Oncolytic virus therapy

Abstract

To provide a method of treating a subject suffering from cancer.SOLUTION: The present invention provides a method comprising the following steps: (a) administering, to a subject, an effective amount of an oncolytic virus to induce the infiltration of one or more T cells into the cancer; (b) isolating the tumor-infiltrated T cells from the cancer of the subject; (c) expanding the tumor-infiltrated T cells ex vivo; and (d) transferring the expanded tumor-infiltrated T cells to the subject suffering from cancer.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Claim of Priority This patent application claims priority based on U.S. Provisional Patent Application No. 62 / 454,526, filed on February 3, 2017, the entire disclosure of which is incorporated herein by reference. Grant Information Not applicable.

[0002] 1. Introduction The subject matter disclosed herein relates to oncolytic viruses, armed oncolytic viruses encoding immunomodulatory factor molecules in an expressible form, and compositions thereof, as well as methods of making and using such oncolytic viruses. The subject matter disclosed herein also relates to tumor-infiltrating T cells induced by oncolytic viruses (“OV-induced T cells”), and methods of making such OV-induced T cells and methods of using such OV-induced T cells for adoptive T cell therapy.

Background Art

[0003] 2. Background of the Invention Tumor-lytic viruses that selectively replicate in cancer cells and kill cancer cells exert their anti-cancer effects through a number of modes (Bartlett DL et al., 2013, Molecular Cancer 12:103-120). First, as their name implies, is cell lysis, which can be achieved through apoptosis, necrosis, pyroptosis, autophagy, or combinations thereof (Guo ZS et al., 2014, Front Oncol 4:74). Additionally, tumor-lytic viruses can attack the blood supply of cancer, thereby causing apoptosis and necrosis of infected as well as non-infected cells. Finally, tumor-lytic viruses induce immunogenic cell death (ICD) of cancer cells, release and present danger signal molecules (signal 0), simultaneously inflammatory cytokines, and cross-present tumor-associated antigens (TAAs) to naive T cells, thereby giving rise to anti-tumor immunity (Guo ZS et al., 2014, Front Oncol 4:74). Potent tumor-lytic viruses not only give rise to a strong and systemic adaptive anti-tumor immunity but also promote the transport of tumor-specific CD8+ T cells into tumor tissue (Bartlett DL et al., 2013, Mol Cancer 12:103; Guo ZS et al., 2017, 8:555).

[0004] This immune response against cancer is not limited to infected cancer cells but also extends to metastatic lesions. The FDA approval of T-VEC, the first drug in this class for treating advanced melanoma in 2015, introduced the potential of this new type of cancer treatment (Andtbacka RH et al., 2015, J Clin Oncol 33:2780-8). Despite their advantages, oncolytic viruses have faced difficulties to date, including gaining access to cancer cells within tumor nodules with an appropriate viral number, the antiviral immune response, and the highly immunosuppressive tumor environment (Zou W., 2005, Nat Rev Cancer 5:263-74). In addition, the infiltration of tumor-specific T cells faces obstacles when they are actually generated and activated, the main one being their infiltration into tumor tissues where they exert cytotoxicity against cancer cells and associated stromal cells.

[0005] These difficulties would preferably be improved by agents that modulate the immune system, such as agents that increase anti-cancer immunity and / or reduce antiviral immunity. However, agents capable of such immune modulation (such as cytokines) can have extensive and potentially dangerous effects on the subject being treated when diffused systemically.

Prior Art Documents

Non-Patent Documents

[0006]

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[0007] 3. Summary of the Invention The subject matter disclosed herein relates to compositions and methods for promoting anti-cancer immunity related to oncolytic virus therapy. In certain embodiments, adoptive T cell therapy is used as part of an anti-cancer regimen that uses oncolytic viruses to promote an immune response against cancer cells, and the oncolytic virus is optionally administered together with an immunomodulatory factor. In certain embodiments, the immunomodulatory factor is linked to the oncolytic virus.

[0008] In certain embodiments, the subject matter disclosed herein provides a method for generating tumor-infiltrating oncolytic virus-induced T cells, comprising the following steps: (a) administering an effective amount of an oncolytic virus to a subject having cancer to induce infiltration of one or more T cells into the cancer; (b) isolating tumor-infiltrating T cells from the subject's cancer; and (c) expanding the tumor-infiltrating T cells ex vivo.

[0009] In certain embodiments, the subject matter disclosed herein provides a method of treating a subject suffering from cancer, comprising administering to the subject one or more tumor-infiltrating oncolytic virus-induced T cells disclosed herein. In certain embodiments, the subject matter disclosed herein provides a method of treating a subject suffering from cancer, comprising the following steps: (a) administering an effective amount of an oncolytic virus to the subject to induce infiltration of one or more T cells into the cancer; (b) Step of isolating tumor-infiltrating T cells from the cancer of the subject; (c) Step of proliferating the tumor-infiltrating T cells ex vivo; and (d) Step of transplanting the proliferated tumor-infiltrating T cells into a subject suffering from cancer.

[0010] In certain embodiments, the oncolytic virus is a vaccinia virus. In certain embodiments, the oncolytic virus is a recombinant vaccinia virus having an inactivated mutation in its thymidine kinase gene, vaccinia growth factor gene, or both. In certain embodiments, the oncolytic virus is a herpes simplex virus. In certain embodiments, the oncolytic virus is an adenovirus.

[0011] In certain embodiments, the tumor-infiltrating T cells are proliferated ex vivo in the presence of IL-2 and IL-7. In certain embodiments, the step of proliferating the tumor-infiltrating T cells includes co-culturing the tumor-infiltrating T cells with dendritic cells and cancer cells. In certain embodiments, the step of proliferating the tumor-infiltrating T cells includes culturing the tumor-infiltrating T cells with cytokines and / or agents. In certain embodiments, the cytokines and / or agents include one or more of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, IL-27, IFN-α, IFN-α2, IFN-β, or IFN-γ, TNF-α, TNF-β, and GM-CSF. In certain embodiments, the step of proliferating the tumor-infiltrating T cells includes culturing the tumor-infiltrating T cells with IL-2, IL-7, and / or GSK3b.

[0012] In certain embodiments, a subject afflicted with cancer is treated with cancer therapy prior to transplanting tumor-infiltrating T cells into the subject afflicted with cancer. In certain embodiments, a subject afflicted with cancer is further treated with cancer therapy. In certain embodiments, a subject afflicted with cancer is further treated with one or more exogenous cytokines and / or agents. In certain embodiments, a subject afflicted with cancer is further administered exogenous IL-2 after the step of transplanting tumor-infiltrating T cells.

[0013] In certain embodiments, the tumor-infiltrating cells are transplanted intraperitoneally or intratumorally. In certain embodiments, the subject matter disclosed herein is directed to the use of oncolytic viruses for treating a subject having cancer.

[0014] In certain embodiments, the subject matter disclosed herein is directed to the use of oncolytic viruses for generating tumor-infiltrating oncolytic virus-induced T cells. In certain embodiments, the subject matter disclosed herein is directed to the use of tumor-infiltrating oncolytic virus-induced T cells for treating a subject having cancer. In certain embodiments, tumor-infiltrating oncolytic virus-induced T cells are generated by a method comprising the following steps: (a) administering an effective amount of an oncolytic virus to a subject having cancer to induce infiltration of one or more T cells into the cancer; (b) isolating tumor-infiltrating T cells from the subject's cancer; and (c) expanding the tumor-infiltrating T cells ex vivo.

[0015] In various embodiments, the subject matter disclosed herein provides oncolytic viruses, induced tumor-infiltrating T cells, therapeutic compositions comprising the viruses and T cells, and methods of treating subjects in a state that would benefit from immunomodulation, including patients afflicted with various cancers.

[0016] In certain embodiments, the subject matter disclosed herein provides an oncolytic virus encoding a membrane-bound protein comprising an immunomodulatory factor molecule linked to an anchor peptide. In certain embodiments, the subject matter disclosed herein provides an oncolytic virus comprising in its genome a nucleic acid encoding a membrane-bound protein comprising an immunomodulatory factor molecule linked to an anchor peptide.

[0017] In certain embodiments, the oncolytic virus is a vaccinia virus. In certain embodiments, the oncolytic virus is a recombinant vaccinia virus having an inactivating mutation in its thymidine kinase gene, vaccinia growth factor gene, or both. In certain embodiments, the nucleic acid encoding the immunomodulatory molecule linked to the anchor peptide is operably linked to the p7.5 e / l promoter.

[0018] In certain embodiments, the anchor peptide comprises a GPI-anchor acceptor peptide or the PD-L1 transmembrane domain. In certain embodiments, the immunomodulatory factor molecule is linked to the anchor peptide via a linker peptide. In certain embodiments, the linker is a flexible linker or a rigid linker. In certain embodiments, the immunomodulatory factor molecule is interleukin-2 and / or interferon-γ and / or tumor necrosis factor-α.

[0019] In certain embodiments, the oncolytic virus is administered as a therapeutic composition. In certain embodiments, the subject matter disclosed herein provides a therapeutic composition comprising the oncolytic virus together with a physiological buffer. In certain embodiments, the therapeutic composition is in lyophilized form. In certain embodiments, the subject matter disclosed herein provides a syringe comprising an effective amount of the therapeutic composition.

[0020] In certain embodiments, the subject matter disclosed herein provides a method of treating a subject afflicted with cancer, comprising administering to the subject an effective amount of a oncolytic virus encoding an immunomodulatory factor molecule. In certain embodiments, the immunomodulatory factor molecule is interleukin-2. In certain embodiments, the cancer is locally invasive. In certain embodiments, the cancer is metastatic. In certain embodiments, the oncolytic virus is administered intratumorally. In certain embodiments, a therapeutically effective amount of an immunomodulator is further administered to the subject afflicted with cancer. In certain embodiments, the immunomodulator is an anti-PD-1 or anti-PD-L1 antibody. In certain embodiments, additional therapy is provided to the subject afflicted with cancer. Brief Description of the Drawings

[0021]

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[0022] 5. Detailed Description of the Invention For clarity, and not by way of limitation, the detailed description of the subject matter disclosed herein is divided into the following subsections: (i) Oncolytic viruses; (ii) Immunomodulatory factor molecules; (iii) Armed oncolytic viruses; a. Secreted immunomodulatory factor molecules; b. Membrane-bound immunomodulatory factor molecules; i. Anchor peptides; ii. Linkers; iii. Non-limiting embodiments; (iv) Administration of oncolytic viruses; (v) Methods of manufacture; a. Isolation and preparation of tumor-infiltrating T cells induced by oncolytic viruses (“OV-induced T cells”); (vi) Methods of treatment a. Adoptive T cell therapy; b. Treatment using armed oncolytic viruses; (vii) Pharmaceutical compositions; and (viii) Kits.

[0023] 5.1 Oncolytic Virus The subject matter disclosed herein can be applied to any oncolytic virus known in the art. An “oncolytic virus” is a virus that exhibits increased replication in cancer cells and lysis of cancer cells as compared to comparable non-cancerous cells; see, for example, Bartlett DL et al., 2013, Molecular Cancer 12:103-120; Kaufman HL et al., 2015, Nature Reviews Drug Discovery 14:642-662 and Chiocca EA and Rabkin SD, 2014, Cancer Immunol Res; 2; 295-300. In certain embodiments, the oncolytic virus exhibits selective replication in cancer cells and less or essentially no replication in non-cancerous cells. In certain embodiments, less replication means that replication in cancer cells compared to equivalent non-cancerous cells is at least about 30% more, or at least about 50% more, or at least about 80% more.

[0024] The term “about” or “substantially” as used herein can mean within an acceptable error range for a particular value as measured by one of ordinary skill in the art, which will depend in part on how the value is measured or determined (e.g., the limitations of the measuring system). For example, “about” can mean within one or more standard deviations of a given value for an implementation. When particular values are recited in the present application and claims, unless otherwise stated, the term “about” can mean an acceptable error range for the particular value, such as ±10% of the value modified by the term “about”.

[0025] Non-limiting examples of oncolytic viruses include the following types: (i) adenovirus ("Ad") (e.g., hTERT-Ad); (ii) herpes simplex virus ("HSV") (e.g., G207, HSV-1716, T-VEC, and HSV-2 ΔPK mutant); (iii) poxvirus, e.g., vaccinia virus (e.g., vSP and vvDD (tk- / vgf-); and see below); (iv) alphavirus; (v) paramyxovirus (e.g., measles virus, mumps virus, and Newcastle disease virus); (vi) rhabdovirus (e.g., vesicular stomatitis virus); (vii) picornavirus (e.g., coxsackievirus, Seneca Valley Virus, and poliovirus); (viii) reovirus; (ix) parvovirus; and (x) recombinant / gene-manipulated versions of any of the foregoing.

[0026] In certain embodiments, the oncolytic virus is an oncolytic virus that has been approved by the U.S. Food and Drug Administration (FDA) or is in clinical trials. For example, but not limited to, the oncolytic virus is talimogene laherparepvec (Talimogene laherparepvec), also known as T-VEC (Imlygic) TM;it can be Amgen, Inc.). In certain embodiments, the oncolytic virus can be pelareorep (Reolysin®; Oncolytics Biotech, Inc.). In certain embodiments, the oncolytic virus can be DNX-2401 (DNAtrix Therapeutics). In certain embodiments, the oncolytic virus can be H101 (Oncorine®; Shanghai Sunway Biotech Co., Ltd.). In certain embodiments, the oncolytic virus can be pexastimogene devacirepvec (JX-594; SillaJen Inc.). In certain embodiments, the oncolytic virus can be CG0070 (Cold Genesys, Inc.). In certain embodiments, the oncolytic virus can be G47Δ (Daiichi-Sankyo Company, Limited).

[0027] In certain embodiments, the oncolytic virus is a vaccinia virus. In certain non-limiting embodiments, the oncolytic virus is a genetically engineered (also referred to as “recombinant”) vaccinia virus. In certain non-limiting embodiments, the virus is a recombinant vaccinia virus based on the Western Reserve (“WR”) strain of vaccinia, e.g., the WR strain commercially available as ATCC number VR1354 from the American Type Culture Collection. Other vaccinia virus strains suitable for genetic engineering include, but are not limited to, the Wyeth strain (ATCC VR-1536), the Lederle-Chorioallantoic strain (ATCC VR-325), and the CL strain (ATCC VR-117).

[0028] In certain non-limiting embodiments, the oncolytic virus is a genetically engineered vvDD vaccinia virus construct that includes, for example, a modified form of a virus described in U.S. Patent Nos. 7,208,313, 8,506,947, and U.S. Patent Application Publication Nos. 2003 / 0031681 and 2007 / 0154458, McCart et al., 2001, Cancer Research 61:8751-8757 and / or Thorne S et al., 2007, J. Clin. Invest. 117:3350-3358 (all of which are incorporated herein by reference in their entirety). For example, without limitation, the vaccinia virus can have deletions in the thymidine kinase (tk) and / or vaccinia growth factor (vgf) genes.

[0029] In certain non-limiting embodiments, the vaccinia virus has inactivating mutations in one or more genes where the product of the gene(s) functions in viral replication. For example, without limitation, one or more of the following genes can carry inactivating mutations: the gene encoding the large subunit of ribonucleotide reductase, the gene encoding the small subunit of ribonucleotide reductase, the gene encoding thymidylate kinase, the gene encoding DNA ligase, the gene encoding dUTPase, the tk gene, and the vaccinia virus growth factor (vgf) gene. In certain embodiments, the inactivating mutation is a mutation that reduces or eliminates the activity of the gene product. In certain embodiments, gene activation can be achieved by mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis).

[0030] Alternatively, or in addition, in certain embodiments, a nucleic acid can be inserted into one or more of the above genes to achieve inactivation. In certain non-limiting embodiments, a nucleic acid encoding a protein can be inserted into one or more of the above genes to achieve inactivation and further to achieve expression of the nucleic acid. In certain embodiments, a nucleic acid encoding an immunomodulatory factor molecule can be inserted into the interior of one of the above genes to achieve inactivation. In certain embodiments, a nucleic acid encoding an immunomodulatory factor molecule (e.g., an ANCHIM protein) linked to an anchor peptide can be inserted into the interior of one of the above genes to achieve inactivation and optionally to express the ANCHIM protein.

[0031] In certain non-limiting embodiments, the oncolytic virus is a vaccinia virus having an inactivating mutation (a mutation that reduces or eliminates the activity of the gene product) in the tk gene. For example, but not limited to, inactivation of the thymidine kinase gene can be generated by insertion of the cytosine deaminase (fcy1) gene within the locus of the thymidine kinase gene of the vaccinia virus genome, thereby resulting in expression of the fcy1 gene rather than the tk gene. In another non-limiting embodiment, a nucleic acid encoding a detectable protein, such as a fluorescent protein (e.g., yellow fluorescent protein (“yfp”)), can be inserted into the tk gene, thereby inactivating the gene. In certain embodiments, a nucleic acid encoding an immunomodulatory factor can be inserted into the tk gene. In certain embodiments, a nucleic acid encoding an ANCHIM protein can be inserted into the tk gene.

[0032] In additional or alternative embodiments, the recombinant vaccinia virus can have an inactivating mutation in the vaccinia growth factor gene. For example, without limitation, insertion of the lacZ gene into the locus of the vgf gene results in expression of the lacZ gene rather than vgf. In certain embodiments, a nucleic acid encoding an immunomodulatory factor can be inserted into the vgf gene. For example, without limitation, a nucleic acid encoding the ANCHIM protein can be inserted into the vgf gene.

[0033] 5.2 Immunomodulatory Factor Molecule Any immunomodulatory factor molecule known in the art can be utilized according to the subject matter disclosed herein. Some cytokines have immunomodulatory activity and would be considered immunomodulatory factor molecules (or simply "immunomodulatory factors") herein.

[0034] In certain embodiments, the immunomodulatory factor molecule can include a cytokine. In certain embodiments, the immunomodulatory factor molecule can include a chemokine. Non-limiting examples of immunomodulatory factor molecules that can be used according to the subject matter disclosed herein include interleukins ("IL"), such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24 or IL-27, C-X-C motif chemokine 11 (CXCL11), chemokine (C-C motif) ligand 5 (CCL5), interferons ("IFN"), such as IFN-α, IFN-α2, IFN-β, or IFN-γ; or tumor necrosis factor ("TNF"), such as TNF-α or TNF-β; and granulocyte macrophage colony-stimulating factor (GM-CSF).

[0035] The immunomodulatory factor molecule can be a human or non-human immunomodulatory factor molecule. Nucleic acids encoding such immunomodulatory factor molecules and the encoded protein sequences are well known in the art. Examples of non-human species include non-human primates, rodents, rabbits, dogs, cats, horses, pigs, sheep, cows, etc.

[0036] In certain non-limiting embodiments, the immunomodulatory factor molecule for use in the subject matter disclosed herein is IL-2. In certain embodiments, human IL-2 comprises at least the immunologically activating portion of the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 13; GenBank accession number CAA25742.1, residues 21-153), or a conservative substitution thereof, e.g., a sequence having one or two amino acid mutations.

[0037] In certain non-limiting embodiments, the immunomodulatory factor molecule for use in the subject matter disclosed herein is TNF-α. In certain non-limiting embodiments, human TNF-α comprises at least the immunologically activating portion of the sequence MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL (SEQ ID NO: 15; NCBI reference sequence number NM_000594.3), or a conservative substitution thereof, e.g., a sequence having one or two amino acid mutations.

