PROLIFERATIVE DISORDERS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/681,857 filed June 7, 2018 and U.S. Provisional Application No. 62/682,216 filed June 8, 2018. The entirety of each of these applications is hereby incorporated by reference for all purposes.

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The Sequence Listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is l6l6lPCT_ST25.txt. The text file is 13 KB, was created on June 7, 2019, and is being submitted electronically via EFSWeb.

BACKGROUND

Triple-negative breast cancer (TNBC) is characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2/neu). Although targeted therapies against hormone receptor-positive and HER2-positive breast cancer have been efficacious, the absence of these molecules on TNBC cells has limited treatment to cytotoxic chemotherapy, radiotherapy, and surgery. This raises a need for targeted therapeutics against this type of cancer.

Activating mutations of the proto-oncogene Ras is common in human tumors. It is reported that human reovirus requires an activated Ras signaling pathway for infection of cultured cells. Coffey et al., Science. 1998, 282(5392):l332-l334. Attempts have been made to utilize modified reoviruses for the treatment of cancer. See Coffey et al. US. Published Patent Application 2018/0344853. US Patent No. 7,727,534 to Lee reports treatment of neoplasms by administration of reovirus. See also US Patent Nos. 8,137,663, 7,163,678, 7,476,382, 7,163,678, 6,756,234. A significant limitation is viral neutralization by the host antibody response. Thus, there is a need to identify improved reoviruses and methods. Rajani et al. report combination therapy with reovirus and anti -PD- 1 blockade controls tumor growth through innate and adaptive immune responses. Mol Ther, 2016, 24: 166-74.

Mahalingam et al. report pelareorep (REOLYSIN ^® ), an isolate of reovirus Type 3 Dearing, as a live, replication-competent reovirus. Cancers (Basel). 2018, 10(6): 160.

Thete et al. report a reassortant type 1 reovirus with a type 3 M2 gene (T1L/T3DM2) establishes infection with greater efficiency than the parental T1L strain. J Virol. 2016, 90(23): 10951-10962.

References cited herein are not an admission of prior art.

SUMMARY

This disclosure relates to modified reovirus and reovirus particles for use in treating or preventing cancer or other proliferative disorders. In certain embodiments, this disclosure relates to using reovirus particles to delivery chemotherapy agents by conjugation a chemotherapy agent to proteins on the exterior of virion particles. In certain embodiments, this disclosure relates to reoviruses having modified nucleic acid and polypeptide sequences. In certain embodiment, modified reovirus comprise a mixture of genes from different stains and mutations therein.

In certain embodiments, this disclosure relates to reoviruses and particles comprising a gene from a T1L reovirus strain and a gene from a T3D reovirus strain, wherein the reovirus has an M2 gene from the T3D reovirus strain. In certain embodiments, the reovirus is replicative and infective of mammalian cells. In certain embodiments, the reovirus has a nucleic acid encoding L3 wherein the amino acid at position 160 is Threonine (T). In certain embodiments, the reovirus has a nucleic acid encoding S3 wherein the amino acid at position 161 is Threonine (T). In certain embodiments, the reovirus has a nucleic acid encoding S3 wherein the amino acid at position 250 is Valine (V). In certain embodiments, the reovirus has a nucleic acid encoding S4 wherein the amino acid at position 49 is Isoleucine (I).

In certain embodiments, this disclosure relates to reovirus particles comprises a reovirus or reassortant reovirus disclosed herein. In certain embodiments, a reovirus particle is conjugated to a chemotherapy agent.

In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of a reovirus or particle as disclosed herein to a subject in need thereof. In certain embodiments, the subject is diagnosed with cancer. In certain embodiments, the subject is diagnosed with triple-negative breast cancer. In certain embodiments, the reovirus particle or reovirus is administered in combination with a second anti-cancer agent. In certain embodiments, the reovirus particle or reovirus is administered before, during, or after radiation therapy.

In certain embodiments, the oncolytic reoviruses disclosed herein are conjugated to an anti cancer agent such as, but not limited to, bevacizumab, gefitinib, erlotinib, temozolomide, docetaxel, cisplatin, 5-fluorouracil, gemcitabine, tegafur, raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea, adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin, mithramycin, vincristine, vinblastine, vindesine, vinorelbine taxol, taxotere, etoposide, teniposide, amsacrine, topotecan, camptothecin, bortezomib, anagrelide, tamoxifen, toremifene, raloxifene, droloxifene, idoxifene fulvestrant, bicalutamide, flutamide, nilutamide, cyproterone, goserelin, leuprorelin, buserelin, megestrol, anastrozole, letrozole, vorozole, exemestane, finasteride, marimastat, trastuzumab, cetuximab, dasatinib, imatinib, combretastatin, thalidomide, and/or lenalidomide or combinations thereof.

In certain embodiments, this disclosure relates to modified reoviruses comprising genome segments and corresponding proteins of these viruses contain point mutations that differentiate them from wild-type genome sequences. In certain embodiments, genome segments are produced by heterologous expression. In certain embodiments, genome segments are produced in a host organism, which does not naturally have this gene or gene fragment. Insertion of the gene in the heterologous host is performed by recombinant technology.

In certain embodiments, this disclosure relates to a recombinant vector comprising and reovirus gene disclosed herein in operable combination with a heterologous promoter.

In certain embodiments, this disclosure relates to pharmaceutical compositions comprising a reovirus or reovirus particle as disclosed herein and a pharmaceutically acceptable excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A illustrates the reassortment of reovirus gene segments after serial passage in MDA-MB-231 cells. Triple-negative breast cancer MDA-MB-231 cells were co-infected with prototypic strains from three reovirus serotypes (Type 1, Type 2, and Type 3) and serially passaged ten times. Viruses with several combinations of reassorted gene segments were obtained from this serial passage. Two reassortant viruses, rlReovirus (rl) and r2Reovirus (r2), were chosen because of phenotypic characteristics and deep sequenced using Illumina next-generation sequencing.

Figure 1B shows polyacrylamide gel electrophoresis of reovirus prototypic strains T1L, T2J, and T3D and reassortant viruses rlReovirus (rl) and r2Reovirus (r2). Strains are differentiated by migration patterns of three large (L), three medium (M), and four small (S) gene segments.

Figure 2 illustrates certain embodiments of this disclosure. It was identified that rlReovirus and r2Reovirus have gene segments from parental strains T1L and T3D. rlReovirus has seven gene segments from T1L and three from T3D (S2, M2, L2), whereas r2Reovirus has nine gene segments from T1L and one from T3D (M2). In addition, rlReovirus has four non- synonymous point mutations in gene segments S2, S3, S4, and L3. r2Reovirus has three non- synonymous point mutations found in S4, L2, and L3.

Figure 3A shows data indicating rlReovirus and r2Reovirus viruses attach to MDA-MB- 231 cells with similar efficiency as T1L. T3D and pelareorep (T3C$) and have enhanced infectivity in MDA-MB-231 cells compared to parental reoviruses. MDA-MB-231 cells were adsorbed with A6334abeled reovirus at an MOI of 5xl0 ⁴ particles/cell for lh at room temperature. Reovirus attachment was assessed by flow cytometry.

Figure 3B shows data on infectivity of MDA-MB-231 cells adsorbed with reovirus at an MOI of 100 PFU/ml and 5 PFU/ml respectively. Infectivity was assessed 18 hpi by indirect immunofluorescence.

Figure 3C shows data with L929 cells.

Figure 4A shows data indicating certain reassortant viruses have faster cell killing kinetics than other reovirus strains tested in MDA-MB-231 cells at an MOI of 500 PFU/ml and viability assessed days 0 through 7 post-adsorption using Presto Blue reagent.

Figure 4B shows data indicating reassortant viruses have similar cell killing kinetics as parental reoviruses in L929 cells.

Figure 5 shows data on cell viability of MDA-MB-231 cells in the presence of small molecule inhibitor drugs or in combination with r2Reovirus 3 days post infection (dpi). MDA- MB-231 cells were treated for 1 h with 1 :500 DMSO or 0.2, 2.0, or 20 mM of doxorubicin, epirubicin, or topotecan. Cells were infected with MOI 200 PFU/cell of r2Reovirus by adding equal volume of virus in media and incubated at 37°C for lh. Presto Blue was added to cells on 0, 1, 2, and 3 days post infection (dpi), and cell viability was measured as mean fluorescence intensity (MFI).

Figure 6 illustrates conjugation of chemotherapy agents to reovirus as an effective method of targeting and killing cancer cells.

Figure 7A shows data indicating the attachment of reovirus and reo-dox400 on MDA-MB-

231 cells. MDA-MB-231 cells were pre-chilled for 1 h at 4°C and infected with reovirus, reo- dox400 with increasing MOIs, or reovirus at increasing MOIs plus exogenous 5 mM doxorubicin for one hour at 4°C with increasing MOIs, stained with reovirus-specific antiserum, and analyzed for cell surface reovirus. Flow cytometry was performed to determine percent of reovirus positive cells

Figure 7B shows mean fluorescent intensity of reovirus (B).

