T cell repertoire dynamics and oncolytic viral therapy

Abstract

To provide methods of treating cancer in a subject.SOLUTION: The methods include: administering to a subject one or more doses of an oncolytic virus (e.g., in an initial round of treatment); selecting a subject with a T-cell population exhibiting high peripheral clonality; and administering, to the subject having a T-cell population exhibiting high peripheral clonality, one or more subsequent doses of the oncolytic virus (e.g., in a second round of treatment).SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 809,190, filed February 22, 2019, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Cancer is one of the leading causes of death. Although cancer has long been the focus of medical research, the main cancer treatments remain surgery, radiation therapy, and chemotherapy. Each of these treatments has limitations, such as the different effects of the same treatment on subjects with similar types of cancer. Summary of the Invention [Means for solving the problem]

[0003] Described herein is a method for treating cancer in a subject, more specifically, a method for treating cancer in a subject selected for its susceptibility to treatment by determining whether the subject shows a T cell population with high peripheral clonality after a first round of treatment.The method includes administering one or more doses of an oncolytic virus, such as a reovirus, to a subject with cancer, selecting a subject with a T cell population with high peripheral clonality (e.g., greater than 0.06) after one or more doses of the oncolytic virus treatment, and administering one or more subsequent doses of the oncolytic virus to the subject with the T cell population with high peripheral clonality.Optionally, the oncolytic virus is administered in combination with one or more additional agents.

[0004] The details of one or more embodiments are set forth in the description below. Other features, objects, and advantages will be apparent from the description, and from the claims. [Brief description of the drawings]

[0005] [Figure 1]FIG. 13 is a graph showing the trend toward decreased peripheral clonality over the treatment period using the pairwise Wilcoxon rank sum test. [Diagram 2] Graph showing trend of increasing peripheral diversity over treatment period using pairwise Wilcoxon rank sum test. [Diagram 3] Graph showing high peripheral clonality and low diversity in C1D1 (day 1 of treatment cycle 1) and C2D1 (C2D1, day 1 of treatment cycle 2) correlated with progression-free survival. [Figure 4] FIG. 1 is a graph showing high peripheral clonality and low diversity in C1D1 and C2D1, which correlates with overall survival. [Diagram 5] 1 is a graph showing the fraction of peripheral T cells over time. [Figure 6] 1 is a graph showing the Morishita index for C1D1. [Figure 7] 1 is a graph showing the proliferation of peripheral clones in C2D1. [Figure 8] 1 is a graph showing that the majority of peripherally expanded clones identified by C2D1 originate from new clones. [Figure 9] Graphs showing that higher peripheral clonality correlates with greater change in CelTIL score in breast cancer patients treated with reovirus and letrozole (A) and in breast cancer patients treated with reovirus and the checkpoint inhibitor atezolizumab (B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] Detailed Description of the Invention Provided herein is a method for treating cancer in a subject by selecting a subject having one or more markers that indicate the subject is responsive to treatment, for example by showing improved overall survival and / or improved progression-free survival. The method includes administering one or more doses of oncolytic virus to the subject (i.e., a first round of treatment with oncolytic virus), selecting a subject having a T cell population that shows high peripheral clonality after administration of one or more doses of oncolytic virus, and administering one or more subsequent doses of oncolytic virus to the selected subject (i.e., a second round of treatment with oncolytic virus). Optionally, the subject also has a T cell population that is low in diversity. By selecting a subject having a T cell population that has high peripheral clonality and low diversity, a subject that shows longer progression-free survival and / or overall survival upon subsequent treatment with oncolytic virus (i.e., a second round of treatment) is selected compared to a subject that is not selected or compared to a subject that does not have a T cell population that has high peripheral clonality and low diversity after administration of one or more doses of oncolytic virus.

[0007] As used herein, clonality refers to the quantification of the degree of monoclonal or oligoclonal expansion by measuring the shape of the clonal frequency distribution. Clonality values ​​range from 0 to 1, with values ​​close to 1 indicating a nearly monoclonal population. Generally, as used herein, high clonality refers to a value of about 0.06 or greater. The term diversity refers to the number of unique rearrangements. Generally, as used herein, low diversity refers to a diversity of a T cell population of less than about 1800 rearrangements.

[0008] Clonality and diversity can be calculated in different ways. As an example, the following formula can be used:

[0009]

number

[0010] Clonality can also be calculated using Simpson's clonality.

number

[0011] As used herein, the term "cancer" refers to any type of cancer, proliferative disease, neoplasm, or malignant tumor found in a mammal, including lymphoma, leukemia, blastoma, germ cell tumor, carcinoma, and sarcoma. Exemplary cancers include brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian, sarcoma, gastric cancer, uterine cancer, and medulloblastoma. Optionally, the cancer is a neoplasm. Optionally, the cancer is head and neck cancer. Optionally, the cancer is lung cancer, liver cancer, lymphoma, pancreatic cancer, melanoma, kidney cancer, or ovarian cancer. Optionally, the cancer is an adenocarcinoma. Optionally, the cancer is a pancreatic adenocarcinoma.

[0012] In some cases, the cancer is metastatic. As used herein, the terms "metastasis", "metastatic" and "metastatic cancer" can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ to another non-adjacent organ or part of the body. Cancer originates at a site of origin, e.g., the pancreas, and the site is called a primary tumor, e.g., primary pancreatic cancer. Some of the cancer cells of the primary tumor or site of origin acquire the ability to penetrate and invade the surrounding normal tissues in the local area and / or penetrate the walls of the lymphatic or vascular system to circulate to other sites and tissues in the body. A clinically detectable secondary tumor formed from the cancer cells of the primary tumor is called a metastatic tumor or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are considered to be similar to those of the original tumor. Thus, when pancreatic cancer metastasizes to the lungs, the secondary tumor at the lung site is composed of abnormal pancreatic cancer cells rather than abnormal lung cells. The secondary tumor in the lung is called metastatic pancreatic cancer. Therefore, the term metastatic cancer refers to the disease in which the subject has or has had a primary tumor, and has one or more secondary tumors.The term non-metastatic cancer, or the subject with non-metastatic cancer, refers to the disease in which the subject has a primary tumor, but does not have secondary tumors.For example, metastatic pancreatic cancer refers to the disease in which the subject has or has a history of a primary pancreatic tumor, and has one or more secondary tumors in a second location or multiple locations, such as the lung.

[0013] Oncolytic viruses for use in the provided methods and kits include, but are not limited to, Reoviridae, Myoviridae, Siphoviridae, Podoviridae, Tesiviridae, Corticoviridae, Plasmaviridae, Liposthrixviridae, Fuselloviridae, Poxyiridae, Iridoviridae, Phycodnaviridae, Baculoviridae, Herpesviridae, Adenoviridae, Papovaviridae, Polydnaviridae, Inoviridae, Microviridae, Geminiviridae, Circoviridae, and the like. Oncolytic viruses include those that are members of the following families: Parvoviridae, Hepadnaviridae, Retroviridae, Cyctoviridae, Birnaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Leviviridae, Picornaviridae, Sequiviridae, Comoviridae, Potyviridae, Caliciviridae, Astroviridae, Nodaviridae, Tetraviridae, Tombusviridae, Coronaviridae, Graviviridae, Togaviridae, and Barnaviridae. Immunoprotected viruses, as well as reassorted or recombinant viruses of these and other oncolytic viruses, are also included in the provided methods. Thus, the oncolytic viruses used in the provided methods are selected from the group consisting of, for example, reovirus, Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), adenovirus, vaccinia virus, parapox orf virus, sindbis virus, and herpes simplex virus. Furthermore, a combination of at least two oncolytic viruses can be used to carry out the provided methods. Although several oncolytic viruses are described below, a person skilled in the art can also carry out the methods of the present invention using other oncolytic viruses by following the disclosure herein and the knowledge available in the art.

