Oncolytic viruses encoding recombinant transforming growth factor (TGF)-beta monomers and their use

Abstract

Oncolytic viruses encoding recombinant TGF-β engineered to prevent homodimerization and recruitment of TGF-β receptor I are described. The engineered TGF-β minimonomer functions as a dominant-negative TGF-β inhibitor. Oncolytic viruses encoding TGF-β minimonomers can be used for cancer immunotherapy to inhibit the immunosuppressive tumor microenvironment. Provided are oncolytic viruses encoding recombinant TGF-β monomers, such as human recombinant TGF-β monomers.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the interests of U.S. Provisional Application No. 63 / 070,965, filed on 27 August 2020, which is incorporated herein by reference in its entirety.

[0002] field This disclosure relates to an oncolytic virus encoding a recombinant TGF-β monomer that functions as an inhibitor of TGF-β signaling. This disclosure further relates to the use of an oncolytic virus encoding a TGF-β monomer for cancer immunotherapy. Approval of government support This invention was made possible with government support from grants awarded by the National Institutes of Health (NIH) under grant numbers GM058670 and CA172886. The government has specific rights to this invention. [Background technology]

[0003] background TGF-β is a multifunctional cytokine with diverse biological effects on cellular processes, including cell proliferation, migration, differentiation, and apoptosis. The three mammalian TGF-β isoforms, TGF-β1, TGF-β2, and TGF-β3, exert their function through cell surface receptor complexes composed of type I (TβRI) and type II (TβRII) serine / threonine kinase receptors. Receptor activation induces a comprehensive TGF-β response by inducing both SMAD proteins and other downstream targets, including Ras, RhoA, TAK1, MEKK1, PI3K, and PP2A (Roberts and Wakefield, Proc Natl Acad Sci USA 100: pp. 8621-8623, 2003; Derynck and Zhang, Nature 425: pp. 577-584, 2003; Massague, Cell 134: pp. 215-230, 2008). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Roberts and Wakefield, Proc Natl Acad Sci USA 100: 8621 - 8623, 2003 [Non-Patent Document 2] Derynck and Zhang, Nature 425: 577 - 584, 2003 [Non-Patent Document 3] Massague, Cell 134: 215 - 230, 2008 [Summary of the Invention] [Means for Solving the Problems]

[0005] Abstract Disclosed is an oncolytic virus encoding a transforming growth factor (TGF)-β monomer that has been engineered to prevent homodimer formation and signal transduction. The recombinant engineered monomeric TGF-β functions as an inhibitor of TGF-β signal transduction by preventing the recruitment of TβRI and thereby blocking downstream signal transduction. Oncolytic viruses such as vaccinia virus encoding monomeric TGF-β can be used, for example, as cancer immunotherapeutic agents.

[0006] Provided is an oncolytic virus encoding a recombinant TGF-β monomer, such as a human recombinant TGF-β monomer. The TGF-β monomer comprises a substitution of cysteine to serine (or alternatively, cysteine to arginine) at the amino acid residue corresponding to residue 77 of human TGF-β2 (described herein as SEQ ID NO: 2) and a deletion of the α3 helix corresponding to amino acid residues 52 - 71 of human TGF-β2. The TGF-β monomer can be, for example, a human TGF-β1, human TGF-β2, or human TGF-β3 monomer.

[0007] In some embodiments, the TGF-β monomer is a human TGF-β2 monomer further comprising a substitution of leucine to arginine at the amino acid residue corresponding to residue 51 of human TGF-β2, and / or a substitution of alanine to lysine at the amino acid residue corresponding to residue 74 of human TGF-β2. In other embodiments, the TGF-β monomer is a human TGF-β1 (described herein as SEQ ID NO: 1) monomer further comprising a substitution of isoleucine to arginine at the amino acid residue corresponding to residue 52 of human TGF-β1; a substitution of alanine to lysine at the amino acid residue corresponding to residue 74 of human TGF-β1; and / or a substitution of alanine to serine at the amino acid residue corresponding to residue 75 of human TGF-β1. In other embodiments, the TGF-β monomer is a human TGF-β3 (described herein as SEQ ID NO: 3) monomer further comprising a substitution of leucine to glutamate at the amino acid residue corresponding to residue 51 of human TGF-β3; a substitution of alanine to glutamate at the amino acid residue corresponding to residue 72 of human TGF-β3; and / or a substitution of alanine to aspartic acid at the amino acid residue corresponding to residue 74 of human TGF-β3. In some examples, the TGF-β monomer further comprises at least one amino acid substitution that enhances the monomer's affinity for TβRII. In some examples, the monomer further comprises at least one amino acid substitution that reduces monomer aggregation. In some examples, the monomer further comprises at least one amino acid substitution that improves monomer folding.

[0008] In alternative embodiments, provided is an oncolytic virus encoding a human recombinant TGF-β2 monomer modified to include the cystine knot region of the protein related to Dan and Cerubus (PRDC). In some examples, the amino acid sequence of the TGF-β2 monomer comprises or consists of SEQ ID NO: 11.

[0009] In some embodiments, the oncolytic virus is a vaccinia virus (VV), herpes simplex virus (HSV), or adenovirus.

[0010] Also provided is a composition comprising an oncolytic virus encoding the human recombinant TGF-β monomer disclosed herein and a pharmaceutically acceptable carrier.

[0011] Also provided are methods for treating cancer in a subject by administering a therapeutically effective dose of the oncolytic virus disclosed herein to the subject, and methods for inhibiting tumor growth or metastasis in a subject having cancer. In some embodiments, the cancer is melanoma, head and neck cancer, or pancreatic cancer.

[0012] The aforementioned and other objects, features, and advantages of the present invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings. [Brief explanation of the drawing]

[0013] [Figure 1A-1B] Figures 1A-1E: Single-cell RNA sequencing (scRNA-seq) reveals that oncolytic viruses induce dramatic immune infiltration, but they succumb to metabolic and immunological suppression. (Figure 1A) Experimental setup. Mice with Pten-deficient, BrafV600E clone 24 melanoma were treated with either PBS or oncolytic double deletion (TK and VGF deletion) VV. Tumor growth curves are shown as an example. CD45+ cells were subjected to scRNA-seq on day 12, when the tumors had not yet regressed. (Figure 1B) Uniform Manifold Approximation and Projection (UMAP) clustering of all sequenced cells from both treatment groups. (Figure 1C) UMAP of cells destroyed by treatment, revealing that VV induces dramatic remodeling of the tumor microenvironment. Circles indicate novel tumor-infiltrating T cells. (Figure 1D) TGF-β response genes in novel T cell infiltration. (Figure 1E) Mitochondrial mass measurements in PBS or VV-treated tumors, demonstrating that CD8+ T cells still succumb to metabolic dysfunction. [Figure 1C-1E]Figures 1A-1E: Single-cell RNA sequencing (scRNA-seq) reveals that oncolytic viruses induce dramatic immune infiltration, but they succumb to metabolic and immunological suppression. (Figure 1A) Experimental setup. Mice with Pten-deficient, BrafV600E clone 24 melanoma were treated with either PBS or oncolytic double deletion (TK and VGF deletion) VV. Tumor growth curves are shown as an example. CD45+ cells were subjected to scRNA-seq on day 12, when the tumors had not yet regressed. (Figure 1B) Uniform Manifold Approximation and Projection (UMAP) clustering of all sequenced cells from both treatment groups. (Figure 1C) UMAP of cells destroyed by treatment, revealing that VV induces dramatic remodeling of the tumor microenvironment. Circles indicate novel tumor-infiltrating T cells. (Figure 1D) TGF-β response genes in novel T cell infiltration. (Figure 1E) Mitochondrial mass measurements in PBS or VV-treated tumors, demonstrating that CD8+ T cells still succumb to metabolic dysfunction.