Figure 8A show infectivity and cytotoxicity of reovirus and reo-dox in MDA-MB-231 cells. MDA-MB-231 cells were pretreated with doxorubicin (10 mM for“No Wash”, 5 pM for “Wash”), then infected with reovirus or reo-dox400 at an MOI 100 PFU/cell for 1 h.“Wash” cells in the reovirus infection with exogenous doxorubicin treatment received fresh 5 pM doxorubicin with inoculum and media spiked with 5 pM doxorubicin after washing out inoculum. After 20 h, cells were scored for infectivity by indirect immunofluorescence assay with reovirus-specific antiserum and DAPI to visualize nuclei.

Figure 8B shows data where MDA-MB-231 cells were infected with reovirus or reo-dox with increasing concentrations of doxorubicin at an MOI of 100 PFU/cell or treated with 5 pM doxorubicin. Cell viability was measured using Presto Blue reagent over a 4 day time course.

Figure 9 shows data from a comet assay for DNA double-strand break damage induced by reo-dox400 in MDA-MB-231 cells. MDA-MB-231 cells were infected with 100 PFU/cell reovirus, reo-dox400, mock, or treated with 5 pM doxorubicin for 0, 1, or 2 days. Cells were embedded in agarose on microscope slides, lysed overnight in neutral lysis solution, electrophoresed, and stained with DAPI. Samples were imaged by fluorescent microscope. Comet tail lengths were measured, and interquartile ranges were compared for each sample. Sampling number is indicated to the right of each dataset. Representative images of comets on day 2 presented for each infection or treatment. Figure 10 shows a group of first S2 gene segments containing amino acid alternatives at position 357, (I) SEQ ID NO: 5, or (V) SEQ ID NO: 6, for reassortant reoviruses rlReovirus- prevalent(rl-p), rlReovirus (rl), and r2Reovirus (r2) when compared to known strains.

Figure 11 shows a group of second S2 gene segments containing amino acid alternatives at position 386, (N) SEQ ID NO: 7, or (H) SEQ ID NO: 8, for reassortant reoviruses rlReovirus- prevalent(rl-p), rlReovirus (rl), and r2Reovirus (r2) when compared to known strains.

Figure 12 shows a group of first S3 gene segments containing amino acid alternatives at position 161, (P) SEQ ID NO: 9, or (T) SEQ ID NO: 10, for reassortant reoviruses rlReovirus (rl) and r2Reovirus (r2) when compared to known strains.

Figure 13 shows a group of second S3 gene segments containing amino acid alternatives at position 250, (I) SEQ ID NO: 11, or (V) SEQ ID NO: 12, for reassortant reoviruses rlReovirus (rl) and r2Reovirus (r2) when compared to known strains.

Figure 14 shows a group of L3 gene segments containing amino acid alternatives at position 160, (A) SEQ ID NO: 13, or (T) SEQ ID NO: 14, for reassortant reoviruses rlReovirus (rl) and r2Reovirus (r2) when compared to known strains.

Figure 15 shows a group of S4 gene segments containing amino acid alternatives at position 49, (V) SEQ ID NO: 15, or (I) SEQ ID NO: 16, for reassortant reoviruses rlReovirus (rl), and r2Reovirus (r2) when compared to known strains.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of immunology, medicine, organic chemistry, biochemistry, molecular biology, pharmacology, physiology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appended claims, the singular forms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

As used herein, a“reassortant” virus refers to a viral genome containing at least one gene that is from a different viral strain such entire sequence of the viral genome is not naturally occurring.

As used herein, "subject" refers to any animal, preferably a human patient, livestock, or domestic pet.

As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g. patient) is cured and the disease is eradicated. Rather, embodiments of the present disclosure also contemplate treatment that merely reduces symptoms, and/or delays disease progression. As used herein, the term "combination with" when used to describe administration with an additional treatment means that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof.

As used herein, the term“conjugate” refers to the joining of two compounds by covalent bonding. In certain embodiments, this disclosure relates to a method in which a drug is bonded to a succinimidyl ester, and the drug-ester conjugate is then bonded to the surface of a virus particle. In certain embodiments, this disclosure relates to the virus produced by this method.

As used herein, the term“triple-negative breast cancer” or“TNBC” refers to breast cancer in which cancer cells do not highly express estrogen receptors (ERs), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER2) on their surface.

As used herein, a“non-naturally occurring” reovirus is a reovirus that has at least one nucleic acid or amino acid modification as compared to wild type sequences derived from, for example, a field isolate (e.g., a patient). Such manipulated or modified reoviruses include laboratory strains, mutagenized, or reassortant versions.

A modification generally occurs at the nucleic acid level, which may or may not manifest itself in the encoded polypeptide. A modification in a nucleic acid can result in one or more conservative or non-conservative amino acid substitutions in the encoded polypeptide, a shift in the reading frame of translation (“frame-shift”) resulting in an entirely different polypeptide encoded from that point on, a premature stop codon resulting in a truncated polypeptide (“truncation”), or a modification in a reovirus nucleic acid may not change the encoded polypeptide at all (“silent” or“nonsense”).

Reovirus particles are reconstituted using methods known in the art. Reoviruses are cultured in, for example, mouse L929 cells or neoplastic cells (e.g., MCF7 (ATCC Accession No. HTB-22), SKBR3 (ATCC Accession No. HTB-30), or MDA MB 468 cells (ATCC Accession No. HTB 132)), and selected based on any number of characteristics that may indicate, for example, a growth advantage over a reovirus that does not contain one or more modifications. Reoviruses are selected following culturing in a cell line (neoplastic or otherwise) and/or following infection of an animal model system.

Inducible promoter systems using heterologous components from other organisms are advantageous for recombinant production of proteins. Promoters are DNA sequences located upstream of coding regions which are specific binding sites for proteins involved in the initiation and regulation of transcription. Promoters of protein-encoding genes are often a region located about 40 bp upstream of the transcriptional initiation site. Some promoter contain a TATA box. Proximal and distal regions of the promoter contain different regulatory sequences such as enhancers, silencers, insulators, and cis-elements that contribute to the fine regulation of gene expression.

In certain embodiments, the disclosure relates to vectors comprising a nucleic acid encoding a peptide disclosed herein or chimeric protein thereof. In certain embodiments, this disclosure relates to expression systems, e.g., in vitro or in vivo cells, comprising a nucleic acid vector in operable combination with a heterologous or autologous promoter encoding a peptide disclosed herein or chimeric protein thereof.

In certain embodiments, the vector optionally comprises a mammalian, human, insect, viral, bacterial, bacterial plasmid, yeast associated origin of replication or gene such as a gene or retroviral gene or lentiviral LTR, TAR, RRE, PE, SLIP, CRS, and INS nucleotide segment or gene selected from tat, rev, nef, vif, vpr, vpu, and vpx or structural genes selected from gag, pol, and env.

In certain embodiments, the vector optionally comprises a gene vector element (nucleic acid) such as a promoter, selectable marker region, lac operon, a CMV promoter, a hybrid chicken B-actin/CMV enhancer (CAG) promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerase promoter, SV40 promoter, internal ribosome entry site (IRES) sequence, cis-acting woodchuck post regulatory element (WPRE), scaffold-attachment region (SAR), inverted terminal repeats (ITR), FLAG tag coding region, c-myc tag coding region, metal affinity tag coding region, streptavidin binding peptide tag coding region, polyHis tag coding region, HA tag coding region, MBP tag coding region, GST tag coding region, polyadenylation coding region, SV40 polyadenylation signal, SV40 origin of replication, Col El origin of replication, fl origin, pBR322 origin, or pUC origin, TEV protease recognition site, loxP site, Cre recombinase coding region, or a multiple cloning site such as having 5, 6, or 7 or more restriction sites within a continuous segment of less than 50 or 60 nucleotides or having 3 or 4 or more restriction sites with a continuous segment of less than 20 or 30 nucleotides. In certain embodiments, the disclosure relates to peptides comprising sequences disclosed herein or variants or fusions thereof wherein the amino terminal end or the carbon terminal end of the amino acid sequence are optionally attached to a heterologous amino acid sequence, label, or reporter molecule. A "label" refers to a detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein or reovirus particle disclosed herein, to facilitate detection. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. In one example, a "label receptor" refers to incorporation of a heterologous polypeptide. A label includes the incorporation of a radiolabeled amino acid or the covalent attachment of biotinyl moieties to a polypeptide that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as ¹⁸ F, ³⁵ S, or ¹³¹ I) fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

Reovirus (Orthoreovirus)

Reovirus infections often occurs during childhood and are cleared without significant health risks to the host. Reoviruses have a double-stranded, segmented RNA genome. The virions measure about 60-80 nm in diameter and possess two capsid shells. The mammalian reovirus genome consists of double-stranded RNA in 10 discrete segments with a total genome size of about 23.5 kbp. Three serologically distinct but related types of reovirus have been recovered from mammalian species: type 1 (representative strains include, for example, Lang (T1L)), type 2 (representative strains include, for example, Jones (T2J)) and type 3 (representative strains include, for example, Dealing or Abney (T3D or T3 A, respectively)).

