 is shown with Figure 1 Figure 2 is a graph showing the residual HCDNA results for the same test material after two overnight storages at 2-8°C following clarification. Figure 3 It is shown that for the same extended storage at 2-8°C after clarification, increasing Benzonase concentration reduces residual HC DNA levels to levels obtained with Triton X-100 cell lysis.

[0010] Figure 4 is shown with Figure 1 (after clarification; solid line), and Figure 2 (after clarification, 1x O / N storage at 2-8°C; dotted line) and Figure 3 (Stored 2xO / N at 2-8°C after clarification; dashed line) Graph of total viral particle titer results for the same test material. Included as controls are results for test material lysed by incubation with 1% Triton X-100 for 1 hour and lysed by incubation with 1% QL300 grade Tween 20 in 20mM Na2HPO4 pH 8.0 lysis buffer for 2 hours, both treated with 10U / mL of Benzonase for 60 minutes. For overnight storage at 2-8°C for up to 2 days prior to freezing at -80°C, increasing the Benzonase concentration from 10 to 20U / mL or varying the Benzonase treatment duration from 60 to 240 minutes had no negative effect on total reovirus titer (HPLC). QL: quality level.

[0011] Figure 5 is a schematic diagram showing the extraction and purification scheme using Triton X-100 (left) and the extraction and purification scheme described in this application (right). DETAILED DESCRIPTION

[0012] Virology remains an intensive field of research due to the vast array of diseases caused by viruses. Efficient virus production is essential for isolating and purifying viral proteins, generating vaccines, or providing infectious viruses for laboratory research. Recently, the development of virotherapy has further necessitated efficient production of infectious viruses.

[0013] Described herein is an improved method for extracting viruses from cell culture using detergent conditions, which can be applied to both small-scale and large-scale virus production. The method includes an extraction step in which a detergent is added to the cell culture. Thereafter, cell debris can be removed from the extraction mixture by, for example, filtration or centrifugation. The resulting viral suspension can be further concentrated and / or enriched by chromatography. Viruses prepared according to the present invention can be used for any purpose, including purification of viral proteins, vaccination, infection of host cells, and clinical administration. The methods provided herein are advantageous for the production of viral drug substances because they result in generally higher yields, and the detergents provided herein are excellent alternatives to Triton X-100. The methods also result in a reduction in the amount of residual host cell DNA (HC DNA).

[0014] As used herein, "adherent cells" refers to cells that adhere to the culture vessel in a cell culture. Examples of adherent cells include monolayer cells, which are cells that form a monolayer of cells on the surface of a culture vessel. "Suspension cells" or "suspended cells" refers to cells that do not adhere to the culture vessel in a cell culture. Suspension cells can be grown in "spinner cultures," which are cultures in which the culture medium is continuously stirred during the culture process.

[0015] As used herein, "ambient temperature" refers to temperatures between about 10°C and about 30°C. Ambient temperature is preferably between about 15°C and about 30°C, more preferably between about 20°C and about 25°C, and most preferably about 25°C.

[0016] As used herein, "cell-associated" virus refers to a virus that is attached to or trapped in a portion of the cell in which the virus has been produced. Thus, before the host cell is lysed, the virus is cell-associated. When cell lysis begins, the virus may still be attached to or trapped in a portion of the disrupted cells and remain cell-associated. However, when the virus is freely released into the culture medium, it is no longer associated with the cell. "Cell-free virus" is a virus that is not associated with the cell.

[0017] As used herein, "cell culture" or "culture of cells" refers to a population of cultured cells under their culture conditions. In particular, a cell culture includes cells and culture medium. Cells that have been pelleted are not considered a cell culture unless they are placed back in culture medium under culture conditions.

[0018] As used herein, "cell lysis" refers to the disruption of the cell membrane of a cell and the subsequent release of all or part of the contents of the cell.

[0019] As used herein, "clinical administration" of a substance refers to contacting any part of the body of a living organism with the substance to improve or maintain the health of the organism.

[0020] As used herein, "harvesting" virus refers to the act of isolating virus produced from a cell culture that has been previously infected with virus. Virus is typically harvested by separating cellular debris from the virus and harvesting the portion containing the virus. Optionally, the virus can be further isolated from soluble material, for example by centrifugation.

[0021] As used herein, "culture conditions" refers to conditions used in cell culture, including but not limited to temperature, type of culture vessel, humidity, concentration of C02or any other gas used in the culture vessel, type of medium, initial density of cells cultured, and (if the cells are infected with virus) initial multiplicity of infection.

[0022] As used herein, "cytopathic effect" is damage to an infected host cell. Cytopathic effect can be indicated by the appearance of cells becoming swollen and granular and the disruption of cell clumps. Cells showing cytopathic effect can also take up a staining dye in a live cell count.

[0023] As used herein, "detergent" is a substance having a hydrophilic portion and a hydrophobic portion. The detergent is preferably a synthetic compound, more preferably a biodegradable synthetic compound. The detergent used in the present application enhances the disruption of cell membranes to facilitate the release of disrupted cell contents.

[0024] As used herein, a cell is "disrupted" when the cell membrane is broken and at least some of the cell contents are released from the cell. Cells can be disrupted, for example, by freeze-thawing, sonication, or detergent treatment.

[0025] As used herein, "extracting" virus refers to the act of converting cell-associated virus to cell-free virus.

[0026] As used herein, "HEK 293 cells" refers to the human embryonic kidney cell line designated 293 (ATCC Accession No. CRL-1573) or derivatives thereof. For example, 293 / SF cells (ATCC Accession No. CRL-1573.1) are HEK 293 cells that have been adapted to grow in serum-free medium. The present application also contemplates HEK 293 cells adapted to grow under other culture conditions, or any variety of HEK 293 cells or derivatives transformed with exogenous DNA, provided that such transformation does not impair the ability of the cells to support efficient reovirus production as described herein.

[0027] As used herein, "incubation" after the addition of a detergent to a cell culture refers to the act of allowing the cell culture to mix with the detergent for a period of time.

[0028] As used herein, "multiplicity of infection" or "MOI" refers to the ratio of the number of viruses to the number of cells when the virus is used to contact the cells.

[0029] As used herein, a "non-enveloped virus" is a virus that does not have an envelope. For example, a non-enveloped virus can be any virus belonging to the Adenoviridae family (e.g., adenovirus), Picornaviridae family (e.g., poliovirus), Reoviridae family (e.g., reovirus), Papovaviridae family (e.g., papillomavirus), Parvoviridae family (e.g., Kurchatov rat virus), or Iridoviridae family (e.g., Goliath iridovirus).

[0030] As used herein, "viability of cells" or "percentage of cells that remain viable" is the percentage of cells in a population that do not show cytopathic effects.

[0031] As used herein, "viral infection" refers to the entry of a virus into a cell and subsequent replication of the virus in the cell.

[0032] As used herein, the term "about" can be used in recognition of experimental error, measurement error, and variations expected by one of ordinary skill in the art. For example, "about" can mean plus or minus 10% of the indicated value, or plus or minus 5%.

[0033] Provided herein is a method of producing a virus from a culture of host cells. The method includes providing a culture of host cells that have been infected with a virus, and contacting the host cells with a first buffer comprising a detergent, and incubating the host cells in the presence of the first buffer for a first period of time, thereby producing a cell lysate. The cell lysate is then contacted with a second buffer comprising an endonuclease for a second period of time to degrade host cell nucleic acids, followed by collecting the virus.

[0034] The detergent can be a non-ionic detergent or an anionic detergent. The non-ionic detergent can be 20 (polysorbate 20), octyl β-D-glucopyranoside (OGP), Tergitol TM 15-S-9 (C12-14 secondary alcohol ethoxylate, also known as polyethylene glycol trimethyl nonyl ether) or 80 (polysorbate 80). The anionic detergent can be sodium deoxycholate. The detergent can be present at a concentration between 0.5% and 2.0% (v / v), or any amount between 0.5% and 2.0% (v / v). Thus, for example, the concentration of the detergent in the buffer can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% (v / v). Optionally, the detergent is Tween 20, and is present in the buffer at a concentration of 0.5%, 1.0%, or 2.0% (v / v).

[0035] In the provided methods, the first buffer containing a detergent can also include a phosphate salt. The first buffer can include a phosphate salt, for example, at a concentration of between 10 mM and 25 mM phosphate salt, 10 mM and 20 mM phosphate salt, 15 mM and 25 mM phosphate salt, 10 mM and 15 mM phosphate salt, 15 mM and 20 mM phosphate salt, or 10 mM and 25 mM phosphate salt. Optionally, the first buffer contains a phosphate salt at a concentration of at least 20 mM phosphate salt. The phosphate salt can be, for example, sodium phosphate or potassium phosphate.