[0014] When a virus invades a cell, double-stranded RNA kinase (PKR) is activated, inhibiting protein synthesis and preventing the virus from replicating in the cell. Some viruses have developed a system that inhibits PKR and promotes viral protein synthesis and viral replication. For example, adenovirus produces large amounts of VA1 RNA, a small RNA. VA1 RNA has an extensive secondary structure and binds to PKR in competition with double-stranded RNA (dsRNA), which normally activates PKR. VA1 RNA does not activate PKR because a minimum length of dsRNA is required for PKR activation. Instead, VA1 RNA sequesters PKR because of its large amount. As a result, protein synthesis is not inhibited and adenovirus can replicate in the cell. Thus, if the PKR inhibitors of adenovirus, vaccinia virus, herpes simplex virus, or parapoxvirus orf virus are mutated so that they no longer inhibit PKR function, the resulting viruses will not infect normal cells due to the inhibition of protein synthesis by PKR, but will replicate in cancer cells that lack PKR activity. In some cases, 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 γ134.5 gene, a parapoxvirus orf virus mutated in the OV20.0L gene, or an influenza virus mutated in the NS-1 gene.

[0015] Vaccinia viruses with mutated viral thymidine kinase (TK) genes cannot make the nucleotides required for DNA replication. In normal cells, cellular TK levels are usually very low and the virus cannot replicate. In tumors, loss of the tumor suppressor Rb or increased 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.

[0016] The vaccinia growth factor (VGF) gene is a homolog of mammalian epidermal growth factor (EGF) and can bind to and activate the EGF receptor (EGFR). The proliferation of vaccinia viruses with mutated VGF genes is restricted to cells with activated EGF pathways, which are commonly mutated in cancer.

[0017] Viruses can be modified or mutated according to the known structure-function relationships of viral PKR inhibitors. For example, the amino-terminal region of E3 protein interacts with the carboxy-terminal domain of PKR, so deletion or point mutation of the carboxy-terminal domain prevents 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 pseudosubstrate of PKR. Truncation or point mutations within the C-terminal portion of the K3L protein, which is homologous to residues 79-83 of eIF-2, abolish PKR inhibitory activity (Kawagishi-Kobayashi et al., Mol. Cell. Biology 17:4146-4158 (1997)).

[0018] Another example is the Delta 24 virus, a mutant adenovirus with a 24-base pair deletion in the E1A region (Fueyo et al., Oncogene 19(1):2-12(2000)). This region serves to bind and inhibit the function of the cellular tumor suppressor Rb, thereby allowing the cellular proliferation machinery, and thus viral replication, to proceed in an uncontrolled manner. Delta 24 does not bind to Rb due to the deletion in the Rb-binding region. Thus, the replication of this mutant virus is inhibited by Rb in normal cells. However, when Rb is inactivated and the cells become neoplastic, Delta 24 is no longer inhibited. Instead, the mutant virus replicates efficiently and lyses Rb-deficient cells.

[0019] Furthermore, vesicular stomatitis virus (VSV) selectively kills tumor cells. Herpes simplex virus 1 (HSV-1) mutant hrR3, defective in ribonucleotide reductase expression, replicates in colon cancer cells but not in normal liver cells (Yoon et al., FASEB J. 14:301-311 (2000)). Newcastle disease virus (NDV) selectively replicates in malignant cells, with the most commonly used strain being 73-T (Reichard et al., J. Surgical Research 52:448-453 (1992); Zorn et al., Cancer Biotherapy 9(3):22-235 (1994); Bar-Eli et al., J. Cancer Res. Clin. Oncol. 122:409-415 (1996)). Vaccinia virus grows in several malignant tumor cell lines. Encephalitis viruses have oncolytic effects on murine sarcoma tumors, but may require attenuation to reduce infectivity in normal cells. Tumor regression has been reported in tumor patients infected with varicella zoster, hepatitis viruses, influenza, chickenpox, and measles viruses (for review, see Nemunaitis, J. Invest. New Drugs 17:375-386 (1999)).

[0020] Optionally, the oncolytic virus is a modified non-reovirus virus that includes a reovirus sigma 1 protein, where the reovirus sigma 1 protein replaces the native attachment protein of the non-reovirus virus, and the modified virus does not include any portion of the native attachment protein of the non-reovirus virus. In this modified non-reovirus virus, the reovirus sigma 1 protein attaches to a carrier cell that protects the virus from neutralizing antibodies during in vivo delivery to a tumor, for example, during systemic delivery. The non-reovirus virus can be, but is not limited to, an adenovirus, a vaccinia virus, a herpes simplex virus, a sindbis virus, or a parapox virus. Optionally, the full-length sequence of the native attachment protein of the non-reovirus virus is replaced with the reovirus sigma 1 protein. The replacement of the native attachment protein of the virus with the reovirus sigma 1 protein allows the virus to attach to a carrier cell that protects the virus from neutralizing antibodies during in vivo delivery. The reovirus sigma 1 protein is described, for example, in WO2008 / 11004, which is incorporated by reference in its entirety.

[0021] In some cases, 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. The virion has a diameter of 60-80 nm and has two concentric capsid shells, each of which is icosahedral. The genome is composed of 10-12 individual segments of double-stranded RNA with a total genome size of 16-27 kbp. The individual RNA segments vary in size. Three different but related types of reoviruses have been recovered from many species. Thus, a reovirus may be a mammalian reovirus or a human reovirus. All three types share a common complement-fixing antigen.

[0022] Human reoviruses include three serotypes: type 1 (Lang strain or T1L), type 2 (Jones strain, T2J), and type 3 (Dearing strain or Abney strain, T3D). The three serotypes can be readily distinguished based on neutralization and hemagglutinin inhibition assays. A reovirus according to the present disclosure can be a type 3 mammalian orthoreovirus. Type 3 mammalian orthoreoviruses include, but are not limited to, the Dearing and Abney strains (T3D or T3A, respectively). See, e.g., ATCC Accession Nos. VR-232 and VR-824. See, e.g., U.S. Patent 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.

[0023] In some cases, the provided methods include the use of reoviruses having mutations. For example, the mutant reoviruses described herein can include a mutation that reduces or essentially eliminates expression of a sigma3 polypeptide, or results in the deletion of a functional sigma3 polypeptide, as described in U.S. Publication No. 2008 / 0292594, the entire contents of which are incorporated herein by reference. The mutation that eliminates expression of a sigma3 polypeptide, or results in the deletion of a functional sigma3 polypeptide, can be in a nucleic acid that encodes a sigma3 polypeptide (i.e., S4 gene) or in a nucleic acid that encodes a polypeptide that modulates expression or function of a sigma3 polypeptide.

[0024] As used herein, a mutation that reduces expression of a sigma 3 polypeptide refers to a mutation that results in a reduction in the amount of sigma 3 polypeptide of at least 30% (e.g., at least 40%, 50%, 60%, 70%, 80%, 90%, or 95%) compared to a reovirus expressing wild-type levels of sigma 3 polypeptide. As used herein, a mutation that essentially eliminates expression of a sigma 3 polypeptide refers to a mutation that results in a reduction in the amount of sigma 3 polypeptide of at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) 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 elimination of functional sigma 3 polypeptide refers to a mutation that results in a sigma 3 polypeptide that allows expression of the sigma 3 polypeptide but is unable to assemble or incorporate into the viral capsid. It will be appreciated that it may be desirable or necessary for the sigma 3 polypeptide to maintain other functions (e.g., the ability to bind RNA) in order for the mutant reovirus to maintain its ability to propagate.