In certain embodiments, this disclosure relates to reassortant reoviruses comprising a gene from a T1L reovirus strain and a gene from a T3D reovirus strain, wherein the reovirus has an M2 gene from the T3D reovirus strain. In certain embodiments, the reovirus is replicative and infective of mammalian cells. In certain embodiments, the reovirus has a nucleic acid encoding L3 wherein the amino acid at position 160 is Threonine (T). In certain embodiments, the reovirus has a nucleic acid encoding S2 wherein the amino acid at position 357 is Valine (V). In certain embodiments, the reovirus has a nucleic acid encoding S2 wherein the amino acid at position 357 is Isoleucine (I). In certain embodiments, the reovirus has a nucleic acid encoding S2 wherein the amino acid at position 386 is Asparagine (N). In certain embodiments, the reovirus has a nucleic acid encoding S2 wherein the amino acid at position 386 is Histidine (H).

In certain embodiments, the reovirus has a nucleic acid encoding S3 wherein the amino acid at position 161 is Threonine (T). In certain embodiments, the reovirus has a nucleic acid encoding S3 wherein the amino acid at position 250 is Valine (V).

In certain embodiments, the reovirus has a nucleic acid encoding S4 wherein the amino acid at position 49 is Isoleucine (I).

In certain embodiments, this disclosure relates to reovirus particles comprises a reovirus or reassortant reovirus disclosed herein. In certain embodiments, the reovirus particle is conjugated to a chemotherapy agent.

The reovirus genome contains ten double-stranded RNA segments, each encoding a single protein, which are classified as large (Ll, L2, L3 encode lambdal - lambda3), medium (Ml, M2, M3 encode mul - mu3), or short (Sl, S2, S3, S4 encode sigmal - sigma4) according to their nucleotide length. The reovirus particle comprises an inner capsid of sigma2 and lambdal, an outer capsid of sigma3 and mul, coated in lambda2 spike proteins, which terminate in sigmal attachment proteins.

Ll encodes Lambda 3, a minor core protein (e.g., GeneBank Reference M24734.1, 1267 amino acids, T3D reovirus strain).

L2 encodes Lambda 2, a core-spike protein (e.g„ GeneBank Reference J03488.1, 1289 amino acid, T3D reovirus strain).

L3 encodes Lambda 1, a major core protein (e.g., GeneBank Reference AF129822.1, 1275 amino acid, T3D reovirus strain). The first 168 amino acids are provided below in SEQ ID NO: 1. Amino acid position 160 is highlighted in bold:

MKRIPRKTKGK S S GKGND S TER ADDGS S QLRDKQNNK AGP ATTEPGT SNREQ Y KARPGIASVQRATESAEMPMKNNDEGTPDKKGNTKGDLVNEHSEAKDEADEATKKQA KDTDK SK AQ VT Y SDTGINN ANEL SRS GNVDNEGGSN QKPMS TRI AE AT S AI V SKHP AR

(SEQ ID NO: 1). Ml encodes Mu2, a minor core protein (e.g., GeneBank Reference AF461684.1, 736 amino acids, T3D reovirus strain).

M2 encodes Mul(MulCa), an outer capsid protein (e.g., GeneBank Reference M20161.1, 708 amino acids, T3D reovirus strain).

M3 encodes MuNS (MuNSCb), a non-structural protein (e.g., GeneBank Reference AF 174384.1, 721 amino acids, T3D reovirus strain).

51 encodes Sigma l/Sigma l sb, an outer capsid protein (e.g., GeneBank Reference M10262.1, 455 amino acids, T3D reovirus strain).

52 encodes Sigma 2, an inner capsid protein e.g., (e.g., GeneBank Reference M25780.1, 418 amino acids, T3D reovirus strain). The 418 amino acids are provided below in SEQ ID NO:

2. Amino acid positions 357 and 386 are highlighted in bold:

MARAAFLFKT VGF GGLQNVPINDELS SHLLRAGN SPWQLTQFLDWISLGRGL AT SALVPTAGSRYYQMSCLLSGTLQIPFRPNHRWGDIRFLRLVWSAPTLDGLVVAPPQVLA QP ALQ AQ ADRVYDCDD YPFLARDPRFKHRVY QQL S AVTLLNLT GF GPIS YVRVDEDM WSGDVNQLLMNYFGHTFAEIAYTLCQASANRPWEYDGTYARMTQIVLSLFWLSYVGVI HQQNTYRTFYFQCNRRGDAAEVWILSCSLNHSAQIRPGNRSLFVMPTSPDWNMDVNLI L S S TLT GCLC S GS QLPLIDNN S VP A V SRNIHGWT GR AGN QLHGF Q VRRM VTEF CDRLRR DGVMTQAQQNQVEALADQTQQFKRDKLETWAREDDQYNQAHPNSTMFRTKPFTNAQ W GRGNT GAT S A AI A ALI (SEQ ID NO: 2).

53 encodes Sigma NS, a non-structural protein (e.g., GeneBank Reference Ml 8389.1, 366 amino acids, T1L reovirus strain). The first 270 amino acids are provided below in SEQ ID NO:

3. Amino acid positions 161 and 250 and highlighted in bold:

MAS SLRAAISKIKRDD VGQQ VCPNYVMLRSS VTTKVVRNVVEY QIRTGGFF SCL AMLRPLQYAKRERLLGQRNLERISTRDILQTRDLHSLCMPTPDAPMSNHQAATMRELIC SYFKVDHTDGLKYIPMDERYSPSSLARLFTMGMAGLHITTEPSYKRVPIMHLAADLDCM TLALPYMITLDGDTVVPVAPTLSAEQLLDDGLKGLACMDISYGCEVDASNRSAGDQSM D S SRCINEL Y CEET AE AICILKT CL VLN CMQFKLEMDDL A (SEQ ID NO: 3)

54 encodes Sigma 3, an outer capsid protein (e.g., GeneBank Reference K02739.1 with 365 amino acids, T3D reovirus strain). The first 102 amino acids are provided below in SEQ ID NO: 4. Amino acid position 49 is highlighted in bold: ME V CLPN GHQ VVDLINN AFEGR V SI Y S AQEGWDKTIS AQPDMM V C GGA V V CM HCLGV V GSLQRKLKHLPHHRCNQQIRHQD YVD V QF ADR VT AHWKRGML SF (SEQ ID NO: 4).

In certain embodiments, this disclosure relates to reassortant genotypes of reovirus (mammalian orthoreovirus). These viruses contain genome segments, and the corresponding proteins encoded by those segments, from two different strain backgrounds, known in the art as T1L and T3D. These viruses are generated by reverse genetics, in which ten plasmids, each containing the cloned DNA sequence corresponding to the RNA sequence of a genome segment, are transfected into baby hamster kidney (BEK) cells that stably express T7 RNA polymerase (BHK-T7 cells). By combining plasmids encoding RNA segments from different strains, it is possible to generate a reassortant virus comprising the desired combination of genome segments and the proteins encoded by said segments.

In certain embodiments, this disclosure relates to reassortant genotypes of mammalian reovirus with point mutations that distinguish the RNA sequence of their segments from that of wild-type T1L or T3D. These point mutations can be introduced to the reverse genetics plasmids described above by means of site-directed mutagenesis.

In certain embodiments, this disclosure relates to a DNA vector comprising a plasmid encoding the L3 protein of reovirus T1L, wherein the L3 protein contains the mutations A160T.

In certain embodiments, this disclosure relates to a DNA vector comprising a plasmid encoding the S3 protein of reovirus T1L, wherein the S3 protein contains the mutation P161T.

In certain embodiments, this disclosure relates to a DNA vector comprising a plasmid encoding the S2 protein of reovirus T3D, wherein the S2 protein contains the mutation V357I.

In certain embodiments, this disclosure relates to a DNA vector comprising a plasmid encoding the S4 protein of reovirus T1L, wherein the S4 protein contains the mutation V49I.

In certain embodiments, this disclosure relates to a composition comprising: liposomes, a DNA vector encoding the Ll protein of reovirus T1L, a DNA vector encoding the L2 protein of reovirus T3D, a DNA vector encoding the L3 protein of reovirus T1L, a DNA vector encoding the L4 protein of reovirus T1L, a DNA vector encoding the Ml protein of reovirus T1L, a DNA vector encoding the M2 protein of reovirus T3D, a DNA vector encoding the M3 protein of reovirus T1L, a DNA vector encoding the Sl protein of reovirus T1L, a DNA vector encoding the S2 protein of reovirus T3D, and a DNA vector encoding the S3 protein of reovirus T1L, wherein the DNA vectors are contained within the liposomes.