[0036] In the provided methods, the host cells can be incubated in the presence of the first buffer for a period of time. Optionally, the first period of time is 60 minutes, 120 minutes, 180 minutes, or 240 minutes. Additionally, incubating the host cells in the presence of the first buffer can occur at a pH of 7.0 to 8.0. Optionally, incubating the host cells in the presence of the first buffer occurs at a pH of 8.0. The incubation of the host cells can occur at a particular temperature. For example, the incubation of the host cells can occur at a temperature of 30 °C to 40 °C. Optionally, the incubation of the host cells occurs at a temperature of 35 °C to 39 °C. Optionally, the incubation of the host cells occurs at a temperature of 36 °C to 38 °C. Optionally, the incubation of the host cells occurs at a temperature of 37 °C.

[0037] In the provided methods, the contacting of the host cells with the first buffer can occur under conditions of agitation. For example, contacting the host cells with the first buffer can occur under agitation at 120 rpm.

[0038] In the provided methods, the cell lysate is then contacted with a second buffer comprising an endonuclease for a second time period to degrade host cell nucleic acids. Optionally, the second time period is 60 minutes, 90 minutes, 120 minutes, 180 minutes, or 240 minutes. Optionally, the endonuclease is Benzonase (a miscellaneous endonuclease that cleaves all forms of DNA and RNA, including single-stranded, double-stranded, linear, and circular) or DNase I (an endonuclease that cleaves single- and double-stranded DNA). The endonuclease can be contacted with the cell lysate at a concentration of 10 to 20 U / mL or any amount between 10 and 20 U / mL. For example, the concentration of the endonuclease can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 U / mL. The cell lysate can be contacted with the endonuclease at a particular temperature. Optionally, contacting the cell lysate with the endonuclease occurs at a temperature of 30 °C to 40 °C. Optionally, contacting the cell lysate with the endonuclease occurs at a temperature of 35 °C to 39 °C. Optionally, contacting the cell lysate with the endonuclease occurs at a temperature of 37 °C. Optionally, contacting the cell lysate with the endonuclease can occur under stirring conditions. For example, contacting the cell lysate with the endonuclease can occur at 120 rpm.

[0039] In the provided methods, the second buffer can comprise other components. For example, the second buffer can comprise MgCl2.

[0040] The methods provided herein can also include removing cell debris. Optionally, the cell debris is removed by filtration. Thus, the virus can be purified based on, for example, size or density differences between the virus and other components in the extract. In particular, filtration or centrifugation can be employed to separate the cell debris from the virus. The cell debris can be removed in a single step or using a stepwise filtration protocol. For example, large chunks can first be removed from the extraction mixture using a pre-filter having a relatively large pore size (e.g., 5 pm or 8 pm), followed by a filter having a small pore size, such as a combination filter unit comprising a 3 pm filter and a 0.8 pm filter. Optionally, following the 5 pm or 8 pm pre-filtration step, a filter having a single pore size of 0.8 pm can be used.

[0041] In the provided methods, the cell can be a mammalian cell. Optionally, the cell is a human embryonic kidney 293 (HEK293) cell. Other cells suitable for use in the provided methods include, but are not limited to, mouse L929 cells, African green monkey kidney cells (Vero cells), and Chinese hamster ovary (CHO) cells. The mammalian cell is optionally grown in suspension.

[0042] The virus can be purified using any method suitable for purification of the virus. For example, the virus can be purified based on its surface charge. Optionally, the virus can be purified by ion exchange chromatography or by size exclusion chromatography. Optionally, the method further comprises purifying the virus by a combination of ion exchange and size exclusion chromatography. Optionally, the ion exchange is performed prior to the size exclusion chromatography. Optionally, the ion exchange is performed after the size exclusion chromatography. The ion exchange can be performed using an anion exchanger. Optionally, a phosphate buffer is used in the ion exchange and can be, for example, a concentration of 100 mM sodium phosphate or monosodium phosphate. Optionally, a phosphate buffer is used in the size exclusion chromatography and can be, for example, a concentration between 10-15 mM sodium phosphate, such as 10, 11, 12, 13, 14, or 15 mM sodium phosphate. Optionally, the size exclusion chromatography is performed at a pH of 7.0 to 7.5, such as pH 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5.

[0043] The virus can also be purified based on size differences, for example using size exclusion chromatography. Optionally, an anion exchange column can be used prior to the size exclusion chromatography. Other chromatographic methods can also be used where appropriate, such as those based on affinity or hydrophobic interactions.

[0044] Also provided herein is a method of producing a reovirus, comprising providing a culture of HEK 293 cells that have been infected with a reovirus; contacting the cells with a first buffer comprising Tween 20 and a phosphate, and incubating the HEK 293 cells in the presence of the first buffer for a first period of time, thereby producing a cell lysate, wherein the incubating is performed at a temperature of about 30 °C to about 40 °C and a pH of about 7.0 to about 8.0; contacting the cell lysate with a second buffer comprising an endonuclease for a second period of time to degrade host cell nucleic acids; removing cell debris by filtration; concentrating the filtrate by ultrafiltration and diafiltration; purifying the reovirus by a combination of ion exchange and size exclusion chromatography; and collecting the reovirus. Optionally, the first buffer comprises 15 mM to 25 mM phosphate. Optionally, the first buffer comprises at least 20 mM phosphate. The phosphate can be, for example, sodium phosphate. Optionally, the first buffer comprises 0.5%, 1.0%, or 2.0% (v / v) Tween 20. The first period of time can be 60 minutes, 120 minutes, 180 minutes, or 240 minutes. The second period of time can be 60 minutes, 90 minutes, 120 minutes, 180 minutes, or 240 minutes. The endonuclease can be Benzonase. The endonuclease can be contacted with the cell lysate at a concentration of 10 to 20 U / mL. Optionally, contacting the cell lysate with the endonuclease occurs at a temperature of 30 °C to 40 °C. Optionally, contacting the cell lysate with the endonuclease occurs at a temperature of 35 °C to 39 °C. Optionally, contacting the cell lysate with the endonuclease occurs at a temperature of 37 °C. Optionally, incubating the host cells occurs at a temperature of 35 °C to 39 °C. Optionally, incubating the host cells occurs at a temperature of 37 °C. Optionally, incubating the host cells in the presence of the first buffer occurs at a pH of 7.0 to 8.0 or at pH 8.0. In these methods, the second buffer can also comprise MgCl2.

[0045] The oncolytic viruses used in the provided methods and kits include, but are not limited to, oncolytic viruses of the following families of members: Reoviridae, Myoviridae, Siphoviridae, Podoviridae, Tectiviridae, Corticoviridae, Plasmaviridae, Lipothrixviridae, Fuselloviridae, Poxyiridae, Iridoviridae, Phycodnaviridae, Baculoviridae, Herpesviridae, Adenoviridae, Papovarviridae, Polydnaviridae, Inoviridae, Microviridae, Geminiviridae, Circoviridae, Parvoviridae, Hepadnaviridae, Retroviridae, Cystoviridae, Birnaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Leviviridae, Picornaviridae, Sequiviridae, Comoviridae, Potyviridae, Caliciviridae, Astroviridae, Nodaviridae, Tetraviridae, Tombusviridae, Coronaviridae, Flaviviridae, Togaviridae, and Barnaviridae. The provided methods also encompass immunoprotective viruses as well as reassortant or recombinant viruses of these and other oncolytic viruses.Thus, the oncolytic viruses used in the provided methods are, for example, selected from the group consisting of reovirus, Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), adenovirus, vaccinia virus, paravaccinia virus, Sindbis virus, and herpes simplex virus. Further, the provided methods can also be practiced using a combination of at least two oncolytic viruses. Several oncolytic viruses are discussed below, and one of ordinary skill in the art can also use other oncolytic viruses to practice the present methods based on the disclosure herein and the knowledge available in the art.

[0046] The viruses used in the provided methods and kits can be non-enveloped viruses. Optionally, the virus is a reovirus, such as a mammalian reovirus, a human reovirus, a serotype 3 virus, a serotype 3 reovirus Dearing strain, or a recombinant reovirus.

[0047] When a virus enters a cell, double-stranded RNA kinases (PKRs) are activated, blocking protein synthesis, and the virus cannot replicate in that cell. Some viruses have developed systems to inhibit PKR and promote viral protein synthesis and viral replication. For example, adenoviruses produce a large amount of small RNA, VA1 RNA. VA1 RNA has extensive secondary structure and competes for binding to PKR with double-stranded RNA (dsRNA) that normally activates PKR. Because activation of PKR requires a minimum length of dsRNA, VA1 RNA does not activate PKR. Instead, it sequesters PKR by its sheer quantity. Thus, protein synthesis is not blocked, and adenoviruses can replicate in the cell. Thus, if the PKR inhibitors in adenovirus, vaccinia virus, herpes simplex virus, or paravaccinia virus are mutated so as to no longer block PKR function, the resulting viruses will not infect normal cells due to the inhibition of protein synthesis by PKR, but they replicate in cancer cells that lack PKR activity. Optionally, the oncolytic virus is an adenovirus mutated in the VA1 region, a vaccinia virus mutated in the K3L and / or E3L region, a vaccinia virus mutated in the thymidine kinase (TK) gene, a vaccinia virus mutated in the vaccinia growth factor (VGF) gene, a herpes virus mutated in the gamma 134.5 gene, a paravaccinia virus mutated in the OV20.0L gene, or an influenza virus mutated in the NS-1 gene.