[0014] [Figure 2A-2B] Figures 2A-2B: Manipulated minimonomers of TGF-β2 act as dominant-negative inhibitors of TGFβR signaling. (Figure 2A) Schematic diagram of how minimonomer TGF-β functions. Unmodified TGF-β acts as a dimer, recruiting TβRI and TβRII to transmit signals (left). Mutation of the cysteine ​​residue that maintains the disulfide bridge and removal of the crucial "heel" helix that contacts the RI, based on structural information, produces a minimonomer TGF-β that retains the ability to bind TGFβRII but inhibits RI recruitment (right). (Figure 2B) Luciferase assays in HEK-293 TGF reporter cell lines treated with various concentrations of TGF-β1, TGF-β3, or minimonomer molecules (also known as mmTGF-β2-7M or dnTGFβ2mm), revealing that dnTGFβ2mm can inhibit the activity of TGF-β1, TGF-β2, and TGF-β3.

[0015] [Figure 3A-3C] Figures 3A-3C: dnTGFβ2mm can be delivered by oncolytic virus and possesses excellent antitumor activity. (Figure 3A) Immunoblots of TGF-β in B16 melanoma cells infected with control virus or VV-dnTGFβ2mm for 24, 48, or 72 hours. The non-reducing gel shows a band at 10 kDa, which is the approximate size of the minimonomer. (Figure 3B) TGF-β reporter assay using recombinant TGF-β and supernatant from control (C) or dnTGFβ2mm-expressing virus-infected cells in a 10-fold dilution. (Figure 3C) Tumor growth curves of B16 melanoma treated with 2.5 × 10⁶ PFU of VVctrl or manipulated dnTGFβ2mm-expressing VV.

[0016] [Figure 4A-4B] Figures 4A-4E: Sequence comparison of the manipulated TGF-β monomer mmTGF-β2-7M (SEQ ID NO: 7) with TGF-β2 (SEQ ID NO: 2) (Figure 4A), mmTGF-β2-7M2R (SEQ ID NO: 9) (Figure 4B), mmTGF-β2-2M-Del8_17 (SEQ ID NO: 10) (Figure 4C), mmTGF-β2-7M-PRDC (SEQ ID NO: 11) (Figure 4D), and mmTGF-β2-7M2R-Del8-17 (SEQ ID NO: 12) (Figure 4E). The difference in sequences is indicated by the numbers below the two aligned sequences, and the identity of the numbers indicates the nature of the difference. Sequence identity is indicated by an asterisk. Below the sequences in Figure 4A are the structures of the TGF-β3-(TβRII)2-(TβRI)2 complex (PDB 2PJY) (left) and the mmTGF-β2-7M-TβRII complex (PDB 5TX4) (right), illustrating some of their main structural features. [Figure 4C-4D]Figures 4A-4E: Sequence comparison of the manipulated TGF-β monomer mmTGF-β2-7M (SEQ ID NO: 7) with TGF-β2 (SEQ ID NO: 2) (Figure 4A), mmTGF-β2-7M2R (SEQ ID NO: 9) (Figure 4B), mmTGF-β2-2M-Del8_17 (SEQ ID NO: 10) (Figure 4C), mmTGF-β2-7M-PRDC (SEQ ID NO: 11) (Figure 4D), and mmTGF-β2-7M2R-Del8-17 (SEQ ID NO: 12) (Figure 4E). The difference in sequences is indicated by the numbers below the two aligned sequences, and the identity of the numbers indicates the nature of the difference. Sequence identity is indicated by an asterisk. Below the sequences in Figure 4A are the structures of the TGF-β3-(TβRII)2-(TβRI)2 complex (PDB 2PJY) (left) and the mmTGF-β2-7M-TβRII complex (PDB 5TX4) (right), illustrating some of their main structural features. [Figure 4E] Figures 4A-4E: Sequence comparison of the manipulated TGF-β monomer mmTGF-β2-7M (SEQ ID NO: 7) with TGF-β2 (SEQ ID NO: 2) (Figure 4A), mmTGF-β2-7M2R (SEQ ID NO: 9) (Figure 4B), mmTGF-β2-2M-Del8_17 (SEQ ID NO: 10) (Figure 4C), mmTGF-β2-7M-PRDC (SEQ ID NO: 11) (Figure 4D), and mmTGF-β2-7M2R-Del8-17 (SEQ ID NO: 12) (Figure 4E). The difference in sequences is indicated by the numbers below the two aligned sequences, and the identity of the numbers indicates the nature of the difference. Sequence identity is indicated by an asterisk. Below the sequences in Figure 4A are the structures of the TGF-β3-(TβRII)2-(TβRI)2 complex (PDB 2PJY) (left) and the mmTGF-β2-7M-TβRII complex (PDB 5TX4) (right), illustrating some of their main structural features.

[0017] [Figures 5A-5F]Figures 5A-5F: Amide 1H-15N 1-bond shift correlation NMR spectra of mmTGF-β2-7M2R (Figures 5A-5C) compared with the parent protein, mmTGF-β2-7M (Figures 5D-5F). Spectra were recorded at 37°C in 10 mM phosphate buffer at pH 4.6 (Figures 5A and 5D) or pH 7.2, either in the absence of CHAPS in the buffer (Figures 5B and 5E) or with CHAPS added to a final concentration of 10 mM (Figures 5C and 5F).

[0018] [Figure 6A-6C] Figures 6A-6C: amide 1H-15N 1-bond shift correlation NMR spectra of mmTGF-β2-2M-Del8-17. Spectra were recorded in 10 mM phosphate buffer at pH 4.6 (Figure 6A) or pH 7.2 at 37°C, either in the absence of CHAPS in the buffer (Figure 6B) or with CHAPS added to a final concentration of 10 mM (Figure 6C).

[0019] [Figures 7A-7D] Figures 7A–7D: Amide 1H-15N 1-bond shift-correlated NMR spectra of mmTGF-β2-7M-PRDC (Figures 7A–7C), and binding to TβRII detected by undenatured gel electrophoresis (Figure 7D). Spectra were recorded at 37°C in 10 mM phosphate buffer at pH 4.8 (Figure 7A) or pH 6.0 (Figures 7B–7C). Figures 7B and 7C differ only in the contour levels at which the signals are plotted (Figure 7B plots at contour levels closer to noise compared to the panel in Figure 7C). The undenatured gels shown in Figure 7D were performed by electrophoresis with either 2 μg of TβRII alone (leftmost lane) or manipulated TGF-β monomers added in specified molar ratios (+A and +B indicate either a 1:1 or 2:1 molar ratio of TβRII:manipulated TGF-β monomers, respectively).