In certain embodiments, the DNA vector encoding the L3 protein of T1L contains the mutation A160T, the DNA vector encoding the S2 protein of T3D contains the mutation V357I, the DNA vector encoding the S3 protein of T1L contains the mutation P161T, and the DNA vector encoding the S4 protein of T1L contains the mutation V49I.

In certain embodiments, this disclosure relates to a composition comprising composition comprising: liposomes, a DNA vector encoding the Ll protein of reovirus T1L, a DNA vector encoding the L2 protein of reovirus T1L, a DNA vector encoding the L3 protein of reovirus T1L, a DNA vector encoding the L4 protein of reovirus T1L, a DNA vector encoding the Ml protein of reovirus T1L, a DNA vector encoding the M2 protein of reovirus T3D, a DNA vector encoding the M3 protein of reovirus T1L, a DNA vector encoding the Sl protein of reovirus T1L, a DNA vector encoding the S2 protein of reovirus T1L, and a DNA vector encoding the S3 protein of reovirus T1L, wherein the DNA vectors are contained within the liposomes.

In certain embodiments, this disclosure relates to virus-chemotherapeutic agent conjugates and method of producing the same. In certain embodiments, a chemotherapeutic agent of interest is bonded to a linker molecule. In certain embodiments, this linker molecule is a succinimidyl ester. In certain embodiments, the succinimidyl ester is succinimidyl 4-[N- maleimidomethyl]cyclohexane-l-carboxylate (SMCC). To conjugate the chemotherapeutic agent of interest to the virus surface, the agent is first incubated with SMCC at room temperature for about 30 minutes to join an amine group on the agent at the carbonyl adjoining the hexane of SMCC, forming an amide, and the SMCC-agent complex is separated from excess SMCC by desalting. The SMCC-agent complex is then incubated with the virus at room temperature for about 30 minutes, to join a sulfhydryl or amino group on a viral protein to the succinimidyl ring, yielding a virus-agent conjugate. This virus-agent conjugate is typically purified by dialysis before use. When the chemotherapeutic agent is doxorubicin and the virus is reovirus, the resultant virus- agent conjugate is referred to as“reo-dox400.” Enhanced Killing of Triple-Negative Breast Cancer Cells by Reassortant Reovirus and Topoisomerase Inhibitors

Triple-negative breast cancer constitutes a subset of breast cancer that is associated with higher rates of relapse, decreased survival, and limited therapeutic options. Mammalian orthoreovirus (reovirus) selectively infects and kills transformed cells and a serotype 3 reovirus is being assessed for its efficacy as an oncolytic agent against several cancers. It is unclear if reovirus serotypes differentially infect and kill triple-negative breast cancer cells and if reovirus-induced cytotoxicity of breast cancer cells can be enhanced by modulating the activity of host molecules and pathways. Reassortant reoviruses were generated by forward genetics with enhanced infective and cytotoxic properties in triple-negative breast cancer cells. From a high-throughput screen of small molecule inhibitors, topoisomerase inhibitors were identified as a class of drugs that enhance reovirus infectivity and cytotoxicity of triple-negative breast cancer cells. Treatment of triple- negative breast cancer cells with topoisomerase inhibitors activates DNA damage response pathways and reovirus infection induces robust production of Type III, but not Type I, interferon. Together, these data show that reassortant viruses with a genetic composition generated by forward genetics in combination with topoisomerase inhibitors more efficiently infect and kill triple- negative breast cancer cells.

Reovirus has a segmented genome with three large (L), three medium (M), and four small (S) dsRNA gene segments. There are three different reovirus serotypes (Type 1, 2, and 3) based on the neutralization ability of antibodies raised against the sΐ attachment protein that is encoded by the Sl gene segment. Reoviruses infect most mammals, and although humans are infected during childhood, infection seldom results in disease. Reovirus induces programmed cell death in vitro and in vivo. Although both Type 1 and Type 3 reovirus can induce apoptosis, Type 3 reoviruses induce apoptosis and necroptosis more efficiently in most cells. Serotype-dependent differences in apoptosis induction segregate with the Sl and M2 gene segments.

Co-infection and serial passaging of parental reoviruses in TNBC cells yields reassortant viruses with enhanced oncolytic capacities compared to parental reoviruses. Reassortant reoviruses have a predominant Type 1 genetic composition with some Type 3 gene segments as well as synonymous and non-synonymous point mutations. Reassortant reoviruses have enhanced infective and cytotoxic capacities in TNBC cells compared to parental viruses. To further enhance the oncolytic properties of these reassortant viruses, a high-throughput screen of small molecule inhibitors was used. DNA- damaging topoisomerase inhibitors were identified as a class of drugs that reduces TNBC cell viability while enhancing reovirus infectivity. Infection of TNBC cells in the presence of topoisomerase inhibitors results in induction of DNA damage, increased levels of Type III but not Type I interferon, and enhanced cell killing. Together, reassortant reo viruses with a genetic composition have enhanced oncolytic properties and pairing of topoisomerase inhibitors with reovirus potentiates TNBC cell killing.

During cell entry, reovirus traverses to endosomes where cathepsin proteases cleave outer capsid protein s3, forming an infectious subvirion particle (ISVP). Certain reassortants disclosed herein have a nonsynonymous mutation in the s3 -encoding S4 gene segment that results in a V49I substitution. This mutation has not been identified to impact reovirus disassembly kinetics, but it is possible it could expedite viral cell entry kinetics. However, reassortant viruses were equally sensitive to E64-d treatment as parental viruses. Although reassortant viruses infected MDA-MB-231 cells more efficiently than T1L, T3D, and pelareorep (T3C$), replication kinetics of the reassortant viruses were similar except for T3D, which had slower replication kinetics. These data indicate that Type 1 reoviruses replicate with enhanced kinetics compared to T3D, but that genetic differences between T3D and T3C$ are sufficient to allow T3C$ to replicate as efficiently as Type 1 viruses. These data also suggest that the enhanced cytotoxic properties of the reassortant viruses over parental viruses are not due to enhanced replication kinetics in MDA-MB-231 cells.

The reovirus L3, S2, and S3 gene segments have distinct roles in reovirus replication. The L3- encoded lΐ protein is a major inner-capsid protein that has phosphohydrolase activity and participates in viral transcription. The S2-encoded s2 protein is essential for the assembly of viral cores. The S3- encoded nonstructural protein aNS is required for viral factory formation. The similarity in replication efficiency observed between T1L and the reassortant viruses suggests the A160T mutation in L3 and I250V mutation in S3 (found in both reassortants) and P161T in S3 (in rlReovirus only) do not impact overall replication efficiency. However, it is possible that point mutations in these gene segments in the reassortant viruses impact the activity of the viral proteins that result in enhanced infectivity or cytotoxicity in the context of TNBC cells.

Of the viruses tested in MDA-MB-231 cells, rlReovirus and r2Reovirus impaired cell viability with the fastest kinetics, and only T3D was severely deficient in killing these cells. The poor induction of cell death by T3D may be related to its dampened replication in these cells. Differences in the induction of apoptosis by reovirus strains segregate with the M2 and Sl gene segments. Apoptosis is activated by fragments of the M2-encoded mΐ protein generated during reovirus cell entry. The mΐ protein impacts reovirus infectivity by enhancing reovirus attachment to cells. Sl is genetically linked to reo virus induction of apoptosis through the activities of both sΐ and s 1 s, although it is unclear if the effects of s I s on the induction of cell death are independent of its ability to regulate viral protein synthesis and induce cell cycle arrest. Significant levels of cell cycle arrest were not observed in MDA- MB-231 cells infected with reassortant reoviruses. It is possible that the enhanced cytopathic properties of reassortant viruses in the context of TNBC cells maps to the T3D M2 gene segment, the various nonsynonymous changes, or a combination of both.

Six microtubule-inhibiting drugs, digoxin, and two serotonin antagonists affected reovirus infectivity, corroborating the role of microtubules, the sodium-potassium ATPase pump, and serotonin receptors in reovirus infection. Of the 17 molecules that enhanced infectivity, 4 are topoisomerase I (topotecan) or II (doxorubicin, epirubicin, and etoposide) inhibitors. Treatment of cells with topoisomerase inhibitors resulted in increased infectivity, with no effect on virus attachment, and no significant increase in viral RNA levels at 8 and 12 h post infection and slight increases in viral RNA at 24 and 48 h post infection. Topoisomerase inhibitors promote DNA double-strand breaks leading to cell death. Reovirus infection does not induce DNA double-strand breaks and promotes cell death through the induction of extrinsic and intrinsic apoptosis or necroptosis.