[0048] A vaccinia virus mutated in the viral thymidine kinase (TK) gene is unable to make the nucleotides required for DNA replication. In normal cells, the cellular TK levels are usually very low and the virus is unable to replicate. In tumors, the loss of the tumor suppressor Rb or an increase in cyclin activity leads to activation of the E2F pathway and high levels of TK expression. Thus, cancer cells have high TK levels and the mutated vaccinia virus can replicate and spread.

[0049] The vaccinia growth factor (VGF) gene is a homolog of the mammalian epidermal growth factor (EGF) and can bind and activate the EGF receptor (EGFR). Vaccinia viruses mutated in the VGF gene are restricted in their growth to cells with an activated EGF pathway, which is often mutated in cancer.

[0050] Viruses can be modified or mutated according to known structure-function relationships of viral PKR inhibitors. For example, because the amino-terminal region of the E3 protein interacts with the carboxy-terminal domain of PKR, deletion or point mutations in the carboxy-terminal domain prevent anti-PKR function (Chang et al., PNAS 89:4825-4829 (1992); Chang et al., Virology 194:537-547 (1993); Chang et al., J. Virol. 69:6605-6608 (1995); Sharp et al., Virol. 250:301-315 (1998); and Romano et al., Mol. and Cell. Bio. 18:7304-7316 (1998)). The K3L gene of vaccinia virus encodes pK3, a pseudo-substrate of PKR. Truncation or point mutations in the C-terminal portion of the K3L protein, homologous to residues 79 to 83 in eIF-2, abolish PKR inhibitory activity (Kawagishi-Kobayashi et al., Mol. Cell. Biology 17:4146-4158 (1997)).

[0051] Another embodiment is the Delta24 virus, a mutant adenovirus carrying a 24 base pair deletion in the El A region. (Fueyo et al., Oncogene 19(1):2-12 (2000)). This region is responsible for binding to the cellular tumor suppressor Rb and inhibiting Rb function, thus allowing the cell proliferation machinery and, by extension, viral replication to proceed in an uncontrolled manner. Delta24 has a deletion in the Rb binding region and does not bind to Rb. Thus, replication of the mutant virus is inhibited by Rb in normal cells. However, if Rb is inactivated and the cell becomes neoplastic, Delta24 is no longer inhibited. Instead, the mutant virus replicates efficiently and lyses the Rb-deficient cell.

[0052] In addition, vesicular stomatitis virus (VSV) selectively kills neoplastic cells. Herpes simplex virus 1 (HSV-1) mutant hrR3, which is defective in ribonucleotide reductase expression, replicates in colon cancer cells rather than in normal hepatocytes (Yoon et al., FASEB J. 14: 301-311 (2000)). Newcastle disease virus (NDV) preferentially replicates in malignant cells, and the most commonly used strain is 73-T (Reichard et al., J. Surgical Research 52: 448-453 (1992); Zom et al., Cancer Biotherapy 9 (3): 22-235 (1994); Bar-Eli et al., J. Cancer Res. Clin. Oncol. 122: 409-415 (1996)). Vaccinia virus propagates in several malignant tumor cell lines. Encephalitis virus has an oncolytic effect in mouse sarcoma tumors, but may need to be attenuated to reduce its infectivity in normal cells. Tumor regression has been described in patients with tumors infected with herpes zoster, hepatitis, influenza, varicella, and measles viruses (for review, see Nemunaitis, J. Invest. New Dmgs 17:375-386 (1999)).

[0053] Optionally, the oncolytic virus is a modified non-reovirus comprising a reovirus σ1 protein, wherein the reovirus σ1 protein replaces the natural attachment protein of the non-reovirus, and wherein the modified virus does not include any portion of the natural attachment protein of the non-reovirus. In the modified non-reovirus, the reovirus σ1 protein is attached to a carrier cell, and during in vivo delivery to a tumor, for example, during systemic delivery, the carrier cell protects the virus from the effects of neutralizing antibodies. The non-reovirus can be, but is not limited to, adenovirus, vaccinia virus, herpes simplex virus, sindbis virus, or parapoxvirus. Optionally, the full-length sequence of the natural attachment protein of the non-reovirus is replaced by the reovirus σ1 protein. Replacing the natural attachment protein of the virus with the reovirus σ1 protein causes the virus to attach to a carrier cell, which protects the virus from the effects of neutralizing antibodies during in vivo delivery. The reovirus σ-1 protein is described in, for example, WO 2008 / 11004, which is incorporated herein by reference in its entirety.

[0054] Optionally, the oncolytic virus is a reovirus. Reovirus refers to any virus classified in the genus Reovirus, whether naturally occurring, modified, or recombinant. Reoviruses are viruses with a double-stranded segmented RNA genome. Virions are 60 to 80 nm in diameter and have two concentric capsid shells, each of which is icosahedral. The genome consists of double-stranded RNA in 10 to 12 discrete segments, with a total genome size of 16 to 27 kbp. The individual RNA segments vary in size. Three different but related types of reovirus have been recovered from many species. Thus, the reovirus can be a mammalian reovirus or a human reovirus. All three types share a common complement-fixing antigen.

[0055] Human reoviruses include three serotypes: type 1 (virus strain Lang or T1L), type 2 (virus strain Jones, T2J), and type 3 (virus strain Dealing or virus strain Abney, T3D). The three serotypes are readily distinguished based on neutralization and hemagglutinin inhibition assays. The reovirus according to the present disclosure can be a type 3 mammalian orthoreovirus. Type 3 mammalian orthoreoviruses include, but are not limited to, the Dealing and Abney strains (T3D or T3A, respectively). See, e.g., ATCC Accession Nos. VR-232 and VR-824. See, e.g., U.S. Pat. Nos. 6,110,461; 6,136,307; 6,261,555; 6,344,195; 6,576,234; and 6,811,775, which are incorporated by reference herein in their entireties.

[0056] Optionally, the provided methods include use of a reovirus having a mutation. For example, the mutant reovirus as described herein can contain a mutation that reduces or substantially eliminates expression of a sigma 3 polypeptide or results in a loss of functional sigma 3 polypeptide, as described in U.S. Publication No. 2008 / 0292594, which is incorporated by reference herein in its entirety. The mutation that eliminates expression of a sigma 3 polypeptide or results in a loss of functional sigma 3 polypeptide can be in the nucleic acid encoding the sigma 3 polypeptide (i.e., the S4 gene) or in a nucleic acid encoding a polypeptide that regulates expression or function of the sigma 3 polypeptide.

[0057] As used herein, a mutation that reduces expression of a sigma 3 polypeptide refers to a mutation that results in at least a 30% reduction (e.g., at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or at least a 95%) in the amount of sigma 3 polypeptide as compared to a reovirus expressing wild-type levels of sigma 3 polypeptide. As used herein, a mutation that substantially eliminates expression of a sigma 3 polypeptide refers to a mutation that results in at least a 95% reduction (e.g., at least a 96%, at least a 97%, at least a 98%, at least a 99%, or 100%) in the amount of sigma 3 polypeptide relative to the amount of sigma 3 polypeptide produced by a wild-type reovirus. As used herein, a mutation that results in a reduction or absence of functional sigma 3 polypeptide refers to a mutation that allows for expression of a sigma 3 polypeptide but results in a sigma 3 polypeptide that is unable to assemble or incorporate into a viral capsid. It will be appreciated that it can be desirable or necessary for a mutant reovirus to retain other functions (e.g., the ability to bind RNA) in order for the mutant reovirus to retain the ability to propagate.

[0058] A mutation in a sigma 3 polypeptide as described herein can result in a sigma 3 polypeptide being incorporated into a capsid at a reduced level relative to a sigma 3 polypeptide that does not contain the mutation (e.g., a wild-type sigma 3 polypeptide). A mutation in a sigma 3 polypeptide as described herein can also result in a sigma 3 polypeptide being unable to incorporate into a viral capsid. Without being bound by any particular mechanism, a sigma 3 polypeptide can have reduced or lack function, for example, due to an inability of the sigma 3 polypeptide and mu 1 polypeptide to properly associate, or due to a conformational change that reduces or prohibits incorporation of the sigma 3 polypeptide into a capsid.

[0059] In addition to mutations that eliminate or reduce expression of a sigma 3 polypeptide or result in a non-functional or reduced-function sigma 3 polypeptide, a mutant reovirus as described herein can contain one or more additional mutations (e.g., a second, third, or fourth mutation) in one or more other reovirus coat polypeptides (e.g., mu 1, lambda 2, and / or sigma 1). Reoviruses containing mutations affecting a sigma 3 polypeptide and, optionally, additional mutations in any or all of the other outer coat proteins can be screened for their ability to infect and cause cytolysis of cells. For example, neoplastic cells that are resistant to lysis by a wild-type reovirus can be used to screen for effective mutant reoviruses described herein.