[0020] [Figure 8]Figure 8: Binding of manipulated TGF-beta monomers (mmTGF-β2-7M-left, mmTGF-β2-7M2R-center, and mmTGF-β2-2M-Del8-17-right) to the TGF-βII receptor, TβRII, as detected by isothermal titration calorimetry (ITC). The upper panel shows the raw thermograms of three repeated titrations, and the lower panel shows the integrated calorimetry (data points) for the three repeated titrations fitted to a 1:1 coupled isotherm (smooth curve). The fitted parameters are provided in the table below.

[0021] [Figures 9A-9D] Figures 9A-9D: HEK-293 cell-based CAGA-Luc TGF-β reporter assay for comparative evaluation of the inhibitory activity of manipulated TGF-β monomers. HEK-293 cells stably transfected with the TGF-β CAGA-Luc reporter were treated for 30 minutes with the specified concentrations of the manipulated TGF-β monomers shown, and then stimulated with the addition of 10 pM TGF-β3. Cells were harvested after 14 hours and assayed for luciferase activity. (Figure 9A) mmTGF-β2-7M (SEQ ID NO: 7), IC50 58.23 nM. (Figure 9B) mmTGF-β2-7M2R (SEQ ID NO: 9), IC50 53.29 nM. (Figure 9C) mmTGF-β2-2M-Del8-17 (SEQ ID NO: 10), IC50 111.0 nM. (Figure 9D) mmTGF-β2-7M-PRDC (SEQ ID NO: 11), IC50 282.5 nM. The data points and error bars shown correspond to the mean and standard deviation of the three measurements. The smoothed curve corresponds to the fit to a standard dose-response inhibition isotherm. The fitted IC50 value is shown.

[0022] [Figure 10]Figure 10: Vaccinia virus (VV) expressing mmTGFβ variant 1 (mmTGF-β2-7M2R-Del8-17) shows superior efficacy in drug-resistant cancer models. (Top) C57 / BL6J mice were inoculated with the head and neck squamous cell carcinoma (HNSCC) MEER subclone. On day 7, the mice were injected intratumorically with 2.5 × 10⁵ PFU of either control VV (VVCtrl) or VV engineered to express mmTGF-β2-7M2R-Del8-17 (referred to as VVmmTGFβ(var1) or VVmmTGFβi) (SEQ ID NO: 12). The control virus showed only a slight curative effect, but half of the mice treated with VVmmTGFβ(var1) showed a complete response and demonstrated a long-lasting survival benefit. (Center) C57 / BL6J mice were inoculated with melanoma clone 24 (CL24). On day 7, mice were administered 2.5 × 10⁵ PFU of either VV control (VVCtrl) or VV expressing mmTGFβi. Administration of VVmmTGFβi resulted in a complete response in 40% of the treated animals. (Below) Addition of anti-PD1 enhanced the tumor inhibitory effect of VVmmTGFβi in the CL24 model. [Modes for carrying out the invention]

[0023] Sequence List The nucleic acid and amino acid sequences listed in the attached sequence listing are indicated using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids, as defined in 37C.FR1.822. Only one strand of each nucleic acid sequence is shown, although complementary strands are understood to be included with reference to the shown strand. The sequence listing is submitted as a 10.8KB ASCII text file created on August 27, 2021, and is incorporated herein by reference. In the attached sequence listing: Sequence ID 1 is the amino acid sequence of wild-type human TGF-β1. Sequence ID 2 is the amino acid sequence of wild-type human TGF-β2. Sequence ID 3 is the amino acid sequence of wild-type human TGF-β3. Sequence ID 4 is the amino acid sequence of an engineered human TGF-β1 monomer called mmTGF-β1. Sequence ID 5 is the amino acid sequence of an engineered human TGF-β2 monomer called mmTGF-β2. Sequence ID 6 is the amino acid sequence of an engineered human TGF-β3 monomer called mmTGF-β3. Sequence ID 7 is the amino acid sequence of an engineered human TGF-β2 monomer called mmTGF-β2-7M. Sequence ID 8 is the amino acid sequence of the human IL-2 signaling sequence. Sequence ID 9 is the amino acid sequence of an engineered human TGF-β2 monomer called mmTGF-β2-7M2R. Sequence ID 10 is the amino acid sequence of an engineered human TGF-β2 monomer called mmTGF-β2-2M-Del8-17. Sequence ID 11 is the amino acid sequence of an engineered human TGF-β2 monomer called mmTGF-β2-7M-PRDC. Sequence ID 12 is the amino acid sequence of an engineered human TGF-β2 monomer called mmTGF-β2-7M2R-Del8-17.

[0024] Detailed explanation I. Abbreviations CHAPS 3-[(3-Colamidopropyl)dimethylammonio]-1-propanesulfonate CKGF (Cystine Knot Growth Factor Fold) HNSCC head and neck squamous cell carcinoma HSQC heteronuclide single quantum correlation NMR nuclear magnetic resonance oVV (oncolytic vaccinia virus) PRDC Dan and Cerubus-related proteins scRNA-seq single-cell RNA sequencing TGF-β Transforming Growth Factor β TβRI Transforming Growth Factor-β1 Receptor TβRII (Transforming Growth Factor-β2 Receptor) TK Thymidine Kinase VGF (Viral Growth Factor) VV vaccinia virus

[0025] II. Terminology Unless otherwise specified, technical terms are used according to their conventional usage. Definitions of general terms in molecular biology can be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; and other similar references.

[0026] Where used herein, the singular forms “a,” “an,” and “the” refer to both singular and plural unless the context explicitly indicates otherwise. For example, the term “antigen” can be considered equivalent to the phrase “at least one antigen,” including one or more antigens. Where used herein, the term “includes” means “includes.” Unless otherwise noted, all given base sizes or amino acid sizes, and all molecular weight or molecular mass values ​​for nucleic acids or polypeptides are approximate and provided for illustrative purposes only. Many methods and materials similar or equivalent to those described herein may be used, but certain preferred methods and materials are described herein. In case of any conflict, this specification, including the explanation of terms, shall prevail. Furthermore, materials, methods, and examples are illustrative and not intended to be limiting.

[0027] To facilitate an overview of various embodiments, a definition of terms is provided below:

[0028] Administration: Providing or administering a therapeutic agent (e.g., an oncolytic virus encoding TGF-β monomer) or other drug to a subject via any effective route. Exemplary routes of administration include, but are not limited to, injection or infusion (intratumor, subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intravenous, intraprostatic, intraventricular, striatal, intracranial, and spinal cord), oral, intratubal, sublingual, rectal, percutaneous, intranasal, vaginal, and inhalation routes.

[0029] Different species: Derived from a separate genetic source or species.