[0038] In certain non-limiting embodiments, the immunomodulatory factor molecule for use in the subject matter disclosed herein is IL-23, which consists of two subunits, IL-23A and IL-12B. In certain non-limiting embodiments, the human IL-23A protein comprises at least the immunostimulatory portion of the sequence MLGSRAVMLLLLLPWTAQGRAVPGGSSPAWTQCQQLSQKLCTLAWSAHPLVGHMDLREEGDEETTNDVPHIQCGDGCDPQGLRDNSQFCLQRIHQGLIFYEKLLGSDIFTGEPSLLPDSPVGQLHASLLGLSQLLQPEGHHWETQQIPSLSPSQPWQRLLLRFKILRSLQAFVAVAARVFAHGAATLSP (SEQ ID NO: 18; GenBank accession number XXX, residues 1-189 or amino acid residues 28-184 of SEQ ID NO: 18), or a sequence having one amino acid mutation therein.

[0039] In certain non-limiting embodiments, the subject matter disclosed herein can be applied using a non-immunomodulatory cytokine, where the cytokine is preferably localized to the cancer cell environment, e.g., the cytokine exhibits toxic effects when administered systemically.

[0040] As used herein, the terms "conservative amino acid substitution" and "conservative modification" mean amino acid modifications that do not significantly affect or change the function and / or activity of the proteins disclosed herein that contain the amino acid sequences. Such conservative modifications include amino acid substitutions, additions, and deletions. The modifications can be introduced into the proteins of the present disclosure by standard techniques known in the art (site-directed mutagenesis and PCR-mediated mutagenesis). Amino acids can be classified into groups according to their physicochemical properties such as charge and polarity.

[0041] The conserved amino acid substitutions are those in which the amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; and neutral charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In certain embodiments, 1 or fewer, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer residues within the recited sequences are altered. Exemplary conserved amino acid substitutions are shown in Table 1 below.

[0042] [Table 1]

[0043] 5.3 Armed Oncolytic Virus The subject matter disclosed herein provides an oncolytic virus comprising a nucleic acid encoding an immunomodulatory factor molecule as described above. In certain embodiments, the immunomodulatory factor molecule encoded by the nucleic acid can be secreted (e.g., secreted from cells infected with the oncolytic virus comprising the nucleic acid). In certain embodiments, the immunomodulatory factor molecule can be linked to an anchor peptide to be membrane-bound.

[0044] 5.3.1 Secreted Immunomodulatory Factor Molecule The subject matter disclosed herein provides an oncolytic virus comprising a nucleic acid encoding an immunomodulatory factor molecule that is a secreted target (e.g., secreted from a cell infected with an oncolytic virus containing a nucleic acid). Suitable oncolytic viruses and immunomodulatory factor molecules are discussed in the above sections. In certain embodiments, the immunomodulatory factor molecule can be of the same species as the subject intended to be treated (e.g., a human immunomodulatory factor molecule for treating a human subject), or can be of a different species (e.g., a murine immunomodulatory factor molecule for treating a human subject).

[0045] In certain non-limiting embodiments, the subject matter disclosed herein provides an oncolytic virus that contains, in its genome, a nucleic acid encoding an immunomodulatory factor molecule. In certain embodiments, the oncolytic virus is herpes simplex virus, vaccinia virus, adenovirus or vesicular stomatitis virus.

[0046] In certain embodiments, the immunomodulatory factor molecule can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, IL-27, CXCL11, CCL5, IFN, IFN-α, IFN-α2, IFN-β, IFN-γ, TNF, TNF-α, TNF-β, GM-CSF or a combination thereof. In certain embodiments, the immunomodulatory factor molecule is IL-2. In certain embodiments, the immunomodulatory factor molecule is IL-23. In certain embodiments, the immunomodulatory factor molecule is TNF-α.

[0047] In certain embodiments, the nucleic acid encoding the immunomodulatory factor molecule can be placed under the control of a promoter that is active or activatable in oncolytic virus-infected cells (e.g., a promoter of an oncolytic virus). The coding nucleic acid can be DNA, RNA or cDNA to be compatible with the nucleic acid of the viral genome into which it is inserted.

[0048] In certain embodiments, the oncolytic virus is herpes simplex virus. In certain embodiments, the herpes virus contains, in its genome, a nucleic acid encoding an immunomodulatory factor molecule as described above. In certain embodiments, the nucleic acid encoding the immunomodulatory factor molecule can be placed under the control of a promoter (e.g., a herpes simplex virus promoter) that is active or activatable in herpes simplex virus-infected cells. The coding nucleic acid can be DNA in order to be compatible with the nucleic acids of the herpes simplex virus genome. In certain embodiments, the oncolytic virus of the present disclosure can be a herpes simplex virus containing a nucleic acid encoding IL-2. In certain embodiments, the oncolytic virus of the present disclosure can be a herpes simplex virus containing a nucleic acid encoding IL-23. In certain embodiments, the oncolytic virus of the present disclosure can be a herpes simplex virus containing a nucleic acid encoding TNF-α.

[0049] In certain embodiments, the oncolytic virus is vaccinia virus. In certain embodiments, the nucleic acid encoding the immunomodulatory factor molecule is operably linked to a promoter (e.g., a vaccinia virus promoter) that is active or activatable in vaccinia virus-infected cells. In certain non-limiting embodiments, the nucleic acid encoding the immunomodulatory factor molecule is operably linked to a vaccinia promoter (e.g., the p7.5 e / l promoter, or the pSe / l promoter) to create a promoter / immunomodulatory factor molecule coding construct. In certain non-limiting embodiments, the promoter / immunomodulatory factor molecule coding construct can be inserted into the tk gene. In certain other non-limiting embodiments, the promoter / immunomodulatory molecule coding construct can be inserted into the vgf gene. In certain embodiments, the oncolytic virus of the present disclosure can be a vaccinia virus containing a nucleic acid encoding IL-2. In certain embodiments, the oncolytic virus of the present disclosure can be a vaccinia virus containing a nucleic acid encoding IL-23. In certain embodiments, the oncolytic virus of the present disclosure can be a vaccinia virus containing a nucleic acid encoding TNF-α.

[0050] In certain non-limiting embodiments, the subject matter disclosed herein provides an oncolytic vaccinia virus that contains, in its genome, a nucleic acid that is a deoxyribonucleic acid encoding IL-2. In a specific non-limiting embodiment, the nucleic acid can encode human IL-2 (e.g., including a portion of the amino acid sequence of at least SEQ ID NO: 13 or a conservative substitution thereof). In certain embodiments, the nucleic acid encoding IL-2 is operably linked to a promoter that is active or activatable in vaccinia virus-infected cells (e.g., a vaccinia virus promoter). For example, but not by way of limitation, the nucleic acid encoding IL-2 can be operably linked to a vaccinia promoter (e.g., the p7.5 e / l promoter, or the pSe / l promoter) to create a promoter / IL-2 encoding construct. In certain non-limiting embodiments, the promoter / IL-2 encoding construct can be inserted into the tk gene. In certain other non-limiting embodiments, the promoter / IL-2 encoding construct can be inserted into the vgf gene.

[0051] In certain non-limiting embodiments, the subject matter disclosed herein provides an oncolytic vaccinia virus that contains, in its genome, a nucleic acid that is a deoxyribonucleic acid encoding TNF-α. In a specific non-limiting embodiment, the nucleic acid can encode human TNF-α (e.g., including the amino acid sequence of SEQ ID NO: 15 or a conservative substitution thereof). In certain embodiments, the nucleic acid encoding TNF-α is operably linked to a promoter that is active or activatable in vaccinia virus-infected cells (e.g., a vaccinia virus promoter). For example, but not by way of limitation, the nucleic acid encoding TNF-α can be operably linked to a vaccinia promoter (e.g., the p7.5 e / l promoter, or the pSe / l promoter) to create a promoter / TNF-α encoding construct. In certain non-limiting embodiments, the promoter / TNF-α encoding construct can be inserted into the tk gene. In certain other non-limiting embodiments, the promoter / IL-2 encoding construct can be inserted into the vgf gene.

[0052] In certain non-limiting embodiments, the subject matter disclosed herein provides an oncolytic vaccinia virus that contains in its genome a nucleic acid that is a deoxyribonucleic acid encoding subunits of IL-23 (e.g., IL-23p19 and IL-23p40). In certain embodiments, the nucleic acids encoding IL-23p19 and IL-23p40 are operably linked to a promoter (e.g., a vaccinia virus promoter) that is active or activatable in vaccinia virus-infected cells. In certain non-limiting embodiments, the oncolytic vaccinia virus contains in its genome a nucleic acid that is a deoxyribonucleic acid encoding a human homolog of IL-23p19 (e.g., IL-23A), and a human homolog of IL-23p40 (e.g., IL-12B), and produces IL-23. In specific non-limiting embodiments, the nucleic acid can encode IL-23p19 or its human homolog (e.g., IL-23A) and / or IL-23p40 or its human homolog (e.g., IL-12B), and is further operably linked to a vaccinia promoter (e.g., the p7.5 e / l promoter, or the pSe / l promoter), to create a promoter / IL-23p19 / IL-23p40 coding construct or a promoter / IL-23A / IL-12B coding construct. In certain non-limiting embodiments, the promoter / IL-23p19 / IL-23p40 coding construct or the promoter / IL-23A / IL-12B coding construct can be inserted into the tk gene. In certain other non-limiting embodiments, the promoter / IL-23p19 / IL-23p40 coding construct or the promoter / IL-23A / IL-12B coding construct can be inserted into the vgf gene.

[0053] 5.3.2 Membrane-Bound Immunomodulatory Factor Molecule The subject matter disclosed herein provides an oncolytic virus encoding a membrane-bound protein comprising an immunomodulatory factor molecule as described above linked to an anchoring peptide, wherein the immunomodulatory factor molecule linked to the anchoring peptide is referred to herein as "ANCHIM". In certain embodiments, the membrane-binding domain is heterologous. In certain embodiments, the membrane-binding domain is autologous. Suitable oncolytic viruses and immunomodulatory factor molecules are discussed in the above sections. The anchoring peptide is discussed in the following section.

[0054] In certain embodiments, the immunomodulatory factor molecule can be of the same species as the subject to be treated (e.g., a human immunomodulatory factor molecule for treating a human subject), or can be of a different species (e.g., a mouse immunomodulatory factor molecule for treating a human subject).

[0055] In certain non-limiting embodiments, the subject matter disclosed herein provides an oncolytic virus comprising, in its genome, a nucleic acid encoding a membrane-bound protein comprising an immunomodulatory factor molecule linked to an anchoring peptide. In certain embodiments, the oncolytic virus is herpes simplex virus, vaccinia virus, adenovirus or vesicular stomatitis virus. In certain embodiments, the immunomodulatory factor molecule can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, IL-27, CXCL11, CCL5, IFN, IFN-α, IFN-α2, IFN-β, IFN-γ, TNF, TNF-α, TNF-β, GM-CSF or a combination thereof. In certain embodiments, the immunomodulatory factor molecule is IL-2. In certain embodiments, the immunomodulatory factor molecule is IL-23. In certain embodiments, the immunomodulatory factor molecule is TNF-α.

[0056] 5.3.2.1 Anchor Peptide In certain embodiments, an anchoring peptide is any protein that, when fused to an immunomodulatory factor molecule, anchors the immunomodulatory factor molecule to the host cell membrane. In certain embodiments, an anchoring peptide can be used to modify an immunomodulatory factor molecule to bind to the membrane of a host cell according to the subject matter disclosed herein (e.g., by adding a GPI or other molecule). In certain non-limiting embodiments, the anchoring peptide is about 10 to about 50 amino acids, or about 15 to about 30, or about 20 amino acids in length.

[0057] In certain embodiments, the anchoring peptide is a glycosylphosphatidylinositol (GPI)-anchor acceptor peptide. In certain non-limiting embodiments, the GPI-anchor acceptor peptide comprises at least a portion of the C-terminus of its native peptide, e.g., comprises a portion of at least 100 C-terminal amino acids of the native peptide, or comprises a portion of at least 50 C-terminal amino acids of the native peptide, or comprises a portion of at least 30 C-terminal amino acids of the native peptide.

[0058] In certain embodiments, the anchoring peptide can include sequences of various proteins and portions of cell membrane proteins that would perform essentially the same function. As exemplified by GPI, the anchoring function can be achieved by modification of the underlying peptide or protein containing non-peptidic elements such as, but not limited to, carbohydrates, lipids, etc.

[0059] In certain embodiments, the anchor peptide can comprise a glycosylphosphatidylinositol (GPI)-anchor acceptor sequence of a human CD16b anchor acceptor peptide. In certain non-limiting embodiments, the anchor is a protein-derived GPI-anchor acceptor peptide as described in Ferguson et al., “Chapter 11: Glycosylphosphatidyl Anchors” in Glycobiology, 2nd Edition, Varki et al., editors, Cold Spring Harbor Press, 2009, such as, but not limited to, alkaline phosphatase, CD58, CD14, NCAM-120, and TAG-1 (the content of which is incorporated herein by reference in its entirety).

[0060] In certain non-limiting embodiments, the GPI-anchor acceptor peptide functions during GPI addition and contains a signal peptide portion ("SPP") that is cleaved during the process. In certain embodiments, the SPP comprises three domains: (1) a first domain comprising three relatively small amino acids (e.g., but not limited to, Gly (G), Ala (A), Ser (S), Asn (N), Asp (D), or Cys (C) or any combination thereof) ω, (ω + 1), and (ω + 2), where ω is linked to the GPI anchor and (ω + 1) and (ω + 2) are the first two residues of the cleaved peptide; (2) a relatively polar domain spacer of about 5-10 amino acid residues; and (3) a hydrophobic domain of about 15-20 amino acids. In certain embodiments, the anchor peptide can further comprise a sequence that targets it to the endoplasmic reticulum to facilitate addition of the GPI anchor when it is not a critical feature viral infection (see Mayor S and Riezman H, 2004, Nature Reviews Molecular Cell Biology 5, 110-120; the content of which is incorporated herein by reference in its entirety). This signal peptide portion extending from the C-terminus of the protein can be illustrated as follows: ω-(ω + 1)-(ω + 2)-polar spacer region-~hydrophobic domain (see Galian C et al., 2012, J. Biol. Chem. 287(20):16399-16409). In certain non-limiting embodiments, the ω-(ω + 1)-(ω + 2)-polar spacer region can comprise a sequence of about 10 amino acids in which at least about 2 or at least about 3 of the residues are G and at least about 2 or at least about 3 or at least about 4 or at least about 5 of the residues are S. In certain non-limiting embodiments, the sequence can be NSTGSGSSGS (SEQ ID NO: 17; Galian, supra).In certain non-limiting embodiments, the hydrophobic domain can comprise from about 15 to about 20 amino acids, which comprise at least about 10 residues selected from the group consisting of A, Leu (L), Val (V), Phe (F), and combinations thereof. Non-limiting examples of SPPs are provided in Table 1 or Figure 8 of Galian et al. (supra), and include the following sequences:. GGALQSTASLFVVSLSLLHLYS (SEQ ID NO: 2; from human CD24); NNSCSSPGGCRLFLSTIPVLWTLL (SEQ ID NO: 3; from human EFNA2); DAAHPGRSVVPALLPLLAGTLLLLETAT (SEQ ID NO: 4; from human PPB1); SAAPRLFPLAWTVLLLPLLLLQT (SEQ ID NO: 5; from human EFNA1); NGSISLAVPLWLLAASLLCLLSCK (SEQ ID NO: 6; from human LSAMP); SGAPTLSPSLLGLLLPAFGILVYLEF (SEQ ID NO: 7; from human CNTN1); NGTSRRAGCIWLLPLLVLHLLLKF (SEQ ID NO: 8; from rat NTRI); NSTGSGSSGSAAAAVAAAAVAAAAVAAAA (SEQ ID NO: 9) or NSTGSGSSGSAAAAVVFVFVFVFVVAAAA (SEQ ID NO: 10).

[0061] In certain embodiments, the GPI anchor acceptor peptide sequence comprises any one of the peptides having SEQ ID NOs: 2-10, which retain from about one or about two amino acid substitutions, insertions, or deletions, or from about one, about two, or about three conservative amino acid substitutions.

[0062] In certain non-limiting embodiments, the GPI anchor acceptor peptide comprises at least a portion (e.g., at least about 10 or at least about 20 or at least about 25 contiguous residues) of the GPI anchor acceptor peptide sequence of CD16b having the sequence SSFSPPGYQVSFCLVMVLLFAVDTGLYFSVKTNI (SEQ ID NO: 1), or a sequence that retains about one or about two amino acid substitutions, insertions or deletions, or about one, about two or about three conservative amino acid substitutions (see Simmons D and Seed B, 1988, Nature 333:568-570).

[0063] In certain non-limiting embodiments, the GPI anchor acceptor peptide comprises at least a portion (e.g., at least about 10 or at least about 20 or at least about 25 contiguous residues) of the GPI anchor acceptor peptide sequence of CD16b having the sequence VSTISSFSPPGYQVSFCLVMVLLFAVDTGLYFSVKTNI (SEQ ID NO: 11), or a sequence that retains about one or about two amino acid substitutions, insertions or deletions, or about one, about two or about three conservative amino acid substitutions (see Simmons D and Seed B, 1988, Nature 333:568-570). In certain non-limiting embodiments, the anchoring peptide does not function via a GPI. In certain non-limiting embodiments, the anchoring peptide can anchor its linked protein to the cell membrane via a fatty acid / diacylglycerol. In certain other non-limiting embodiments, the anchoring peptide comprises a region that can form a transmembrane domain (e.g., a region that is substantially hydrophobic and / or a region predicted to be a transmembrane domain by standard software such as TMpred, although not limited thereto). In certain embodiments, the anchoring peptide is the PD-L1 transmembrane domain.

[0064] In certain non-limiting embodiments, the nucleic acid encoding an immunomodulatory factor molecule linked (optionally via a linker) to an anchoring peptide can be placed under the control of a promoter that is active or activatable in oncolytic virus-infected cells (e.g., a promoter of an oncolytic virus). The coding nucleic acid can be DNA, RNA, or cDNA to be compatible with the nucleic acids of the viral genome into which it is inserted.

[0065] 5.3.2.2 Linker In certain embodiments, the immunomodulatory factor molecule can be directly linked to the anchoring peptide. Alternatively, the immunomodulatory factor molecule can be linked to the anchoring peptide via a linker. In certain embodiments, the linker is selected from a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, a non-helical linker, or a combination thereof. In certain embodiments, the immunomodulatory factor molecule can be linked to the anchoring peptide via one or more linkers (e.g., two or more, three or more, or four or more).

[0066] In certain embodiments, the linker is a peptide linker. In certain embodiments, the peptide linker contains Gly and Ser. In certain embodiments, the peptide linker can be, for example, but not limited to, about 1 to about 25 or about 5 to about 20 or about 5 to about 15 amino acids in length. Non-limiting examples of linkers for use in the subject matter disclosed herein are disclosed in International Publication WO2017 / 165464 (e.g., SEQ ID NOs: 42, 44, 45, 75, 76, 77, and 78), the contents of which are incorporated herein by reference in their entirety.