[0060] For example, other mutations can reduce or substantially eliminate expression of a mu1 polypeptide or result in the absence of a functional mu1 polypeptide. The mu1 polypeptide, encoded by the M2 gene, can be involved in cell penetration and can play a role in the activation of the transcriptase. Each virion contains about 600 copies of the mu1 polypeptide, which exists as a 1 : 1 complex with the sigma 3 polypeptide. The mu1 polypeptide is myristoylated at its N-terminus, and then the myristoylated N-terminus is cleaved 42 residues from the C-terminus, resulting in a C-terminal fragment (mu1C). Additionally or alternatively, further mutations can reduce or substantially eliminate expression of a lambda 2 polypeptide or result in the absence of a functional lambda 2 polypeptide, and / or further mutations can reduce or substantially eliminate expression of a sigma 1 polypeptide or result in the absence of a functional sigma 1 polypeptide. The lambda 2 polypeptide, encoded by the L2 gene, is involved in virion assembly and exhibits guanylyltransferase and methyltransferase activities. The sigma 1 polypeptide, encoded by the S1 gene, is involved in cell attachment and acts as a viral hemagglutinin.

[0061] Optionally, the reovirus comprises a lambda-3 polypeptide having one or more amino acid modifications, a sigma-3 polypeptide having one or more amino acid modifications, a mu-1 polypeptide having one or more amino acid modifications, a mu-2 polypeptide having one or more amino acid modifications, or any combination thereof. For example, the reovirus has: a lambda-3 polypeptide having one or more amino acid modifications; a sigma-3 polypeptide having one or more amino acid modifications; a mu-1 polypeptide having one or more amino acid modifications; and / or a mu-2 polypeptide having one or more amino acid modifications, as described in U.S. Serial No. 12 / 046,095, which is incorporated herein by reference in its entirety. By way of example, the one or more amino acid modifications in the lambda-3 polypeptide is Val at residue 214, Ala at residue 267, Thr at residue 557, Lys at residue 755, Met at residue 756, Pro at residue 926, Pro at residue 963, Leu at residue 979, Arg at residue 1045, Val at residue 1071, or any combination thereof, numbered relative to GenBank Accession No. M24734.1 (SEQ ID NO: 23). Note that when the amino acid sequence is Val at residue 214 or Val at residue 1071, the amino acid sequence further includes at least one additional change in the amino acid sequence. Optionally, the lambda-3 polypeptide includes the sequence set forth in SEQ ID NO: 19. By way of further example, the one or more amino acid modifications in the sigma-3 polypeptide is Leu at residue 14, Lys at residue 198, or any combination thereof, numbered relative to GenBank Accession No. K02739 (SEQ ID NO: 25). Note that when the amino acid sequence is Leu at residue 14, the amino acid sequence further includes at least one additional change in the amino acid sequence. Optionally, the sigma-3 polypeptide includes the sequence set forth in SEQ ID NO: 15. By way of further example, the one or more amino acid modifications in the mu-1 polypeptide is Asp at residue 73, numbered relative to GenBank Accession No. M20161.1 (SEQ ID NO: 27). Optionally, the mu-1 polypeptide includes the sequence set forth in SEQ ID NO: 17. By way of further example, the amino acid modification in the mu-2 polypeptide is Ser at residue 528, numbered relative to GenBank Accession No. AF461684.1 (SEQ ID NO: 29). Optionally, the mu-1 polypeptide includes the sequence set forth in SEQ ID NO: 17. The reovirus having one or more modifications as described herein can further include a reovirus sigma-2 polypeptide.Such sigma-2 polypeptides have a Cys at one or more of positions 70, 127, 195, 241, 255, 294, 296, or 340, numbered relative to GenBank Accession No. NP_694684.1 (SEQ ID NO: 30). Optionally, the sigma-2 polypeptide comprises the sequence shown in SEQ ID NO: 12.

[0062] Optionally, the reovirus comprises: an LI genomic segment comprising one or more nucleic acid modifications, an S4 genomic segment comprising one or more nucleic acid modifications, an Ml genomic segment comprising one or more nucleic acid modifications, an M2 genomic segment comprising one or more nucleic acid modifications, or any combination thereof. Optionally, the reovirus has: an LI genomic segment having one or more nucleic acid modifications; an S4 genomic segment having one or more nucleic acid modifications; an Ml genomic segment having one or more nucleic acid modifications; and / or an M2 genomic segment having one or more nucleic acid modifications, as described in WO 2008 / 110004, which is incorporated herein by reference in its entirety. For example, the one or more nucleic acid modifications in the LI genomic segment is a T at position 660, a G at position 817, an A at position 1687, a G at position 2283, ATG at positions 2284-2286, a C at position 2794, a C at position 2905, a C at position 2953, an A at position 3153, or a G at position 3231, numbered relative to GenBank Accession No. M24734.1 (SEQ ID NO: 22). Optionally, the LI genomic segment comprises the sequence set forth in SEQ ID NO: 8. For further example, the one or more nucleic acid modifications in the S4 genomic segment is an A at position 74 and an A at position 624, numbered relative to GenBank Accession No. K02739 (SEQ ID NO: 24). Optionally, the S4 genomic segment comprises the sequence set forth in SEQ ID NO: 4. For further example, the nucleic acid modification in the M2 genomic segment can be a C at position 248, numbered relative to GenBank Accession No. M20161.1 (SEQ ID NO: 26). The M2 genomic segment, for example, comprises the sequence set forth in SEQ ID NO: 6. For additional example, the nucleic acid modification in the Ml genomic segment is a T at position 1595, numbered relative to GenBank Accession No. AF461684.1 (SEQ ID NO: 28). Optionally, the Ml genomic segment comprises the sequence set forth in SEQ ID NO: 5. The reovirus as described herein can comprise any modification or combination of modifications disclosed herein. Optionally, the reovirus as described herein comprises a genomic segment having any one or both of the sequences set forth in SEQ ID NOs: 1-10 or the polypeptides set forth in SEQ ID NOs: 11, 12, and 16-21 and the sequences set forth in SEQ ID NOs: 13 and 14.Optionally, the reovirus disclosed herein is identified as IDAC Accession No. 190907-01, which was deposited with the International Depositary of Canada (IDAC, National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington St., Winnipeg, Manitoba Canada R3E 3R2, on September 19, 2007).

[0063] Sindbis virus (SIN) can be used in the methods described herein. Sindbis virus is a member of the Alphavirus genus of the Togaviridae family. The Sindbis virus genome is a 11703 nucleotide single-stranded RNA, capped at the 5' end and polyadenylated at the 3' end. The genome consists of a 49S untranslated region (UT), nonstructural proteins nsPl, nsP2, nsP3, and nsP4, followed by a promoter. The promoter is followed by a 26S UT, structural proteins C, E3, E2, 6K, and El, and finally a 3' UT and polyadenylated end. The genomic 49S RNA is positive-sense, infectious, and serves as mRNA in infected cells.

[0064] Sindbis vectors systemically and specifically infect / detect and kill metastatic tumors in vivo, significantly inhibiting tumor growth and prolonging survival (Hurtado et al., Rejuvenation Res. 9(1): 36-44 (2006)). Sindbis virus infects mammalian cells using a Mr 67,000 laminin receptor, which is higher in tumor cells than in normal cells. Tumor overexpression of the laminin receptor can explain the specificity and efficacy shown by Sindbis vectors against tumor cells in vivo. Sindbis does not need to be genetically manipulated to target cancer cells or injected directly into tumors. Sindbis injected anywhere in the body travels to the target area through the bloodstream (Tseng et al., Cancer Res. 64(18):6684-92 (2004)). Sindbis can also be genetically engineered to carry one or more genes that suppress the immune response to the virus and / or stimulate the immune response against tumors, such as anti-tumor cytokine genes, such as interleukin-12 and interleukin-15 genes.

[0065] The virus can be chemically or biochemically pre-treated (e.g., by treatment with a protease, such as chymotrypsin or trypsin) prior to administration to neoplastic cells. Pre-treatment with a protease removes the coat or capsid of the virus and can increase infectivity of the virus. The virus can be coated with a liposome or micelle (Chandran and Nibert, J. of Virology 72(1): 467-75 (1998)) to reduce or prevent an immune response by a mammal that has developed immunity to the virus. For example, virions can be treated with chymotrypsin in the presence of an alkyl sulfate detergent at a micelle-forming concentration to produce new, infectious subvirions. The oncolytic virus can also be a reassortant virus or ISVP.