[0030] Isolated: “Isolated” biological components, such as nucleic acids, proteins (including antibodies), organelles, or recombinant viruses, are substantially separated or purified from other biological components in their environment (e.g., cells), i.e., other chromosomal DNA and RNA as well as extrachromosomal DNA and RNA, proteins, and organelles. “Isolated” nucleic acids and proteins include those purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acids or proteins. “Isolated” does not require absolute purity and may include proteins, peptides, nucleic acid molecules, or viruses that are at least 50% isolated, for example, at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated.

[0031] Melanoma: A form of cancer originating from melanocytes (cells that produce the pigment melanin). Melanocytes are primarily found in the skin, but are also present in the intestines and eyes. As used herein, “melanoma” refers to melanoma at any stage, or any subtype of melanoma, such as superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, lentigo malignant melanoma, intraepidermal melanoma, mucosal melanoma, and uveal melanoma.

[0032] Modification: A change in a nucleic acid sequence or protein sequence. For example, amino acid sequence modifications include, for example, substitutions, insertions, and deletions, or combinations thereof. Insertions include amino and / or carboxyl-terminated fusions and intrasequence insertions of one or more amino acid residues. Deletions are characterized by the removal of one or more amino acid residues from a protein sequence. In some embodiments herein, modifications (such as substitutions, insertions, or deletions) result in changes in function, such as a reduction or enhancement of a particular activity of a protein. Modification by substitution is a modification in which at least one residue is removed and a different residue is inserted in its place. Amino acid substitutions are typically single-residue substitutions, but can occur simultaneously at multiple different locations. A combination of substitutions, deletions, insertions, or any combination thereof may be used to arrive at a final mutant sequence. These modifications can be prepared by modifying nucleotides in the DNA encoding the protein, thereby producing DNA encoding the modification. Techniques for producing insertion, deletion, and substitution mutations at predetermined sites in DNA with known sequences are known. A “modified” protein, nucleic acid, or virus is one that has one or more modifications outlined above.

[0033] Monomer: A single molecular unit (such as a protein) that can combine with other molecular units to form a dimer or polymer. In the context of this disclosure, “TGF-β monomer” is a single TGF-β polypeptide chain in which the wild-type version can combine with other TGF-β monomers to form a dimer. In some embodiments described herein, recombinant TGF-β monomers are manipulated to prevent dimerization.

[0034] Neoplasm, malignant tumor, cancer, or tumor: A neoplasm is an abnormal proliferation of tissue or cells resulting from excessive cell division. The proliferation of neoplasms can lead to a tumor. The amount of tumor in an individual is called the “tumor load,” which can be measured as the number, volume, or weight of tumors. Tumors that do not metastasize are called “benign.” Tumors that invade surrounding tissue and / or can metastasize are called “malignant.”

[0035] Examples of blood tumors include leukemias, which include acute leukemias (such as 11q23-positive acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin lymphomas (low-grade and high-grade), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and myelodysplasia.

[0036] Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synoviomas, mesotheliomas, Ewing's tumors, leiomyosarcomas, rhabdomyosarcomas, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer (including basement membrane carcinoma, ductal carcinoma, and lobular carcinoma), lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, and medullary thyroid cancer. Examples include cancer, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular cancer, seminoma, bladder cancer, and CNS tumors (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma). In some cases, the tumor is a head and neck cancer, such as squamous cell carcinoma of the head and neck, which can occur in the oral cavity, pharynx, larynx, sinuses, nasal cavity, and salivary glands. In some cases, the head and neck cancer is positive for human papillomavirus, such as HPV type 16.

[0037] Oncolytic viruses: Any viruses that preferentially replicate in tumor cells and kill them. This term includes naturally occurring oncolytic viruses as well as recombinant viruses designed to target and kill tumor cells. Exemplary oncolytic viruses include, but are not limited to, vaccinia viruses, adenoviruses, reoviruses, herpes simplex viruses, measles viruses, coxsackieviruses, parvoviruses, rhinoviruses, polioviruses, and varicella-stomatitis viruses (see, for example, Raja et al., J Immunother Cancer 6: 140, 2018).

[0038] Pancreatic cancer: Cancer that originates in the pancreatic tissue. Pancreatic cancer typically spreads rapidly, is rarely detected in its early stages, and has a poor prognosis in most patients who are diagnosed. The most common type of pancreatic cancer is pancreatic adenocarcinoma, which accounts for approximately 85% of all pancreatic cancer cases.

[0039] Peptides or polypeptides: polymers in which monomers are linked together by amide bonds, consisting of amino acid residues. When the amino acids are alpha amino acids, either L-optical isomers or D-optical isomers may be used, with L-isomers being preferred. The terms “peptide,” “polypeptide,” or “protein,” as used herein, encompass any amino acid sequence and are intended to include modified sequences, including modified globulin proteins. The terms “peptide” and “polypeptide” are particularly intended to include naturally occurring proteins and those produced by recombinant or synthetic means.

[0040] Conservative amino acid substitutions are those that, when made, do the least to disrupt the properties of the original protein; that is, the protein's structure and especially its function are preserved and not significantly altered by such substitutions. Examples of conservative substitutions are given below.

[0041] [Table 3]

[0042] Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the substitution area, for example, as a sheet or helix structure, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

[0043] Generally, substitutions that are expected to bring about the greatest change in protein properties are non-conservative substitutions, such as (a) a hydrophilic residue, e.g., serine or threonine, being replaced by (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine, or alanine; (b) cysteine ​​or proline being replaced by (or by) any other residue; (c) a residue with a positively charged side chain, e.g., lysine, arginine, or histidine, being replaced by (or by) a negatively charged residue, e.g., glutamine or aspartic acid; or (d) a residue with a bulky side chain, e.g., phenylalanine, being replaced by one without a side chain, e.g., glycine.

[0044] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers (mediums) useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st Edition (2005) describes compositions and formulations suitable for the pharmaceutical delivery of one or more therapeutic compounds, molecules, or drugs (e.g., oncolytic viruses).

[0045] Generally, the properties of the carrier depend on the specific method of administration used. For example, parenteral formulations typically contain an injectable fluid that includes pharmaceutically and physiologically acceptable fluids such as water, saline, equilibrium salt solutions, aqueous dextrose, glycerol, etc., as the medium. With respect to solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the administered pharmaceutical composition may contain trace amounts of non-toxic auxiliary substances such as wetting agents or emulsifiers, preservatives, pH buffers, etc. (e.g., sodium acetate or sorbitan monolaurate).

[0046] Preventing, treating, or ameliorating a disease: "Preventing" a disease refers to inhibiting the complete onset of the disease. "Treatment" refers to therapeutic interventions that improve the signs or symptoms of a disease or condition after the disease or condition has begun to develop, such as reducing the tumor burden (e.g., a decrease in tumor volume or size) or a decrease in the number of metastases. "Ameliorating" refers to reducing the number or severity of the signs or symptoms of the disease.

[0047] Recombinant: Recombinant nucleic acids or proteins have sequences that do not exist in nature, or sequences created by the artificial combination of two otherwise separate sequence segments. This artificial combination is often achieved by the chemical synthesis or artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. The term recombinant includes nucleic acids, proteins, and viruses that have been modified by the addition, substitution, or deletion of parts of a native nucleic acid molecule or protein.