[0067] In certain embodiments, the peptide linker is a flexible linker. In certain embodiments, the flexible linker can be (G4S)3, which corresponds to GGGGSGGGGSGGGGS (SEQ ID NO: 12). In certain non-limiting embodiments, the linker linked to the anchoring peptide can include the amino acid sequence GPAGGGGSGGGGSGGGGSVSTISSFSPPGYQVSFCLVMVLLFAVDTGLYFSVKTNI (SEQ ID NO: 16) or a sequence retaining about one or about two amino acid substitutions, insertions or deletions, or about one, about two or about three conservative amino acid substitutions. Non-limiting examples of certain GPI-anchor type cytokines are provided in U.S. Patent Application Publication No. 2003 / 0105054 (e.g., paragraphs

[0045] and

[0075] ) and U.S. Patent No. 6,277,368, the contents of which are hereby incorporated by reference in their entirety.

[0068] In certain embodiments, the peptide linker includes a rigid linker. In certain embodiments, the rigid linker can be (A(EA3K)4AAA) (SEQ ID NO: 36), which corresponds to AEAAAKEAAAKEAAAKEAAAKAAA. In certain embodiments, the linker includes the amino acid sequence of SEQ ID NO: 36 or a sequence retaining about one or about two amino acid substitutions, insertions or deletions, or about one, about two or about three conservative amino acid substitutions.

[0069] 5.3.2.3 Non-Limiting Embodiments In certain non-limiting embodiments, the subject matter disclosed herein provides an oncolytic virus encoding a membrane-bound protein comprising an immunomodulatory factor molecule as described above linked to an anchoring peptide, optionally via a linker.

[0070] In certain embodiments, the oncolytic virus is herpes simplex virus, vaccinia virus, adenovirus or vesicular stomatitis virus. In certain embodiments, the immunomodulatory factor molecule can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, IL-27, CXCL11, CCL5, IFN, IFN-α, IFN-α2, IFN-β, IFN-γ, TNF, TNF-α, TNF-β, GM-CSF or combinations thereof. In certain embodiments, the immunomodulatory factor molecule is IL-2. In certain embodiments, the immunomodulatory factor molecule is IL-23. In certain embodiments, the immunomodulatory factor molecule is TNF-α.

[0071] In certain non-limiting embodiments, the anchoring peptide comprises at least a portion of any one of SEQ ID NOs: 1-11 (e.g., the portion comprising at least about 10 or at least about 20 or at least about 25 contiguous amino acid residues). In specific non-limiting embodiments, the anchoring peptide comprises the amino acid sequence of SEQ ID NO: 11, or a sequence retaining about one or about two amino acid substitutions, insertions or deletions, or about one, about two or about three conservative amino acid substitutions.

[0072] In certain non-limiting embodiments, the immunomodulatory factor molecule is linked to the anchoring peptide by a peptide linker about 1 to about 25 or about 5 to about 15 amino acids in length. In certain non-limiting embodiments, the linker comprises at least a portion of any one of SEQ ID NOs: 12 and 36 (e.g., the portion comprising at least about 10 or at least about 20 or at least about 25 contiguous amino acid residues). In certain non-limiting embodiments, the linker comprises the sequence (G4S)3 (SEQ ID NO: 12) or a conservative substitution thereof. In certain non-limiting embodiments, the linker comprises the sequence (A(EA3K)4AAA) (SEQ ID NO: 36) or a conservative substitution thereof.

[0073] In certain embodiments, the oncolytic virus can comprise a gene encoding a membrane-bound fusion protein comprising an immunomodulatory factor molecule fused to an anchoring peptide (e.g., a GPI anchor (e.g., the GPI anchor acceptor sequence of human CD16b)) via a rigid linker (alternatively referred to herein as "RGPI" or "RG"). In certain embodiments, the oncolytic virus can comprise a gene encoding a membrane-bound fusion protein comprising an immunomodulatory factor molecule fused to an anchoring peptide (e.g., a GPI anchor (e.g., the GPI anchor acceptor sequence of human CD16b)) via a flexible linker (alternatively referred to herein as "FGPI" or "FG"). In certain embodiments, the GPI anchor sequence can be replaced with the PD-L1 transmembrane domain.

[0074] In certain embodiments, the oncolytic virus can comprise a gene encoding a membrane-bound fusion protein comprising an immunomodulatory factor fused to the PD-L1 transmembrane domain via a rigid linker. In certain embodiments, the oncolytic virus can comprise a gene encoding a membrane-bound fusion protein comprising an immunomodulatory factor fused to the PD-L1 transmembrane domain via a (G4S)3 linker. In certain embodiments, the oncolytic virus can comprise a gene encoding a membrane-bound fusion protein (alternatively referred to herein as "FPTM") comprising an immunomodulatory factor fused to the PD-L1 transmembrane domain via a flexible linker.

[0075] In certain embodiments, the subject matter disclosed herein provides an oncolytic virus (e.g., vaccinia virus) that contains in its genome a nucleic acid that is a deoxyribonucleic acid encoding IL-2. In certain non-limiting embodiments, the IL-2 is human IL-2. In certain non-limiting embodiments, the human IL-2 comprises at least a portion of the amino acid sequence of SEQ ID NO: 13 or a conservative substitution thereof, or the sequence having a mutation of one amino acid or two amino acids. In certain embodiments, the IL-2 is optionally linked to an anchoring peptide and contains a linker peptide between the IL-2 and the anchoring peptide. In certain non-limiting embodiments, the anchoring peptide comprises at least a portion of any one of SEQ ID NOs: 1-11, 17, and 37 (e.g., the portion containing at least about 10 or at least about 20 or at least about 25 contiguous amino acid residues). In certain non-limiting embodiments, the IL-2 is linked to the anchoring peptide by a peptide linker that is about 1 to about 25 or about 5 to about 15 amino acids in length. In certain embodiments, the nucleic acid encoding IL-2 is operably linked to a promoter that is active or activatable in oncolytic virus-infected cells, e.g., a promoter of the oncolytic virus (e.g., a vaccinia virus promoter).

[0076] In certain non-limiting embodiments, the nucleic acid present in the genome of the oncolytic virus can encode human IL-2 (e.g., comprising the amino acid sequence of SEQ ID NO: 13), linked via a peptide linker (e.g., comprising the amino acid sequence of SEQ ID NO: 12) to an anchoring peptide (e.g., comprising the amino acid sequence of SEQ ID NO: 11). For example, without limitation, the nucleic acid can encode APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLTGPAGGGGSGGGGSGGGGS VSTISSFSPPGYQVSFCLVMVLLFAVDTGLYFSVKTNI (SEQ ID NO: 14) or a sequence retaining about one or about two amino acid substitutions, insertions or deletions, or about one, about two or about three conservative amino acid substitutions. In certain non-limiting embodiments, the encoding nucleic acid can be operably linked to an oncolytic virus promoter. In certain embodiments, the encoding nucleic acid can be operably linked to a vaccinia promoter (e.g., the p7.5 e / l promoter, or the pSe / l promoter) to create a promoter / ANCHIM encoding construct. In certain non-limiting embodiments, the promoter / ANCHIM encoding construct can be inserted into the tk gene. In certain other non-limiting embodiments, the promoter / ANCHIM encoding construct can be inserted into the vgf gene.

[0077] In certain non-limiting embodiments, the nucleic acid can encode human IL-2 (e.g., comprising the amino acid sequence of SEQ ID NO: 13), linked via a peptide linker (e.g., comprising the amino acid sequences of SEQ ID NOs: 12 and 36) to an anchoring peptide (e.g., comprising the amino acid sequences of SEQ ID NOs: 11 and 37). In certain non-limiting embodiments, the coding nucleic acid can be operably linked to a vaccinia promoter (e.g., the p7.5 e / l promoter, or the pSe / l promoter), to create a promoter / ANCHIM coding construct. In certain non-limiting embodiments, the promoter / ANCHIM coding construct can be inserted into the tk gene. In certain other non-limiting embodiments, the promoter / ANCHIM coding construct can be inserted into the vgf gene.

[0078] In certain non-limiting embodiments, the subject matter disclosed herein provides an oncolytic vaccinia virus that contains in its genome a nucleic acid that is a deoxyribonucleic acid encoding TNF-α. In certain embodiments, TNF-α is optionally linked to an anchoring peptide through a linker peptide. In certain embodiments, the nucleic acid is operably linked to a promoter (e.g., a vaccinia virus promoter) that is active or activatable in vaccinia virus-infected cells. In certain non-limiting embodiments, TNF-α is human TNF-α. In certain non-limiting embodiments, human TNF-α has at least the immunostimulatory portion of SEQ ID NO: 15 or a sequence having one or two amino acid mutations in that sequence. In certain non-limiting embodiments, the anchoring peptide contains at least a portion of any one of SEQ ID NOs: 1-11 (e.g., the portion containing at least about 10 or at least about 20 or at least about 25 contiguous amino acid residues). In a specific non-limiting embodiment, the anchoring peptide contains SEQ ID NO: 11 or a sequence having about one or about two amino acid substitutions, insertions, or deletions, or about one, about two, or about three conservative amino acid substitutions in that sequence. In certain non-limiting embodiments, TNF-α is linked to the anchoring peptide by a peptide linker that is about 1 to about 25 or about 5 to about 15 amino acids in length. In certain non-limiting embodiments, the linker contains the sequence (G4S)3 (SEQ ID NO: 12). In certain non-limiting embodiments, the encoding nucleic acid is operably linked to a vaccinia promoter (e.g., the p7.5 e / l promoter, or the pSe / l promoter), and a promoter / ANCHIM encoding construct can be made. In certain non-limiting embodiments, the promoter / ANCHIM encoding construct can be inserted into the tk gene. In certain other non-limiting embodiments, the promoter / ANCHIM encoding construct can be inserted into the vgf gene. In certain non-limiting embodiments, the linker contains the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 12). In certain non-limiting embodiments, the linker contains the sequence of a rigid linker (SEQ ID NO: 36).

[0079] In certain non-limiting embodiments, the subject matter disclosed herein provides an oncolytic vaccinia virus that contains in its genome a nucleic acid that is a deoxyribonucleic acid encoding IL-23p19 and IL-23p40. In certain embodiments, IL-23p19 and IL-23p40 are optionally linked to an anchor peptide and include a linker peptide between the IL-23p40 and the anchor peptide. In certain embodiments, the nucleic acid encoding IL-23p19 is operably linked to a promoter (e.g., a vaccinia virus promoter) that is active or activatable in vaccinia virus-infected cells. In certain non-limiting embodiments, the oncolytic vaccinia virus contains in its genome a nucleic acid that is a deoxyribonucleic acid encoding a human homolog of IL-23p19 (e.g., IL-23A), and a human homolog of IL-23p40 (e.g., IL-12B), and produces IL-23. In certain non-limiting embodiments, the human IL-23A protein includes at least the immunologically activating portion of the sequence of SEQ ID NO: 18 or a sequence having one amino acid mutation in said sequence.

[0080] In specific non-limiting embodiments, the nucleic acid can encode IL-23p19 or a human homolog thereof (e.g., IL-23A) (e.g., including the amino acid sequence of SEQ ID NO: 18), and can further encode IL-23p40 or a human homolog thereof (e.g., IL-12B), linked via a peptide linker (e.g., including the amino acid sequences of SEQ ID NOs: 12 and 36) to an anchor peptide (e.g., including the amino acid sequences of SEQ ID NOs: 11 and 37). In certain non-limiting embodiments, the encoding nucleic acid is operably linked to a vaccinia promoter (e.g., the p7.5 e / l promoter, or the pSe / l promoter), and a promoter / ANCHIM encoding construct can be produced. In certain non-limiting embodiments, the promoter / ANCHIM encoding construct can be inserted into the tk gene. In certain other non-limiting embodiments, the promoter / ANCHIM encoding construct can be inserted into the vgf gene.

[0081] In certain non-limiting embodiments, the subject matter disclosed herein, as described above, optionally includes a linker peptide between the immunomodulatory factor molecule and the anchor peptide, and provides a oncolytic herpes simplex virus that contains in its genome a nucleic acid encoding an immunomodulatory factor molecule linked to the anchor peptide. In certain embodiments, the nucleic acid encoding the immunomodulatory factor molecule linked to the anchor peptide, optionally including a linker peptide between the immunomodulatory factor molecule and the anchor peptide, can be placed under the control of a promoter that is active or activatable in oncolytic herpes simplex virus-infected cells (e.g., a promoter of oncolytic herpes simplex virus). The coding nucleic acid linked to the anchor peptide, optionally including a linker peptide between the immunomodulatory factor molecule and the anchor peptide, can be DNA, RNA, or cDNA in order to be compatible with the nucleic acid of the viral genome into which it is inserted.

[0082] The subject matter disclosed herein further provides a pharmaceutical composition comprising, for example, one or more of the above-described genetically engineered (recombinant) oncolytic viruses in a physiological buffer, and such therapeutic compositions in solid, liquid, frozen, or lyophilized form. The subject matter disclosed herein further provides a delivery device (e.g., a syringe) containing a therapeutically effective amount of such a pharmaceutical composition. Non-limiting examples of pharmaceutical compositions comprising one or more of the oncolytic viruses described herein are disclosed in Section 5.7 below.

[0083] 5.4 Administration of Oncolytic Virus The oncolytic viruses disclosed herein (e.g., armed oncolytic viruses) can be administered according to any known method in the art. For example, without limitation, methods for delivering an oncolytic virus (e.g., vaccinia virus) as described herein, or a pharmaceutical composition thereof, or a composition containing antitumor T cells isolated from cancer tissue, to cancer or tumor cells can be via intratumoral injection. In certain embodiments, alternative administration methods, such as intravenous administration via infusion, parenteral administration, intravenous administration, intradermal administration, intramuscular administration, transdermal administration, rectal administration, intraurethral administration, intravaginal administration, intranasal administration, intrathecal administration, or intraperitoneal administration can also be used. The route of administration can vary depending on the location and nature of the tumor. In certain embodiments, the route of administration can be intradental administration, transdermal administration, parenteral administration, intravenous administration, intramuscular administration, intranasal administration, subcutaneous administration, topical administration (e.g., in the vicinity of a tumor with or particularly near a vasculature associated with the tumor), percutaneous administration, intrathecal administration, intratracheal administration, intraperitoneal administration, intraarterial administration, intravesical administration, intratumoral administration, inhalation, perfusion, administration by lavage, or oral administration. In certain embodiments, the modified virus can be administered to the patient from a source transplanted into the patient's body.

[0084] In certain embodiments, administration of the modified virus can be by continuous infusion over a selected period. In certain embodiments, an oncolytic vaccinia virus as described herein, or a pharmaceutical composition containing the same, can be administered by infusion at a therapeutically effective dose over a period of about 15 minutes, about 30 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 100 minutes, or about 120 minutes or more.

[0085] The oncolytic vaccinia virus or pharmaceutical composition of the present disclosure can be administered as a liquid dosage form, in which case the total administration volume is about 1 mL to about 5 mL, about 5 mL to 10 mL, about 15 mL to about 20 mL, about 25 mL to about 30 mL, about 30 mL to about 50 mL, about 50 mL to about 100 mL, about 100 mL to 150 mL, about 150 mL to about 200 mL, about 200 mL to about 250 mL, about 250 mL to about 300 mL, about 300 mL to about 350 mL, about 350 mL to about 400 mL, about 400 mL to about 450 mL, about 450 mL to 500 mL, about 500 mL to 750 mL or about 750 mL to 1000 mL.

[0086] In certain embodiments, a single dose of the virus can mean the amount administered to a subject or tumor over 1, 2, 5, 10, 15, 20 or 24 hours. In certain embodiments, the dose can be administered over time or by separate injections. In certain embodiments, multiple doses (e.g., 2, 3, 4, 5, 6 or more doses) of the vaccinia virus can be administered to a subject, for example, in which case the second treatment can be performed within 1, 2, 3, 4, 5, 6, 7 days or weeks of the first treatment. In certain embodiments, multiple doses of the modified virus can be administered to a subject over 1, 2, 3, 4, 5, 6, 7 or more days or weeks. In certain embodiments, the oncolytic vaccinia virus or pharmaceutical composition as described herein can be administered over a period of about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks or about 52 weeks, or more. The frequency of administration of the oncolytic vaccinia virus or pharmaceutical composition as described herein can, in certain cases, be once a day, twice a day, once a week, once every 3 weeks, once every 4 weeks (or once a month), once every 8 weeks (or once every 2 months), once every 12 weeks (or once every 3 months), or once every 24 weeks (or once every 6 months).

[0087] As used interchangeably herein, the term "therapeutically effective amount" or "effective amount" can mean an amount of oncolytic virus that, when administered, may be sufficient to prevent or to some extent reduce the onset of one or more symptoms of a disorder, disease, or condition in a subject being treated. The term "therapeutically effective amount" can also mean an amount of oncolytic virus that is sufficient to elicit a biological or medical response in a cell, tissue, system, animal, or human. An effective amount in such methods can include an amount that reduces the cancer growth rate or spread, or that extends the survival period in a subject. The present disclosure provides a method of reducing tumor growth that can include administering to a tumor an effective amount of a modified virus as described above. In certain embodiments, an effective amount of the modified virus or its pharmaceutical composition can include an amount sufficient to induce a delay, inhibition, or reduction in tumor growth or size, and can include eradication of the tumor. Reducing tumor growth can be manifested, for example, by a decrease in the growth rate or an extension of the survival of a subject bearing the tumor. In certain embodiments, a "therapeutically effective amount" or "effective amount" can include an amount sufficient to induce infiltration of T cells into the tumor and / or cancer.

[0088] The effective amount of the virus can be determined by methods known in the art. In certain embodiments, the virus can be administered in an amount sufficient to induce tumor lysis in at least about 20% of the cells in the tumor, at least about 30% of the cells in the tumor, at least about 40% of the cells in the tumor, at least about 50% of the cells in the tumor, at least about 60% of the cells in the tumor, at least about 70% of the cells in the tumor, at least about 80% of the cells in the tumor, or at least about 90% of the cells in the tumor.

[0089] In certain embodiments, the amount of virus administered is about 1×10 7 ~1×10 10Individual infectious virus particles or plaque - forming units (pfu), or about 1×10 7 ~1×10 9 pfu per square meter of body surface area of the subject to be treated m 2 may be. In certain embodiments, the virus can be administered at a dose that can contain about 1×10 8 pfu. In certain embodiments, the amount of virus administered is about 1×10 3 ~1×10 12 individual virus particles or pfu, or about 1×10 5 ~1×10 10 pfu, or about 1×10 5 ~1×10 8 pfu, or about 1×10 8 ~1×10 10 pfu may be. In certain embodiments, the virus is about 1×10 3 pfu / dose to about 1×10 4 pfu / dose, about 1×10 4 pfu / dose to about 1×10 5 pfu / dose, about 1×10 5 pfu / dose to about 1×10 6 pfu / dose, about 1×10 7 pfu / dose to about 1×10 8 pfu / dose, about 1×10 9 pfu / dose to about 1×10 10 pfu / dose, about 1×10 10 pfu / dose to about 1×10 11 pfu / dose, about 1×10 11 pfu / dose to about 1×10 12 pfu / dose, about 1×10 12 pfu / dose to about 1×10 13 pfu / dose, about 1×10 13 pfu / dose to about 1×10 14 pfu / dose, or about 1×10 14 pfu / dose to about 1×10 15 pfu / dose can be administered at a dose that can contain. In certain embodiments, the oncolytic vaccinia virus of the subject matter disclosed herein is about 1×10 3 virus particles / dose to about 1×10 4Viral particles / dosage, about 1×10 4 Viral particles / dosage ~ about 1×10 5 Viral particles / dosage, about 1×10 5 Viral particles / dosage ~ about 1×10 6 Viral particles / dosage, about 1×10 7 Viral particles / dosage ~ about 1×10 8 Viral particles / dosage, about 1×10 9 Viral particles / dosage ~ about 1×10 10 Viral particles / dosage, about 1×10 10 Viral particles / dosage ~ about 1×10 11 Viral particles / dosage, about 1×10 11 Viral particles / dosage ~ about 1×10 12 Viral particles / dosage, about 1×10 12 Viral particles / dosage ~ about 1×10 13 Viral particles / dosage, about 1×10 13 Viral particles / dosage ~ about 1×10 14 Viral particles / dosage, or about 1×10 14 Viral particles / dosage ~ about 1×10 15 It can be administered at a dosage that can include a viral particles / dosage.