Methods of Use

In certain embodiments, this disclosure relates to methods of treating cancer or other proliferative disorders comprising administering an effective amount of a reovirus or particle as disclosed herein to a subject in need thereof. In certain embodiments, the subject is diagnosed with cancer. In certain embodiments, the subject is diagnosed with triple-negative breast cancer. In certain embodiments, the reovirus particle or reovirus is administered in combination with a second anti-cancer agent. In certain embodiments, the reovirus particle or reovirus is administered before, during, or after radiation therapy.

"Cancer" refers any of various cellular diseases with malignant neoplasms characterized by the proliferation of cells. It is not intended that the diseased cells must actually invade surrounding tissue and metastasize to new body sites. Cancer can involve any tissue of the body and have many different forms in each body area. Within the context of certain embodiments, whether "cancer is reduced" may be identified by a variety of diagnostic manners known to one skill in the art including, but not limited to, observation the reduction in size or number of tumor masses or if an increase of apoptosis of cancer cells observed, e.g., if more than a 5 % increase in apoptosis of cancer cells is observed for a sample compound compared to a control without the reovirus or reovirus particle. It may also be identified by a change in relevant biomarker or gene expression profile, such as PSA for prostate cancer, HER2 for breast cancer, or others.

Contemplated malignancies are located in the colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, hypophysis, testicles, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax and genito-urinary apparatus. Also contemplated are childhood acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adrenocortical carcinoma, adult (primary) hepatocellular cancer, adult (primary) liver cancer, adult acute lymphocytic leukemia, adult acute myeloid leukemia, adult Hodgkin's disease, adult Hodgkin's lymphoma, adult lymphocytic leukemia, adult non-Hodgkin's lymphoma, adult primary liver cancer, adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-related malignant tumors, anal cancer, astrocytoma, cancer of the biliary tract, cancer of the bladder, bone cancer, brain stem glioma, brain tumors, breast cancer, cancer of the renal pelvis and ureter, primary central nervous system lymphoma, central nervous system lymphoma, cerebellar astrocytoma, brain astrocytoma, cancer of the cervix, childhood (primary) hepatocellular cancer, childhood (primary) liver cancer, childhood acute lymphoblastic leukemia, childhood acute myeloid leukemia, childhood brain stem glioma, childhood cerebellar astrocytoma, childhood brain astrocytoma, childhood extracranial germ cell tumors, childhood Hodgkin's disease, childhood Hodgkin's lymphoma, childhood visual pathway and hypothalamic glioma, childhood lymphoblastic leukemia, childhood medulloblastoma, childhood non-Hodgkin's lymphoma, childhood supratentorial primitive neuroectodermal and pineal tumors, childhood primary liver cancer, childhood rhabdomyosarcoma, childhood soft tissue sarcoma, childhood visual pathway and hypothalamic glioma, chronic lymphocytic leukemia, chronic myeloid leukemia, cancer of the colon, cutaneous T-cell lymphoma, endocrine pancreatic islet cells carcinoma, endometrial cancer, ependymoma, epithelial cancer, cancer of the oesophagus, Ewing's sarcoma and related tumors, cancer of the exocrine pancreas, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic biliary tract cancer, cancer of the eye, breast cancer in women, Gaucher's disease, cancer of the gallbladder, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal tumors, germ cell tumors, gestational trophoblastic tumor, head and neck cancer, hepatocellular cancer, Hodgkin's disease, Hodgkin's lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancers, intraocular melanoma, islet cell carcinoma, islet cell pancreatic cancer, Kaposi's sarcoma, cancer of kidney, cancer of the larynx, cancer of the lip and mouth, cancer of the liver, cancer of the lung, lymphoproliferative disorders, macroglobulinemia, breast cancer in men, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, occult primary metastatic squamous neck cancer, primary metastatic squamous neck cancer, metastatic squamous neck cancer, multiple myeloma, multiple myeloma/plasmatic cell neoplasia, myelodysplastic syndrome, myelogenous leukemia, myeloid leukemia, myeloproliferative disorders, paranasal sinus and nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma during pregnancy, non melanoma skin cancer, non-small cell lung cancer, metastatic squamous neck cancer with occult primary, buccopharyngeal cancer, malignant fibrous histiocytoma, malignant fibrous osteosarcoma/histiocytoma of the bone, epithelial ovarian cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, paraproteinemias, purpura, parathyroid cancer, cancer of the penis, hypophysis tumor, neoplasia of plasmatic cells/multiple myeloma, primary central nervous system lymphoma, primary liver cancer, prostate cancer, rectal cancer, renal cell cancer, cancer of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, cancer of the salivary glands, sarcoidosis, sarcomas, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous neck cancer, stomach cancer, pineal and supratentorial primitive neuroectodermal tumors, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, transitional renal pelvis and ureter cancer, trophoblastic tumors, cell cancer of the renal pelvis and ureter, cancer of the urethra, cancer of the uterus, uterine sarcoma, vaginal cancer, optic pathway and hypothalamic glioma, cancer of the vulva, Waldenstrom's macroglobulinemia, Wilms' tumor and any other hyperproliferative disease, as well as neoplasia, located in the system of a previously mentioned organ.

The cancer to be treated in the context of the present disclosure may be any type of cancer or tumor. These tumors or cancer include, and are not limited to, tumors of the hematopoietic and lymphoid tissues or hematopoietic and lymphoid malignancies, tumors that affect the blood, bone marrow, lymph, and lymphatic system. Hematological malignancies may derive from either of the two major blood cell lineages: myeloid and lymphoid cell lines. The myeloid cell line normally produces granulocytes, erythrocytes, thrombocytes, macrophages and mast cells; the lymphoid cell line produces B, T, NK and plasma cells. Lymphomas, lymphocytic leukemias, and myeloma are from the lymphoid line, while acute and chronic myelogenous leukemia, myelodysplastic syndromes and myeloproliferative diseases are myeloid in origin.

In certain embodiments, reovirus or particles disclosed herein are combined with another anti-cancer agent. In certain embodiments, the anti-cancer agent selected from abemaciclib, abiraterone acetate, methotrexate, paclitaxel, adriamycin, acalabrutinib, brentuximab vedotin, ado- trastuzumab emtansine, aflibercept, afatinib, netupitant, palonosetron, imiquimod, aldesleukin, alectinib, alemtuzumab, pemetrexed disodium, copanlisib, melphalan, brigatinib, chlorambucil, amifostine, aminolevulinic acid, anastrozole, apalutamide, aprepitant, pamidronate disodium, exemestane, nelarabine, arsenic trioxide, ofatumumab, atezolizumab, bevacizumab, avelumab, axicabtagene ciloleucel, axitinib, azacitidine, carmustine, belinostat, bendamustine, inotuzumab ozogamicin, bevacizumab, bexarotene, bicalutamide, bleomycin, blinatumomab, bortezomib, bosutinib, brentuximab vedotin, brigatinib, busulfan, irinotecan, capecitabine, fluorouracil, carboplatin, carfilzomib, ceritinib, daunorubicin, cetuximab, cisplatin, cladribine, cyclophosphamide, clofarabine, cobimetinib, cabozantinib-S-malate, dactinomycin, crizotinib, ifosfamide, ramucirumab, cytarabine, dabrafenib, dacarbazine, decitabine, daratumumab, dasatinib, defibrotide, degarelix, denileukin diftitox, denosumab, dexamethasone, dexrazoxane, dinutuximab, docetaxel, doxorubicin, durvalumab, rasburicase, epirubicin, elotuzumab, oxaliplatin, eltrombopag olamine, enasidenib, enzalutamide, eribulin, vismodegib, erlotinib, etoposide, everolimus, raloxifene, toremifene, panobinostat, fulvestrant, letrozole, filgrastim, fludarabine, flutamide, pralatrexate, obinutuzumab, gefitinib, gemcitabine, gemtuzumab ozogamicin, glucarpidase, goserelin, propranolol, trastuzumab, topotecan, palbociclib, ibritumomab tiuxetan, ibrutinib, ponatinib, idarubicin, idelalisib, imatinib, talimogene laherparepvec, ipilimumab, romidepsin, ixabepilone, ixazomib, ruxolitinib, cabazitaxel, palifermin, pembrolizumab, ribociclib, tisagenlecleucel, lanreotide, lapatinib, olaratumab, lenalidomide, lenvatinib, leucovorin, leuprolide, lomustine, trifluridine, olaparib, vincristine, procarbazine, mechlorethamine, megestrol, trametinib, temozolomide, methylnaltrexone bromide, midostaurin, mitomycin C, mitoxantrone, plerixafor, vinorelbine, necitumumab, neratinib, sorafenib, nilutamide, nilotinib, niraparib, nivolumab, tamoxifen, romiplostim, sonidegib, omacetaxine, pegaspargase, ondansetron, osimertinib, panitumumab, pazopanib, interferon alfa- 2b, pertuzumab, pomalidomide, mercaptopurine, regorafenib, rituximab, rolapitant, rucaparib, siltuximab, sunitinib, thioguanine, temsirolimus, thalidomide, thiotepa, trabectedin, valrubicin, vandetanib, vinblastine, vemurafenib, vorinostat, zoledronic acid, or combinations thereof such as cyclophosphamide, methotrexate, 5-fluorouracil (CMF); doxorubicin, cyclophosphamide (AC); mustine, vincristine, procarbazine, prednisolone (MOPP); sdriamycin, bleomycin, vinblastine, dacarbazine (ABVD); cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP); rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone (RCHOP); bleomycin, etoposide, cisplatin (BEP); epirubicin, cisplatin, 5-fluorouracil (ECF); epirubicin, cisplatin, capecitabine (ECX); methotrexate, vincristine, doxorubicin, cisplatin (MV AC).