[0048] Target: Living multicellular organisms, including vertebrates, which is a category that includes both humans and non-human mammals.

[0049] Therapeutic effective dose: The amount of a compound or composition, such as an oncolytic virus encoding recombinant TGF-β monomer, sufficient to obtain the desired effect in the subject being treated. For example, this may be the amount necessary to inhibit or block TGF-β signaling in cells. In other examples, this may be the amount necessary to inhibit or suppress tumor growth. In one embodiment, the therapeutic effective dose is the amount necessary to eliminate a tumor, reduce the size of a tumor, or prevent tumor metastasis, for example, to reduce the tumor size and / or volume by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% compared to the size / volume / number before treatment, and / or to reduce the number and / or size / volume of metastases by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or further 100%. In one embodiment, the therapeutically effective dose is the amount necessary to extend the survival of a subject with the same cancer, compared to the survival of a subject without treatment with a recombinant TGF-β monomer-encoding oncolytic virus, for example, by at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, or at least 5 years. When administered to a subject, a dose is generally used that achieves a target tissue concentration (e.g., in the tumor) that has been shown to achieve the desired in vitro effect.

[0050] Transgene: A gene inserted into the genome of a different organism (such as an oncolytic virus). Transgenes may also be called heterogenes. In the context of this disclosure, transgenes may encode, for example, chemokines, cytokines, tumor-associated antigens, immune costimulatory molecules, immune checkpoint inhibitors, suicide genes, tumor suppressor genes, pro-apoptotic proteins, or anti-angiogenic proteins.

[0051] Transforming growth factor-β (TGF-β): A secreted, multifunctional protein that regulates proliferation, cell differentiation, and several other cellular functions. Many cells synthesize TGF-β, and almost all cells express TGF-β receptors. The term "TGF-β" refers to three distinct protein isoforms, TGF-β1, TGF-β2, and TGF-β3, encoded by the genes TGFB1, TGFB2, and TGFB3, respectively.

[0052] TGF-β signaling pathway: A signaling pathway involved in many cellular processes, including cell proliferation, differentiation, and apoptosis. Members of the TGF-β pathway include, but are not limited to, TGF-β1, TGF-β2, TGF-β3, TGF-β receptor type I, and TGF-β receptor type II.

[0053] TGF-β receptor: The term "TGF-β receptor" includes TGF-β receptor type I (TβRI, encoded by TGFBR1) and TGF-β receptor type II (TβRII, encoded by TGFBR2). TGF-β receptors are serine / threonine protein kinases. When type I and type II TGF-β receptors bind to TGF-β, they form heterodimer complexes that transmit TGF-β signals from the cell surface to the cytoplasm.

[0054] Vaccinia virus: A large enveloped virus with a double-stranded DNA genome (approximately 190 kb). Vaccinia virus is a member of the Poxviridae family. In some embodiments herein, vaccinia virus is a Western Reserve strain of vaccinia virus.

[0055] III. Overview of Some Embodiments Immunotherapy has dramatically changed the landscape of cancer treatment (Ribas and Wolchok, Science 359(6382):1350-1355, 2018). Most notably, the use of monoclonal antibody-mediated blockade of co-inhibitory "checkpoint" molecules on T cells has yielded impressive clinical results, leading to FDA approval in multiple indications and the 2018 Nobel Prize in Medicine. These drugs act by reactivating tumor-infiltrating T cells, differentiating them productively and lysing tumor cells. However, the success of these drugs depends on patients with a smoldering immune response dormant against cancer cells, including tumor mutagenesis (Hellmann et al., N Engl J Med 378(22):2093-2104, 2018), pre-existing T cell infiltrations, and high PD-L1 expression. Therefore, only a small number of patients benefit from these treatments. However, the vast majority of patients have immunologically "cold" tumors with little or no immune infiltration. In these patients, other modalities must be used to stimulate immune infiltration and antigen release in order to reactivate the immune response.

[0056] Oncolytic viruses represent an attractive means of stimulating the tumor microenvironment and stimulating anti-tumor immunity (Bommareddy et al., Nat Rev Immunol 18(8):498-513, 2018; Ribas et al., Cell 170(6):1109-1119, 2017). The fundamental concept behind oncolytic viruses is that it may be necessary to manipulate the lytic virus by removing viral genes typically used to promote a proliferative transformation-like state, thereby causing the transformed cells to replicate (Raja et al., J Immunother Cancer 6(1):140, 2018). Genetic engineering strategies can be used to selectively infect tumor cells, induce replication, and manipulate the tumor cells to immunogenically lyse them (Raja et al., J Immunother Cancer 6(1):140, 2018). Thus, these agents also have the ability to elicit a strong antiviral immune response as well as the ability to vaccinate patients against their own tumors. More importantly, these viruses offer an opportunity to deliver gene cargo to the tumor microenvironment because they selectively replicate in tumor cells. In fact, the FDA-approved oncolytic virus T-vec contains a gene encoding GM-CSF to stimulate dendritic cell invasion and maturation (Ott and Hodi, Clin Cancer Res 22(13):3127-3131, 2016). Oncolytic viruses provide immune infiltration, novel anti-tumor immunity, and delivery of novel gene-coding drugs to enhance therapeutic responses. Thus, oncolytic viruses provide a means to direct tumor cytotoxicity, immunotherapy, and gene therapy for cancer.

[0057] This disclosure describes an oncolytic virus encoding a monomeric form of TGF-β that functions as a dominant-negative inhibitor of TGF-β signaling. Provided are oncolytic viruses encoding recombinant TGF-β monomers having a cysteine-to-serine substitution or a cysteine-to-arginine substitution at the amino acid residue corresponding to residue 77 of human TGF-β2 as described in SEQ ID NO: 2; and a deletion of the α3 helix corresponding to amino acid residues 52-71 of human TGF-β2 as described in SEQ ID NO: 2. These modifications prevent the TGF-β monomer from forming a dimer. In some embodiments, the TGF-β monomer is a human, mouse, rat, or other mammalian TGF-β monomer. In certain examples, the TGF-β monomer is a human TGF-β monomer.

[0058] In some embodiments, the TGF-β monomer is a human TGF-β2 monomer. In some examples, the human TGF-β2 monomer further includes a leucine-to-arginine substitution at the amino acid residue corresponding to residue 51 of SEQ ID NO: 2; and / or an alanine-to-lysine substitution at the amino acid residue corresponding to residue 74 of SEQ ID NO: 2. These substitutions increase the net charge of the monomer.

[0059] In some embodiments, the human TGF-β2 monomer further comprises at least one amino acid substitution that enhances the monomer's affinity for TβRII. In some examples, the at least one amino acid substitution that enhances the monomer's affinity for TβRII includes: a lysine-to-arginine substitution at the amino acid residue corresponding to residue 25 of SEQ ID NO: 2; an arginine-to-lysine substitution at the amino acid residue corresponding to residue 26 of SEQ ID NO: 2; a leucine-to-valine substitution at the amino acid residue corresponding to residue 89 of SEQ ID NO: 2; an isoleucine-to-valine substitution at the amino acid residue corresponding to residue 92 of SEQ ID NO: 2; a lysine-to-arginine substitution at the amino acid residue corresponding to residue 94 of SEQ ID NO: 2; a threonine-to-lysine substitution at the amino acid residue corresponding to residue 95 of SEQ ID NO: 2; and / or an isoleucine-to-valine substitution at the amino acid residue corresponding to residue 98 of SEQ ID NO: 2.