[0090] 5.5 Manufacturing Method 5.5.1 Isolation and Preparation of Oncolytic Virus-Induced Tumor-Infiltrating T Cells (「OV-Induced T Cells」) In certain embodiments, the subject matter disclosed herein relates to oncolytic viruses that promote the infiltration of immune cells into the tumor microenvironment and thus result in tumor-infiltrating immune cells induced by oncolytic viruses.

[0091] In certain embodiments, the subject matter disclosed herein relates to oncolytic viruses that give rise to a systemic potent anti-tumor immune cell, where the anti-tumor cells can be isolated from tumor tissue, expanded ex vivo, and administered to cancer patients (e.g., in the manner of adoptive T cell transfer) for cancer treatment.

[0092] In certain embodiments, the subject matter disclosed herein relates to oncolytic viruses that promote the infiltration of T cells into the tumor microenvironment and thus result in oncolytic virus-induced T cells (also referred to herein as "tumor-infiltrating T cells" or "TILs"). In certain embodiments, the oncolytic virus promotes the infiltration of tumor-specific CD8+ and CD4+ T cells. In certain embodiments, the oncolytic virus promotes the infiltration of activated innate immune cells. In certain embodiments, the activated innate immune cells include natural killer (NK) cells.

[0093] In certain embodiments, the subject matter disclosed herein relates to a method of generating oncolytic virus-induced T cells, comprising administering to a subject an effective amount of an oncolytic virus and inducing and trafficking T cells to the tumor tissue of the subject. Non-limiting examples of oncolytic viruses are disclosed in Sections 5.1 and 5.3 above. In certain embodiments, the oncolytic virus can be vaccinia virus. For example, without limitation, the vaccinia virus can be vvDD. In certain embodiments, the oncolytic virus (e.g., vvDD) encodes an immunomodulatory factor molecule in an expressible form. For example, without limitation, the immunomodulatory factor molecule can be IL-2, IL-15, CXC11, and / or CCL5. In certain embodiments, the subject matter disclosed herein relates to an oncolytic vaccinia virus (e.g., vvDD) that encodes at least one secreted and / or at least one membrane-bound immunomodulatory factor molecule as described above in an expressible form. In certain non-limiting embodiments, the oncolytic virus encodes IL-2, e.g., secreted or membrane-bound IL-2, in an expressible form.

[0094] In certain embodiments, a method of generating OV-induced T cells further comprises isolating OV-induced T cells from a subject (e.g., from a subject's tumor). In certain embodiments, OV-induced T cells can be isolated from lymphoid and non-lymphoid tissues or from peripheral blood. In certain embodiments, OV-induced T cells can be isolated from cancerous tissue. For example, T cells can be isolated from tissue by digesting the tissue and using density gradient centrifugation (e.g., using a Percoll density gradient).

[0095] In certain embodiments, a method of generating OV-induced T cells further comprises expanding ex vivo isolated OV-induced T cells. In certain embodiments, during ex vivo expansion, the isolated OV-induced T cells can be treated with one or more cytokines, lymphokines, and / or one or more agents (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, IL-27, IFN-α, IFN-α2, IFN-β, IFN-γ, TNF-α, TNF-β, GM-CSF or combinations thereof). For example, without limitation, the isolated OV-induced T cells can be treated with IL-7, IL-2, and / or a GSK3b inhibitor. In certain embodiments, OV-induced T cells can be treated with IL-2. In certain embodiments, OV-induced T cells can be treated with IL-7. In certain embodiments, OV-induced T cells can be treated with IL-2 and IL-7. In certain non-limiting examples, the isolated T cells can be treated for about 2 to about 10 days (e.g., about 3 days).

[0096] In certain embodiments, isolated OV-induced T cells are co-cultured with dendritic cells and cancer cells. In certain embodiments, OV-induced T cells can be co-cultured with cancer cells infected with a oncolytic virus (e.g., an oncolytic virus disclosed herein). In certain embodiments, the cancer cells can be infected with an oncolytic virus different from the oncolytic virus used to generate the OV-induced T cells. In certain embodiments, the isolated OV-induced T cells are co-cultured with dendritic cells and cancer cells for about 2 to about 10 days (e.g., about 2 days). In certain embodiments, the co-cultured T cells are subsequently treated with one or more cytokines and / or one or more agents.

[0097] In certain embodiments, to quantify the ability of an oncolytic virus to promote T cell infiltration in the tumor microenvironment, isolated OV-induced T cells can be analyzed using antibodies against CD3, CD8, CD4, and 4-1BB. In certain embodiments, the oncolytic virus promotes infiltration of CD3 + , CD8 + , CD4 + , and 4-1BB + T cells. In certain embodiments, the isolated OV-induced T cells are CD3 + , CD8 + , CD4 + and / or 4-1BB + . In certain embodiments, the OV-induced T cells are CD3 + . In certain embodiments, the OV-induced T cells are CD3 + . In certain embodiments, the OV-induced T cells are CD4 + . In certain embodiments, the OV-induced T cells are CD8 + . In certain embodiments, the OV-induced T cells are CD4 + 4-1BB + . In certain embodiments, the OV-induced T cells are CD8 + 4-1BB + .

[0098] In certain embodiments, the OV-induced T cells can be used for cancer therapy via adoptive T cell transfer, as described in Section 5.6.1 below. In certain embodiments, the subject matter disclosed herein relates to adoptive immunotherapy, in which case tumor-infiltrating T cells are isolated from tumor tissue, expanded ex vivo, and can be administered to cancer patients for cancer treatment (e.g., in the manner of adoptive T cell transfer). In certain embodiments, the OV-induced T cells can exert their functions of killing cancer cells and associated stromal cells. In certain embodiments, the OV-induced T cells can be transplanted into the subject from whom the cells were isolated. Alternatively, and / or in addition thereto, the OV-induced T cells can be transplanted into a different subject. In certain embodiments, the OV-induced T cells are allogeneic.

[0099] In certain embodiments, prior to transplantation of the isolated OV-induced T cells into a subject, the isolated OV-induced T cells can be analyzed for tumor specificity in a co-culture assay. In certain embodiments, the isolated OV-induced T cells are co-cultured with target cancer cells, irrelevant cancer cells, or splenocytes for about 1 to about 10 days (e.g., about 1 day). In a specific embodiment, the cancer cells are gamma-irradiated. In certain embodiments, the isolated OV-induced T cells are co-cultured with target cancer cells, irrelevant cancer cells, or splenocytes. In certain embodiments, the isolated OV-induced T cells for transplantation can exhibit specific reactivity against target tumor cells. In certain embodiments, the specific reactivity includes an increase in IFN-γ expression by the isolated OV-induced T cells when co-cultured with target cancer cells compared to when co-cultured with irrelevant target cancer cells or splenocytes.

[0100] 5.6 Treatment Method The present disclosure provides a method for the treatment of a subject having cancer. In particular, the present disclosure provides a method for the treatment of a subject having cancer, which comprises, as a mode of adoptive T cell immunotherapy, administering to the subject having cancer an isolated OV-induced T cell. The present disclosure further provides a method for the treatment of a subject having cancer, which comprises administering a oncolytic virus armed with an immunomodulatory factor molecule (e.g., secreted or membrane-bound).

[0101] Non-limiting examples of oncolytic viruses for use in the methods disclosed herein are disclosed in Sections 5.1, 5.3, and 5.5 above. Methods of administering oncolytic viruses are disclosed in Section 5.4 above. The subject can be a human or non-human subject. Non-limiting examples of non-human subjects include non-human primates, dogs, cats, horses, pigs, cows, mice, rats, hamsters, and other rodents, rabbits, and the like.

[0102] Non-limiting examples of cancers that can be treated by the disclosed methods include gastrointestinal cancer, colon cancer, colorectal cancer, ovarian cancer, mesothelioma, melanoma, breast cancer, brain cancer (e.g., glioblastoma), prostate cancer, cervical cancer, non-small cell lung cancer, kidney cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, adenocarcinoma of the liver, gastric cancer, liver cancer, peritoneal cancer, pleural cancer, hematopoietic cell cancer, and metastatic cancer.

[0103] In certain embodiments, treatment by administration of isolated OV-induced T cells or by using a modified virus can be used alone or in combination with one or more immunomodulatory agents. Immunomodulatory agents include any compound, molecule or substance capable of suppressing antiviral immunity associated with a tumor or cancer. In certain embodiments, the immunomodulatory agent may be capable of suppressing innate immunity or adaptive immunity against a modified virus. Non-limiting examples of immunomodulatory agents include anti-CD33 antibodies or variable regions thereof, anti-CD11b antibodies or variable regions thereof, COX2 inhibitors (e.g., celecoxib), cytokines such as IL-12, GM-CSF, IL-2, IFNβ and IFNγ, and chemokines such as MIP-1, MCP-1 and IL-8. In certain embodiments, immunomodulatory agents include, but are not limited to, immune checkpoint inhibitors such as anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PDL1 antibodies and TLR agonists (e.g., Poly I:C). In certain embodiments, checkpoint inhibitors include molecules and / or compounds that inhibit and / or reduce PD-1, PD-L1 and / or CTLA4 activity and / or function. In certain embodiments, the immunomodulatory agent can be administered systemically or locally.

[0104] As used herein, "in combination with" means that a virus such as an oncolytic vaccinia virus or a pharmaceutical composition thereof as described herein, and an additional therapy, are administered to a subject as part of a treatment regimen or treatment plan. In certain embodiments, being used in combination does not require that the oncolytic virus and one or more agents be physically combined prior to administration or that they be administered over the same time frame. For example, without limitation, the oncolytic virus and one or more agents can be administered to the subject being treated simultaneously, or can be administered sequentially at the same time or in any order, or at different times. In certain embodiments, the methods of the disclosure can include administration of an immunomodulatory agent prior to administration of an oncolytic virus (e.g., an oncolytic virus that includes a nucleic acid encoding an immunomodulatory factor molecule (e.g., IL-2)). For example, without limitation, the immunomodulatory agent can be, e.g., IL-2, administered systemically or locally. In certain embodiments, the methods of the subject matter disclosed herein can include promoting an initial immune response in a subject being treated by administration of an oncolytic virus (e.g., an oncolytic virus that includes a nucleic acid encoding an immunomodulatory factor molecule (e.g., IL-2)) as disclosed herein, and / or administration of an immunomodulatory agent (e.g., IL-2).

[0105] In certain embodiments, the methods of the subject matter disclosed herein can include administering one or more isolated OV-induced T cells, a virus or pharmaceutical composition thereof as disclosed herein, and one or more additional therapies that follow, precede, or are combined therewith. Non-limiting examples of such therapies include chemotherapy, radiation, oncolytic virus therapy using additional viruses, therapy using immunomodulatory factor proteins, anti-cancer agents, or any combination thereof. Anti-cancer agents include, but are not limited to, chemotherapeutic agents, radiation therapy agents, cytokines, immune checkpoint inhibitors, angiogenesis inhibitors, apoptosis inducers, anti-cancer antibodies, and / or cyclin-dependent kinase inhibitors. In certain embodiments, cancer therapies include chemotherapy, biological therapy, radiation therapy, immunotherapy, hormone therapy, anti-vascular therapy, cryotherapy, toxin therapy, and / or surgery, or any combination thereof.

[0106] Certain non-limiting embodiments of the subject matter disclosed herein provide methods of treating cancer where the cancer is early-stage cancer. Certain non-limiting embodiments of the subject matter disclosed herein provide methods of treating cancer as described above where the cancer is not early-stage cancer. For example, but not limited to, the cancer can be advanced cancer. In certain embodiments, advanced cancer can mean cancer that is no longer limited to the primary organ (e.g., it may locally spread beyond the primary organ or may metastasize to another location in the body). In certain embodiments, advanced cancer can be grade III cancer. In certain non-limiting embodiments, the subject matter disclosed herein can be used to treat locally invasive or metastatic cancer.

[0107] In certain embodiments, the subject matter disclosed herein can be applied to the treatment of colon cancer that is stage T3 or higher and / or M1 or higher. In certain embodiments, the subject matter disclosed herein can be applied to the treatment of ovarian cancer that is stage 1C or higher. In another specific non-limiting embodiment, the subject matter disclosed herein can be applied to the treatment of mesothelioma that is stage 2 or stage 3 or higher. In certain embodiments, the subject matter disclosed herein can be applied to the treatment of melanoma that is stage III or higher.

[0108] 5.6.1 Adoptive T Cell Immunotherapy The subject matter disclosed herein provides a method of treating a subject afflicted with cancer using isolated OV-induced T cells. In certain embodiments, the OV-induced T cells are generated as described in section 5.5 above. In certain embodiments, the OV-induced T cells can be transplanted into the subject from whom the cells were isolated. Alternatively, and / or in addition thereto, the OV-induced T cells can be transplanted into a different subject.

[0109] The subject matter disclosed herein provides adoptive T cell immunotherapy comprising the steps of isolating OV-induced T cells, expanding them ex vivo, and transplanting them back into a subject afflicted with cancer. Methods for adoptive T cell transfer are disclosed in U.S. Patent Application Publication No. 2003 / 0170238, the contents of which are incorporated herein by reference.

[0110] In certain embodiments, a method of treating a subject afflicted with cancer comprises administering to the subject a therapeutically effective amount of OV-induced T cells. In certain embodiments, the OV-induced T cells are isolated from a different subject. In certain embodiments, the OV-induced T cells are isolated from the subject to be treated with the OV-induced T cells.

[0111] In certain embodiments, a method of treating a subject afflicted with cancer comprises administering to the subject an effective amount of an oncolytic virus, inducing the generation of potent anti-tumor T cells, promoting the transport of the induced potent anti-tumor T cells into tumor tissue to generate OV-induced T cells, isolating the OV-induced T cells, and administering the isolated OV-induced T cells to the subject.

[0112] In certain embodiments, a method of treating a subject afflicted with cancer comprises: (a) administering to the subject an effective amount of an oncolytic virus; (b) isolating OV-induced T cells; (c) expanding tumor-infiltrating T cells; and (d) transplanting the tumor-infiltrating T cells into the subject afflicted with cancer.

[0113] In certain non-limiting embodiments, as described above, isolated OV-induced T cells that have been treated with one or more cytokines and / or one or more agents and expanded ex vivo are subsequently introduced, for example, intraperitoneally (i.p.) into cancer patients. In certain embodiments, the isolated OV-induced T cells are administered intratumorally to cancer patients.

[0114] Non-limiting examples of oncolytic viruses for use in the disclosed methods are described above. For example, without limitation, the oncolytic virus can be herpes simplex virus, vaccinia virus, adenovirus or vesicular stomatitis virus. In certain embodiments, the oncolytic virus can be the Western Reserve strain of vaccinia virus. In certain embodiments, the oncolytic virus is the vvDD vaccinia virus. In certain embodiments, the oncolytic virus is vaccinia virus (e.g., vvDD). In certain non-limiting embodiments, the oncolytic virus can contain a nucleic acid encoding an immunomodulatory factor molecule in its genome. In certain embodiments, the subject matter disclosed herein relates to an oncolytic vaccinia virus encoding, in an expressible form, at least one secreted and / or at least one membrane-bound immunomodulatory factor molecule, comprising an immunomodulatory factor molecule linked to a membrane-binding domain (e.g., an anchor peptide) as described above. In certain embodiments, the immunomodulatory factor molecule can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, IL-27, CXCL11, CCL5, IFN, IFN-α, IFN-α2, IFN-β, IFN-γ, TNF, TNF-α, TNF-β, GM-CSF or a combination thereof. In certain embodiments, the immunomodulatory factor molecule is IL-2. In certain embodiments, the immunomodulatory factor molecule is IL-23. In certain embodiments, the immunomodulatory factor molecule is TNF-α. In certain embodiments, the immunomodulatory factor molecule is CXC11. In certain embodiments, the immunomodulatory factor molecule is CCL5. In certain embodiments, the immunomodulatory factor molecule is IL-15. In certain embodiments, the oncolytic virus (e.g., vaccinia virus) can contain a nucleic acid encoding IL-2. In certain embodiments, the oncolytic virus is the Western Reserve strain of vaccinia virus containing a nucleic acid encoding IL-2. In certain embodiments, the oncolytic virus is the vvDD vaccinia virus containing a nucleic acid encoding IL-2. Certain In an embodiment, the oncolytic virus (e.g., vaccinia virus) can comprise a nucleic acid encoding IL-2 linked to an anchoring peptide. In certain embodiments, the oncolytic virus is the Western Reserve strain of vaccinia virus comprising a nucleic acid encoding IL-2 linked to an anchoring peptide. In certain embodiments, the oncolytic virus is the vvDD vaccinia virus comprising a nucleic acid encoding IL-2 linked to an anchoring peptide. In certain embodiments, IL-2 is linked to the anchoring peptide via a rigid linker. For example, without limitation, the oncolytic virus is the Western Reserve strain of vaccinia virus comprising a nucleic acid encoding IL-2 linked to an anchoring peptide via a rigid linker (e.g., a linker comprising the (A(EA3K)4AAA) linker). In certain embodiments, the oncolytic virus is the vvDD vaccinia virus comprising a nucleic acid encoding IL-2 linked to an anchoring peptide via a rigid linker (e.g., a linker comprising the (A(EA3K)4AAA) linker).

[0115] In certain non-limiting embodiments, prior to T cell transplantation, another cancer therapy disclosed above can be administered to a cancer patient. In certain non-limiting embodiments, after T cell transplantation, another cancer therapy disclosed above can be administered to a cancer patient. For example, but not limited to, cancer therapies can include chemotherapy, biological therapy, radiation therapy, immunotherapy, hormone therapy, anti-vascular therapy, cryotherapy, toxin therapy and / or surgery or combinations thereof. In certain non-limiting embodiments, one or more exogenous cytokines and one or more agents (e.g., immunomodulators) can be administered to a cancer patient prior to T cell transplantation. In certain non-limiting embodiments, one or more exogenous cytokines and one or more agents can be administered to a cancer patient after T cell transplantation. In certain embodiments, the cytokine and / or agent can be administered locally or systemically. In certain embodiments, the cytokine and / or agent is administered locally. For example, but not limited to, a cancer patient can be administered exogenous IL-2 before and / or after T cell transplantation. For example, but not limited to, a cancer patient can be administered exogenous IL-2 after T cell transplantation.