[0025] The mutations of the sigma 3 polypeptides described herein can result in the sigma 3 polypeptide being incorporated into the capsid at a reduced level compared to a sigma 3 polypeptide not containing the mutation (e.g., a wild-type sigma 3 polypeptide). The mutations of the sigma 3 polypeptides described herein can also result in the sigma 3 polypeptide being unable to be incorporated into the viral capsid. Without being bound to any particular mechanism, the sigma 3 polypeptide may be reduced or lose function, for example, due to the inability of the sigma 3 polypeptide and the mu 1 polypeptide to properly bind, or due to a conformational change that reduces or prevents the incorporation of the sigma 3 polypeptide into the capsid.

[0026] In addition to mutations that abolish or reduce expression of a sigma3 polypeptide, or result in a dysfunctional or reduced functional sigma3 polypeptide, the mutant reoviruses described herein can have one or more additional mutations (e.g., a second, third, or fourth mutation) in one of the other reovirus capsid polypeptides (e.g., mu1, lambda2, and / or sigma1). Reoviruses containing mutations that affect a sigma3 polypeptide, and optionally additional mutations in any or all of the other outer capsid proteins, can be screened for the ability of such mutant reoviruses to infect cells and cause lysis of the cells. For example, neoplastic cells that are resistant to lysis by wild-type reovirus can be used to screen for effective mutant reoviruses described herein.

[0027] For example, further mutations can reduce or essentially eliminate expression of the mu1 polypeptide or result in the absence of functional mu1 polypeptide. The mu1 polypeptide encoded by the M2 gene is believed to be involved in cell penetration and is believed to play a role in transcriptase activation. Each virion contains approximately 600 copies of the mu1 polypeptide, which is present in a 1:1 complex with the sigma3 polypeptide. The mu1 polypeptide is myristoylated at its N-terminus and then cleaved 42 residues from the myristoylated N-terminus to generate a C-terminal fragment (mu1C). Additionally or alternatively, further mutations can reduce or essentially eliminate expression of the lambda2 polypeptide or result in the absence of functional lambda2 polypeptide and / or further mutations can reduce or essentially eliminate expression of the sigma1 polypeptide or result in the absence of functional sigma1 polypeptide. The lambda2 polypeptide is encoded by the L2 gene, is involved in particle assembly, and exhibits guanylyltransferase and methyltransferase activity. The sigma 1 polypeptide is encoded by the S1 gene and is involved in cell attachment and functions as the viral hemagglutinin.

[0028] Optionally, a reovirus has a lambda 3 polypeptide with one or more amino acid modifications, a sigma 3 polypeptide with one or more amino acid modifications, a mu 1 polypeptide with one or more amino acid modifications, a mu 2 polypeptide with one or more amino acid modifications, or any combination thereof. For example, as described in U.S. Ser. No. 12 / 046,095, the entire contents of which are incorporated by reference herein, a reovirus has a lambda 3 polypeptide with one or more amino acid modifications, a sigma 3 polypeptide with one or more amino acid modifications, a mu 1 polypeptide with one or more amino acid modifications, and / or a mu 2 polypeptide with one or more amino acid modifications. By way of example, the one or more amino acid modifications of the lambda 3 polypeptide are 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, as numbered relative to GenBank Accession No. M24734.1 (SEQ ID NO: 23). Note that if residue 214 of the amino acid sequence is Val or residue 1071 is Val, the amino acid sequence further comprises at least one further alteration within the amino acid sequence. Optionally, the lambda 3 polypeptide comprises the sequence shown in SEQ ID NO: 19. By way of further example, the one or more amino acid modifications of the sigma 3 polypeptide are Leu at residue 14, Lys at residue 198, or any combination thereof, as numbered relative to GenBank Accession No. K02739 (SEQ ID NO: 25). Note that if residue 14 of the amino acid sequence is Leu, the amino acid sequence further comprises at least one additional alteration within the amino acid sequence. Optionally, the sigma3 polypeptide comprises the sequence set forth in SEQ ID NO: 15. As a further example, the one or more amino acid alterations of the mu1 polypeptide are Asp at residue 73 as numbered relative to GenBank Accession No. M20161.1 (SEQ ID NO: 27). Optionally, the mu1 polypeptide comprises the sequence set forth in SEQ ID NO: 17.As a further example, the amino acid modification of the mu 1 polypeptide is a Ser at residue 528, as numbered relative to GenBank Accession No. AF461684.1 (SEQ ID NO: 29). Optionally, the mu 1 polypeptide comprises the sequence set forth in SEQ ID NO: 17. A reovirus described herein having one or more modifications can further comprise a reovirus sigma 2 polypeptide. Such a sigma 2 polypeptide has a Cys at one or more of positions 70, 127, 195, 241, 255, 294, 296, or 340, as numbered relative to GenBank Accession No. NP_694684.1 (SEQ ID NO: 30). Optionally, the sigma 2 polypeptide comprises the sequence set forth in SEQ ID NO: 12.

[0029] Optionally, the reovirus comprises an L1 genome segment comprising one or more nucleic acid modifications, an S4 genome segment comprising one or more nucleic acid modifications, an M1 genome segment comprising one or more nucleic acid modifications, an M2 genome segment comprising one or more nucleic acid modifications, or any combination thereof. Optionally, the reovirus comprises an L1 genome segment with one or more nucleic acid modifications, an S4 genome segment with one or more nucleic acid modifications, an M1 genome segment with one or more nucleic acid modifications, and / or an M2 genome segment with one or more nucleic acid modifications, as described in WO2008 / 110004, which is incorporated herein by reference in its entirety. By way of example, the one or more nucleic acid modifications in the L1 genome segment are T at position 660, G at position 817, A at position 1687, G at position 2283, ATG at positions 2284-2286, C at position 2794, C at position 2905, C at position 2953, A at position 3153, or G at position 3231, when numbered relative to GenBank Accession No. M24734.1 (SEQ ID NO: 22). Optionally, the L1 genome segment comprises the sequence set forth in SEQ ID NO: 8. By way of further example, the one or more nucleic acid modifications in the S4 genome segment are A at position 74 and A at position 624, when numbered relative to GenBank Accession No. K02739 (SEQ ID NO: 24). Optionally, the S4 genome segment comprises the sequence set forth in SEQ ID NO: 4. By way of further example, the nucleic acid modification in the M2 genome segment can be a C at position 248, as numbered relative to GenBank Accession No. M20161.1 (SEQ ID NO: 26). The M2 genome segment comprises, for example, the sequence set forth in SEQ ID NO: 6. Also by way of example, the nucleic acid modification in the M1 genome segment is a T at position 1595, as numbered relative to GenBank Accession No. AF461684.1 (SEQ ID NO: 28). Optionally, the M1 genome segment comprises the sequence set forth in SEQ ID NO: 5. The reoviruses described herein can comprise any modification or combination of modifications disclosed herein.Optionally, a reovirus described herein comprises a genome segment having a sequence set forth in SEQ ID NOs: 1-10, or a polypeptide set forth in one or both of SEQ ID NOs: 11, 12, and 16-21, and SEQ ID NOs: 13 or 14. Optionally, a reovirus disclosed herein is identified as IDAC Accession No. 190907-01, deposited on September 19, 2007 at the International Depository Center of Canada (IDAC, Public Health Agency of Canada, National Laboratory of Microbiology, 1015 Arlington St., Winnipeg, Manitoba Canada R3E 3R2).