[0060] In some cases, the human TGF-β2 monomer includes: a cysteine-to-serine substitution at the amino acid residue corresponding to residue 77 of human TGF-β2 as described in SEQ ID NO: 2; a deletion of the α3 helix corresponding to amino acid residues 52-71 of human TGF-β2 as described in SEQ ID NO: 2; a lysine-to-arginine substitution at the amino acid residue corresponding to residue 25 of SEQ ID NO: 2; an arginine-to-lysine substitution at the amino acid residue corresponding to residue 26 of SEQ ID NO: 2; a leucine-to-arginine substitution at the amino acid residue corresponding to residue 51 of SEQ ID NO: 2; an alanine-to-lysine substitution at the amino acid residue corresponding to residue 74 of SEQ ID NO: 2; a leucine-to-valine substitution at the amino acid residue corresponding to residue 89 of SEQ ID NO: 2; an isoleucine-to-valine substitution at the amino acid residue corresponding to residue 92 of SEQ ID NO: 2; a lysine-to-arginine substitution at the amino acid residue corresponding to residue 94 of SEQ ID NO: 2; a threonine-to-lysine substitution at the amino acid residue corresponding to residue 95 of SEQ ID NO: 2; and an isoleucine-to-valine substitution at the amino acid residue corresponding to residue 98 of SEQ ID NO: 2.

[0061] In certain cases, the amino acid sequence of the human TGF-β2 monomer is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5 or SEQ ID NO: 7. In certain non-limiting cases, the amino acid sequence of the human TGF-β2 monomer includes or consists of SEQ ID NO: 5 or SEQ ID NO: 7. In some cases, the human TGF-β2 monomer further includes an N-terminal methionine residue.

[0062] In some embodiments, the human TGF-β2 monomer includes, or further includes, at least one amino acid substitution that reduces monomer aggregation and / or improves folding. In some examples, the at least one amino acid substitution that reduces monomer aggregation and / or improves folding includes a cysteine-to-valine substitution at the amino acid residue corresponding to residue 7 of SEQ ID NO: 2; a cysteine-to-alanine substitution at the amino acid residue corresponding to residue 16 of SEQ ID NO: 2; a cysteine-to-arginine substitution at the amino acid residue corresponding to residue 77 of SEQ ID NO: 2; and / or a valine-to-arginine substitution at the amino acid residue corresponding to residue 79 of SEQ ID NO: 2.

[0063] In certain cases, the human TGF-β2 monomer includes the deletion of the α3 helix corresponding to amino acid residues 52-71 of human TGF-β2 as described in SEQ ID NO: 2; the substitution of lysine to arginine at the amino acid residue corresponding to residue 25 of SEQ ID NO: 2; the substitution of arginine to lysine at the amino acid residue corresponding to residue 26 of SEQ ID NO: 2; the substitution of leucine to arginine at the amino acid residue corresponding to residue 51 of SEQ ID NO: 2; the substitution of alanine to lysine at the amino acid residue corresponding to residue 74 of SEQ ID NO: 2; the substitution of cysteine ​​to arginine at the amino acid residue corresponding to residue 77 of SEQ ID NO: 2; the substitution of valine to arginine at the amino acid residue corresponding to residue 79 of SEQ ID NO: 2; the substitution of leucine to valine at the amino acid residue corresponding to residue 89 of SEQ ID NO: 2; the substitution of isoleucine to valine at the amino acid residue corresponding to residue 92 of SEQ ID NO: 2; the substitution of lysine to arginine at the amino acid residue corresponding to residue 94 of SEQ ID NO: 2; the substitution of threonine to lysine at the amino acid residue corresponding to residue 95 of SEQ ID NO: 2; and the substitution of isoleucine to valine at the amino acid residue corresponding to residue 98 of SEQ ID NO: 2. In certain non-limiting cases, the amino acid sequence of the human TGF-β2 monomer includes or consists of SEQ ID NO: 9. In some cases, the human TGF-β2 monomer further includes an N-terminal methionine residue.

[0064] In other specific examples, the human TGF-β2 monomer includes a cysteine-to-serine substitution at the amino acid residue corresponding to residue 77 of SEQ ID NO: 2; a deletion of the α3 helix corresponding to amino acid residues 52-71 of human TGF-β2 as described in SEQ ID NO: 2; a cysteine-to-valine substitution at the amino acid residue corresponding to residue 7 of SEQ ID NO: 2; a cysteine-to-alanine substitution at the amino acid residue corresponding to residue 16 of SEQ ID NO: 2; a lysine-to-arginine substitution at the amino acid residue corresponding to residue 25 of SEQ ID NO: 2; a leucine-to-arginine substitution at the amino acid residue corresponding to residue 51 of SEQ ID NO: 2; an alanine-to-lysine substitution at the amino acid residue corresponding to residue 74 of SEQ ID NO: 2; and a lysine-to-arginine substitution at the amino acid residue corresponding to residue 94 of SEQ ID NO: 2. In certain non-limiting examples, the amino acid sequence of the human TGF-β2 monomer includes or consists of SEQ ID NO: 10. In some examples, the human TGF-β2 monomer further includes an N-terminal methionine residue.

[0065] Other specific examples include: the deletion of the α3 helix corresponding to amino acid residues 52-71 of human TGF-β2 as described in SEQ ID NO: 2; the substitution of cysteine ​​to valine at the amino acid residue corresponding to residue 7 of SEQ ID NO: 2; the substitution of cysteine ​​to alanine at the amino acid residue corresponding to residue 16 of SEQ ID NO: 2; the substitution of lysine to arginine at the amino acid residue corresponding to residue 25 of SEQ ID NO: 2; the substitution of arginine to lysine at the amino acid residue corresponding to residue 26 of SEQ ID NO: 2; the substitution of leucine to arginine at the amino acid residue corresponding to residue 51 of SEQ ID NO: 2; and the substitution of alanine at the amino acid residue corresponding to residue 74 of SEQ ID NO: 2. This includes substitutions from to lysine; substitutions from cysteine ​​to arginine at the amino acid residue corresponding to residue 77 of SEQ ID NO: 2; substitutions from valine to arginine at the amino acid residue corresponding to residue 79 of SEQ ID NO: 2; substitutions from leucine to valine at the amino acid residue corresponding to residue 89 of SEQ ID NO: 2; substitutions from isoleucine to valine at the amino acid residue corresponding to residue 92 of SEQ ID NO: 2; substitutions from lysine to arginine at the amino acid residue corresponding to residue 94 of SEQ ID NO: 2; substitutions from threonine to lysine at the amino acid residue corresponding to residue 95 of SEQ ID NO: 2; and substitutions from isoleucine to valine at the amino acid residue corresponding to residue 98 of SEQ ID NO: 2. In certain non-limiting examples, the amino acid sequence of the human TGF-β2 monomer includes or consists of SEQ ID NO: 12. In some examples, the human TGF-β2 monomer further includes an N-terminal methionine residue.