[0116] In certain embodiments, a subject administered with the OV-induced T cells of the subject matter disclosed herein can receive radiation therapy before and / or after administration of the OV-induced T cells. For example, but not limited to, the radiation dose can be about 100 Rad (1 Gy) to about 500 Rad (5 Gy), about 5,000 Rad (50 Gy) to about 100,000 Rad (1000 Gy), or about 50,000 Rad (500 Gy), or other suitable doses within the described ranges. "Gy" can mean the unit for the specific absorbed dose of radiation exposure equal to 100 Rad as used herein. Gy is an abbreviation of "Gray".

[0117] 5.6.2 Treatment Using Armed Oncolytic Virus The subject matter disclosed herein further provides a method of treating a subject afflicted with cancer, comprising administering to the subject an effective amount of a oncolytic virus (e.g., an armed oncolytic virus) as disclosed herein. Non-limiting examples of oncolytic viruses (e.g., armed oncolytic viruses) for use in the methods disclosed herein are disclosed in Sections 5.1, 5.3, and 5.5 above. Methods of administering the oncolytic virus are disclosed in Section 5.4 above.

[0118] In certain embodiments, the oncolytic virus can be a herpes simplex virus, vaccinia virus, adenovirus, or vesicular stomatitis virus that contains in its genome a nucleic acid encoding an immunomodulatory factor molecule as described herein. In certain embodiments, the oncolytic virus can be the Western Reserve strain of vaccinia virus that contains in its genome a nucleic acid encoding an immunomodulatory factor molecule as described herein. In certain embodiments, the oncolytic virus is the vvDD vaccinia virus that contains in its genome a nucleic acid encoding an immunomodulatory factor molecule as described herein. In certain embodiments, the oncolytic virus encodes at least one secreted immunomodulatory factor molecule and / or at least one membrane-bound immunomodulatory factor molecule as described above in an expressible form. In certain embodiments, the immunomodulatory factor molecule can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, IL-27, CXCL11, CCL5, IFN, IFN-α, IFN-α2, IFN-β, IFN-γ, TNF, TNF-α, TNF-β, GM-CSF, or a combination thereof. In certain embodiments, the immunomodulatory factor molecule is IL-2. In certain embodiments, the immunomodulatory factor molecule is IL-23. In certain embodiments, the immunomodulatory factor molecule is TNF-α. In certain embodiments, the immunomodulatory factor molecule is CXC11. In certain embodiments, the immunomodulatory factor molecule is CCL5. In certain embodiments, the immunomodulatory factor molecule is IL-15. In certain embodiments, the oncolytic virus (e.g., vaccinia virus) can contain a nucleic acid encoding IL-2. In certain embodiments, the oncolytic virus is the Western Reserve strain of vaccinia virus that contains a nucleic acid encoding IL-2. In certain embodiments, the oncolytic virus is the vvDD vaccinia virus that contains a nucleic acid encoding IL-2. In certain embodiments, the oncolytic virus (e.g., vaccinia virus) can contain a nucleic acid encoding IL-2 linked to an anchor peptide. In certain embodiments, the tumor The oncolytic virus is a Western Reserve strain of vaccinia virus that contains a nucleic acid encoding IL-2 linked to an anchoring peptide. In certain embodiments, the IL-2 is linked to the anchoring peptide via a rigid linker. For example, without limitation, the oncolytic virus is a Western Reserve strain of vaccinia virus that contains a nucleic acid encoding IL-2 linked to an anchoring peptide via a rigid linker (e.g., a linker containing the (A(EA3K)4AAA) linker). In certain embodiments, the oncolytic virus is a vvDD vaccinia virus that contains a nucleic acid encoding IL-2 linked to an anchoring peptide via a rigid linker (e.g., a linker containing the (A(EA3K)4AAA) linker).

[0119] The subject matter disclosed herein provides a method of treating a subject afflicted with cancer, the method comprising administering to the subject an effective amount of an armed oncolytic virus (e.g., an oncolytic virus encoding an immunomodulatory factor molecule in an expressible form as described above). In certain embodiments, administration of the oncolytic virus results in secretion of the immunomodulatory factor molecule from virus-infected cells. In certain embodiments, the immunomodulatory factor molecule is IL-2.

[0120] The subject matter disclosed herein provides a method of treating a subject afflicted with cancer, the method comprising administering to the subject an effective amount of an oncolytic virus encoding a membrane-bound protein comprising an immunomodulatory factor molecule linked to an anchoring peptide in an expressible form as described above. In certain embodiments, the immunomodulatory factor molecule is IL-2.

[0121] In certain embodiments, the subject matter disclosed herein provides a method of inhibiting the growth and / or proliferation of cancer cells of a subject and / or promoting the death of cancer cells, the method comprising administering to the subject a therapeutically effective amount of the oncolytic vaccinia virus described herein.

[0122] In certain embodiments, the subject matter disclosed herein provides a method of inhibiting the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of the oncolytic vaccinia virus described herein.

[0123] The present invention further provides a method of reducing or inhibiting the growth of a tumor, comprising administering to the tumor and / or contacting the tumor with a therapeutically effective amount of the oncolytic vaccinia virus described herein.

[0124] In certain embodiments, the subject matter disclosed herein provides a method for extending the survival period of a subject having cancer, comprising administering to the subject a therapeutically effective amount of the oncolytic vaccinia virus described herein. In certain embodiments, the survival period of a subject having cancer is extended by about 1 month, about 2 months, about 4 months, about 6 months, about 8 months, about 10 months, about 12 months, about 14 months, about 18 months, about 20 months, about 2 years, about 3 years, about 5 years or more.

[0125] In certain embodiments, the subject matter disclosed herein further provides a method of treating a subject afflicted with cancer, comprising administering to the subject an effective amount of an oncolytic virus encoding an immunomodulatory factor molecule in an expressible form that can be secreted from infected cells, and inducing the generation of potent anti-tumor T cells.

[0126] The subject matter disclosed herein also provides a method of treating a subject afflicted with cancer, comprising administering to the subject an effective amount of an oncolytic virus encoding a membrane-bound protein comprising an immunomodulatory factor molecule linked to a heterologous anchor peptide as described above, and promoting the trafficking of induced potent anti-tumor T cells to tumor tissue where the T cells will exert their cytotoxicity against cancer cells and associated stromal cells, i.e., exhibit an anti-tumor function.

[0127] The subject matter disclosed herein also provides a method of treating a subject afflicted with cancer, comprising administering to the subject an effective amount of an oncolytic virus that can encode in an expressible form a membrane-bound protein comprising an immunomodulatory factor molecule linked to an anchor peptide as described above, inducing the generation of potent anti-tumor T cells, and promoting the trafficking of the induced potent anti-tumor T cells to tumor tissue where the T cells will exert their cytotoxicity against cancer cells and associated stromal cells, i.e., exhibit an anti-tumor function.

[0128] Certain non-limiting embodiments of the subject matter disclosed herein provide a method of treating cancer as described above, wherein the cancer is early-stage cancer. Certain non-limiting embodiments of the subject matter disclosed herein provide a method of treating cancer as described above, wherein the cancer is not early-stage cancer. Certain non-limiting embodiments of the subject matter disclosed herein provide a method of treating cancer as described above, wherein the cancer is late-stage cancer.

[0129] In certain embodiments, the subject matter disclosed herein provides a method of treating cancer when there is an increased tumor burden and / or edema in the liver and / or kidney, and / or an increased presence of immunosuppressive CD4 + Foxp3 + CD4 + PD-1 + and CD8 + PD-1 + T cells, G-MDSC and PD-L1 + cells, and / or a reduced presence of NK cells, or when there is increased expression of PD-1, PD-L1, TGF-β and VEGF in the tumor microenvironment.

[0130] In certain embodiments, administration of an effective amount of an oncolytic virus results in an increase in the levels of IFN-γ, granzyme B, perforin, and TGF-β, IL-10 and / or a decrease in the levels of angiogenesis markers (e.g., CD105 and VEGF) in tumors administered an oncolytic virus encoding a membrane-bound protein capable of expressing an immunomodulatory factor molecule that can be secreted, compared to tumors administered an oncolytic virus encoding a membrane-bound protein comprising an immunomodulatory factor molecule linked to an anchoring peptide as described above.

[0131] In certain embodiments, the subject matter disclosed immediately above provides a method of treating a subject afflicted with cancer, comprising administering to the subject an effective amount of an oncolytic virus as described herein, optionally in combination with a checkpoint inhibitor (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody).

[0132] In certain embodiments, the subject matter disclosed immediately above provides a method of treating a subject afflicted with cancer, comprising administering to the subject an effective amount of an oncolytic virus as described herein, optionally in combination with an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody. For example, but not limited to, the methods disclosed herein may comprise administering to the subject an effective amount of an oncolytic virus comprising a nucleic acid encoding IL-2, in combination with an anti-PD-1 antibody or an anti-PD-L1 antibody. In certain embodiments, the methods disclosed herein may comprise administering to the subject an effective amount of an oncolytic virus comprising a nucleic acid encoding IL-2, in combination with an anti-CTLA-4 antibody. In certain embodiments, a method of treating a subject afflicted with cancer comprises administering to the subject an effective amount of the Western Reserve strain of vaccinia virus comprising a nucleic acid encoding IL-2, in combination with an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody.

[0133] In certain embodiments, the subject matter disclosed herein also provides a method of treating a subject afflicted with cancer, comprising administering to the subject an effective amount of an oncolytic virus capable of expressing a membrane-bound protein comprising an immunomodulatory factor molecule linked to an anchoring peptide as described above, in combination with an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody. For example, without limitation, the methods disclosed herein can comprise administering to the subject an effective amount of an oncolytic virus comprising a nucleic acid encoding a membrane-bound protein comprising IL-2 linked to an anchoring peptide, in combination with an anti-PD-1 antibody or an anti-PD-L1 antibody. In certain embodiments, the methods disclosed herein can comprise administering to the subject an effective amount of an oncolytic virus comprising a nucleic acid encoding a membrane-bound protein comprising IL-2 linked to an anchoring peptide, in combination with an anti-CTLA-4 antibody. In certain embodiments, a method of treating a subject afflicted with cancer can comprise administering to the subject an effective amount of the Western Reserve strain of vaccinia virus comprising a nucleic acid encoding IL-2 linked to an anchoring peptide, in combination with an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody. In certain embodiments, IL-2 is linked to the anchoring peptide via a rigid linker. For example, without limitation, the oncolytic virus is the Western Reserve strain of vaccinia virus comprising a nucleic acid encoding IL-2 linked to an anchoring peptide via a rigid linker (e.g., a linker comprising the (A(EA3K)4AAA) linker).

[0134] In certain embodiments, the methods of the subject matter disclosed herein can comprise depletion of natural killer (NK) cells, CD8+ T cells, and / or CD4+ T cells. In certain embodiments, the methods of the subject matter disclosed herein can comprise neutralization of circulating IFN-γ. For example, without limitation, the methods of the subject matter disclosed herein can comprise administering to the subject an oncolytic virus as disclosed herein, and CD8+ T cells, NK cells and / or CD4 + It can include depletion of T cells and / or neutralization of circulating IFN-γ in the subject's body. In certain embodiments, depletion and / or neutralization can be obtained by using antibodies (e.g., antibodies against CD8, CD4, NK1.1 and / or IFN-γ). In certain embodiments, a method of treating a subject suffering from cancer can include administering to the subject an effective amount of an oncolytic virus comprising a nucleic acid encoding IL-2, and can include deletion of NK cells. For example, but not limited to, the method can include administering to the subject an effective amount of the Western Reserve strain of vaccinia virus comprising a nucleic acid encoding IL-2, and can include deletion of NK cells (e.g., by administration of an anti-NK1.1 antibody). In certain embodiments, a method of treating a subject suffering from cancer can include administering to the subject an effective amount of an oncolytic virus comprising a nucleic acid encoding a membrane-bound protein comprising an immunomodulatory factor linked to an anchor peptide, and can include deletion of NK cells. In certain embodiments, the methods disclosed herein can include administering to the subject an effective amount of an oncolytic virus comprising a nucleic acid encoding a membrane-bound protein comprising IL-2 linked to an anchor peptide, and can include deletion of NK cells. For example, but not limited to, the method can include administering to the subject an effective amount of the Western Reserve strain of vaccinia virus comprising a nucleic acid encoding a membrane-bound protein comprising IL-2 linked to an anchor peptide, and can include deletion of NK cells (e.g., by administration of an anti-NK1.1 antibody). In certain embodiments, the subject is suffering from early-stage cancer. In certain embodiments, the cancer is not early-stage cancer. In certain embodiments, the subject is suffering from late-stage cancer.

[0135] 5.7 Pharmaceutical Composition The present disclosure further provides a pharmaceutical composition comprising one or more isolated OV-induced T cells isolated from cancer tissue.

[0136] The present disclosure further provides a pharmaceutical composition comprising a modified virus disclosed herein. In certain embodiments, a pharmaceutical composition containing a modified virus (such as an oncolytic vaccinia virus) as described herein is prepared as a solution, a dispersion in glycerol, liquid polyethylene glycol, and / or any combination thereof, a solid dosage form, an inhalation dosage form, a nasal administration dosage form, a liposomal formulation, a dosage form containing nanoparticles, a dosage form containing microparticles, a polymeric dosage form, or any combination thereof.

[0137] The pharmaceutical composition is formulated in relation to a particular route of administration. For example, but not limited to, pharmaceutical compositions that can be administered parenterally, intravenously, intradermally, intramuscularly, transdermally, or intraperitoneally are described in U.S. Patent Nos. 5,543,158, 5,641,515, and 5,399,363 (the contents of these documents are incorporated herein by reference in their entirety).

[0138] In certain embodiments, a pharmaceutical composition as described herein can include a pharmaceutically acceptable carrier (e.g., an excipient). "Pharmaceutically acceptable" as used herein includes any carrier that does not interfere with the effectiveness of the biological activity of the active ingredient and / or is not toxic to the patient to whom it is administered. Excipients can be the excipients described in Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986). Non-limiting examples of suitable excipients include buffers, preservatives, stabilizers, binders, compressants, lubricants, chelating agents, dispersion promoters, disintegrants, flavors, sweeteners, and coloring agents.

[0139] In certain embodiments, the excipient can be a buffering agent. Non-limiting examples of suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate. As buffering agents, sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium glucomate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide and other calcium salts or combinations thereof can be used in pharmaceutical formulations.

[0140] 5.8 Kit The present disclosure further provides a kit comprising one or more of the disclosed oncolytic viruses described herein. In embodiments, the present disclosure provides a kit for administering a modified virus as described herein. In certain embodiments, the kit of the present disclosure can comprise a modified virus or a pharmaceutical composition comprising a modified virus as described above. In embodiments, the present disclosure provides a kit comprising one or more OV-induced T cells. In certain embodiments, the kit of the present disclosure can further comprise one or more components such as instructions for use, devices and additional reagents, and components such as test tubes, containers and syringes for performing the methods disclosed above.

[0141] In certain embodiments, the kits of the present disclosure can include instructions for use, a device for administering the modified virus to a subject, or a device for administering an additional agent or compound to a subject. For example, without limitation, the instructions can include the modified virus, and optionally, other components included in the kit, methods for determining the appropriate condition of the subject, appropriate dosages, and methods of administration, including the appropriate method of administering the modified virus. The instructions can also include guidance for monitoring the subject over the duration of the treatment.

[0142] In certain embodiments, the kits of the present disclosure can include a device for administering the modified virus to a subject. Any of a variety of devices known in the art for administering pharmaceuticals and pharmaceutical compositions can be included in the kits provided herein. For example, without limitation, such devices include hypodermic needles, intravenous needles, catheters, needleless injection devices, inhalers, and liquid dispensers (such as eye droppers). In certain embodiments, for example, a modified virus intended to be delivered systemically by intravenous infusion can be included in the kit with a hypodermic needle and syringe.

[0143] In certain embodiments, the present disclosure provides a kit for isolating tumor-infiltrating T cells induced by oncolytic virus ( "OV-induced T cells") from a subject with cancer after administration of a modified virus as described herein. The present disclosure further provides a kit comprising isolated OV-induced T cells expanded according to the methods described herein. In certain embodiments, the OV-induced T cells are cryopreserved. Alternatively, and / or in addition thereto, the OV-induced T cells are provided in a culture medium. In certain embodiments, the kit can provide isolated T cells and one or more cytokines and / or one or more agents (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, IL-27, IFN-α, IFN-α2, IFN-β, or IFN-γ, TNF-α, TNF-β, and GM-CSF) for treating dendritic cells and cancer cells and / or T cells.

[0144] In certain embodiments, the kits of the present disclosure can include one or more additional agents that can be administered in combination with the oncolytic virus and / or the isolated OV-induced T cells. For example, but not limited to, the kit can include cytokines (e.g., IL-2), and / or anti-PD-1 antibodies and / or anti-PD-L1 antibodies.

[0145] In certain embodiments, the kits of the present disclosure can provide a device for isolating T cells from a subject after administering a modified virus to the subject, or a device for transplanting processed T cells into a subject. For example, without limitation, the kits of the present disclosure can include instructions for the method of isolating OV-induced T cells, as well as methods for determining the appropriate condition of the subject, appropriate dosage, and appropriate administration methods for transplanting the processed T cells into the patient, including the appropriate administration method for transplanting the processed T cells into the patient. In certain embodiments, the kits of the present disclosure can include instructions for use, a device for administering a modified virus to a subject, and / or a device for administering an additional agent or compound to a subject, and / or a device for isolating OV-induced T cells. For example, without limitation, the instructions can include descriptions of the modified virus, and optionally other components included in the kit, methods for determining the appropriate condition of the subject, appropriate dosage, and appropriate administration methods for administering the modified virus, including the appropriate administration method for administering the modified virus. The instructions can also include descriptions of the steps for isolating OV-induced T cells, methods for processing the isolated T cells, and methods for transplanting the processed T cells into the patient. The instructions can also include guidance for monitoring the subject over the duration of the treatment.

Example

[0146] 6. Example 1: Oncolytic Vaccinia Virus Expressing Membrane-Bound IL-2 Exhibited Potent Antitumor Efficacy and Reduced Toxicity The virus "vvDD-mIL-2" was generated by genetically engineering vvDD (or VVDD), a tumor-lytic vaccinia virus as described in U.S. Patent No. 7,208,313 (the disclosure of which is incorporated herein by reference in its entirety), to express murine IL-2 ("mIL-2") by inserting an mIL-2-encoding nucleic acid under the control of the vaccinia p7.5e / l promoter into the viral tk gene, resulting in secretion of mIL-2 from infected cells. The antitumor efficacy of this IL-2 armed virus was tested in mouse models of colon cancer, ovarian cancer, and mesothelioma generated by inoculating mice with MC38-luc, ID8-luc, or AB12-luc tumor cells, respectively, which were labeled with luciferase ("luc") to enable monitoring of tumor development. Using the experimental scheme shown in Figure 1A, 5.0e5 tumor cells were inoculated subcutaneously on day 0, and then on day 5, mice were administered either PBS or 1.0e8 pfu of vvDD-IL-2 or, as a control, vvDD not armed with mIL-2. The number of mice in each test group was 10 ± 4. Mouse survival was monitored, and the results are shown in Figures 1B - D; in each case, vvDD-mIL-2 substantially extended the survival period compared to the control.