[0066] The oncolytic virus can be a recombinant oncolytic virus. For example, a recombinant oncolytic virus is produced by reassortment of genomic segments from two or more genetically distinct oncolytic viruses, also referred to herein as reassortants. Reassortment of oncolytic virus genomic segments can occur after a host organism is infected with at least two genetically distinct oncolytic viruses. Recombinant viruses can also be produced in cell culture, for example, by co-infecting a permissive host cell with genetically distinct oncolytic viruses. Optionally, the method includes use of a recombinant oncolytic virus produced by reassortment of genomic segments from two or more genetically distinct oncolytic viruses, wherein at least one parent virus is genetically engineered to comprise one or more chemically synthesized genomic segments, has been treated with a chemical or physical mutagen, or is itself the result of a recombination event. Optionally, the method includes use of a recombinant oncolytic virus that is recombined in the presence of a chemical mutagen, including but not limited to dimethyl sulfate and ethidium bromide, or a physical mutagen, including but not limited to ultraviolet light and other forms of radiation.

[0067] Optionally, the method includes use of an oncolytic virus that includes a mutation (insertion, substitution, deletion, or duplication) in one or more genomic segments. Such a mutation can comprise additional genetic information that recombines with the host cell genome, or comprise a synthetic gene, such as a gene encoding an agent that suppresses an anti-viral immune response.

[0068] Optionally, the oncolytic virus is a mutant oncolytic virus. For example, the oncolytic virus can be modified by incorporating a mutant coat protein, e.g., in the virion outer capsid. The mutant oncolytic virus is optionally a mutant reovirus. Mutant reoviruses as described herein can contain mutations that reduce or substantially eliminate expression of a sigma 3 polypeptide or mutations that result in the absence of a functional sigma 3 polypeptide, as described in U.S. Publication No. 2008 / 0292594, which is incorporated herein by reference in its entirety. Optionally, the mutant reovirus used in the provided methods is mutated as described in U.S. Patent No. 7,803,385, which is incorporated herein by reference in its entirety.

[0069] Mutations referred to herein can be a substitution, insertion, or deletion of one or more nucleotides. Point mutations include, for example, single nucleotide transitions (purine to purine or pyrimidine to pyrimidine) or transversions (purine to pyrimidine or vice versa) and single or multiple nucleotide deletions or insertions. Mutations in a nucleic acid can result in one or more conservative or non-conservative amino acid substitutions in the encoded polypeptide, which can result in a conformational change or loss or partial loss of function, a shift in the translational reading frame (frame shift) resulting in a completely different polypeptide being encoded from the point of the shift, a premature stop codon resulting in a truncated polypeptide (truncation), or a mutation in a viral nucleic acid can not change the encoded polypeptide at all (silent or nonsense). For disclosure regarding conservative or non-conservative amino acid substitutions, see, e.g., Johnson and Overington, 1993, J. Mol. Biol. 233:716-38; Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89:10915-19; and U.S. Patent No. 4,554,101.

[0070] A variety of methods known in the art can be used to generate mutations in the nucleic acid of the oncolytic virus. For example, site-directed mutagenesis can be used to modify the reovirus nucleic acid sequence. One of the most common methods of site-directed mutagenesis is oligonucleotide-directed mutagenesis. In oligonucleotide-directed mutagenesis, an oligonucleotide encoding the desired sequence change is annealed to one strand of the DNA of interest and serves as a primer for the initiation of DNA synthesis. In this way, the oligonucleotide containing the sequence change is incorporated into the newly synthesized strand. See, e.g., Kunkel, 1985, Proc. Natl. Acad. Sci. USA 82:488; Kunkel et al., 1987, Meth. Enzymol. 154:367; Uewis and Thompson, 1990, Nucl. Acids Res. 18:3439; Bohnsack, 1996, Meth. Mol. Biol. 57:1; Deng and Nickoloff, 1992, Anal. Biochem. 200:81; and Shimada, 1996, Meth. Mol. Biol. 57:157. Other methods are routinely used in the art to modify the sequence of a protein or polypeptide. For example, a nucleic acid containing a mutation can be generated using PCR or chemical synthesis, or a polypeptide with the desired sequence change in amino acids can be chemically synthesized. See, e.g., Bang and Kent, 2005, Proc. Natl. Acad. Sci. USA 102:5014-9 and references therein.

[0071] Also provided are compositions comprising viruses collected according to the methods provided herein, and such compositions can comprise, for example, a pharmaceutically acceptable excipient. The compositions provided herein are administered in vitro or in vivo in a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be a solid, semisolid, or liquid material that can serve as a vehicle, a diluent, or a medium for the reovirus. Thus, the compositions containing the oncolytic virus and / or one or more of the provided agents can be in the form of tablets, pills, powders, lozenges, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solid or in 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.

[0072] Optionally, the composition containing the oncolytic virus is suitable for infusion. For intravenous infusion, there are two types of commonly used fluids: crystalloids and colloids. Crystalloids are aqueous solutions of mineral salts or other water-soluble molecules. Colloids contain larger insoluble molecules, such as gelatin; blood itself is a colloid. The most commonly used crystalloid fluid is normal saline, a 0.9% solution of sodium chloride in water, which approximates the concentration in blood (isotonic). Ringer’s lactate or Ringer’s acetate are other isotonic solutions commonly used for large-volume fluid replacement. If the patient is at risk for having low blood sugar or high sodium, a 5% dextrose in water solution, sometimes called D5W, is often used instead.

[0073] Some embodiments of suitable carriers include phosphate buffered saline or another physiologically acceptable buffer, lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. The pharmaceutical compositions additionally include, but are not limited to, lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives; sweetening agents; and flavoring agents. The pharmaceutical compositions can be formulated to provide quick, sustained, or delayed release of the mutant reovirus after administration by employing procedures known in the art. In addition to the representative formulations described below, other suitable formulations for use in pharmaceutical compositions can be found in Remington: The Science and Practice of Pharmacy, 22ndEd., Loyd V. Allen, Jr. et al. eds., Pharmaceutical Press (2012). To prepare a solid composition (e.g., a tablet), the mutant reovirus can be mixed with a pharmaceutical carrier to form a solid composition. Optionally, the tablets or pills can be coated or otherwise compounded to provide a dosage form affording the release of the active ingredient in a particular location or which provides a sustained action over an extended period of time. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope surrounding the former. The two components can be separated by an enteric layer that serves to resist dissolution in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0074] Liquid formulations including the reovirus and / or pharmaceutical agents for oral administration or for injection generally include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored, emulsions with edible oils such as corn oil, cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0075] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions can contain suitable pharmaceutically acceptable excipients as described supra. Such compositions can be administered either orally or nasally as an aerosol or by inhalation, and are acted on in either the topical or systemic areas. Compositions in pharmaceutically acceptable solvents can be nebulized by use of inert gases. Nebulized solutions can be inhaled directly from the nebulizing device or the nebulizing device can be attached to a face mask tent, or an intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered orally or nasally from devices that deliver the formulation in an appropriate manner.

[0076] Another formulation optionally employed in the methods of the disclosure includes a transdermal delivery device (e.g., a patch). Such transdermal patches can be used to provide continuous or discontinuous infusion of viruses and agents as described herein. The construction and use of transdermal patches for delivery of agents is well known in the art. See, e.g., U.S. Patent No. 5,023,252. Such patches can be constructed to provide continuous, pulsatile, or on demand delivery of the mutant reovirus.

[0077] As described above, the viruses and / or other agents can be encapsulated in liposomes or micelles, if necessary, to reduce or prevent an immune response in a mammal that has developed an immunity to the virus or agent. Such compositions are referred to as immunoprotected viruses or agents. See, e.g., U.S. Patent Nos. 6,565,831 and 7,014,847.

[0078] In the provided methods, the oncolytic virus is administered in a manner such that it can ultimately contact the target tumor or tumor cells, e.g., systemically. The route of administration of the virus, as well as the formulation, carrier, or vehicle, depends on the location and type of the target cells. A variety of routes of administration can be employed. For example, for accessible solid tumors, the virus can be administered by direct injection into the tumor. For example, for hematopoietic tumors, the virus can be administered intravenously or intravascularly. For tumors that are not easily accessible within the body (e.g., metastases), the virus is administered in a manner such that it can be transported systemically through the body of the mammal, e.g., intravenously or intramuscularly, to reach the tumor. Alternatively, the virus can be administered directly to a single solid tumor, which then carries systemically through the body to metastases. The virus can also be administered subcutaneously, intraperitoneally, intrathecally or intraventricularly (e.g., for brain tumors), locally (e.g., for melanoma), orally (e.g., for oral or esophageal cancer), rectally (e.g., for colorectal cancer), vaginally (e.g., for cervical or vaginal cancer), nasally, by inhalation spray, or by aerosol formulation (e.g., for lung cancer).