[0066] In alternative embodiments, the provided is an oncolytic virus encoding a human recombinant TGF-β2 monomer modified to include the cystine knot region of PRDC. In some examples, the amino acid sequence of the TGF-β2 monomer is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11. In certain examples, the amino acid sequence of the TGF-β2 monomer includes or consists of SEQ ID NO: 11. In some examples, the human TGF-β2 monomer further includes an N-terminal methionine residue.

[0067] In some embodiments, the TGF-β monomer is a human TGF-β1 monomer. In some examples, the human TGF-β1 monomer further includes an isoleucine-to-arginine substitution at the amino acid residue corresponding to residue 52 of SEQ ID NO: 1; an alanine-to-lysine substitution at the amino acid residue corresponding to residue 74 of SEQ ID NO: 1; and / or an alanine-to-serine substitution at the amino acid residue corresponding to residue 75 of SEQ ID NO: 1. These substitutions increase the net charge of the monomer. In some examples, the human TGF-β1 monomer further includes at least one amino acid substitution that increases the monomer's affinity for TβRII.

[0068] In some cases, the amino acid sequence of the human TGF-β1 monomer is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4. In certain non-limiting cases, the amino acid sequence of the human TGF-β1 monomer includes or consists of SEQ ID NO: 4. In some cases, the human TGF-β1 monomer further includes an N-terminal methionine residue.

[0069] In some embodiments, the TGF-β monomer is a human TGF-β3 monomer. In some examples, the human TGF-β3 monomer further includes a leucine-to-glutamic acid substitution at the amino acid residue corresponding to residue 51 of SEQ ID NO: 3; an alanine-to-glutamic acid substitution at the amino acid residue corresponding to residue 72 of SEQ ID NO: 3; and / or an alanine-to-aspartic acid substitution at the amino acid residue corresponding to residue 74 of SEQ ID NO: 3. These substitutions increase the net charge of the monomer. In some examples, the human TGF-β3 monomer further includes at least one amino acid substitution that increases the monomer's affinity for TβRII.

[0070] In some cases, the amino acid sequence of the human TGF-β3 monomer is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6. In certain non-limiting cases, the amino acid sequence of the human TGF-β3 monomer includes or consists of SEQ ID NO: 6. In some cases, the human TGF-β3 monomer further includes an N-terminal methionine residue.

[0071] In some embodiments, the TGF-β monomer further comprises a signal sequence, such as a heterologous signal sequence. In some examples, the heterologous signal sequence is an IL-2 signal sequence. In specific examples, the IL-2 signal sequence contains or consists of the amino acid sequence of SEQ ID NO: 8. In other examples, the heterologous signal sequence is a signal sequence of albumin, trypsinogen-2, immunoglobulin kappa, CD33, or human secreted alkaline phosphatase (SEAP).

[0072] Oncolytic viruses can be any natural or engineered oncolytic viruses. In some embodiments, the oncolytic virus is a vaccinia virus, herpes simplex virus, or adenovirus. In some examples, the oncolytic virus is a vaccinia virus, e.g., a Western Reserve strain of vaccinia virus. In specific examples, the oncolytic virus is a vaccinia virus having modifications to the gene encoding thymidine kinase (TK) and the gene encoding viral growth factor (VGF). For example, the modifications may be a complete deletion of the gene, a partial deletion of the gene, insertion of a heterologous nucleic acid sequence into the gene, or substitution of a portion of the gene with a heterologous nucleic acid sequence (see, for example, U.S. Patent No. 7,208,313, incorporated herein by reference). In some examples, a nucleic acid sequence encoding a TGF-β monomer is inserted into the gene encoding TK or the gene encoding VGF. In specific examples, the nucleic acid sequence encodes a TGF-β monomer having a signal sequence, e.g., an IL-2 signal sequence.

[0073] Also provided herein are compositions comprising the oncolytic viruses disclosed herein and pharmaceutically acceptable carriers, diluents, and / or excipients. Also provided herein are tumor cells containing the oncolytic viruses disclosed herein, such as cancer cells, such as pancreatic cancer cells, melanoma cells, or head and neck cancer cells (such as HNSCC). Furthermore, methods are provided for treating cancer in a subject by administering a therapeutically effective amount of the oncolytic viruses or compositions disclosed herein to the subject. Also provided are methods for inhibiting tumor growth or tumor metastasis in a subject with cancer by administering a therapeutically effective amount of the oncolytic viruses or compositions disclosed herein to the subject. In some embodiments, the oncolytic viruses or compositions are administered by subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intraprostatic, or intratumoral injection. In some embodiments, cancer is breast cancer, brain cancer, pancreatic cancer, prostate cancer, skin cancer, bladder cancer, liver cancer, ovarian cancer, kidney cancer, endometrial cancer, colorectal cancer, stomach cancer, skin cancer (such as malignant melanoma), head and neck cancer, or thyroid cancer. In specific examples, cancer is melanoma, head and neck cancer, or pancreatic cancer.

[0074] IV. Oncolytic viruses Oncolytic viruses are naturally occurring or modified viruses capable of targeting and killing tumor cells. Often, oncolytic viruses designed for immunotherapy are genetically modified to enhance tumor targeting, reduce virulence to normal cells, and / or express one or more transgenes to promote an antitumor response (Raja et al., J Immunother Cancer 6:140, 2018; Zheng et al., Mol Ther Oncolytics 15:234-247, 2019). For example, oncolytic viruses can stimulate cytokines (GM-CSF, IFN-α, IFN-β, IFN-γ, IL-2, IL-12, ILK-15, IL-18, IL-21, and IL-24, etc.), chemokines (CCL5, CCL20, CCL21, DCXCL4L1, and CXCL10, etc.), tumor-associated antigens (CEA, PSA, hDCT, and CLND6, etc.), immune costimulatory molecules (e.g., CD28, ICOS, OX40, CD30, CD40, and 4-1BB), and immune checkpoint inhibitors (PD-1, It is possible to manipulate the expression of genes such as CTLA4, LAG3, and TIM3, suicide genes (e.g., HSV-TK, CD, nitroreductase, and cytochrome P450), tumor suppressor genes (e.g., p53, PTEN, p16, Rb, and MnSOD), pro-apoptotic proteins (e.g., apoptin, lactaptin, TRAIL, and SMAC), or anti-angiogenic proteins (VEGI, VEGFR-I-Ig, anti-VEGF antibodies, baculostatin, and FGFR) (for an overview, see Zheng et al., Mol Ther Oncolytics 15:234-247, 2019; also see U.S. Patent Application Publications 2019 / 0330655 and 2020 / 0000862, which are incorporated herein by reference). The oncolytic viruses disclosed herein may encode one or more of the above-mentioned transgenes, or other transgenes designed to enhance the antitumor response.