[0147] However, when tumors were allowed to progress in the mouse body prior to virus treatment, the survival of vvDD-mIL-2-treated mice was inferior to that of the control. As shown in Figure 2A, when the mIL-2 armed virus was administered 9 days after inoculation with MC38-luc (colon cancer) tumor cells, recipient mice died within one week (Figure 2B). Since the tumor burden in these mice would be equivalent to that of control mice in this study, it is speculated that the animals died due to the toxic effects of the secreted IL-2.

[0148] For the purpose of reducing the possible toxic effects of IL-2, a tumor-lytic vaccinia virus was genetically engineered to express membrane-anchored IL-2 in infected tumor cells. Figure 3A shows a schematic diagram of a part of the genetically engineered vvDD virus, in which the nucleic acid encoding the (G4S)3 linker having the sequence GPAGGGGSGGGGSGGGGSVSTISSFSPPGYQVSFCLVMVLLFAVDTGLYFSVKTNI (SEQ ID NO: 16) and the GPI-anchor peptide, all functionally linked to the p7.5e / l promoter, is fused in-frame to the mIL-2-encoding nucleic acid inserted into the tk gene, and a virus designated "vvDD-mIL-2-GPI" is produced. For the purpose of detecting virus-infected cells, a gene encoding yellow fluorescent protein ("YFP") that is functionally linked to the pSe / l promoter and transcribed in the direction opposite to that of mIL-2 also disrupts the tk gene. When tested in the MC38 colon cancer cell line and the B16 melanoma cell line, as demonstrated by flow cytometry, vvDD-mIL-2-GPI expressed mIL-2 that was mostly retained on the membrane of infected cells, while the mIL-2 expressed by vvDD-mIL-2 was not retained on the membrane (Figures 4A - B). Virus-infected cells were gated by YFP, and subsequently, the IL-2 expression on the gated YFP+ cells was analyzed. Figures 5A - B represent the expression intensity of IL-2 on the cell membrane. These data clearly demonstrated that vvDD-mIL-2-GPI expressed much more membrane-anchored IL-2 on infected cancer cells.

[0149] Figure 6 shows the results of an experiment comparing the "disarmed" vvDD (parent virus) with vvDD-mIL-2 and vvDD-mIL-2-GPI. Mice were administered either PBS or (disarmed) vvDD, vvDD-mIL-2, or vvDD-mIL-2-GPI (at a dose of 1.0e8 pfu) five days after tumor cell inoculation. As shown in Figure 6, PBS control-treated mice died within approximately 20 days after tumor cell inoculation, and the majority of mice treated with disarmed vvDD died within approximately 30 days of tumor inoculation (all mice in this group died by day 43), but after 50 days, all mice treated with vvDD-mIL-2 survived, and more than 80% of mice treated with vvDD-mIL-2-GPI survived, indicating that the "anchor-type" IL-2 retains beneficial IL-2 function (n = 8).

[0150] Figure 7 compares the IL-2-related toxic effects in mice treated with either 2.0e8 pfu (2.0×10 8 pfu) of vvDD-mIL-2 or vvDD-mIL-2-GPI nine days after inoculation with MC38-luc cells. Five days after virus treatment, more than 80% of the mice treated with vvDD-mIL-2 died, while all vvDD-mIL-2-GPI-treated mice survived (n = 13 - 15).

[0151] 7. Example 2: Adoptive Transfer of Oncolytic Virus-Induced Tumor-Infiltrating T Cells (「OV-Induced T Cells」) Resulted in a Marked Therapeutic Effect in Syngeneic C57BL / 6 Mice Bearing Intraperitoneal MC38 Tumors In this example, a study of oncolytic vaccinia viruses expressing membrane-bound immunostimulatory cytokines is provided.

[0152] 5×10 5 individual MC38-luc cancer cells were intraperitoneally inoculated into B6 mice, and based on in vivo animal IVIS imaging seven days after tumor cell injection, the mice were divided into groups as required according to tumor growth conditions. The grouped mice were given 5×10 6Individual T cells or 100 μL of PBS as a control were injected intraperitoneally. T cells were generated by intratumoral injection of vvDD-IL-2-GPI (1.0e8 pfu) into MC38 subcutaneous tumor-bearing mice. Ten days after virus treatment, tumors were harvested and T cells were isolated using CD90.2 beads (Miltenyl Biotec, CA, USA). T cells were co-cultured with αDC-MC38 for 2 days. On day 2, IL-2 (4 ng / mL) and IL-7 (5 ng / mL) or additionally a GSK3b inhibitor (7 μM) were added. On day 5, T cells were transplanted into MC38-luc tumor-bearing mice by intraperitoneal (i.p.) injection. Prior to T cell transplantation, treatment mice were irradiated with a sub-lethal dose of 5 Gy to mimic lymphodepletion similar to the clinical protocol. All treatment mice were given exogenous cytokine assistance with IL-2 (100,000 IU / mouse, intraperitoneal, every 12 hours for 3 days). Figure 8A shows the timeline of the experimental setup.

[0153] As shown in Figure 8B, when OV-induced T cells treated ex vivo with IL-2, IL-7, and a GSK3b inhibitor were administered, 100% of the tumor-bearing mice survived for at least 100 days after inoculation. When OV-induced T cells treated ex vivo with IL-2 and IL-7 were administered, approximately 50% of the tumor-bearing mice survived for at least 100 days after inoculation. Bioluminescence images of mice with MC38-luc tumors on day 21 after treatment are shown in Figure 8C, and images on day 28 after treatment are shown in Figure 8D. As shown in Figure 8, oncolytic vaccinia viruses expressing novel forms of immunostimulatory cytokines (such as membrane-bound forms) can induce potent tumor-specific T cells and promote the transport of such T cells into tumor tissue where they can exert their function against tumor growth in situ. In addition, OV-induced T cells can be isolated and expanded ex vivo under appropriate conditions. When these cultured and expanded tumor-specific T cells were reinjected into second tumor-bearing mice, the T cells showed potent antitumor activity in the syngeneic mouse MC38 colon tumor model. This approach demonstrated that antitumor T cells generated and enriched by oncolytic viruses can be used for cancer therapy.

[0154] 8. Example 3: Presentation of Rigid Membrane-Bound Interleukin-2 with Potent Efficacy and High Safety 8.1. Introduction Interleukin-2 (IL-2), a multifunctional cytokine, is an established cancer therapeutic agent, but its clinical application is limited due to severe life-threatening side effects that follow the systemic use of high doses of IL-2. 1-4 Considerable efforts have been devoted to developing IL-2 fusion proteins, IL-2 / anti-IL-2 antibody complexes, and "supercytokines," or IL-2 variants such as chemically modified IL-2, to extend the in vivo half-life and improve biological activity and safety. 5-13。This example discloses a new form of IL-2 immunotherapy via local delivery of cell membrane-bound IL-2 in the tumor bed by a tumor-targeted oncolytic vaccinia virus. Presenting IL-2 on the cell membrane, either with flexibility or rigidity, cured mice with established early peritoneal colon cancer, which was similar to the effect of delivery of secreted IL-2 by vaccinia virus. While secreted IL-2 delivered by vaccinia virus resulted in high lethality in a late-stage tumor model during the viral replication period, presenting IL-2 on the cell membrane with rigidity significantly increased the survival rate of mice with late-stage peritoneal colon cancer. Presenting IL-2 on the cell membrane with rigidity brought about a major change in the immune state of the tumor microenvironment, which enabled the combination of anti-PD-1 / PD-L1 Abs to cure the majority of mice with late-stage peritoneal colon cancer. These findings indicate that the new form of IL-2 immunotherapy may be clinically applicable for cancer treatment.

[0155] Virus-delivered secreted IL-2 has been suggested to have the ability to treat established tumors in a mouse model (Figure 1). 14 。To reduce the severe toxic side effects caused by systemic use of high doses of IL-2 and to treat pleural, peritoneal, hematopoietic, or metastatic cancers, oncolytic vaccinia virus vvDD was used. Treating pleural, peritoneal, hematopoietic, or metastatic cancers by intratumoral virus injection can be difficult. Since membrane-bound cytokines have been suggested to be retained on the membrane without apparently defective cytokine function 15-17 ,the oncolytic vaccinia virus, which has been recognized as safe, was used to deliver membrane-bound IL-2 for the purpose of treating peritoneal colon cancer. The approach using oncolytic vaccinia virus vvDD can result in the use of fewer viruses and may cause fewer side effects.

[0156] 8.2 Methods Mice and Cell Lines: Female C57BL / 6 (abbreviated as B6) mice and BALB / c mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed under specific pathogen-free conditions in the animal facility of the University of Pittsburgh. All animal studies were approved by the facility's Animal Care and Use Committee. Mouse colon cancer MC38-luc, ovarian cancer ID8-luc, and mesothelioma AB12-luc were generated as previously described. 31 . Mouse melanoma B16 was obtained from ATCC. All cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and penicillin / streptomycin (Invitrogen, Carlsbad, CA) in a 5% CO2 incubator at 37 °C.

[0157] Virus Production : The vSC20, vgf gene-deleted Western Reserve (WR) strain VV was used as the parental virus for homologous recombination. The plasmid pCMS1-IRES, which has two multiple cloning sites separated by the IRES sequence from pLVX-IRES-ZsGreen, was constructed from the shuttle plasmid pSEM-1. 32 Next, a fragment containing the GPI anchor sequence amplified from human CD16b by flexible linker or rigid linker + PCR was inserted into pCMS1-IRES, thereby obtaining plasmids pCMS1-IRES-FG or pCMS1-IRES-RG, respectively. Mouse IL-2 cDNA was inserted into pCMS1-IRES, pCMS1-IRES-FG, or pCMS1-IRES-RG to obtain shuttle plasmids pCMS1-IL-2, pCMS1-IL-2-FG, or pCMS1-IL-2-RG. The GPI anchor sequence in pCMS1-IL-2-FG was further replaced with the mouse PD-L1 transmembrane domain to obtain the shuttle plasmid pCMS1-IL-2-FPTM. All these shuttle vectors were used for homologous recombination of mouse IL-2 variants into the tk locus of the vaccinia virus genome. Primers for plasmid cloning based on PCR are listed in Table 2.

[0158]

Table 2

[0159] To generate the novel viruses vvDD-IL-2, vvDD-IL-2-FG, vvDD-IL-2-RG and vvDD-IL-2-FPTM, CV-1 cells were infected with vSC20 at a multiplicity of infection (MOI) of 0.1, followed by transfection with a shuttle plasmid to generate virus seeds. Selection of the novel recombinant viruses was based on the expression of yellow fluorescent protein in CV1 cells 24 hours after seed virus infection. vvDD-YFP, or vvDD for short (a double-virus gene deletion type (tk- and vgf-) VV carrying yfp cDNA at the tk locus), is the control virus in this study.

[0160] In Vitro Virus Replication Assay : Tumor cells were seeded at 1.0×10 5 cells / well in a 6-well plate, and the next day, were infected for 2 hours at an MOI of 0.1, 1.0, or 10 with the indicated virus in 1 mL of medium containing 2% fetal bovine serum. After infection, 3 mL of medium containing 10% fetal bovine serum was added to the cells, and the cells were cultured until harvested 24 hours, 48 hours, and 72 hours after virus infection. The cell pellet was homogenized using a FastPrep Cell Disrupter (model number FP120; Qbiogene, Carlsbad, CA) to release virions, and the resulting cell lysate was titrated using CV-1 cells and the virus amount was measured by plaque assay.

[0161] In Vitro MTS Cytotoxicity Assay : Tumor cells were seeded at 1.0×10 4Cells / wells were seeded and infected with the indicated viruses at MOI 0.05, 0.1, 0.5, 1.0, and 5.0 the next day. Cell viability was determined 48 and 72 hours post-infection by the CellTiter 96 Aqueous Nonradioactive cell proliferation assay, or MTS assay (Promega, Madison, WI).

[0162] Virus-Delivered IL-2 Expression In Vitro : MC38-luc (3×10 5 ) cells, B16 (2×10 5 ) cells or AB12-luc (3×10 5 ) cells were seeded in 24-well plates overnight and infected with vvDD, vvDD-IL-2, vvDD-IL-2-FG, vvDD-IL-2-RG or vvDD-IL-2-FPTM at MOI = 1 in 0.15 mL of DMEM containing 2% FBS for 2 hours. 0.35 mL of DMEM containing 10% FBS was added to the cells and cultured until harvested 24 hours post-viral infection. Culture supernatants were harvested to measure IL-2 by ELISA (BD Bioscience, San Jose, CA), and cell pellets were used to extract RNA for measuring membrane-bound IL-2 by flow cytometry or IL-2 expression by RT-qPCR.

[0163] Rodent Tumor Model : Separately, 5×10 5 MC38-luc cancer cells, 3.5×10 6 ID8-luc cancer cells were intraperitoneally inoculated into B6 mice, or 4×10 5Individual AB12-luc cells were inoculated intraperitoneally, and according to the tumor size based on in vivo animal IVIS imaging performed using the Xenogen IVIS 200 Optical In Vivo Imaging System (Caliper Life Sciences, Hopkinton, MA), they were divided into the necessary groups 5 or 9 days after tumor cell inoculation. The indicated virus, antibody, combination, or PBS was injected intraperitoneally into the grouped mice, respectively. In some experiments, α-CD8 Ab (250 μg / injection; clone 53-6.7; Bio X Cell), α-CD4 Ab (clone GK1.5, Bio X Cell; 150 μg / injection), α-NK1.1 (clone PK136, Bio X Cell; 300 μg / injection), or α-IFN-γ Ab (clone XMG1.2, Bio X Cell; 200 μg / injection) was injected intraperitoneally into the mice to deplete CD8 + T cells, CD4 + T cells, NK1.1 + cells or neutralize circulating IFN-γ. In some experiments, the mice were sacrificed to collect tumor tissue and spleen at the indicated time points.

[0164] MC38-luc tumor-bearing B6 mice that were treated with the indicated vaccinia virus and survived for more than 60 days were re-inoculated subcutaneously with 5×10 5 individual MC38-luc cells / mouse. Naïve B6 mice were inoculated with the same dose of tumor as a control. The primary tumor size was measured using vernier calipers at two vertical diameters.

[0165] Evaluation of Treatment-Related ToxicityVirus-treated mice were sacrificed 4 - 5 days after treatment for the collection of blood, lungs, kidneys, and spleens. Blood samples were maintained at room temperature for 2 hours and serum was separated by centrifugation to measure IL-2 and TNF-α using commercially available kits (BD Biosciences and BioLegend, respectively) according to the vendor's instructions. Tissue edema was monitored using water content. Briefly, the weight of the wet tissue was measured and dehydrated overnight at 90 °C in a chemical hood. The weight difference between the wet and dry tissues was calculated.

[0166] Flow Cytometry: The weight of the harvested tumor tissue was measured and incubated at 37°C for 1 - 2 hours in RPMI 1640 medium containing 2% FBS, 1 mg / mL collagenase, 0.1 mg hyaluronidase, and 200 U DNase I (all enzymes were obtained from Sigma, St. Louis, MO) to prepare single cells. Single cells derived from in vitro virus-infected cells or tumor tissues were blocked with α-CD16 / 32 Ab (clone 93), and then stained with antibodies against mouse CD45 (APC or PerCP-Cy5.5, clone: 30-F11), CD11b (PE, clone: M1 / 70), Ly6G (APC, clone: 1A8), Ly6C (clone: HK1.4), F4 / 80 (FITC, clone: BM8, e-Bioscience), CD4 (APC or FITC, clone: RM4-5, BD Biosciences), Foxp3 (PE, clone: FJK-16s, e-Bioscience), CD8 (APC or PE, clone: 53-6.7), CD44 (FITC, clone: IM7), PD-1 (PE, clone: J43, e-Bioscience), IFN-γ (APC, clone: XMG1.2, e-Bioscience), CD3 (FITC, clone: 17A2, eBioscience), NK1.1 (PE, clone: PK136), PD-L1 (APC, clone: 10F.9G2), IL-2 (APC-SA + biotin-IL-2, clone: JES6-5H4). All antibodies without specified suppliers were purchased from BioLegend. Samples were collected using a BD Accuri C6 cytometer and data were analyzed using BD Accuri C6 cytometer software.

[0167] RT-qPCR: Total RNA was extracted from virus-infected cells or tumor tissues using the RNeasy kit (Qiagen, Valencia, CA). One microgram of RNA was used for cDNA synthesis, and mRNA expression analysis was performed by TaqMan analysis on a StepOnePlus system (Life Technologies, Grand Island, NY) using 25 - 50 ng of the resulting cDNA. All primers for the analysis were purchased from Thermo Fisher Scientific (Waltham, MA). Gene expression was normalized to the housekeeping gene HPRT1 and represented as the fold change (2 -ΔCT ), where ΔCT = CT (標的遺伝子) - CT (HPRT1) .

[0168] Statistics : Statistical analysis was performed using the Student's t-test (GraphPad Prism version 7). Animal survival rates were represented using Kaplan - Meyer survival curves and statistically analyzed using the log-rank test (GraphPad Prism version 7). Values of P < 0.05 were considered statistically significant, and all P-values were two-sided. In the figures, standard symbols were used: * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001.

[0169] 8.3 Results Viruses vvDD-IL-2, vvDD-IL-2-FPTM, vvDD-IL-2-FG, and vvDD-IL-2-RG were generated based on vvDD (Figure 9). Briefly, vvDD-IL-2 produced secreted mouse IL-2, and vvDD-IL-2-FPTM, vvDD-IL-2-FG, and vvDD-IL-2-RG presented mouse IL-2 on the cell membrane after infection. vvDD-IL-2-FPTM produced IL-2 fused with the mouse PD-L1 transmembrane domain and the flexible linker (G4S)3 in between. vvDD-IL-2-FG and vvDD-IL-2-RG produced IL-2 fused with the glycosylinositol lipid (GPI) anchor sequence of human CD16b and the flexible linker (G4S)3 and the rigid linker (A(EA3K)4AAA) in between, respectively. These four viruses had similar replication ability and cytotoxicity in tumor cells compared to the parental virus vvDD (Figure 10). vvDD-IL-2 produced significantly more IL-2 in the supernatant than the other viruses but did not produce membrane-bound IL-2. vvDD-IL-2-FPTM, vvDD-IL-2-FG, and vvDD-IL-2-RG produced only a small amount of IL-2 in the supernatant but produced significantly more membrane-bound IL-2 than VVDD-IL-2. The amount of membrane-bound IL-2 in vvDD-IL-2-RG was more than that in vvDD-IL-2-FG, and the amount of membrane-bound IL-2 in vvDD-IL-2-FG was more than that in vvDD-IL-2-FPTM (Figure 11A).

[0170] Next, the mRNA levels of IL-2 and the viral marker gene A34R were determined in tumor cells after in vitro virus infection. The data showed that the viral gene A34R mRNA was similar, but the IL-2 mRNA pattern was similar to the pattern of the above-mentioned amount of membrane-bound IL-2. Without being bound to a particular theory, this indicates that the exact components of the chimeric protein can affect mRNA stability and further affect the amount of IL-2 presented on the cell membrane (Figure 12).