[0030] 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 single-stranded RNA of 11703 nucleotides that is capped at the 5' end and polyadenylated at the 3' end. The genome consists of a 49S untranslated region (UT), nonstructural proteins nsP1, nsP2, nsP3, and nsP4, followed by a promoter. The promoter is followed by the 26S UT, structural proteins C, E3, E2, 6K, and E1, and finally the 3'UT and polyadenylated end. The 49S RNA of the genome is positive-sense, infectious, and functions as an mRNA in infected cells.

[0031] Sindbis vectors infect / detect and kill metastatic tumors systemically and specifically in vivo, significantly suppressing tumor growth and increasing survival rates (Hurtado et al., Rejuvenation Res. 9(1):36-44(2006)). Sindbis virus infects mammalian cells by utilizing the Mr 67,000 laminin receptor, which is elevated in tumor cells versus normal cells. Overexpression of laminin receptors in tumors may explain the specificity and efficacy of Sindbis vectors against tumor cells in vivo. Sindbis does not need to be genetically engineered to target cancer cells, nor does it need to be injected directly into tumors. Sindbis travels to the target area via the bloodstream, regardless of where it is injected into a subject (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 viruses and / or genes that stimulate an immune response to tumors, such as anti-tumor cytokine genes, e.g., interleukin 12 and interleukin 15 genes.

[0032] The virus can be chemically or biochemically pretreated (e.g., by treatment with a protease such as chymotrypsin or trypsin) before administration to the neoplastic cells. Pretreatment with a protease can remove the viral coat or capsid to increase the infectivity of the virus. The virus can be coated with liposomes or micelles to reduce or prevent immune responses from mammals that have developed immunity to the virus (Chandran and Nibert, J. of Virology 72(1):467-75(1998)). For example, virions can be treated with chymotrypsin in the presence of micelle-forming concentrations of alkyl sulfate detergents to generate new infectious subvirion particles. The oncolytic virus can be a reassortant virus or an ISVP.

[0033] The oncolytic virus may be a recombinant oncolytic virus. For example, the recombinant oncolytic virus results from the reassortment of genome segments from two or more genetically distinct oncolytic viruses, also referred to herein as reassortants. The reassortment of genome segments of the oncolytic virus may occur after infection of a host organism with at least two genetically distinct oncolytic viruses. The recombinant virus may also be produced in cell culture, for example, by co-infection of permissive host cells with genetically distinct oncolytic viruses. Optionally, the method includes the use of a recombinant oncolytic virus resulting from the reassortment of genome segments from two or more genetically distinct oncolytic viruses, where at least one parent virus is genetically engineered, contains one or more chemically synthesized genome segments, has been treated with chemical or physical mutagens, or is itself the result of a recombination event. Optionally, the method includes the use of a recombinant oncolytic virus that has undergone recombination in the presence of chemical mutagens, including but not limited to dimethyl sulfate and ethidium bromide, or physical mutagens, including but not limited to ultraviolet light and other forms of radiation.

[0034] In some cases, the method includes the use of oncolytic viruses having mutations, including insertions, substitutions, deletions, or duplications in one or more genome segments. Such mutations can include additional genetic information as a result of recombination with the host cell genome, or can include synthetic genes, such as, for example, genes encoding agents that suppress antiviral immune responses.

[0035] Optionally, the oncolytic virus is a mutant oncolytic virus. For example, the oncolytic virus can be modified, for example, by incorporating a mutant coat protein into the virion outer capsid. Optionally, the mutant oncolytic virus is a mutant reovirus. The mutant reovirus described herein can include a mutation that reduces or essentially eliminates the expression of sigma3 polypeptide, or results in the loss of functional sigma3 polypeptide, as described in US Publication No. 2008 / 0292594, which is incorporated herein by reference in its entirety. Optionally, the mutant reovirus used in the provided method is mutated as described in US Patent No. 7,803,385, which is incorporated herein by reference in its entirety.

[0036] Mutations as referred to herein may be substitutions, insertions or deletions of one or more nucleotides. Point mutations include, for example, single nucleotide transitions (purine to purine, or pyrimidine to pyrimidine) or inversions (purine to pyrimidine, or vice versa), and deletions or insertions of single or multiple nucleotides. Mutations in nucleic acids may result in one or more conservative or non-conservative amino acid substitutions in the encoded polypeptide, which may result in conformational changes or loss or partial loss of function, a shift in the translational reading frame (frameshift) resulting in a completely different encoded polypeptide from that point on, a premature stop codon (truncation) resulting in a shortened polypeptide, or mutations in viral nucleic acids may not change the encoded polypeptide at all (silent or nonsense). For disclosures regarding conservative and 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. Pat. No. 4,554,101.

[0037] Any number of methods known in the art can be used to generate mutations in the nucleic acid of oncolytic virus. For example, site-directed mutagenesis can be used to modify the nucleic acid sequence of reovirus. One of the most common methods of site-directed mutagenesis is oligonucleotide-directed mutagenesis. In oligonucleotide-directed mutagenesis, an oligonucleotide that sequentially codes for the desired change(s) is annealed to one strand of the DNA of interest and serves as a primer to initiate DNA synthesis. In this way, the oligonucleotide that contains the sequence change is incorporated into the newly synthesized strand. See, for example, Kunkel, 1985, Proc. Natl. Acad. Sci. USA 82:488; Kunkel et al., 1987, Meth. Enzymol. 154:367; Lewis 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 also routinely used in the art to modify the sequence of a protein or polypeptide. For example, nucleic acids containing mutations can be generated using PCR or chemical synthesis, or a polypeptide with the desired changes in amino acid sequence can be chemically synthesized. See, e.g., Bang and Kent, 2005, Proc. Natl. Acad. Sci. USA 102:5014-9 and references therein.

[0038] Viruses can be purified using standard methods, see, for example, Schiff et al., "Orthoreoviruses and Their Replication," Ch 52, in Fields Virology, Knipe and Howley, eds., 2006, Lippincott Williams and Wilkins; Smith, eds., 2006, which are incorporated by reference in their entirety. See, e.g., W. et al., 1969, Virology 39(4):791-810; and U.S. Patent Nos. 7,186,542, 7,049,127, 6,808,916, and 6,528,305. As used herein, purified virus refers to a virus that has been separated from cellular components that naturally accompany the virus. Typically, a virus is considered purified when it is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) by dry weight free from proteins and other cellular components with which it is naturally associated.

[0039] The provided method includes administering one or more additional agents to the subject. Optionally, the additional agent is a chemotherapeutic agent. Optionally, the additional agent is a cancer immunotherapy agent. Chemotherapeutic agents include, but are not limited to, alkylating agents, anthracyclines, taxanes, epothilones, histone deacetylase inhibitors, inhibitors of topoisomerase I, inhibitors of topoisomerase II, kinase inhibitors, monoclonal antibodies, nucleotide and precursor analogs, peptide antibiotics, platinum-based compounds, retinoids, and vinca alkaloids and derivatives. Agents used in cancer immunotherapy are agents that stimulate the immune system to help the subject's immune system fight cancer. Cancer immunotherapy agents include cells, such as dendritic cells and CAR-T cells, cytokines, and antibodies. Optionally, the cancer immunotherapy agent is an immune checkpoint inhibitor. Optionally, the immune checkpoint inhibitor is an antibody. As noted throughout, administration of an additional agent, e.g., a chemotherapeutic agent or a cancer immunotherapeutic agent, can include administration of more than one additional agent to the subject, i.e., administration of a combination of additional agents.