[0079] Optionally, the virus is administered to the individual continuously at least once a day for a period of time in the first or subsequent rounds of treatment, or intermittently or continuously over the course of a day for several consecutive days. Thus, for example, the virus is administered to the individual over a period of time by intravenous administration in any pharmacologically acceptable solution form, or by infusion. For example, the substance can be administered systemically by injection (e.g., IM or subcutaneously) or orally (at least once a day) each day, or by infusion in a manner that results in delivery to the tissue or bloodstream of the individual each day. When the virus is administered by infusion over a period of time, the period of time is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 24 hours, or any time between 1 and 24 hours, inclusive, or longer. Optionally, the period of time is 5, 15, 30, 60, 90, 120, 150, or 180 minutes, or any time between 5 and 180 minutes, inclusive, or longer. Thus, for example, the virus is administered by infusion for 60 minutes. The administration can be repeated each day for 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 21, 28 days, or any number of days between 2 and 28 days, inclusive, or longer.

[0080] The viruses disclosed herein are administered in an amount sufficient to achieve treatment of a cancer or proliferative disorder (i.e., an effective amount). A cancer or proliferative disorder is treated when a treatment regimen comprising the virus administered to proliferating cells affects lysis (e.g., oncolysis) of the affected cells, resulting in a decrease in the number of abnormally proliferating cells, a decrease in the size of a neoplasm, and / or a decrease or elimination of symptoms associated with the proliferative disorder (e.g., pain). As used herein, the term oncolysis means that at least 10% of the proliferating cells are lysed (e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 75% of the cells are lysed). The percentage of lysis can be determined, for example, by measuring a decrease in the size of a neoplasm or a decrease in the number of proliferating cells in a mammal, or by measuring the amount of lysis of cells in vitro (e.g., a biopsy from a proliferating cell). The effective amount of the virus used in the treatment regimen will be based on the individual, and can be determined at least in part on the particular virus used; the size, age, sex of the individual; and the size and other characteristics of the abnormally proliferating cells. For example, for treatment of humans, about 10 3 to 10 12 plaque forming units (PFU) of the virus are used, depending on the type, size, and number of proliferating cells or neoplasms present. The effective amount can be, for example, about 1.0 PFU / kg body weight to about 10 15 PFU / kg body weight (e.g., about 10 2 PFU / kg body weight to about 10 13 PFU / kg body weight). Optionally, the effective amount is about 1 x 10 8 to about 1 x 1012 PFU or TCID50. Optionally, the effective amount is about 3 x 10 10 to about 1 x 10 10 TCID50. For example, the effective amount can be between 3.0 x 10 10 and 4.5 x 10 10 TCID50. Optionally, the effective amount is 4.5 x 10 10 TCID50. Optionally, the vial containing the virus can include, for example, 5 x 10 9 to 1 x 10 11 TCID50 / mL.

[0081] Optimal dosages of the viruses and therapeutic agents, as well as compositions and kits comprising the viruses and agents, will depend on a variety of factors. The exact amount required will vary depending on the species, age, body weight, and general condition of the individual; the severity of the disease being treated; the particular virus and its mode of administration. Thus, it is not possible to specify an exact amount appropriate for every composition or kit. However, one of ordinary skill in the art, with access to this

[0082] Effective dosages and schedules for administering a therapeutic regimen can be determined empirically. For example, animal models of various proliferative disorders are available from Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609 USA. Both direct (e.g., histology of tumors) and functional measures (e.g., survival of individuals or size of tumors) can be used to monitor response to therapy. These methods involve sacrificing representative animals to assess the population, thereby increasing the number of animals required for the experiment. Measuring luciferase activity in tumors provides an alternative method to assess tumor volume without sacrificing animals and allows for longitudinal population-based analysis of therapy. The dosage range of the compositions administered should be sufficient to effect the desired effect on symptoms of the disease. The dosage should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions and anaphylactic reactions. In the event an adverse reaction occurs, the dosage can be adjusted by the individual physician.

[0083] The dosage can vary and can be administered in one or more doses of the administration form, such as once a day for one or several days. The viruses and therapeutic agents provided are administered in a single dose or in multiple doses (e.g., two doses, three doses, four doses, six doses, or more). For example, where administration is by infusion, the infusion can be a single continuous dose or can be delivered by multiple infusions. The treatment can continue for several days to several months or until disease remission is achieved.

[0084] The provided methods can further be combined with other tumor therapies such as chemotherapy, radiation therapy, surgery, hormone therapy, and / or other immunotherapies. Suitable additional therapeutic agents include, but are not limited to, analgesics, anesthetics, stimulants, corticosteroids, anticholinergic agents, anticholinesterase agents, anticonvulsants, antineoplastic agents, allosteric inhibitors, anabolic steroids, antirheumatic agents, psychotherapeutic agents, neuroleptic agents, anti-inflammatory agents, anthelmintics, antibiotics, anticoagulants, antifungal agents, antihistamines, antimuscarinic agents, antimycobacterial agents, antiprotozoal agents, antiviral agents, dopamine, hematologic agents, immunologic agents, muscarinic agents, protease inhibitors, vitamins, growth factors, and hormones. One of skill in the art can readily determine the selection of agents and dosages based on the given disease being treated.

[0085] The provided combinations of viruses and therapeutic agents can be administered simultaneously (e.g., in a mixture), separately but at the same time (e.g., through separate intravenous lines into the same individual), or sequentially (e.g., first administration of one compound or agent, then administration of the second). Thus, the term combination is used to refer to the concomitant, simultaneous, or sequential administration of two or more agents.

[0086] When one compound is administered before another, the first compound is administered minutes, hours, days, or weeks before the second compound is administered. For example, the first compound can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 36, 48, 60, or 72 hours, or any time in between, including the endpoints, before the second compound is administered. Optionally, the first compound is administered more than 72 hours before the second compound. As another example, the first compound can be administered 1, 5, 15, 30, 60, 90, 120, 150, or 180 minutes, or any time in between, including the endpoints, before the second compound is administered. Optionally, the first compound is administered 1, 2, 3, 4, 5, 6, 7, 14, 21, or 28 days, or any time in between, including the endpoints, before the second compound is administered. Optionally, the first compound is administered more than 28 days before the second compound.

[0087] The oncolytic virus or pharmaceutical composition comprising such a virus can be packaged into a kit. The kit further comprises one or more additional agents or pharmaceutical compositions comprising additional agents. The kit can comprise a chemotherapeutic agent or a cancer immunotherapeutic agent. Optionally, the kit comprises an immune checkpoint inhibitor.

[0088] The oncolytic viruses and / or additional agents and pharmaceutical compositions containing the same can be packaged in one or more containers. When the kit contains a pharmaceutical composition, the pharmaceutical composition can be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable for unitary dosing to human subjects and other mammals, each unit containing a predetermined quantity of an oncolytic virus or other agent, such as an immune checkpoint inhibitor, calculated to produce the desired therapeutic effect in association with a suitable pharmaceutically acceptable carrier. Optionally, the kit includes a reovirus and an immune checkpoint inhibitor.

[0089] The oncolytic viruses in the provided kits can be any of the oncolytic viruses described herein. The provided kits can include more than one dose of oncolytic virus. Optionally, each dose of oncolytic virus comprises about 10 3 to 10 12 plaque forming units (PFU) of oncolytic virus. Optionally, each dose comprises about 10 8 to 10 12 PFU of oncolytic virus. Optionally, each dose comprises about 10 8 to 10 12 TCID50of oncolytic virus. Optionally, each dose comprises about 1 x 10 10 to 3 x 10 10 TCID50of oncolytic virus. For example, each dose can be between 3.0 x 10 10 to 4.5 x 10 10 TCID50. Optionally, each dose is 4.5 x 10 10 TCID50. Optionally, the vials containing the virus can include, for example, 5 x 10 9 to 1 x 10 11 TCID50 / mL.

[0090] As used herein, the terms treatment, treat, or treating refer to a method for reducing the impact of a disease or condition or symptoms of a disease or condition. Thus, in the disclosed methods, treatment can refer to a 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% reduction or improvement in the severity of a disease or condition or symptoms of a disease or condition that has been identified. For example, a method of treating cancer is considered a treatment if one or more symptoms of the disease in a subject are reduced by 10% or more as compared to a control. Thus, the reduction can be 10% or greater, 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 100% or any percentage reduction within the range of 10% to 100% as compared to the native or control level. It will be appreciated that treatment does not necessarily refer to a cure or complete elimination of a disease, condition, or symptoms of a disease or condition.

[0091] As used herein, the term individual can be a vertebrate, more particularly a mammal (e.g., a human, equine, porcine, rabbit, canine, ovine, caprine, non-human primate, bovine, feline, guinea pig, or rodent), fish, bird, or reptile or amphibian. The term does not denote a particular age or sex. Thus, adult and newborn individuals, both male and female, are intended to be covered. The terms patient and subject are used interchangeably as used herein, and can refer to a subject having a disease or disorder. The term patient or individual includes human and veterinary individuals.