[0171] To evaluate the antitumor efficacy of four types of viruses, each virus was intraperitoneally injected at a dose of 2×10 8 PFU / mouse to treat B6 mice (early tumor model) that had received MC38-luc mouse colon cancer cell inoculation 5 days earlier. Survival results showed that vvDD-IL-2, vvDD-IL-2-FG, and vvDD-IL-2-RG produced stronger antitumor effects compared to PBS or vvDD, while vvDD-IL-2-FPTM did not (Figure 11B). Without being bound by a specific theory, this can be explained by the low amount of IL-2 presented on the cell membrane after vvDD-IL-2-FPTM infection (Figures 11A and 12). Mice treated with vvDD-IL-2 survived longer than those treated with vvDD-IL-2-FG, but not longer than those treated with vvDD-IL-2-RG (Figure 11B).

[0172] All surviving mice treated with vvDD-IL-2, vvDD-IL-2-FG, and vvDD-IL-2-RG rejected subcutaneous tumor re-inoculation, indicating that a systemic antitumor response had occurred (Figure 13). Within 1 week after vvDD-IL-2 treatment, several mice died (Figure 11B). Without being bound by a specific theory, this could be a sign of toxicity induced by IL-2. To investigate whether the secreted IL-2 produced by vvDD-IL-2 treatment can induce a certain degree of systemic toxicity and whether it can be more toxic to mice with a large tumor burden, using the same virus dose, the safety and antitumor efficacy of the virus were evaluated in a 5-day tumor-bearing mouse model (early tumor model) and a more immunosuppressive tumor model (mice had a larger tumor burden, more immunosuppressive CD4 + Foxp3 + 、CD4 + PD-1 + and CD8 + PD-1 + T cells, G-MDSC, and PD-L1 +Evaluated by switching to a 9-day tumor-bearing mouse model (late tumor model) with cells, fewer NK cells, and higher PD-1, PD-L1, TGF-β, and VEGF expression in the tumor microenvironment (Figures 14 and 15). Late tumor mice also have increased severe edema in the liver and kidneys compared to the early tumor model (Figure 15). Treatment results from the late tumor model showed that vvDD-IL-2 treatment led to high mortality within 1 week after treatment, while treatment with other viruses was shown to be safe. vvDD-IL-2-FG and vvDD-IL-2-RG treatments significantly extended the survival period of the animals compared to vvDD treatment, and vvDD-IL-2-RG treatment resulted in a significantly better survival period (Figures 11C and 16A).

[0173] To further investigate virus-induced toxicity, IL-2 serum levels were measured and found to be 100-fold higher in the serum of mice treated with vvDD-IL-2, reaching approximately 24950 pg / mL, compared to the serum of mice treated with other viruses (Figure 16B). Low levels of IL-2 were also detected in the serum of mice treated with vvDD-IL-2-FG and vvDD-IL-2-RG. Without being bound to any particular theory, this can be explained by the relatively loose binding of GPI-anchored proteins to the cell membrane compared to transmembrane proteins, and that GPI-anchored proteins may be spontaneously released from the cell membrane due to shedding or proteolytic cleavage, and that free GPI-anchored proteins may also translocate to the cell membrane via a process termed "cell surface painting". 18 It is thought that it may be able to migrate to the cell membrane.

[0174] IL-2 can induce an increase in TNF-α serum levels 19 To examine whether, TNF-α serum levels were measured after vvDD-IL-2 treatment, and it was found that vvDD-IL-2 treatment induced a significant increase in TNF-α serum levels (Figure 16C). Mice also had vascular leak syndrome induced by IL-2 20Tissue edema, an indicator for measurement, was also evaluated. Only vvDD-IL-2 treatment induced substantially more pulmonary and hepatic edema, as evidenced by increased water content in the lung and liver (Figures 16D-16E). Tissue edema was also found. In addition, elevated serum IL-2 levels were found in vvDD-IL-2-treated mice even in the early tumor model (Figures 17A-17D). Taken together, these data indicated that vvDD-IL-2-FG treatment and vvDD-IL-2-RG treatment were safer than vvDD-IL-2.

[0175] Treatment with vvDD-IL-2-FG and vvDD-IL-2-RG had similar therapeutic efficacy in the early tumor model. However, although both treatments had a similar safety profile, only vvDD-IL-2-RG treatment resulted in a substantially better survival period in the late tumor model. To explore the reason for this result, the immune cell profiles in the tumor microenvironment and spleen were examined using the late tumor model. The proportion (%) of activated CD4 + Foxp3 - T cells and CD8 + IFN-γ + T cells was higher in tumors treated with vvDD-IL-2-RG compared to tumors treated with other viral treatments (Figures 18A and 18B). To examine whether memory CD8 + T cells and NK cells could readily respond to IL-2 21 , memory CD8 + CD44 hi T cells and CD3 - NK1.1 + cells were examined. The proportion (%) of both types of cells from tumors after viral treatment had the same pattern as the above-activated T cells. Similar results were observed even in the spleen (Figures 18C and 19A-19C).

[0176] Furthermore, the presence of CD4 + Foxp3 + T cells in tumors treated with the virus was examined. The presence of CD4 +The proportion (%) of T regulatory cells was the same as above (Figure 18D). That is, in tumors after vvDD-IL-2-RG treatment, CD8 + T cells were increased compared to tumors treated with other virus treatments, so the CD8 + / Treg ratio in tumors treated with vvDD-IL-2-RG was significantly higher (Figure 18E and Figure 19D).

[0177] Next, the expression of tumor promoting factors and anti-tumor factors was examined in tumors recovered from the late stage tumor model after virus treatment. Compared to tumors treated with other virus treatments, in tumors treated with vvDD-IL-2-RG, the expression of IFN-γ, granzyme B, and perforin increased, and the expression of TGF-β and the angiogenesis markers CD105 and VEGF decreased (Figure 18F - 18K). The expression of IL-10 was also found to be significantly increased in tumors treated with vvDD-IL-2-RG (Figure 18L). Without being bound by a particular theory, although IL-10 can function as both an immune stimulator and an immunosuppressor in cancer, here, IL-10 can be increased by IL-2 and has an inhibitory effect on the expression of TNF-α, which has recently been suggested to be a tumor growth factor for minimal residual tumors, thereby making the vvDD-IL-2-RG treatment safer and more effective (Figure 16C) 19,22-24 。

[0178] To examine whether the anti-tumor effect induced by vvDD-IL-2-RG treatment is IFN-γ and CD8 + T cell-dependent or CD4 + T cell-dependent, IFN-γ, CD4 + and CD8 + T cells as well as NK1.1 + cells were depleted with antibodies after vvDD-IL-2-RG treatment. As a result, the anti-tumor effect induced by vvDD-IL-2-RG treatment is IFN-γ and CD8 + T cell-dependent and CD4 +It was demonstrated that it was not T cell-dependent (Figure 18M). The results also demonstrated that NK cell depletion led to significantly better survival rates compared to vvDD-IL-2-RG treatment alone. Without being bound by a particular theory, the reason may be that NK cells could interfere with viral therapy via the natural cytotoxic receptor NKP46. The expression of NKP46 was significantly increased by vvDD-IL-2-RG treatment, that is, NK cell depletion may have removed this interference (Figure 20). 25 。

[0179] Collectively, these data demonstrated that vvDD-IL-2-RG treatment changed the immune state in tumor-bearing mice from immunosuppressive to immune-favorable, and this ultimately led to better survival rates.

[0180] The combination of oncolytic vaccinia virus and anti-PD-L1 antibody has previously been suggested to act synergistically to enhance the therapeutic efficacy against early tumor models. 26 However, the combination of vvDD and anti-PD-L1 antibody did not function in late tumor models (Figure 21). Both vvDD-IL-2-FG treatment and vvDD-IL-2-RG treatment produced antitumor effects against late tumor models and induced high PD-1, PD-L1 and CTLA-4 expression in tumors (Figure 11C, Figures 22A - 22C, and Figures 23A - 23C). Therefore, to investigate whether the combination could treat tumor-bearing mice for 9 days, the combinations of vvDD-IL-2-FG and vvDD-IL-2-RG with anti-PD-1 / PD-L1 antibody or anti-CTLA-4 antibody were tested. The results showed that vvDD-IL-2-RG combined with anti-PD-1 / PD-L1 antibody cured tumor-bearing mice, while the combination with anti-CTLA-4 antibody did not (Figure 22D). Without being bound by a particular theory, this may be due to different mechanisms for anti-CTLA-4 and anti-PD-1 / PD-L1 checkpoint blockade. Anti-CTLA-4 antibody mainly acts on CD4 in the priming phase +It acts on T cells, but anti-PD-1 / PD-L1 antibodies preferentially act on exhausted T cells within tumors, which may be important for overcoming the more immunosuppressive microenvironment in late-stage large solid tumors. The combination of vvDD-IL-2-FG and anti-PD-1 / PD-L1 antibodies did not improve survival. Without being bound to a particular theory, this suggests that changes in the immune state in tumor-bearing mice, particularly late-stage tumor-bearing mice, are essential for the efficacy of monotherapy and combination therapies. This will be borne out by several recent reports of the efficacy of oncolytic viruses in combination with immune checkpoint blockade in preclinical models and clinical trials (Figures 22D and 24). 27-30 。

[0181] In summary, this example demonstrated that vvDD-IL-2-RG treatment could significantly reduce the systemic toxicity induced by IL-2 utilization, effectively cause changes in the anti-tumor immune state in late-stage tumor-bearing mice, and ultimately result in much better survival. The combination of vvDD-IL-2-RG with anti-PD-1 and PD-L1 antibodies could cure mice with late-stage tumors. That is, this example showed that vvDD-IL-2-RG treatment could be a new form of IL-2 immunotherapy that may be clinically applicable to cancer and immunosuppressive cancers.

[0182] 8.4 References 1. Boyman, O. & Sprent, J. The role of interleukin-2 during homeostasis and activation of the immune system. Nat Rev Immunol 12, 180-190, doi:10.1038 / nri3156 (2012). 2. Liao, W., Lin, J. X. & Leonard, W. J. Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy. Immunity 38, 13-25, doi:10.1016 / j.immuni.2013.01.004 (2013). 3. Lotze, M. T. et al. In vivo administration of purified human interleukin 2. II. Half life, immunologic effects, and expansion of peripheral lymphoid cells in vivo with recombinant IL 2. J Immunol 135, 2865-2875 (1985). 4. Yang, J. C. et al. Randomized study of high-dose and low-dose interleukin-2 in patients with metastatic renal cancer. J Clin Oncol 21, 3127-3132, doi:10.1200 / JCO.2003.02.122 (2003). 5. Hu, P., Mizokami, M., Ruoff, G., Khawli, L. A. & Epstein, A. L. Generation of low-toxicity interleukin-2 fusion proteins devoid of vasopermeability activity. Blood 101, 4853-4861, doi:10.1182 / blood-2002-10-3089 (2003). 6. Melder, R. J. et al. Pharmacokinetics and in vitro and in vivo anti-tumor response of an interleukin-2-human serum albumin fusion protein in mice. Cancer Immunol Immunother 54, 535-547, doi:10.1007 / s00262-004-0624-7 (2005). 7. Puskas, J. et al. Development of an attenuated interleukin-2 fusion protein that can be activated by tumour-expressed proteases. Immunology 133, 206-220, doi:10.1111 / j.1365-2567.2011.03428.x (2011). 8. Vazquez-Lombardi, R. et al. Potent antitumour activity of interleukin-2-Fc fusion proteins requires Fc-mediated depletion of regulatory T-cells. Nat Commun 8, 15373, doi:10.1038 / ncomms15373 (2017). 9. Lazear, E. et al. Targeting of IL-2 to cytotoxic lymphocytes as an improved method of cytokine-driven immunotherapy. Oncoimmunology 6, e1265721, doi:10.1080 / 2162402X.2016.1265721 (2017). 10. Boyman, O., Surh, C. D. & Sprent, J. Potential use of IL-2 / anti-IL-2 antibody immune complexes for the treatment of cancer and autoimmune disease. Expert Opin Biol Ther 6, 1323-1331, doi:10.1517 / 14712598.6.12.1323 (2006). 11. Letourneau, S. et al. IL-2 / anti-IL-2 antibody complexes show strong biological activity by avoiding interaction with IL-2 receptor alpha subunit CD25. Proc Natl Acad Sci U S A 107, 2171-2176, doi:10.1073 / pnas.0909384107 (2010). 12. Levin, A. M. et al. Exploiting a natural conformational switch to engineer an interleukin-2 'superkine'. Nature 484, 529-533, doi:10.1038 / nature10975 (2012). 13. Katre, N. V., Knauf, M. J. & Laird, W. J. Chemical modification of recombinant interleukin 2 by polyethylene glycol increases its potency in the murine Meth A sarcoma model. Proc Natl Acad Sci U S A 84, 1487-1491 (1987). 14. Qin, H. et al. Gene therapy for head and neck cancer using vaccinia virus expressing IL-2 in a murine model, with evidence of immune suppression. Mol Ther 4, 551-558, doi:10.1006 / mthe.2001.0493 (2001). 15. Zeh, H. J. et al. First-in-human study of Western Reserve Strain oncolytic vaccinia virus: safety, systemic spread ad anti-tumor activity. Mol Ther (In press) (2014). 16. Pan, W. Y. et al. Cancer immunotherapy using a membrane-bound interleukin-12 with B7-1 transmembrane and cytoplasmic domains. Mol Ther 20, 927-937, doi:10.1038 / mt.2012.10 (2012). 17. Ji, J. et al. Glycoinositol phospholipid-anchored interleukin 2 but not secreted interleukin 2 inhibits melanoma tumor growth in mice. Mol Cancer Ther 1, 1019-1024 (2002). 18. Paulick, M. G. & Bertozzi, C. R. The glycosylphosphatidylinositol anchor: a complex membrane-anchoring structure for proteins. Biochemistry 47, 6991-7000, doi:10.1021 / bi8006324 (2008). 19. Baluna, R. & Vitetta, E. S. Vascular leak syndrome: a side effect of immunotherapy. Immunopharmacology 37, 117-132 (1997). 20. Rosenstein, M., Ettinghausen, S. E. & Rosenberg, S. A. Extravasation of intravascular fluid mediated by the systemic administration of recombinant interleukin 2. J Immunol 137, 1735-1742 (1986). 21. Boyman, O., Kovar, M., Rubinstein, M. P., Surh, C. D. & Sprent, J. Selective stimulation of T cell subsets with antibody-cytokine immune complexes. Science 311, 1924-1927, doi:10.1126 / science.1122927 (2006). 22. Dennis, K. L., Blatner, N. R., Gounari, F. & Khazaie, K. Current status of interleukin-10 and regulatory T-cells in cancer. Curr Opin Oncol 25, 637-645, doi:10.1097 / CCO.0000000000000006 (2013). 23. Lentsch, A. B. et al. Interleukin-10 inhibits interleukin-2-induced tumor necrosis factor production but does not reduce toxicity in C3H / HeN mice. J Leukoc Biol 60, 51-57 (1996). 24. Kottke, T. et al. Subversion of NK-cell and TNFalpha Immune Surveillance Drives Tumor Recurrence. Cancer Immunol Res 5, 1029-1045, doi:10.1158 / 2326-6066.CIR-17-0175 (2017). 25. Alvarez-Breckenridge, C. A. et al. NK cells impede glioblastoma virotherapy through NKp30 and NKp46 natural cytotoxicity receptors. Nat Med 18, 1827-1834, doi:10.1038 / nm.3013 (2012). 26. Liu, Z., Ravindranathan, R., Kalinski, P., Guo, Z. S. & Bartlett, D. L. Rational combination of oncolytic vaccinia virus and PD-L1 blockade works synergistically to enhance therapeutic efficacy. Nat Commun 8, 14754, doi:10.1038 / ncomms14754 (2017). 27. Saha, D., Martuza, R. L. & Rabkin, S. D. Macrophage Polarization Contributes to Glioblastoma Eradication by Combination Immunovirotherapy and Immune Checkpoint Blockade. Cancer Cell 32, 253-267 e255, doi:10.1016 / j.ccell.2017.07.006 (2017). 28. Samson, A. et al. Intravenous delivery of oncolytic reovirus to brain tumor patients immunologically primes for subsequent checkpoint blockade. Sci Transl Med 10, doi:10.1126 / scitranslmed.aam7577 (2018). 29. Bourgeois-Daigneault, M. C. et al. Neoadjuvant oncolytic virotherapy before surgery sensitizes triple-negative breast cancer to immune checkpoint therapy. Sci Transl Med 10, doi:10.1126 / scitranslmed.aao1641 (2018). 30. Ribas, A. et al. Oncolytic Virotherapy Promotes Intratumoral T Cell Infiltration and Improves Anti-PD-1 Immunotherapy. Cell 170, 1109-1119 e1110, doi:10.1016 / j.cell.2017.08.027 (2017). 31. Liu, Z. et al. CXCL11-Armed oncolytic poxvirus elicits potent antitumor immunity and shows enhanced therapeutic efficacy. OncoImmunology 5 (2016). 32. Rintoul, J. L. et al. A selectable and excisable marker system for the rapid creation of recombinant poxviruses. PloS one 6, e24643, doi:10.1371 / journal.pone.0024643 (2011).

[0183] 9. Example 4: Adoptive Transfer of Tumor-Specific Tumor-Infiltrating T Cells Resulted in a Marked Therapeutic Effect in Syngeneic C57BL / 6 Mice Bearing Peritoneal MC38 Tumors 9.1 Introduction Immunotherapy is rapidly evolving, presenting an attractive treatment strategy for gastrointestinal cancers by reactivating the patient's immune system to fight cancer, in addition to standard treatments such as surgery, chemotherapy, and radiotherapy. Gastrointestinal cancers, including colorectal, gastric, liver, and cholangiocarcinoma, are among the top 10 most frequent malignancies worldwide (1). Immune infiltration affects tumor progression and patient survival, and high lymphocyte infiltration has been reported to be associated with anti-tumor responses and to improve clinical outcomes in several types of GI adenocarcinomas (2-9). Various immunotherapy strategies, such as cancer vaccines, adoptive T cell transfer of autologous T cells, or checkpoint blockade, have been developed, but are not yet available for a wide range of GI tumors due to tumor heterogeneity and poor immune infiltration. Tumor cells express immune escape mechanisms that alter the immune system to avoid detection by effector cells. This includes cell surface expression of immune system checkpoint ligands such as programmed cell death ligand 1 (PD-L1) (10,11), secretion of soluble immunosuppressive factors such as transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), interleukin-10 (IL-10), galectin-1, indoleamine 2,3-dioxygenase (12-14), and downregulation of major histocompatibility complex (MHC) class I expression; overexpression of receptors such as C-X-C chemokine receptor type 4 (CXCR4), basic fibroblast growth factor, and epidermal growth factor (15,16).