[0040] As used herein, immune checkpoint inhibitor refers to any compound that inhibits immune inhibitory checkpoint proteins. Inhibition can be a reduction in the function of the protein or a complete block in function. In some cases, immune checkpoint inhibitors can be antibodies that specifically recognize immune checkpoint proteins. Immune checkpoint inhibitors are well known and include peptides, antibodies, nucleic acid molecules, and small molecules. Immune checkpoint refers to molecules expressed by T cells that enhance signals (stimulatory checkpoint molecules) or reduce signals (inhibitory checkpoint molecules). Immune checkpoint molecules are known to constitute immune checkpoint pathways similar to CTLA-4 and PD-1 dependent pathways (see, for example, Pardoll, 2012 Nature Rev Cancer 12:252-264; Mellman et al, 2011. Nature 480:480-489). Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3, and VISTA.

[0041] Examples of anti-CTLA-4 antibodies are described in U.S. Patent Nos. 5,811,097, 5,811,097, 5,855,887, 6,051,227, 6,207,157, 6,682,736, 6,984,720, and 7,605,238. For example, CTLA-4 antibodies include tremelimumab (ticilimumab, CP-675206) and ipilimumab (10D1, also known as MDX-D010).

[0042] Examples of PD-1 and PD-L1 antibodies are described in U.S. Patent Nos. 7,488,802, 7,943,743, 8,008,449, 8,168,757, and 8,217,149, as well as PCT Patent Application Publication Nos. WO03042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699. PD-1 inhibitors include anti-PD-L1 antibodies and anti-PD-1 antibodies. Examples of PD-1 and PD-L1 inhibitors include nivolumab (MDX1106, BMS936558, ONO4538), lambrolizumab (MK-3475 or SCH900475), pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab.

[0043] Other immune checkpoint inhibitors include lymphocyte activation gene 3 (LAG-3) inhibitors such as IMP321, a soluble Ig fusion protein (Brignone et al, 2007, J. Immunol. 179:4202-4211). Immune checkpoint inhibitors include B7 inhibitors such as B7-H3 and B7-H4 inhibitors. B7 inhibitors include the anti-B7-H3 antibody MGA271 (Loo et al, 2012, Clin. Cancer Res. July 15(18)3834).

[0044] Optionally, the immune checkpoint inhibitor is an IDO inhibitor. Examples of IDO inhibitors are described in WO2014150677. Examples of IDO inhibitors include 1-methyl-tryptophan (IMT), β-(3-benzofuranyl)-alanine, β-(3-benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6-fluoro-tryptophan, 4-methyl-tryptophan, 5-methyltryptophan, 6-methyl-tryptophan, 5-methoxy-tryptophan, 5-hydroxy-tryptophan, indole 3-carbinol, 3,3'-diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9-vinylcarbazole, acemetacin, 5-bromo-tryptophan, 5-bromoindoxyl diacetate, 3-amino-naphthoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole, brassinin derivatives, thiohydantoin derivatives, β-carboline derivatives or brazilexin derivatives. Optionally, the IDO inhibitor is selected from 1-methyl-tryptophan, β-(3-benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3-amino-naphthoic acid, and β-[3-benzo(b)thienyl]-alanine, or a derivative or prodrug thereof.

[0045] Examples of alkylating agents include, but are not limited to, nitrogen mustards, nitrosoureas, tetrazines, aziridines, cisplatin and derivatives, and non-classical alkylating agents. Specific examples of alkylating agents include, but are not limited to, mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide, busulfan, N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, streptozotocin, dacarbazine, mitozolomide, temozolomide, thiotepa, mitomycin, diaziquone, cisplatin, carboplatin, oxaliplatin, procarbazine, and hexamethylmelamine.

[0046] Other exemplary chemotherapeutic agents include, but are not limited to, 5-fluorouracil, mitomycin C, methotrexate, hydroxyurea, mitoxantrone, anthracyclines (e.g., epirubicin and doxorubicin), antibodies against receptors (e.g., herceptin, etoposide, pregnasome), hormone therapy (e.g., tamoxifen and antiestrogens), interferons, aromatase inhibitors, progestational agents, and LHRH analogs. Suitable aromatase inhibitors include, but are not limited to, letrozole, anastrozole, exemestane, vorozole, formestane, fadrozole, testolactone, aminoglutethimide, 1,4,6-androstatriene-3,17-dione, and 4-androstene-3,6,17-trione.

[0047] The present disclosure provides a pharmaceutical composition comprising one or more oncolytic viruses. The present disclosure also provides a pharmaceutical composition comprising one or more chemotherapeutic agents. In some cases, the pharmaceutical composition comprises one or more oncolytic viruses and one or more chemotherapeutic agents. Thus, the provided composition can comprise a single agent or multiple agents.

[0048] The compositions provided herein are administered in vitro or in vivo in a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be a solid, semi-solid, or liquid material that can function as a vehicle, carrier, or 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 solids or in liquid media), ointments containing up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile sealed powders.

[0049] In some cases, the composition containing the oncolytic virus is suitable for infusion. For intravenous infusion, there are two types of fluids commonly used: crystalloids and colloids. Crystalloids are aqueous solutions of mineral salts or other water-soluble molecules. Colloids contain larger insoluble molecules such as gelatin. Blood is itself a colloid. The most commonly used crystalloid is normal saline, a 0.9% sodium chloride solution that is close to the blood concentration (isotonic). Ringer's lactate or Ringer's acetate is another isotonic solution that is often used for large volume replacement. A 5% solution of dextrose in water, often called D5W, is often used instead when patients are at risk of having hypoglycemia or hypernatremia.

[0050] Some examples of suitable carriers include phosphate buffered saline or another physiologically acceptable buffer solution, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. Pharmaceutical compositions can further include, but are not limited to, lubricating agents such as, for example, talc, magnesium stearate, and mineral oil, wetting agents, emulsifying and suspending agents, preserving agents such as, for example, methylbenzoate and propylhydroxybenzoate, sweetening agents, and flavoring agents. Pharmaceutical compositions can be formulated to provide rapid, sustained, or delayed release of mutant reovirus after administration by using procedures well known in the art. Other suitable formulations for use in pharmaceutical compositions other than the representative formulations described below can be found in Remington: The Science and Practice of Pharmacy 22d edition Loyd V. Allen et al, editors, Pharmaceutical Press (2012). To prepare solid compositions such as tablets, the mutant reovirus can be mixed with a pharmaceutical carrier to form a solid composition. Optionally, the tablet or pill can be coated or otherwise compounded to provide a dosage form with the advantage of prolonged action. For example, the tablet or pill can include an inner dosage component and an outer dosage component, the latter being in the form of an envelope that covers the former. These two components can be separated by an enteric layer that functions to resist disintegration in the stomach and allows the inner component to enter the duodenum intact or to be released in a delayed manner, and various materials can be used for such enteric layers or coatings, including many polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0051] Liquid formulations containing the reovirus and / or drugs for oral administration or injection generally include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and emulsions flavored with edible oils such as corn oil, cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0052] Compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. These liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients, as described herein. Such compositions may be administered by the oral or nasal respiratory route for local or systemic effect. Compositions in pharma-ceutically acceptable solvents may be nebulized using inert gases. Nebulized solutions may be inhaled directly from the nebulizing device, or the nebulizing device may be attached to a face mask tent or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered orally or nasally from a device that delivers the formulation in an appropriate form.