In certain embodiments, the anti-cancer agent is an anti-PD-l, anti-CTLA4 antibody or combinations thereof, such as an anti-CTLA4 (e.g., ipilimumab, tremelimumab) and anti -PD 1 (e.g., nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab). In certain embodiments, the method of administration is in a subject with a lymphodepleted environment. In certain embodiments, lymphodepleting agents (e.g., cyclophosphamide and fludarabine).

In certain embodiments, the disclosure contemplates treating or preventing breast cancer using compounds disclosed herein and one more other anti-cancer agents. In certain embodiments, the disclosure contemplates treating or preventing breast cancer using compounds disclosed herein and trastuzumab and/or lapatinib. In certain embodiments, the disclosure contemplates treating or preventing breast cancer using compounds disclosed herein and docetaxel and cyclophosphamide. In certain embodiments, the disclosure contemplates treating or preventing breast cancer using compounds disclosed herein and docetaxel, carboplatin, and trastuzumab. In certain embodiments, the disclosure contemplates treating or preventing breast cancer using compounds disclosed herein and cyclophosphamide, doxorubicin, and 5-fluorouracil (5-FU). In certain embodiments, the disclosure contemplates treating or preventing breast cancer using compounds disclosed herein and docetaxel, doxorubicin, and cyclophosphamide. In certain embodiments, the disclosure contemplates treating or preventing breast cancer using compounds disclosed herein and doxorubicin and cyclophosphamide followed by paclitaxel or docetaxel. In certain embodiments, the disclosure contemplates treating or preventing breast cancer using compounds disclosed herein and 5-FU, epirubicin, and cyclophosphamide followed by docetaxel or paclitaxel.

In certain embodiments, reovirus as disclosed herein generally exhibits a growth advantage over a reovirus that does not contain a corresponding modification. Representative growth advantages include, but are not limited to, an increased rate of lysis; an increased size of plaque formation; an increased rate of RNA replication; an increased rate of RNA transcription; an increased rate of translation; an increased rate of virus assembly and/or packaging; an increased number of viral progeny; an increased ability of a reovirus to be taken up by a host cell; an increased or enhanced ability to uncoat; enhanced cell lysis or inducement to cell death including apoptosis, necrosis or autophagy; an enhanced ability to infect, lyse and kill human neoplastic cells lines; decreased immunogenicity in mammalian cells; differential susceptibility to interferon sensitivity; decreased toxicity toward the host; enhanced drug interaction; enhanced radiotherapy interaction; or the ability to release effective tumor epitopes.

In certain embodiments, this disclosure relates to methods of treating a proliferative disorder in a patient. Such methods generally include administering a modified reovirus or virus particle as described herein or a pharmaceutical composition containing such a modified reovirus or reovirus particle to the patient. Typically, the reovirus is administered in an amount effective to cause oncolysis, and can be administered more than once. Representative routes of administration include, for example, direct injection, intravenously, intravascularly, intramuscularly, subcutaneously, intraperitoneally, topically, orally, rectally, vaginally, nasally, or by inhalation. The methods of treating a proliferative disorder as described herein can be accompanied by one of more procedures such as surgery, chemotherapy, radiation therapy, and immunosuppressive therapy.

A“proliferative disorder” is any cellular disorder in which the cells proliferate more rapidly than normal tissue growth. Thus a“proliferating cell” is a cell that is proliferating more rapidly than normal cells. A proliferative disorder includes, but is not limited to, neoplasms, which are also referred to as tumors. A neoplasm includes, but is not limited to, pancreatic cancer, breast cancer, brain cancer (e.g., glioblastoma), lung cancer, prostate cancer, colorectal cancer, thyroid cancer, renal cancer, adrenal cancer, liver cancer, neurofibromatosis, and leukemia. A neoplasm includes a solid neoplasm (e.g. sarcoma or carcinoma) or a cancerous growth affecting the hematopoietic system (e.g., lymphoma or leukemia). Other proliferative disorders include, but are not limited to neurofibromatosis.

Generally, in proliferative disorders for which reovirus is used as a treatment, at least some of the proliferating cells have a mutation in which the Ras gene (or an element of the Ras signaling pathway) is activated, either directly (e.g., by an activating mutation in Ras) or indirectly (e.g., by activation of an upstream or downstream element in the Ras pathway). Activation of an upstream element in the Ras pathway includes, for example, transformation with epidermal growth factor receptor (EGFR). Activation of a downstream element in the Ras pathway includes, for example, a mutation within B-Raf. In addition, reovirus is useful for treating proliferative disorders caused by mutations or dysregulation of PKR.

In certain embodiments, a reovirus or particle as disclosed herein is administered to a mammal that has a proliferative disorder. As used herein, administration refers to delivery of a reovirus or particle such that the reovirus or particle contacts the proliferating cells. The route by which a reovirus or particle is administered depends on the type of disorder and the location of the proliferating cells. A wide variety of administration routes can be employed. For example, for a solid neoplasm that is accessible, a reovirus or particle is administered by direct injection. For a hematopoietic neoplasm, for example, a reovirus or particle is administered intravenously or intravascularly. For certain neoplasms, e.g., those not easily accessible within the body such as metastases or brain tumors, a reovirus or particle is administered in a manner such that it is transported systemically through the body of the mammal to thereby reach the neoplasm (e.g., intravenously, intramuscularly, subcutaneously, or intra-peritoneally). A reovirus or particle also is administered locally including, for example, topically (e.g., for melanoma), orally (e.g., for oral or esophageal neoplasm), rectally (e.g., for colorectal neoplasm), vaginally (e.g., for cervical or vaginal neoplasm), nasally or by inhalation (e.g., for lung neoplasm). A reovirus or particle is optionally administered by more than one route and/or to more than one location in an individual.

Targeted administration may be used to administer a reovirus or particle. For example, dendritic cells containing a reovirus or particle may be administered to a subject. See, for example, US Publication No. 2008/0014183. In another example of targeted delivery, carrier cells may be used to target cells of a proliferative disorder and prevent immune recognition of a reovirus or particle which they carry.

A reovirus or particle as disclosed herein is administered in an amount that is sufficient to treat the proliferative disorder (e.g., an“effective amount”). A proliferative disorder is“treated” when administration of a reovirus or particle as disclosed herein to proliferating cells affects one or more symptoms or clinical signs of the disorder including, e.g., increasing lysis (e.g., “oncolysis”) of the cells, reducing the number of proliferating cells, reducing the size or progression of a neoplasm, reducing pain associated with the neoplasm, as compared to the signs or symptoms in the absence of the treatment. As used herein, the term“oncolysis” means at least 10% of the proliferating cells are lysed (e.g., at least 20%, 30%, 40%, 50%, or 75% of the cells are lysed). The percentage of lysis can be determined, for example, by measuring the reduction in the size of a neoplasm or in the number of proliferating cells in a mammal, or by measuring the amount of lysis of cells in vitro (e.g., from a biopsy of the proliferating cells).

An effective amount of a reovirus or particle disclosed herein is determined on an individual basis and is based, at least in part, on the particular reovirus or particle used; the individual's size, age, gender; and the size and other characteristics of the proliferating cells. For example, for treatment of a human, approximately 10 ³ to 10 ¹² plaque forming units (PFU) of a reovirus or particle is used, depending on the type, size and number of proliferating cells or neoplasms present. The effective amount can be from about 1.0 PFU/kg body weight to about 10 ¹⁵ PFU/kg body weight (e.g., from about 10 ² PFU/kg body weight to about 10 ³ PFU/kg body weight). A reovirus or particle is administered in a single dose or in multiple doses (e.g., two, three, four, six, or more doses). Multiple doses are administered concurrently or consecutively (e.g., over a period of days or weeks). Treatment with a reovirus or particle as disclosed herein lasts from several days to several months or until diminution of the disease is achieved.