[0184] The immunosuppressive tumor microenvironment presents optimal conditions for the recruitment of immunosuppressive macrophages, MDSCs, and Tregs, which impede an effective anti-tumor T cell response. Inhibitory checkpoint molecules such as CTL-4, PD-1, TIM3, and LAG3 are upregulated in chronically stimulated T cells, further promoting T cell anergy. Strategies for reversing the immunosuppressive tumor microenvironment (TME) and breaking immune tolerance are presented by oncolytic virus therapy. After selectively infecting and replicating in cancer cells and the accompanying endothelial cells, oncolytic viruses kill those cells in the cancer tissue while leaving the unaffected healthy tissue intact (17, 18). Immunogenic cell death (ICD) of stromal and cancer cells induced by OV exposes the natural repertoire of tumor-associated antigens (TAAs) along with danger signals (damage-associated molecular patterns (DAMPs)) and OV-derived pathogen-associated molecular pattern (PAMP) molecules and inflammatory cytokines, thereby generating anti-tumor immunity (19-21). In a randomized phase II clinical trial in patients with advanced hepatocellular carcinoma, the oncolytic vaccinia virus (Pexa-Vec) armed with GM-CSF was associated with an objective response rate of 15% (22). In a phase I clinical trial, the oncolytic vaccinia virus (Western Reserve strain) vvDD was shown to be tumor-selective and to promote an anti-tumor response (23, 24). To improve the immune response, various oncolytic VVs have been genetically engineered to express tumor antigens, T cell co-stimulatory molecules, and inflammatory cytokines (25). Their efficacy and safety have been demonstrated in preclinical trials (26-30). This example demonstrates for the first time that oncolytic vaccinia virus-induced tumor-infiltrating T cells (OV-induced T cells) (also referred to herein as "tumor-infiltrating T cells" or "TILs") can be used for ex vivo expansion and adoptive T cell transfer as a novel immunotherapy approach.

[0185] 9.2 Methods Mice and cell lines: Female C57BL / 6 mice were obtained from The Jackson Laboratory (Bar, Harbor, ME, USA) and housed in the specific pathogen-free conditions of the University of Pittsburgh animal facility. All animal studies were approved by the university's institutional animal care and use committee. Mouse colon cancer cell lines MC38 and B16 were obtained from ATCC. Mouse colon cancer MC38-luc was generated as previously described. All cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1× penicillin / streptomycin solution (Invitrogen, Carlsbad, CA) in a 5% CO2 incubator at 37°C.

[0186] Viruses : Recombinant vaccinia viruses (Western Reserve strain) vvDD, vvDD-CCL5 (31) and vvDD-CXC11 (32) have been previously described. In the subject matter disclosed herein, newly developed vvDD-IL-15 and vvDD-IL-2 are also provided. The viruses were purified in HeLa cells. Virus titers were determined in CV-1 cells using the plaque assay. For intratumoral virus administration, 1e8 pfu / mouse was used.

[0187] Rodent tumor models : For the subcutaneous (s.c.) tumor model, B6 mice were subcutaneously inoculated with 5×10 5 MC38 cancer cells. When the s.c. tumor area reached 5×5 mm 2 vvDD-IL-2, vvDD-IL-15, vvDD-CXC11, vvDD-CCL5, vvDD or PBS was injected intratumorally at 1e8 pfu / tumor. In some experiments, IL-2 was administered at 1×10 6Administered intratumorally in the IU / tumor (Prometheus, San Diego, CA). The primary tumor size was measured using calipers at two perpendicular diameters and then measured every other day. Ten days after viral treatment, tumor tissue or spleen was harvested and processed into a single cell suspension for T cell isolation and further analysis. For the peritoneal (i.p.) tumor model, 5 × 10 5 MC38-luc cells were inoculated intraperitoneally into B6 mice and randomized according to tumor size based on in vivo IVIS imaging of live animals 7 days after tumor cell injection using the Xenogen IVIS 200 Optival In Vivo Imaging System (Caliber Life Sciences, Hopkinton, MA).

[0188] Generation of tumor-reactive T cells for adoptive transfer : 5 × 10 5 MC38 cancer cells were subcutaneously inoculated into B6 mice. The tumor area was 5 × 5 mm 2Once reached, vvDD-IL-2 (1e8 pfu / tumor) was injected intratumorally. Ten days later, the tumors were harvested, incubated at 37 °C in digestion buffer (Miltenyl Biotec, San Diego, CA), and mashed through a 100 μm tissue strainer. Erythrocyte lysis was performed using ACK lysis buffer (Thermo Fisher Scientific, Waltham, MA). For leukocyte isolation, Percoll (GE, Healthcare Life Science, Marlborough, MA) gradient centrifugation was used according to the protocol by Liu et al. (Liu, Y., Chen, K., Wang, C., Gong, W., Yoshimura, T., Wang, J. M. and Liu, M. (2013) Isolation of Mouse Tumor-Infiltrating Leukocytes by Percoll Gradient Centrifugation. Bio-protocol 3(17): e892. DOI: 10.21769 / BioProtoc.892). Leukocytes were recovered at the interface between 40% and 80% discontinuous Percoll gradients and subsequently magnetically separated (CD90.2 beads, Miltenyl Biotec, San Diego, CA). T cells were cultured for 4 days in 24-well plates at a concentration of 1×10 6 cells / well in RPMI complete medium containing 30 IU / mL IL-2 (Miltenyl Biotec, San Diego, CA) and 5 ng / mL IL-7 (Biolegend, San Diego, CA). As control T cells, spleens were harvested from non-tumor-bearing untreated mice and processed into single cell suspensions and subsequent magnetic separation (CD90.2 beads). Naïve T cells were cultured under the same conditions as virus-induced T cells. Prior to adoptive T cell transfer, T cells were analyzed for tumor specificity in co-culture assays. T cells (2×10 4 cells / well) were left unstimulated (medium) or co-cultured with γ-irradiated MC38 tumor cells (2×10 4 cells / well, 96-well plates) or γ-irradiated B16 tumor cells (2×104 (cells / well) or naive splenocytes from non-tumor-bearing B6 mice (2×10 4 Unrelated target cells such as (cells / well) were used for stimulation in duplicate for 24 hours. The plate settings were used for IFN-γ ELISPOT or flow cytometry analysis as described above.

[0189] Adoptive cell transfer and cytokine administration : B6 mice were intraperitoneally inoculated with 5×10 5 MC38-luc cancer cells, and were divided into the required groups according to tumor growth conditions based on in vivo animal IVIS imaging 7 days after tumor cell injection. The grouped mice were administered 1×10 6 vvDD-IL-2-induced T cells, naive T cells or PBS. All treated mice were irradiated with a sub-lethal dose of 5 Gy to mimic lymphopenia according to the clinical protocol, and then cell transfer and exogenous cytokine supplementation of IL-2 (100,000 IU / mouse, intraperitoneally, every 12 hours for 3 days after transplantation) (Prometheus, San Diego, CA) were performed.

[0190] Flow cytometry : After 24 hours, the co-culture assay was performed as described above. T cells were stained with Zombie aqua (Biolegend, San Diego, CA), and then stained with antibodies against mouse CD3, CD8, CD4, 4-1BB (Biolegend, San Diego, CA). Samples were collected by BD Bioscience LSRII Fortessa. Data were analyzed using BD FACS Diva software and FlowJo software (Tree Star Inc., Ashland, OR).

[0191] IFN-γ ELISPOT: The harvested tumor tissue was incubated at 37°C in digestion buffer (Miltenyl Biotec, San Diego, CA) and mashed through a 100 μm tissue strainer. Erythrocyte lysis was performed using ACK lysis buffer (Thermo Fisher Scientific, Waltham, MA), and the cell suspension was passed through a 40 μm filter to obtain a single cell suspension. CD8 + Isolation of CD8 T cells was performed using the negative α mouse CD8 microbead isolation protocol (Miltenl Biotec, San Diego, CA). A 96-well plate (MAHAS4510, Millipore, Burlington, MA) was coated with anti-mouse IFN-γ mAb at 15 mg / mL (clone AN18, Mabtech Inc., Cincinnati, OH). T cells (2×10 4 cells / well) were left unstimulated (medium) or stimulated with γ-irradiated MC38 tumor cells (2×10 4 cells / well, 96-well plate) or γ-irradiated B16 tumor cells (2×10 4 cells / well) or irrelevant target cells such as naive splenocytes from non-tumor-bearing B6 mice (2×10 4 cells / well) in two replicates for 24 hours.

[0192] After appropriate washing, biotinylated secondary antibody (clone R4-6A2-biotin, Mabtech, Inc) was added and incubated at room temperature for 2 hours. Spot color development was performed using the Vectastain Elite ABC and AEC Peroxidase substrate (SK-4200) kit (Vector Laboratories, Inc. Burlingame, CA). The number of IFN-γ spots was analyzed using ImmunoSpot TM (Cellular Technology, Ltd., Shaker Heights, OH).

[0193] Long-term survival of mice: The health status and survival period of the treated mice were carefully monitored. All mice bearing subcutaneous or peritoneal tumors were monitored via caliper measurements for changes in tumor size or abdominal circumference. When the subcutaneous tumor size of the mice exceeded 20 mm in diameter or the abdominal circumference exceeded 1.5 times the original measured value, the mice either died naturally due to the disease or were sacrificed.

[0194] Statistics : Statistical analysis was performed using the Student's t-test (GraphPad Prism version 5). Animal survival rates were represented using the Kaplan–Meier survival curve and statistically analyzed using the log-rank test (GraphPad Prism version 5). Values of p < 0.05 were considered statistically significant, and all p-values were two-sided. In the figures, standard symbols were used: * p < 0.05; ** p < 0.01; *** p < 0.001; and NS: not significant.

[0195] 9.3. Results vvDD-IL-2 is a potent regulator of the TME that attracts a large number of tumor-reactive T cells To test whether various vaccinia virus constructs promote an antitumor immune response and attract T cells into the TME, the T cell immune response in the TME of MC38 subcutaneous tumor-bearing mice was tested using vvDD, vvDD-CXC11, vvDD-CCL5, vvDD-IL-15, vvDD-IL-2, or PBS 10 days after intratumoral virus treatment. CD8 + T cells from virus-treated tumors were analyzed for IFN-γ responses by ELISPOT. Compared to the PBS control, all virus-treated tumors showed a significant increase in reactive CD8 + T cells when tested against γ-irradiated MC38 tumor cells. vvDD-IL-2-treated tumors showed the highest levels of reactive CD8 + T cells compared to vvDD- or vvDD-IL-15-treated tumors (Figure 25A).

[0196] Virus-induced CD8+ To examine whether the T cells are tumor-specific, the IFN-γ responses of CD8 + T cells derived from vvDD-IL-2, vvDD or PBS-treated tumors were measured after co-culture with irrelevant target cells (e.g., γ-irradiated B16 tumor cells, naive splenocytes from non-tumor-bearing B6 mice) and γ-irradiated MC38 tumor cells. Virus-treated tumors showed a significant increase in MC38-reactive CD8 + T cells compared to the PBS control. CD8 + T cells induced by vvDD-IL-2 showed very specific reactivity against MC38 tumor cells compared to irrelevant target cells (e.g., B16 and naive splenocytes). vvDD-induced T cells showed a similar level of MC38 reactivity compared to vvDD-IL-2, but showed a higher background reactivity against non-specific control cells such as B16 and naive splenocytes compared to vvDD-IL-2 (Figure 25B). In summary, vvDD-IL-2-induced T cells are very MC38-specific.

[0197] IL-2 expressed by the virus promotes strong infiltration of tumor-reactive CD8 + T cells in the TME compared to treatment with IL-2 alone To examine the ability of IL-2 alone to attract T cells into the TME compared to IL-2 armed virus therapy, MC38 subcutaneous tumor-bearing mice were treated intratumorally with IL-2, vvDD-IL-2, vvDD or PBS. Ten days later, tumor-infiltrating CD8 + T cells were analyzed for IFN-γ responses by ELISPOT. The IFN-γ responses of CD8 + T cells induced by vvDD-IL-2 were superior compared to IL-2 treatment, control virus or PBS (prior) (Figure 26).

[0198] vvDD-IL-2 promotes T cell infiltration in the TME of MC38 tumor-bearing mice To quantify the ability of vvDD-IL-2 to promote T cell infiltration in the TME, the treated tumors were analyzed for total CD3 + infiltrating cells as well as CD3 + CD8 + and CD3 + CD4 +Analyzed by immunofluorescence staining against T cells (Figures 27 and 28). Both viral constructs promoted a significant increase in the infiltration of total CD3 + cells and CD8 + T cells in the TME compared to the PBS control. Viral treatment showed an increase in CD4 + T cell infiltration compared to the PBS samples. Similar results were observed in other experimental settings when T cells were purified from tumor tissues and quantified per gram of tumor (Figure 28).

[0199] vvDD-IL-2-induced tumor-infiltrating T cells can be expanded and maintain their tumor specificity . To test whether vaccinia virus-induced T cells can present a novel strategy for adoptive T cell transfer, an ex vivo culture protocol for tumor-infiltrating T cells was established for T cell induction in MC38 subcutaneous tumor-bearing mice treated with vvDD-IL-2, vvDD, or PBS (Figure 29A). After 10 days, the tumors were harvested, and infiltrating CD8 + T cells and CD4 + T cells from each tumor were cultured in RPMI complete medium in the presence of IL-2 and IL-7.

[0200] To test whether the cultured T cells maintained their tumor specificity, T cells were tested for their tumor recognition using a co-culture assay containing relevant target cells (irradiated MC38 tumor cells) and irrelevant target cells (such as γ-irradiated B16 tumor cells or naive splenocytes from non-tumor-bearing B6 mice). After 24 hours, the cells were analyzed for tumor specificity using IFN-γ ELISPOT or 4-1BB expression by flow cytometry. According to IFN-γ ELISPOT, T cells from vvDD-IL-2, vvDD, or PBS-treated tumors presented tumor-specific IFN-γ secretion compared to irrelevant target cells (e.g., B16 or naive splenocytes) (Figure 29B).

[0201] CD8 + tumor-specific T cell responses and CD4 +To distinguish tumor-specific T cell responses, flow cytometry results from each sample were summarized in Figure 29C. vvDD-IL-2-induced T cells had a very tumor-specific CD8 with lower non-specific reactivity against irrelevant target cells when compared to the vvDD or PBS groups + and CD4 + T cells were shown (Figure 29C). Representative flow cytometry plots of CD8 + 4-1BB + T cells and CD4 + 4-1BB + T cells from one sample per group are shown in Figure 30.

[0202] Tumor-reactive T cells generated by vvDD-IL-2 present a novel strategy in adoptive T cell transfer . A newly developed approach of oncolytic virus-induced T cells for ACT was tested in MC38 intraperitoneal tumor-bearing mice. Prior to T cell transplantation, treated mice were given sub-lethal 5 Gy irradiation to mimic lymphodepletion similar to the clinical protocol. Grouped mice were injected intraperitoneally with vvDD-IL-2-induced T cells, naive T cells or PBS. All treated mice were given exogenous cytokine assistance with IL-2. To monitor the kinetics of tumor growth over time, the therapeutic response was monitored by in vivo bioluminescence imaging in live animals (Figure 33C). Mice administered vvDD-IL-2-induced T cells showed the strongest tumor regression compared to control mice. Regarding animal survival duration, T cells generated by the virus provided the best overall survival compared to mice administered only irradiation or PBS with naive T cells or IL-2 (Figure 33C). Imaging on day 17 after ACT is shown in Figure 34.

[0203] In summary, in the preclinical model disclosed herein, virus-induced T cells show therapeutic potential. To explore the tumor specificity of the transplanted T cells, an established co-culture assay was performed prior to ACT (Figure 31). Representative flow cytometry plots of CD8 + 4-1BB + T cells and CD4 + 4-1BB +Representative flow cytometry plots of T cells are shown in Figure 32. T cells were analyzed by flow cytometry for 4-1BB expression and by ELISPOT for IFN-γ secretion. Virus-induced T cells presented tumor-specific IFN-γ spots that were mostly non-reactive to irrelevant target cells compared to naive T cells (Figures 31B and 31C). Analyzing T cells from the same samples used for ELISPOT for 4-1BB expression, only virus-induced T cells showed significant tumor-specific CD8+4-1BB+ and CD4+4-1BB expression compared to the control group.

[0204] 9.4 Discussion This example shows that cytokine-armed oncolytic vaccinia virus can promote intratumoral T cell infiltration and generate tumor-specific T cells (OV-induced T cells) for adoptive T cell transfer.

[0205] Strong lymphocyte infiltration has been reported to be associated with improved anti-tumor responses and clinical outcomes. However, many of the patients with gastrointestinal malignancies show pure immune cells infiltrating tumors with a very immunosuppressive microenvironment. The use of oncolytic vaccinia virus, which induces immunogenic cell death, provides an effective strategy to overcome tumors with weak immunogenicity. Virus-mediated cell death results in the release of potential danger signals and cross-presentation of tumor-associated antigens, which leads to anti-tumor innate and adaptive immunity. In this example, the ex vivo expansion of vaccinia virus-induced tumor-infiltrating T cells and their use for adoptive T cell transfer in a preclinical mouse colon cancer model were demonstrated. Intratumoral administration of cytokine-armed oncolytic vaccinia virus promotes T cell infiltration into the TME. Virus-induced T cells are highly tumor-specific, proliferate ex vivo, and maintain their therapeutic capacity when transplanted into tumor-bearing mice. This example presents a strategy for promoting intratumoral T cell infiltration and generating tumor-specific T cells in the tumor microenvironment for adoptive T cell transfer.

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[0208] In addition to the various embodiments shown and claimed, the disclosed subject matter is also directed to other embodiments having other combinations of the features disclosed and claimed herein. Accordingly, the specific features presented herein can be combined with one another in other ways within the scope of the disclosed subject matter so that the disclosed subject matter includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the disclosed subject matter has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to the embodiments disclosed.

[0209] It will be apparent to those skilled in the art that various modifications and alterations can be made to the disclosed subject matter's systems and methods without departing from the spirit or scope of the disclosed subject matter. That is, the disclosed subject matter is intended to cover modifications and variations that fall within the scope of the appended claims and their equivalents. Various references, patents, and patent applications are cited herein, and their contents are hereby incorporated by reference in their entirety.

Claims

1. 1. A composition for treating a subject suffering from melanoma, comprising tumor-infiltrating T cells, the composition comprising tumor-infiltrating T cells, the tumor-infiltrating T cells being administered by the following steps: (a) administering to a subject an effective amount of an oncolytic virus to induce infiltration of one or more T cells into the cancer, wherein the oncolytic virus is a herpes simplex virus (HSV) containing a nucleic acid encoding granulocyte-macrophage colony-stimulating factor (GM-CSF); and (b) isolating the tumor-infiltrating T cells induced in step (a) from the subject's melanoma and expanding them ex vivo. The composition is obtained via a method comprising:

2. The composition of claim 1 , wherein the melanoma is metastatic.

3. 10. The composition of claim 1, wherein the composition is administered after the subject has been treated with a cancer therapy.

4. 10. The composition of claim 1, wherein the composition is administered before the subject is treated with a cancer therapy.

5. The composition of claim 1 , administered in combination with one or more exogenous cytokines.

6. The composition of claim 5 , wherein the one or more exogenous cytokines include exogenous IL-2.

7. 2. The composition of claim 1, wherein the oncolytic virus is Talimogene laherparepvec (T-VEC).

8. 5. The composition of claim 3 or 4, wherein the cancer therapy comprises administration of an immune checkpoint inhibitor.

9. 9. The composition of claim 8, wherein the immune checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

10. The composition of claim 1 , wherein the oncolytic virus is administered via intratumoral injection.

11. Oncolytic virus is approximately 1 × 10 per dose 5 pfu to 1 × 10 8 The composition of claim 1 , administered in pfu.

12. 9. The composition of claim 8, wherein the immune checkpoint inhibitor is administered one or more of before, in combination with, or after administration of the oncolytic virus.

13. The composition of claim 12, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

14. 10. The composition of claim 8, wherein an immune checkpoint inhibitor is administered one or more of before, in combination with, or after administration of the composition.

15. The composition of claim 14, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

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