[0053] Another formulation optionally used in the methods of the present disclosure includes a transdermal delivery device (e.g., a patch). Such transdermal patches can be used to provide continuous or discontinuous infusion of the viruses and agents described herein. The construction and use of transdermal patches to deliver pharmaceutical agents is well known in the art. See, for example, U.S. Patent No. 5,023,252. Such patches can be configured for continuous, pulsatile, or on-demand delivery of mutant reoviruses.

[0054] As mentioned above, viruses and / or other drugs can be optionally coated with liposomes or micelles to reduce or prevent immune responses in mammals that have developed immunity to the virus or drug. Such compositions are called immunoprotected viruses or drugs. See, e.g., U.S. Patent Nos. 6,565,831 and 7,014,847.

[0055] In the provided methods, the oncolytic virus is administered, for example, systemically, in such a way that it can ultimately reach the target tumor or tumor cells. The route by which the virus is administered and the formulation, carrier, or vehicle are determined according to the location and type of the target cells. Various routes of administration can be used. For example, in the case of accessible solid tumors, the virus can be administered by direct injection into the tumor. For example, in the case of hematopoietic tumors, the virus can be administered intravenously or intravascularly. For tumors that are not easily accessible in the body, such as metastases, the virus is administered (e.g., intravenously or intramuscularly) so that it can be transported systemically in the mammalian body and thereby reach the tumor. Alternatively, the virus can be administered directly to a single solid tumor, where it is carried through the body to metastases. The virus can also be administered subcutaneously, intraperitoneally, intrathecally or intraventricularly (e.g., for brain tumors), topically (e.g., for melanoma), orally (e.g., for oral or esophageal cancer), rectally (e.g., for colorectal cancer), intravaginally (e.g., for cervical or vaginal cancer), intranasally, in an inhalation spray or aerosol formulation (e.g., for lung cancer).

[0056] Optionally, the virus is administered to the subject continuously at least once a day for a period of time between the first round or subsequent rounds of treatment, or intermittently or continuously throughout the day on each successive day. Thus, the virus is administered to the subject, for example, by intravenous administration in any pharmacologically acceptable solution, or as an infusion over a period of time. For example, the substance can be administered systemically by injection (e.g., intramuscularly or subcutaneously), or taken orally at least once a day daily, or administered by infusion in such a way that it is delivered daily to the subject's tissue or bloodstream. When the virus is administered by infusion over a period of time, the period of time can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 24 hours, or any time between 1 and 24 hours, or more. Optionally, the period of time can be 5, 15, 30, 60, 90, 120, 150, or 180 minutes, or any time between 5 and 180 minutes, or more. Thus, for example, the virus is administered by a 60 minute infusion, which can be repeated daily for 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 21, 28 days, or any number of days between 2 and 28 days, or longer.

[0057] The viruses disclosed herein are administered in an amount sufficient (i.e., an effective amount) to treat cancer or a proliferative disease. A cancer or a proliferative disease is treated when administration of a therapeutic regimen comprising the virus to proliferative cells causes lysis of the diseased cells (e.g., oncolysis), resulting in a reduction in the number of abnormally proliferative cells, a reduction in the size of the neoplasm, and / or a reduction or elimination of symptoms (e.g., pain) associated with the proliferative disease. As used herein, the term oncolysis means that at least 10% of the proliferative cells are lysed (e.g., at least about 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 the neoplasm or the number of proliferative cells in the mammal, or by measuring the amount of lysis of cells in vitro (e.g., from a biopsy from the proliferative cells). The effective amount of the virus used in the therapeutic regimen is determined on an individual basis and can be based, at least in part, on the particular virus used, the size, age, and sex of the individual, and the size and other characteristics of the abnormally proliferative cells. For example, in human treatment, approximately 10% of patients with pulmonary hypertension may be treated with pulmonary hypertension, depending on the type, size, and number of proliferating cells or neoplasms present. 3 ~10 12 Plaque-forming units (PFU) of the virus are used. An effective amount is, 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) ~ approx. 10 13 In some cases, the effective amount may be about 1×10 PFU / kg body weight. 8 ~Approx. 1×10 12 PFU or TCID50. Optionally, the effective amount is about 3×10 10 ~Approx. 1×10 10 TCID50.

[0058] The optimal dosage of the virus and therapeutic agent, as well as the compositions and kits containing the virus and agent, depend on a variety of factors. The exact amount required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disease being treated, the specific virus and its method of administration. Thus, it is not possible to specify an exact amount for every composition or kit. However, the appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation, taking into account the guidance provided herein.

[0059] Effective dosages and schedules for administering treatment regimens can be empirically determined. For example, animal models of various proliferative diseases can be obtained from Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609 USA. Both direct measurements (e.g., tumor histology) and functional measurements (e.g., subject survival or tumor size) can be used to monitor response to treatment. These methods involve sacrificing representative animals to evaluate populations, which increases the number of animals required for an experiment. Measuring luciferase activity within tumors provides an alternative method to evaluate tumor volume without sacrificing animals, allowing for longitudinal population-based analysis of treatment. The dosage range for administration of the composition is large enough to produce the desired effect affecting the symptoms of the disease. The dosage should not be so large as to cause adverse side effects, such as undesirable cross-reactions and anaphylactic reactions. Dosage can be adjusted by the individual physician if there are contraindications.

[0060] Dosages vary, for example, administered in one or more doses daily for one or several days. The viruses and therapeutic agents provided are administered in a single dose or in multiple doses (e.g., two, three, four, six, or more doses). For example, when administration is by injection, the injection may be a single continuous dose or may be administered by multiple injections. Treatment may continue for days to months, or until alleviation of the disease is achieved.

[0061] The provided method can be further combined with other tumor therapies, such as radiation therapy, surgery, hormone therapy, and / or other immunotherapy. Suitable additional therapeutic agents include, but are not limited to, analgesics, anesthetics, central stimulants, corticosteroids, anticholinergics, anticholinesterases, anticonvulsants, antineoplastics, allosteric inhibitors, anabolic steroids, antirheumatics, psychotherapeutic drugs, neuroleptics, anti-inflammatory drugs, antihelminthic drugs, antibiotics, anticoagulants, antifungal drugs, antihistamines, antimuscarinic drugs, antimycobacterial drugs, antiprotozoal drugs, antiviral drugs, dopaminergic drugs, hemodynamic drugs, immunological drugs, muscarinic drugs, protease inhibitors, vitamins, growth factors, and hormones. The choice of drug and dosage can be easily determined by those skilled in the art based on the specific disease to be treated.

[0062] The provided virus and therapeutic agent combinations may be administered together (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines to the same subject), or sequentially (e.g., one of the compounds or agents is administered first, followed by the second). Thus, the term combination is used to refer to the administration of two or more agents together, simultaneously, or sequentially.

[0063] When one compound is administered before another, the first compound is administered minutes, hours, days, or weeks before the administration of the second compound. 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 before the administration of the second compound, or any time between 1 and 72 hours. 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 before the administration of the second compound, or any time between 1 and 180 minutes. Optionally, the first compound can be administered 1, 2, 3, 4, 5, 6, 7, 14, 21, or 28 days before the administration of the second compound, or any time between 1 and 28 days. Optionally, the first compound is administered more than 28 days before the second compound.