In certain embodiments, a reovirus or particle as disclosed herein is optionally administered in conjunction with surgery or removal of proliferating cells (e.g., a neoplasm). It also is contemplated that a reovirus or particle as disclosed herein is optionally administered in conjunction with or in addition to radiation therapy. It is further contemplated that a reovirus or particle as disclosed herein is optionally administered in conjunction with or in addition to known anticancer compounds, chemotherapeutic agents, and/or immunosuppressive agents. Such agents, include, but are not limited to, 5-fluorouracil, mitomycin C, methotrexate, hydroxyurea, gemcitabine, cyclophosphamide, dacarbazine, mitoxantrone, anthracyclines (Epirubicin, Irinotecan, and Doxorubicin), topoisomerase inhibitors such as etoposide or camptothecin, platinum compounds such as carboplatin and cisplatin, taxanes such as taxol and taxotere, hormone therapies such as tamoxifen and anti-estrogens, interleukins, interferons, aromatase inhibitors, progestational agents, LHRH analogs, mTOR inhibitors (e.g., rapamycin and derivatives thereof, and combinations thereof.

It is further contemplated that a reovirus or particle as disclosed herein is administered in conjunction with an agent that can increase endothelial permeability and/or decrease interstitial fluid pressure. Such agents include, for example, TNF-a. It is contemplated that a reovirus or particle as disclosed herein can be administered in conjunction with any combination of the therapies and agents described herein.

Pharmaceutical compositions

A reovirus or particle as described herein can be included, along with a pharmaceutically acceptable carrier, in a pharmaceutical composition. Such pharmaceutical compositions can include, for example, one or more chemotherapeutic agents and/or one or more immunosuppressive agents.

In addition to one or more reoviruses and/or particles as disclosed herein, a pharmaceutical composition typically includes a pharmaceutically acceptable carrier or excipient. A pharmaceutically acceptable carrier includes a solid, semi-solid, or liquid material that acts as a vehicle, carrier or medium for the reovirus. Thus, for example, compositions containing a reovirus or particle disclosed herein are 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.

Some examples of suitable carriers include phosphate-buffered saline or another physiologically acceptable buffer, 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. A pharmaceutical composition additionally can include, without limitation, 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. Pharmaceutical compositions of the invention can be formulated to provide quick, sustained or delayed release of a reovirus or particle as disclosed herein after administration by employing procedures known in the art.

In a specific embodiment, it may be desirable to administer the pharmaceutical compositions locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as fibers. In some embodiments, the reovirus, viral vectors, or particles as disclosed herein are formulated in liposomes for targeted delivery. Liposomes are vesicles comprised of concentrically ordered phospholipid bilayers which encapsulate an aqueous phase. Liposomes typically comprise various types of lipids, phospholipids, and/or surfactants. The components of liposomes are arranged in a bilayer configuration, similar to the lipid arrangement of biological membranes. Liposomes are particularly preferred delivery vehicles due, in part, to their biocompatibility, low immunogenicity, and low toxicity.

Methods of preparing liposomes with a prolonged serum half-life, i.e., enhanced circulation time can be used to make liposomes-virus vector compositions. Preferred liposomes are not rapidly cleared from circulation, i.e., are not taken up into the mononuclear phagocyte system (MPS). Useful liposomes for use in the disclosed compositions and methods can be generated by reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

For preparing solid compositions such as tablets, a reovirus or particle as disclosed herein is mixed with a pharmaceutical carrier to form a solid composition. Optionally, tablets or pills are coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, a tablet or pill comprises an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components, for example, are separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials are 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.

Liquid formulations that include a reovirus or particle disclosed herein 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 com oil, cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. These liquid or solid compositions optionally contain suitable pharmaceutically acceptable excipients as described herein. Such compositions are administered, for example, by the oral or nasal respiratory route for local or systemic effect. Compositions in pharmaceutically acceptable solvents are nebulized by use of inert gases. Nebulized solutions are inhaled, for example, directly from the nebulizing device, from an attached face mask tent, or from an intermittent positive pressure breathing machine. Solution, suspension, or powder compositions are administered, orally or nasally, for example, from devices which deliver the formulation in an appropriate manner.

Another formulation that is employed in the methods taught herein employs transdermal delivery devices (“patches”). Such transdermal patches are used to provide continuous or discontinuous infusion of a reovirus or particle as disclosed herein. The construction and use of transdermal patches for the delivery of pharmaceutical agents are performed according to methods known in the art. Such patches are constructed for continuous, pulsatile, or on-demand delivery of a reovirus or particle as disclosed herein.

A reovirus or particle as described herein is optionally chemically or biochemically pretreated (e.g., by treatment with a protease such as chymotrypsin or trypsin) prior to administration (e.g., prior to inclusion in the pharmaceutical composition). Pretreatment with a protease removes the outer coat or capsid of the virus and can be used to increase the infectivity of the virus. Additionally or alternatively, a reovirus or particle disclosed herein is coated in a liposome or micelle to reduce or prevent an immune response in a mammal that has developed immunity toward a reovirus.

A reovirus or particle disclosed herein or a pharmaceutical composition comprising such a reovirus or particle can be packaged into a kit. It is contemplated that a kit optionally includes one or more chemotherapeutic agents. A pharmaceutical composition, for example, is formulated in a unit dosage form. The term“unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of a reovirus or particle as disclosed herein calculated to produce the desired therapeutic effect in association with a suitable pharmaceutically acceptable carrier.

EXAMPLES

Generation of reassortant viruses in triple-negative breast cancer cells by forward genetics.

Reovirus serotypes have distinct infective, replicative, and cell killing properties and the segmented nature of the reovirus genome allows the generation of viruses with novel properties through gene reassortment following co-infection. To generate reoviruses with enhanced replicative properties in TNBC cells, MDA-MB-231 cells were co-infected with prototype laboratory strains T1L, T2J, and T3D and serially passaged in these cells ten or twenty times (Fig. 1 A). Following serial passage, individual viral clones were isolated by plaque assay and the gene segment identity for each clone (44 clones following 10 passages, 45 clones following 20 passages) was determined by SDS-gel electrophoresis. Of the 44 isolates analyzed following 10 serial passages, 8 distinct electropherotypes were identified, with 23 isolates (52%) having the same electropherotype (r2Reovirus). Following 20 serial passages, 6 distinct isolates were identified, including two (r9 and rlO) that were not observed after passage 10. The most predominant electropherotypes following 20 serial passages were rlReovirus and r2Reovirus, constituting 33% and 27% respectively of all isolates. Illumina Next-Generation Sequencing™ (NGS) revealed that rlReovirus is composed of seven gene segments from T1L and three from T3D (L2, M2, S2), while r2Reovirus is composed of nine gene segments from T1L and one from T3D (M2) (FIG 2). In addition, both viruses have nonsynonymous point mutations that result in an Ala to Thr substitution at amino acid 160 in L3, an Ile to Val substitution at amino acid in S3, and Val to Ile substitution at amino acid 49 in S4. A Pro to Thr substitution at amino acid 160 are also found in rlReovirus. In addition, rlReovirus and r2Reovirus have several synonymous point mutations. The rlReovirus S2 gene segment, but no other gene segment, has single residue variations that range from 35% to 65%. This suggests that rlReovirus may not be clonal but a mixture of two or more viruses with different genetic signatures at the S2 gene segment or that the virus contains two S2 gene segments. We did not detect single residue variations in gene segments from either parental T1L, T2J, or T3D or r2Reovirus, suggesting this is not an intrinsic property of the S2 gene segment carried from parental viruses. Together, these data indicate that co-infection and serial passaging of reoviruses in MDA-MB-231 cells leads to the generation of reassortant reoviruses with novel genetic compositions.

Reassortant reoviruses attach to cells with similar efficiency but infect MDA-MB-231 cells more efficiently than parental reoviruses

Reovirus attaches to cells via a strength-adhesion mechanism whereby the viral attachment fiber sΐ binds to cell-surface carbohydrate and proteinaceous receptors JAM-A or NgRl . To determine the attachment efficiency of rlReovirus and r2Reovirus in comparison to parental reoviruses, MDA-MB-231 cells were adsorbed with vehicle (mock) or Alexa 633 (A633)-labeled T1L, T3D, pelareorep (T3C$), or reassortant reoviruses at an MOI of 5xl0 ⁴ particles/cell and assessed for cell surface reovirus by flow cytometry (Fig. 3A). Reassortant reoviruses attach to cells with similar efficiency as T1L, but less efficiently than Type 3 reoviruses T3D and T3C$. As reassortant reoviruses contain a T1L Sl gene segment, it is not surprising that they attach to cells to similar levels as parental T1L. These data also indicate that other genetic changes found in rlReovirus and r2Reovirus do not impact the ability of the viruses to attach to cells.