[0092] Disclosed are materials, compositions, and components used in or with the disclosed methods and compositions, or to produce the products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific references of each are not expressly disclosed, each is specifically contemplated and has independent significance from the other combinations and permutations. For example, if a number of inhibitors are disclosed and if a number of modifications to the molecules comprising the inhibitors are discussed, each combination is specifically contemplated regardless of whether the specific reference is explicitly discussed. Additionally, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method step or combination of method steps of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0093] Throughout this application, various publications are cited. The entire disclosures of these publications are incorporated into this application by reference.

[0094] A number of aspects have been described. However, it should be understood that various modifications may be made. Furthermore, when a feature or step is described, it can be combined with any other feature or step described herein, even if that combination is not explicitly stated. Therefore, other aspects are also within the scope of the claims.

[0095] Example

[0096] Example 1. Lysis, extraction and purification of reovirus from host cells.

[0097] 1% Triton X-100 is used in the currently validated reovirus manufacturing process to lyse HEK293 cells to maximize viral particle recovery at the upstream harvest step. However, Triton X-100 is listed on the REACH list and cannot be used for commercial production in the EU due to its environmental impact. Therefore, identification and testing of alternative detergents is needed to avoid any delays in reovirus manufacturing.

[0098] Table 1 is a list of the alternatives tested in this example.

[0099] Table 1. List of detergents.

[0100]

[0101] Alternative candidate detergents were evaluated at varying concentrations (0.5%, 1%, 2%) and durations (1 hour, 2 hours, 3 hours) during the lysis step, followed by incubation with Benzonase to digest host cell DNA (HCDNA). The detergents tested were Deviron C16; ECOSURF EH-9; octyl β-D-glucopyranoside (OGP); sodium deoxycholate (NaDOC); Tween 20; Tween 80, and Tergitol 15-S-9. Samples were analyzed for viral particle recovery (HPLC), infectious viral particles (TCID50), and residual HCDNA. These data were compared to Triton X-100, and cells lysed via freeze / thaw cycles served as a control. The results are shown in Tables 2 and 3.

[0102] Table 2. Alternative Detergents - Virus Titer Results (HPLC)

[0103]

[0104]

[0105] dpi: days post infection; CTL: control; hr: hours; vp: viral particles

[0106] Table 3. Alternative Detergents - Virus Titer Results (HPLC)

[0107]

[0108] dpi: days post infection; CTL: control; hr: hours; vp: viral particles

[0109] Conditions that resulted in >80% viral particle recovery relative to the Triton X-100 control were selected for full downstream analysis. Virus recoveries ranged from 90% to 98% under 0.5% octyl-D-glucopyranoside (OGP) and 2-hour Tergitol 15-S-9 (0.5%, 1%, 2%) conditions. 0.1% sodium deoxycholate (NaDOC) showed the best efficiency, 16% higher than Triton X-100. However, there was a trend that increasing NaDOC concentration resulted in decreased viral titers, with no viral particles detected for the 0.5% NaDOC concentration. Furthermore, because NaDOC is an anionic detergent, it is incompatible with the ion exchange purification step unless it is removed from the sample prior to purification using an ion exchange column. Therefore, it was decided not to continue using this detergent. As described above for NaDOC, OGP and Deviron C-16 also showed decreasing titers with increasing detergent concentration, suggesting that these higher detergent doses may degrade reovirus.

[0110] Tween 20 was tested at different concentrations (0.5%, 1%, 1.5%) and incubation times (1 hour, 2 hours, 3 hours). The results are shown in Table 4.

[0111] Table 4. Tween 20 virus titer results (HPLC)

[0112]

[0113] dpi: days post infection; CTL: control; hr: hours; vp: viral particles

[0114] None of these conditions resulted in >80% recovery of viral particles relative to Triton X-100, therefore further optimization of Tween 20 lysis conditions was required. These results are shown in Table 5.

[0115] Table 5. Tween 20 virus titer results (HPLC).

[0116]

[0117] dpi: days post infection; CTL: control; hr: hours; PO4: sodium phosphate; vp: viral particles

[0118] The 20 mM sodium phosphate (PO4) buffer lysis condition showed a positive trend with increasing pH. For the same pH condition, the results for 1% Tween 20 were better than for 0.5% Tween 20. 1% Tween 20 with 20 mM sodium phosphate at pH 8 showed titers higher than the cutoff value for 80% Triton X-100. However, using 10 mM sodium phosphate buffer at pH 7.0, 7.5, or 8.0 or 50 mM Tris buffer, the Tween 20 lysis condition did not improve relative to Triton X-100.

[0119] The results of further optimization of the 1% Tween 20 lysis condition are shown in Table 6.

[0120] Table 6. Further optimization of Tween 20 - viral titer results (HPLC).

[0121]

[0122] dpi: days post infection; CTL: control; hr: hours; O / N: overnight; vp: viral particles

[0123] The salt in Table 6 refers to 200 mM NaCl, which was included to see if it improved the Tween 20 lysis condition.

[0124] Further optimization of the 1% Tween 20, 20 mM phosphate lysis buffer, pH 8 included longer detergent incubation times (1 hour, 2 hours, 3 hours) and the addition of 200 mM NaCl after Benzonase digestion. For both Tween detergents (Tween 20 and Tween 80), the addition of 200 mM NaCl did not improve viral particle recovery. Incubation with Tween 20 for 2 hours resulted in the highest HPLC recovery. Lysis with Tween 80 relative to Triton X-100 resulted in HPLC recovery >80%. However, since the results with Tween 80 were comparable to Tween 20, it was decided to continue using Tween 20.

[0125] Further analysis of alternative detergents used in place of Triton X-100, including infectious virus titers, is shown in Table 7. Note that while the Triton X-100 control appears to have higher HPLC and TCID50 recoveries than Tween 20, at the larger production scale (1.25 L), this trend is reversed, with Tween 20 resulting in improved HPLC and TCID50 titers relative to Triton X-100.

[0126] Table 7. Triton X-100 Alternative-Detergent Virus Titer (TCID50) Results

[0127]

[0128] dpi: days post infection; CTL: control; PO4: sodium phosphate; O / N: incubation overnight at 4°C

[0129] The TCID50 virus titer results showed a similar trend to the HPLC data. The detergent that resulted in the best recovery of infectious virus titer relative to Triton X-100 was 1% Tergitol 15-S-9, 1% Tween 20, 0.5% OGP, and 0.1% NaDOC in 20 mM PO4 buffer pH 8.

[0130] In summary, the screening of different alternative detergents identified several potential candidates to replace Triton X-100 (OGP, NaDOC, Tween 20, Tergitol 15-S-9).

[0131] Three conditions were used to evaluate the impact of detergents on the 1.25 L scale manufacturing process relative to the current Triton X-100 process (Table 8). The following detergents and conditions were tested: 1% Tween 20 with 20 mM NaPO4 lysis buffer pH 8.0, 2 hours incubation; 1% Tergitol 15-S-9, 1 hour incubation; and 0.5% OGP, 1 hour incubation. For Tergitol 15-S-9, the overall HPLC recovery was 21% higher than that of Triton X-100 (5.28 x 10 13 For 4.35x10 13 ), and the total infectious virus particles (TCID50) were 25% higher than Triton X-100 (3.22x10 12 2.57x10 12 For 0.5% OGP, the total virus particle yield was 8.24×10 13 HPLC, and the total infectious virus particle yield was 2.10x10 12TCID50. Tween 20 gave better results than Triton X-100 for both total virus particle yield (HPLC) and infectious virus particle yield (TCID50). 14 HPLC for 9.94x10 13 HPLC) and TCID50 (3.27x10 12 2.53x10 12 ), Tween 20 gave 54% and 29% higher yields than Triton X-100, respectively. These are the best yields of the three detergents tested in the reovirus production process at the 1.25 L scale.

[0132] Table 8. Summary of bulk purified materials using three different detergents (1.25 L manufacturing scale)

[0133]

[0134]

[0135] hr: hour vp: virus particle

[0136] From Table 8, the data suggest that cell lysis using Tween 20 may be less efficient in reducing HC DNA and host cell proteins (HCPs). Additional small-scale experiments were performed according to Table 9 below to determine optimal Benzonase digestion conditions to ensure that Tween 20 produced HC DNA and HCP levels similar to or lower than those produced by the Triton X-100 process.

[0137] Table 9. Benzonase optimization after cell lysis with 1% Tween 20, 20 mM Na2HPO4, pH 8.0, 2 hours incubation

[0138]

[0139] hr: hours as shown in Table 9 and Figures 1 to 3 As shown in , increasing the Benzonase concentration from 10 to 20 U / mL reduced the residual HCDNA levels to levels obtained with Triton X-100 cell lysis. When used at a concentration of 20 U / mL, the optimal Benzonase digestion duration ranged from 90 to 240 minutes.

[0140] As shown in Table 9 and Figures 1 to 4As shown in , increasing the Benzonase concentration from 10 to 20 U / mL or changing the Benzonase treatment duration from 60 to 240 minutes did not negatively affect total reovirus titers (HPLC) for overnight storage at 2-8°C for up to 2 days after clarification.