[0064] The oncolytic virus or a pharmaceutical composition comprising such a virus can be packaged in a kit. The kit also includes one or more additional agents or a pharmaceutical composition comprising the additional agent. The kit may include a chemotherapeutic agent or a cancer immunotherapeutic agent. Optionally, the kit includes an immune checkpoint inhibitor. The oncolytic virus and / or additional agent and the pharmaceutical composition comprising them can be packaged in one or more containers. When the kit includes a pharmaceutical composition, the pharmaceutical composition can be formulated as a unit dosage form. The term "unit dosage form" refers to a physically discrete unit suitable as a unitary dosage in human subjects and other mammals, each unit containing a predetermined amount of oncolytic virus or other agent, e.g., an immune checkpoint inhibitor, calculated to produce a desired therapeutic effect, together with a suitable pharma- ceutically acceptable carrier. Optionally, the kit includes a reovirus and an immune checkpoint inhibitor.

[0065] The oncolytic virus of the provided kits may be any of the oncolytic viruses described herein. The provided kits may include multiple doses of the oncolytic virus. Optionally, each dose of the oncolytic virus is about 10 3 ~10 12 plaque forming units (PFU) of oncolytic virus. Optionally, each dose contains about 10 8 ~10 12 PFU of oncolytic virus. Optionally, each dose contains about 10 8 ~10 12 TCID50 of oncolytic virus. Optionally, each dose contains about 1×10 10 ~3×10 10 Contains TCID50 of oncolytic virus.

[0066] As used herein, the terms treatment, treat, treating, or ameliorate refer to a method of reducing the effects of a disease or condition, or the symptoms of a disease or condition. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction or improvement of the severity of an established disease or condition, or the symptoms of a disease or condition. For example, a method for treating cancer is considered therapeutic if one or more symptoms of the disease are reduced by 10% in a subject compared to a control. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% reduction, or any percentage reduction between 10-100%, compared to the natural or control levels. It is understood that treatment does not necessarily refer to a cure or complete elimination of a disease, condition, or the symptoms of a disease or condition.

[0067] As used herein, the term subject may be a vertebrate, more specifically a mammal (e.g., human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), fish, bird, or reptile, or amphibian. The term does not indicate a specific age or sex. Thus, adult and neonatal subjects are intended to be encompassed, regardless of whether they are male or female. As used herein, patient or subject may be used interchangeably and may refer to a subject having a disease or disorder. The term patient or subject includes human and animal subjects.

[0068] Disclosed are materials, compositions, and components that can be used in, with, or in preparation for, or are products of, the disclosed methods and compositions. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each of the different individual and collective combinations and permutations of these compounds are specifically contemplated and described herein, even if specific references to them may not be expressly disclosed. For example, when an inhibitor is disclosed and contemplated, and many modifications that can be made to many molecules, including this inhibitor, each and every combination and permutation of the inhibitor and possible modifications is specifically contemplated, unless specifically indicated otherwise. Similarly, any subset or combination of these is specifically contemplated and disclosed. This concept applies to all aspects of the disclosure, including, but not limited to, each step in a method of using the disclosed compositions. Thus, if there are various additional steps that may be performed, it is to be understood that each of these additional steps may be performed with any particular method step or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is to be considered as specifically contemplated and disclosed.

[0069] Throughout this application various publications are referenced. The disclosures of these publications in their entireties are incorporated by reference into this application.

[0070] A number of embodiments have been described. Nevertheless, it will be understood that various modifications may be made. Moreover, when one feature or step is described, that feature or step can be combined with any other feature or step herein, even if the combination is not explicitly described. Thus, other embodiments are within the scope of the claims. EXAMPLES

[0071] Example 1. Analysis of T cell repertoire in patients with pancreatic adenocarcinoma during pancreatic remission and chemotherapy treatment Reovirus serotype 3, Dearing strain (Pelareolep), is a non-enveloped human reovirus that has been shown to induce tumor lysis and innate and adaptive immune responses, resulting in an inflammatory phenotype and antitumor activity. Patients with advanced (unresectable or metastatic) histologically confirmed pancreatic adenocarcinoma that had progressed (did not tolerate treatment) after first-line therapy were studied using Pelareolep and chemotherapy in combination with pembrolizumab. The study features intravenous Pelareolep in combination with pembrolizumab and one of three chemotherapy backbone regimens: gemcitabine, irinotecan, or leucovorin / 5-fluorouracil (5-FU), one of which was repeated every 3 weeks in treatment cycles until disease progression.

[0072] Experimental Design: T cells were analyzed by immunoSEQ assay (Adaptive Biotechnologies®; Seattle, WA) at C1D1 and C2D1 (approximately 3 weeks later) in a group of 9 subjects. More specifically, genomic DNA was obtained from samples of peripheral blood mononuclear cells (PBMCs) at each time point. The table below summarizes this analysis. Values ​​shown are mean values ​​with ranges (min-max) in brackets. [Table 1] Clonality and diversity were calculated using the following formula:

number

[0073] Clonality is robust to a range of sampling depths. Due to differences of two orders of magnitude, all samples were downsampled to a minimum template number of 2581. As shown in Figure 1, there was a trend toward decreased peripheral clonality over the treatment period using pairwise Wilcoxon rank sum tests; PWRS<0.01.

[0074] Clonality is often inversely proportional to diversity, which is the number of unique rearrangements assuming 2581 templates. As shown in Figure 2, using the pairwise Wilcoxon rank sum test, there was a trend toward increased peripheral diversity over the treatment period, although this was less pronounced than for clonality.

[0075] Log-rank (Mantel-Cos) analyses were performed on progression-free survival and overall survival. Clonality was scaled to 0.1 units. Diversity was scaled to 100 units.

[0076] As shown in Figure 3, clonality and diversity correlate with progression-free survival, with stronger p-values ​​for C1D1. Higher peripheral clonality and lower diversity are associated with longer progression-free survival.

[0077] As shown in Figure 4, clonality and diversity correlate with overall survival, with stronger p-values ​​for C2D1. Higher peripheral clonality and lower diversity are associated with better outcomes.

[0078] The peripheral T cell fraction is the number of T cells relative to the total nucleated cells. As shown in Figure 5, there was a slight trend toward an increase in the peripheral T cell fraction, but the overall trend was not significant because there were patients who moved in both directions.

[0079] Differences in clone frequencies were examined using a false discovery rate (FDR) corrected binomial index, which calculates indices of expanded clones and repertoire overlap / similarity. Expanded clones were both novel and pre-existing.

[0080] The Morishita index takes into account both the overlap rate of the repertoire between two samples and the clonal frequency. A completely identical repertoire is scored as 1, and two completely different samples are scored as 0. The normal variation over a month is approximately 0.9-0.95. As shown in Figure 6, the median Morishita index between C2D1-C1D1 is 0.83, which is less than 0.6 for the three samples. This suggests significant peripheral repertoire turnover.

[0081] Peripherally expanded clones were determined between C1D1 and C2D1. Due to the high variability in template numbers, expanded clones per 100,000 cumulative templates were reported. The usual variation over 4 weeks is approximately 5-10 expanded clones. As shown in Figure 7, the median is above 40 in all cases. Only one sample had fewer than 18 expanded clones.

[0082] The peripherally expanded clones were either pre-existing clones or newly identified clones (i.e., not detected at the initial time point). As shown in Figure 8, most peripheral clonal expansion was observed from new clones.

[0083] In summary, peripheral clonality decreases and intrinsic rearrangements increase between C1D1 and C2D1, consistent with a general increase in diversity. High peripheral clonality and low diversity are associated with longer overall survival. High levels of peripheral repertoire turnover occur between C1D1 and C2D1. Repertoire turnover is accompanied by a significant clonal expansion, mainly with an increase in "new" clones (clones not detected in C1D1).