To determine how genetic changes in rlReovirus and r2Reovirus affect reovirus infection of TNBC cells, MDA-MB-231 cells were pretreated with DMSO or the cysteine protease inhibitor E64-d, which blocks reovirus cell entry by preventing proteolysis during endocytic uptake, adsorbed with mock, T1L, T3D, T3C$, or reassortant reoviruses at an MOI of 100 PFU/cell and assessed for infectivity after 18 h by indirect immunofluorescence using reovirus-specific antiserum (Fig. 3B). In contrast to that observed in the attachment assay, rlReovirus and r2Reovirus infect MDA-MB-231 cells more efficiently than parental reoviruses or T3C$, with both reassortant viruses infecting cells over 2-fold more efficiently. Infection with all viruses tested was impaired by E64-d, indicating a similar requirement for proteolytic processing during entry. These data indicate that reassortant reoviruses establish infection more efficiently in MDA-MB- 231 cells than parental reoviruses and that infection of these cells requires proteasomal processing of the virion during cell entry.

To determine if the increased infectivity of the reassortant viruses is limited to MDA-153 MB -231 cells, the infectivity of parental and reassortant reoviruses was assessed on murine L929 fibroblasts, which are highly susceptible to reovirus infection and are used to propagate the virus (Fig. 3C). L929 cells were adsorbed with mock, T1L, T3D, T3C$, or reassortant reoviruses at an MOI of 5 PFU/cell and assessed for infectivity after 18 h by indirect immunofluorescence using reovirus-specific antiserum. In contrast to that observed in MDA-MB-231 cells, reassortant reoviruses infect L929 cells to similar levels as parental T1L, but less efficiently than both T3D and T3C$. These data indicate that rlReovirus and r2Reovirus more efficiently infect TNBC cells, but not L929 cells. This suggests that the genetic changes found in the reassortant viruses confer enhanced infection in the TNBC cells used for serial passage at a step after attachment. Replication kinetics of reassortant reoviruses are similar to TIL but faster than T3D

To determine the replication efficiency of parental and reassortant reoviruses, MDA-MB- 231 cells were adsorbed with mock, T1L, T3D, T3C$ or reassortant reoviruses at an MOI of 10 PFU/cell and assessed for viral replication over a 3 day course of infection. Despite the differences observed in infectivity, all viruses except T3D replicated with similar kinetics, with T3C$ having slightly faster replication kinetics at day 1 post infection than all other viruses tested. T1L, T3C$, rlReovirus, and r2Reovirus had similar replication kinetics at days 2 and 3 post infection. T3D replication kinetics were significantly slower than all other viruses tested. Interestingly, although T3C$ only differs from T3D by 22 amino acids, its replication kinetics are more similar to T1L and the reassortant reoviruses than T3D. These data indicate that although reassortant reoviruses establish infection in MDA-MB-231 cells more efficiently than parental reoviruses, replication kinetics are similar to T1L but significantly enhanced compared to T3D. rlReovirus and r2Reovirus impact cell viability with faster kinetics than parental 178 reoviruses in MDA-MB-231 but not L929 cells

Type 3 reoviruses induce cell death more efficiently than Type 1 reoviruses in vitro and 1 in vivo. To determine the efficacy of viral-induced cytotoxicity in TNBC cells, MDA-MB-231 cells were adsorbed with mock, T1L, T3D, T3C$, rlReovirus and r2Reovirus at an MOI of 500 PFU/cell, or treated with staurosporine as a positive control, and assessed for cell viability for 7 days (Fig. 4A). Compared to mock-infected cells, all reoviruses tested impaired cell viability, with reassortant reoviruses impairing cell viability with the fastest kinetics. In reassortant reovirus- infected cells, cell viability peaked at day 2 post infection, reaching levels similar to staurosporine by day 5 post infection. Cell viability peaked at day 3 post infection in T1L-, T3D-, and T3C$- infected cells reaching staurosporine levels by day 5 with T1L and day 6 with T3C$. T3D-infected cells did not reach staurosporine levels during the time course. Overall, the impact on cell viability by reassortant viruses was 1 day ahead of T1L and T3C$ and 2-3 days ahead of T3D. To determine if similar effects on cell viability could be observed in another TNBC cell line, MDA-MB-436 cells were infected with mock, T1L, T3D, T3C$, or r2Reovirus and assessed for cell viability over 6 days. Similar to that observed in MDA-MB-231 cells, r2Reovirus induced cell death with faster kinetics than either parental T1L or T3D, or T3C$. T3D did not significantly impact MDA-MB- 436 cell viability. These data show that reassortant viruses negatively affect cell viability of TNBC cells more efficiently than parental reoviruses and the oncolytic T3C$ strain. These data also suggest that T3D is not efficient at inducing cell death in at least a subset of TNBC cells.

To determine if rlReovirus and r2Reovirus differ from parental reoviruses in their ability to impair cell viability of non-TNBC cells, L929 cells were adsorbed with mock, T1L, T3D, T3C$, rlReovirus, or r2Reovirus at an MOI of 500 PFU/cell and assessed for cell viability over a 3 day time course (Fig. 4B). In contrast to that observed in MDA-MB-231 cells, all reoviruses tested impaired cell viability with relatively similar kinetics except for T3C$, which impaired L929 cell viability with significantly faster kinetics. These data indicate that reassortant viruses induce cell death with faster kinetics in MDA-MB-231 and MDA-MB-436 cells but not in L929 cells. As r2Reovirus had enhanced infectivity and cytotoxicity in MDA-MB-231 compared to parental viruses and no single nucleotide variants in the S2 gene segment, experiments in the rest of the study were performed with r2Reovirus.

Identification of small molecules that impact reovirus infectivity of MDA-MB-231 cells.

To identify small molecule inhibitors that enhance the oncolytic potential of reovirus, a high-throughput screen to assess the effect of small molecules from the NIH Clinical Collection I and II (NCC) on reovirus infectivity was performed. The NCC is composed of compounds that have been through Phase I-III clinical trials. To test the effects on reovirus infectivity of compounds in the NCC, MDA-MB-231 cells were pre-treated with vehicle (DMSO), 4 mM E64- d, or 10 mM NCC compounds for 1 h. r2Reovirus was added to cells at an MOI of 20 PFU/cell, incubated for 20 h post infection in the presence of DMSO, 2 mM E64-d, or 5 mM NCC compounds, and scored for infectivity by indirect immunofluorescence using reovirus-specific antiserum. Of the 700 compounds in the NCC, 20 increased reovirus infectivity whereas 17 decreased infectivity. Six microtubule-inhibiting compounds impaired reovirus infectivity, corroborating a need for microtubule function in reovirus cell entry. The sodium ATPase pump inhibitor digoxin and two serotonin antagonists also impaired reovirus infection, corroborating a role for the sodium ATPase pump and serotonin receptors in reovirus infection. Four topoisomerase inhibitors, doxorubicin, epirubicin, etoposide (topoisomerase II inhibitors) and topotecan (topoisomerase I inhibitor), significantly enhanced reovirus infectivity. Topoisomerase inhibitors enhance reovirus infection of MDA-MB-231 cells

To determine if topoisomerase inhibitors affect reovirus infection of TNBC cells, MDA- MB-231 cells were treated with increasing concentrations of doxorubicin, epirubicin, and topotecan for 1 h at 37°C, adsorbed with mock or r2Reovirus at an MOI of 100 PFU/cell, and scored for infectivity by indirect immunofluorescence using reovirus-specific antiserum. Reovirus infectivity increased slightly when cells were treated with 0.1 mM and more significantly when treated with 1.0 pM with all three drugs. Treatment of cells with 10 pM doxorubicin or epirubicin decreased infectivity compared to 1.0 pM treatment, likely due to cellular cytotoxicity. In contrast, treatment of cells with 10 pM topotecan enhanced reovirus infectivity more than any other concentration tested. These data indicate that topoisomerase inhibitors augment reovirus infectivity in a concentration-dependent manner.

Topoisomerase inhibitors enhance reovirus-mediated cell killing of MDA-MB-231 cells

To determine if topoisomerase inhibitors confer additive or synergistic effects on reovirus- mediated cytotoxicity, MDA-MB-231 cells were treated with vehicle (DMSO) or increasing concentrations of doxorubicin, epirubicin, or topotecan for 1 h at 37°C, adsorbed with r2Reovirus at an MOI of 200 PFU/cell, and assessed for cell viability over 3 days. Treatment with 0.1 pM of all three drugs did not significantly impact cell viability in the presence or absence of r2Reovirus. In the absence of virus, 1.0 pM doxorubicin and epirubicin impaired cell viability to similar levels as virus alone and moderately enhanced in the presence of reovirus. These effects can be especially observed at day 3 post infection (Fig. 5) Similar results were observed with 10 pM doxorubicin and epirubicin, except that the drugs alone had significant cytotoxic properties. In contrast, 1.0 pM topotecan had modest effects on cell viability in the absence of reovirus, but addition of reovirus had a synergistic effect on the cytotoxic effects of both topotecan and reovirus. Similar results were observed with 10 pM topotecan. Together, these data indicate that the combination of topoisomerase inhibitors with reovirus, especially topotecan, enhances the cytopathic properties of drugs and virus in a TNBC cell line.