[0141] In summary, as shown in the Examples and described throughout the application, cells can be lysed using 1% Tween 20 in 20 mM NaPO4 (pH 8.0) lysis buffer for 2 hours, followed by digestion with 20 U / mL of Benzonase for 1.5 hours. Figure 5 Schematic diagram showing the reovirus purification workflow, including the virus extraction protocol.

[0142] The following is a summary of the production and extraction of viruses as described in the Examples. HEK293S cells were thawed and expanded for approximately 6-8 passages until a viral load of 0.4 x 10 6 The target viable cell density of 1.5 cells / mL was sufficient to inoculate the 1.5 L bioreactor. The bioreactor operating conditions were temperature = 37°C; dissolved oxygen = 40% air saturation; pH = 7.2 ± 0.05; and agitation rate = 150 rpm (ramping). Once the cell density (1.8–2.4 x 10 6 Cultures were infected at a concentration of 100 cells / mL (between 10 and 200 μg / mL). Virus stock was prepared in 24 mL of culture medium at a multiplicity of infection (MOI) of 0.5 viruses / cell. Virus was added to approximately 1.5 L of fresh culture medium to bring the final volume to 3 L. Cell viability was monitored and harvested 3 days post-infection (if viability was below 35%) or 4 days post-infection. Glucose was added as needed during infection.

[0143] For harvest, the culture volume of the bioreactor was split into 2 equal volumes, each of which was distributed in 2 x 3.5 L Chemap bioreactor containers. Triton X-100 (0.91% v / v final) was added to one container and Tween 20 (1% final) in 20 mM NaP04 pH 8.0 lysis buffer was added to the other container. For Triton X-100, after 1 hour incubation at 37°C with agitation at 120 rpm, 500 U / mL Benzonase stock solution in 50 mM MgCl2was added to give a final concentration of 10 U / mL in 1 mM MgCl2. After 1 hour of Benzonase digestion, purification of reovirus from the cell culture was initiated. For Tween 20, 20 mM NaP04 pH 8.0, after 2 hours incubation at 37°C with agitation at 120 rpm, 1000 U / mL Benzonase solution in 50 mM MgCl2was added to give a final concentration of 20 U / mL in 1 mM MgCl2. After 1.5 hours of Benzonase digestion, purification of reovirus from the cell culture was initiated.

Claims

1. A method for producing viruses from a culture of host cells, comprising the steps of: (a) providing a culture of host cells infected with the virus; (b) contacting the host cells with a first buffer comprising a detergent, and incubating the host cells in the presence of the first buffer for a first period of time, thereby producing a cell lysate; (c) contacting the cell lysate with a second buffer comprising an endonuclease for a second period of time to degrade host cell nucleic acids; and (d) collecting the virus.

2. The method of claim 1, wherein the detergent is a nonionic detergent.

3. The method of claim 2, wherein the non-ionic detergent is Tween 20, and wherein the first buffer further comprises phosphate. The method of claim 3 , wherein the first buffer comprises 10 mM to 25 mM phosphate. The method of claim 3 , wherein the first buffer comprises at least 20 mM phosphate.

6. The method of claim 3 or 4, wherein the phosphate is sodium phosphate.

7. The method of any one of claims 1 to 6, wherein incubating the host cells in the presence of the first buffer occurs at a pH of 7.0 to 8.

0.

8. The method of any one of claims 1 to 7, wherein incubating the host cells in the presence of the first buffer occurs at a pH of 8.

0.

9. The method of any one of claims 1 to 8, wherein the first buffer comprises 0.5%, 1.0% or 2.0% (v / v) Tween 20.

10. The method of claim 2, wherein the detergent is octyl β-D-glucopyranoside (OGP), Tergitol 15-S-9, or Tween 80.

11. The method of claim 1, wherein the detergent is sodium deoxycholate.

12. The method of any one of claims 1 to 11, wherein the first period of time is 60, 120, 180 or 240 minutes.

13. The method of any one of claims 1 to 12, wherein the second period of time is 60 minutes, 90 minutes, 120 minutes, 180 minutes, or 240 minutes.

14. The method of any one of claims 1 to 13, wherein the endonuclease is Benzonase.

15. The method of any one of claims 1 to 14, wherein the endonuclease is contacted with the cell lysate at a concentration of 10 to 20 U / mL.

16. The method of any one of claims 1 to 15, wherein contacting the cell lysate with the endonuclease occurs at a temperature of 30°C to 40°C.

17. The method of any one of claims 1 to 15, wherein contacting the cell lysate with the endonuclease occurs at a temperature of 35°C to 39°C.

18. The method of any one of claims 1 to 17, wherein incubating the host cells occurs at a temperature of 30°C to 40°C.

19. The method of any one of claims 1 to 17, wherein incubating the host cells occurs at a temperature of 35°C to 39°C.

20. The method of any one of claims 1 to 19, wherein the second buffer further comprises MgCl2.

21. The method of any one of claims 1 to 20, wherein the method further comprises removing cell debris.

22. The method of claim 21, wherein the cell debris is removed by filtration.

23. The method of any one of claims 1 to 22, wherein the virus is a non-enveloped virus.

24. The method of any one of claims 1 to 22, wherein the virus is a reovirus.

25. The method of claim 24, wherein the reovirus is a mammalian reovirus.

26. The method of claim 25, wherein the mammalian reovirus is a human reovirus.

27. The method of claim 26, wherein the human reovirus is a serotype 3 virus.

28. The method of claim 27, wherein the serotype 3 reovirus is the Dearing strain.

29. The method of claim 24, wherein the reovirus is a recombinant reovirus.

30. The method of any one of claims 1 to 29, wherein the cells are human embryonic kidney 293 (HEK 293) cells.

31. The method of claim 30, wherein the HEK 293 cells are grown in suspension.

32. The method of any one of claims 1 to 31 , further comprising purifying the virus by a combination of ion exchange and size exclusion chromatography.

33. The method of claim 32, wherein the ion exchange is performed using an anion exchanger.

34. The method of claim 32 or 33, wherein the ion exchange is performed before the size exclusion chromatography.

35. The method of any one of claims 32 to 34, wherein a phosphate buffer is used in the ion exchange.

36. The method of claim 35, wherein the phosphate buffer comprises 100 mM sodium phosphate.

37. The method of any one of claims 32 to 36, wherein a phosphate buffer is used in the size exclusion chromatography.

38. The method of claim 37, wherein the phosphate buffer comprises 10 to 15 mM sodium phosphate.

39. The method of any one of claims 32 to 38, wherein the size exclusion chromatography is performed at a pH of 7.

4.

40. A composition comprising the virus collected according to any one of claims 1 to 39.

41. The composition of claim 40, further comprising a pharmaceutically acceptable excipient.

42. A method for producing a reovirus, comprising: (a) providing a culture of HEK 293 cells infected with reovirus; (b) contacting the cells with a first buffer comprising Tween 20 and phosphate, and incubating the HEK 293 cells in the presence of the first buffer for a first period of time, thereby producing a cell lysate, wherein the incubation occurs at a temperature of about 30° C. to about 40° C. and at a pH of about 7.0 to about 8.0; (c) contacting the cell lysate with a second buffer comprising an endonuclease for a second period of time to degrade host cell nucleic acids; (d) removing cell debris by filtration; (e) concentrating the filtrate by ultrafiltration and diafiltration; (f) purifying the reovirus by a combination of ion exchange and size exclusion chromatography; and (g) collecting the reovirus.

43. The method of claim 42, wherein the first buffer comprises 10 mM to 25 mM phosphate.

44. The method of claim 42, wherein the first buffer comprises at least 20 mM phosphate.

45. The method of any one of claims 42 to 44, wherein the phosphate salt is sodium phosphate.

46. ​​The method of any one of claims 42 to 45, wherein the first buffer comprises 0.5%, 1.0% or 2.0% (v / v) Tween 20.

47. The method of any one of claims 42 to 46, wherein the first period of time is 60, 120, 180 or 240 minutes.

48. The method of any one of claims 42 to 47, wherein the second period of time is 60 minutes, 90 minutes, 120 minutes, 180 minutes, or 240 minutes.

49. The method of any one of claims 42 to 48, wherein the endonuclease is Benzonase.

50. The method of any one of claims 42 to 49, wherein the endonuclease is contacted with the cell lysate at a concentration of 10 to 20 U / mL.

51. The method of any one of claims 42 to 50, wherein contacting the cell lysate with the endonuclease occurs at a temperature of 30°C to 40°C.

52. The method of any one of claims 42 to 50, wherein contacting the cell lysate with the endonuclease occurs at a temperature of 35°C to 39°C.

53. The method of any one of claims 42 to 52, wherein incubating the host cells occurs at a temperature of 35°C to 39°C.

54. The method of any one of claims 42 to 53, wherein the second buffer further comprises MgCl2.

55. The method of any one of claims 42 to 54, wherein incubating the host cells in the presence of the first buffer occurs at a pH of 8.0.

Images