[0084] Example 2. Analysis of T cell repertoire in breast cancer patients during percutaneous pulmonary resection and treatment with aromatase inhibitors or checkpoint inhibitors To investigate T cell responses and changes within the tumor microenvironment (TME), women with early stage breast cancer were divided into two groups (six patients each) and administered Pelareolep in combination with letrozole or atezolizumab. Patients were treated with Pelareolep on days 1, 2, 8, and 9. Letrozole was administered daily beginning on day 3, and atezolizumab was administered once on day 3. Tumor biopsies were collected at diagnosis, on day 3, and approximately on day 21. The primary endpoint of the study was the CelTIL score. The CelTIL score is an index to quantify changes in tumor cellularity and TIL infiltration, and increases in CelTILs are associated with a better response to treatment (Nuciforo, et al., Ann. Oncol. 29:170-77 (2018)). Tumor tissue was examined for Pelareolep replication, and changes to the TME were assessed by immunohistochemistry and TCR immunosequencing (ImmunoSEQ assay). Peripheral blood was also examined by TCR immunosequencing.

[0085] Analysis of CelTIL has shown an increase in 4 of 6 patients so far. Productive viral replication was extremely high in day 3 and day 21 biopsies, as measured by in situ detection of viral capsid protein in tumor cells. Immunohistochemistry analysis revealed an increase in CD8+ T cells and elevated expression of PDL1 in day 3 and day 21 biopsies in all patients. Overall, the degree of viral replication was consistent with changes in CelTIL and changes within the TME. TCR-seq of blood showed that the level of T cell clonality correlated with changes in the TME and CelTIL. Thus, higher T cell peripheral clonality correlated with higher CelTIL, indicating that patients were more responsive to treatment. The present invention provides, for example, the following items. (Item 1) 1. A method of treating cancer in a subject, comprising: (i) administering to the subject one or more doses of an oncolytic virus; (ii) selecting subjects having a T cell population that exhibits high peripheral clonality following treatment with the one or more doses of the oncolytic virus; (iii) administering one or more subsequent doses of the oncolytic virus to a subject having a T cell population exhibiting high peripheral clonality. (Item 2) 2. The method of claim 1, wherein the peripheral clonality is greater than 0.06. (Item 3) 3. The method of claim 1 or 2, wherein the subject also has a less diverse T cell population. (Item 4) The method according to claim 3, wherein the diversity of the T cell population is less than 1800 rearrangements. 5. The method according to any one of items 1 to 4, wherein the cancer is an adenocarcinoma. (Item 6) The method according to any one of items 1 to 4, wherein the cancer is breast cancer or pancreatic adenocarcinoma. (Item 7) about 10 3 ~1012 7. The method according to any one of items 1 to 6, wherein plaque forming units (PFU) of the oncolytic virus are administered to the subject. (Item 8) about 10 8 ~10 12 7. The method according to any one of items 1 to 6, wherein a PFU of said oncolytic virus is administered to said subject. (Item 9) about 10 8 ~10 12 7. The method according to any one of items 1 to 6, wherein TCID50 of the oncolytic virus is administered to the subject. (Item 10) 10. The method according to any one of items 1 to 9, wherein the oncolytic virus is administered as an intravenous infusion. (Item 11) 11. The method of any one of items 1 to 10, further comprising administering to the subject one or more additional therapeutic agents. (Item 12) 12. The method of claim 11, wherein the additional therapeutic agent is a chemotherapeutic agent. (Item 13) 12. The method of claim 11, wherein the additional agent is an immune checkpoint inhibitor or an aromatase inhibitor. (Item 14) 14. The method of claim 13, wherein the immune checkpoint inhibitor is a PD-1 or PD-L1 inhibitor. (Item 15) 14. The method of claim 13, wherein the immune checkpoint inhibitor is selected from the group consisting of nivolumab, lambrolizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab. (Item 16) 16. The method according to any one of items 1 to 15, wherein selection of said subject with a T cell population with high peripheral clonality and low diversity results in increased progression-free survival or overall survival of said oncolytic virus-treated subject compared to a subject without selection or compared to a subject without a T cell population with high peripheral clonality and low diversity after administration of one or more doses of said oncolytic virus. (Item 17) 17. The method according to any one of items 1 to 16, wherein the oncolytic virus is selected from the group consisting of reovirus, Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), adenovirus, vaccinia virus, parapox orf virus, Sindbis virus, and herpes simplex virus. (Item 18) 18. The method of claim 17, wherein the reovirus is a mammalian reovirus. (Item 19) 19. The method of claim 18, wherein the reovirus is a human reovirus. (Item 20) 19. The method of claim 18, wherein the reovirus is selected from the group consisting of serotype 1 reovirus, serotype 2 reovirus, and serotype 3 reovirus. (Item 21) 21. The method of claim 20, wherein the reovirus is a serotype 3 reovirus. (Item 22) 22. The method of claim 21, wherein the serotype 3 reovirus is a Dearing strain reovirus. (Item 23) 19. The method of claim 18, wherein the reovirus is deposited under IDAC accession number 190907-01. (Item 24) 19. The method of claim 18, wherein 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. (Item 25) The reovirus comprises a polypeptide comprising: a sigma3 polypeptide having one or more amino acid modifications selected from the group consisting of Leu at residue 14, Lys at residue 198, or any combination thereof, as numbered relative to GenBank Accession K02739, wherein if the amino acid sequence has a Leu at residue 14, the amino acid sequence further comprises at least one additional modification within the amino acid sequence; a mu1 polypeptide having at least one amino acid modification, said at least one amino acid modification including an Asp at residue 73, as numbered relative to GenBank Accession No. M20161.1; a lambda 3 polypeptide having one or more amino acid modifications selected from the group consisting of: 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, as numbered relative to GenBank Accession No. M24734.1, wherein if the amino acid sequence has a Val at residue 214 or a Val at residue 1071, the amino acid sequence further comprises at least one further modification within the amino acid sequence; or 19. The method of claim 18, comprising one or more of the mu2 polypeptides having at least one amino acid modification, wherein the at least one amino acid modification comprises Ser at residue 528 as numbered relative to GenBank Accession No. AF461684.1. (Item 26) The reovirus comprises the following genome segments: an S4 genome segment having one or more nucleic acid modifications, wherein the one or more nucleic acid modifications in the S4 genome segment are selected from the group consisting of A at position 74 and A at position 624, as numbered relative to GenBank Accession No. K02739; an M2 genome segment having at least one nucleic acid modification, said at least one nucleic acid modification comprising a C at position 248, as numbered relative to GenBank Accession No. M20161.1; an L1 genome segment having one or more nucleic acid modifications selected from the group consisting of T at position 660, G at position 817, A at position 1687, G at position 2283, ATG at positions 2284-2286, C at position 2794, C at position 2905, C at position 2953, A at position 3153, and G at position 3231, as numbered relative to GenBank Accession No. M24734.1; or 19. The method of claim 18, comprising one or more of the M1 genome segments having at least one nucleic acid modification, the at least one nucleic acid modification comprising a T at position 1595 as numbered relative to GenBank Accession No. AF461684.1. (Item 27) 20. The method of claim 18, wherein the reovirus is a recombinant reovirus. (Item 28) 19. The method of claim 18, wherein the reovirus is a modified reovirus.

Claims

[Claim 1] The invention described in the specification.

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