[0075] The oncolytic viruses disclosed herein may be based on any one of several different types of viruses that are known to be oncolytic or that have been modified to possess oncolytic properties. Examples of oncolytic viruses include, but are not limited to, poxviruses (such as vaccinia virus, cowpox virus, canarypox virus, and fowlpox virus), herpes simplex viruses (such as HSV-1), adenoviruses, measles viruses, reoviruses, coxsackieviruses, parvoviruses, polioviruses, rhinoviruses, varicella stomatitis virus (VSV), mumps virus, Newcastle disease virus (NDV), retroviruses, Seneca Valley viruses, or their chimeric forms (such as poliovirus / rhinovirus and adenovirus / HSV) (Raja et al., J Immunother Cancer 6:140, 2018; Zheng et al., Mol Ther Oncolytics 15:234-247, 2019).

[0076] In some embodiments of this specification, the oncolytic virus is a vaccinia virus. The vaccinia virus may be any strain, for example, a virus from Elstree, Wyeth, Copenhagen, or the Western Reserve. In some examples, the oncolytic vaccinia virus contains one or more genetic modifications, each of which plays a role in promoting viral replication in normal cells, such as mutations or deletions in the gene encoding thymidine kinase (J2R), the gene encoding ribonucleotide reductase (14L and F4L), and / or the gene encoding vaccinia virus growth factor (VGF). Other vaccinia virus genes that may be modified include the A56R gene (encoding hemagglutinin), one or more interferon regulatory genes, the B13R gene (encoding a caspase-1 inhibitor), or the F2L gene (encoding a viral dUTPase) (see, for example, U.S. Patent Application Publications 2019 / 0330655 and 2020 / 00197457, which are incorporated herein by reference).

[0077] In some embodiments, the oncolytic virus is a Western Reserve strain of vaccinia virus. In certain examples, the Western Reserve strain of vaccinia virus includes modifications to the gene encoding TK and the gene encoding VGF. For example, the modifications may be a complete deletion of the gene, a partial deletion of the gene, insertion of a heterologous nucleic acid sequence into the gene, or substitution of a portion of the gene with a heterologous nucleic acid sequence (see, for example, McCart et al., Cancer Res 61(24):8751-8757, 2001; and U.S. Patent No. 7,208,313, as incorporated herein by reference). In certain non-limiting examples, a nucleic acid sequence encoding a TGF-β monomer having an IL-2 signaling sequence is inserted into the gene encoding TK.

[0078] V. Pharmaceutical compositions and administration of oncolytic viruses Provided herein are compositions comprising oncolytic viruses encoding recombinant TGF-β monomers. The compositions are suitable for formulations and in vitro or in vivo administration. Optionally, the compositions comprise one or more of the oncolytic viruses of this disclosure and a pharmaceutically acceptable carrier. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 22 nd This is described in Edition, Loyd V. Allen et al., editors, Pharmaceutical Press (2012). A pharmaceutically acceptable carrier comprises a material that is not biologically or otherwise undesirable, for example, the material is administered to a subject without causing undesirable biological effects or adverse interactions with other components in the pharmaceutical composition in which it is contained. When administered to a subject, the carrier is selected as necessary to minimize the degradation of the active ingredient and to minimize adverse side effects in the subject.

[0079] Oncolytic viruses or their compositions are administered according to known methods, for example, by intravenous administration, such as a bolus or continuous infusion over a period of time. Administration may be local or systemic. The compositions can be administered via any of several routes of administration, including local, oral, parenteral, intravenous, intra-articular, intraperitoneal, intramuscular, intrathecal, subcutaneous, intracavitary, percutaneous, intrahepatic, intracranial, intraspinal, intraarticular, intra-articular bursa, intratumoral, nebulization / inhalation, intraprostatic, or bronchoscopy-based infusion. Thus, the compositions are administered in multiple ways depending on whether local or systemic treatment is preferred and the area being treated.

[0080] In some embodiments, the composition for administration contains the oncolytic virus described herein in a pharmaceutically acceptable carrier, such as an aqueous carrier. Various aqueous carriers, such as buffered saline, may be used. These solutions are sterile and generally do not contain undesirable substances. These compositions can be sterilized by conventional sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, toxicity adjusters, etc. (e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.) if required to approximate a physiological state. The concentration of the active agent in these formulations can vary considerably and is selected primarily based on fluid volume, viscosity, body weight, etc., according to the specific administration method and the needs of the target population.

[0081] Pharmaceutical formulations of oncolytic viruses can be prepared by mixing oncolytic viruses of a desired degree of purity with pharmaceutically acceptable carriers, excipients, or stabilizers as needed. Such formulations may be lyophilized or aqueous solutions.

[0082] Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used. Acceptable carriers, excipients, or stabilizers may include acetates, phosphates, citrates, and other organic acids; antioxidants (e.g., ascorbic acid), preservatives, low molecular weight polypeptides; proteins (e.g., serum albumin or gelatin), or hydrophilic polymers (e.g., polyvinylpyrrolidone); and amino acids, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents; as well as ionic and nonionic surfactants (e.g., polysorbate); salt-forming counterions (e.g., sodium); metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants. Oncolytic viruses can be formulated at any appropriate concentration of infectious units or viral particles.

[0083] Oncolytic viruses can be prepared, alone or in combination with other suitable components, into aerosol formulations administered by inhalation (i.e., they can be "sprayed"). The aerosol formulations can be contained within a pressurized, acceptable propellant, such as dichlorodifluoromethane, propane, or nitrogen.

[0084] Formulations suitable for parenteral administration, such as intra-articular, intravenous, intramuscular, intratumor, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solvents, concentrators, stabilizers, and preservatives. In the provided methods, the composition can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, intravesically, intratumorally, or intrathecally. In some examples, parenteral administration, intratumor administration, or intravenous administration are the methods of administration. Formulations of the compound may be shown in sealed containers, such as ampoules and vials, in unit doses or multiple doses.

[0085] Injectable solutions and suspensions can be prepared from the sterilized powders, granules, and tablets of the previously described types. The pharmaceutical preparation can be in unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate amounts of the active ingredient. Thus, the pharmaceutical composition can be administered in various unit dosage forms depending on the method of administration. For example, suitable unit dosage forms for oral administration include, but are not limited to, powders, tablets, pills, capsules, and lozenges.

[0086] In therapeutic applications, the oncolytic virus or its composition is administered to a subject in an effective amount or dose. One or multiple administrations of the composition may be administered as needed. "Patient" or "subject" includes both humans and other animals, particularly mammals. Thus, the method is applicable to both human therapy and veterinary applications.

[0087] The effective amount of the oncolytic virus is determined on a case-by-case basis and is at least partially based on the specific oncolytic virus used; the build, age, gender, and general health of the individual. For example, when administered to a human, depending on the type, size, and number of the existing proliferating cells or neoplasms, at least 10 3 plaque-forming units (PFU), such as at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , or at least 10 12 PFU of the oncolytic virus, such as approximately 10 3 ~10 12 PFU of the oncolytic virus is used. The effective amount is about 1.0 pfu / kg body weight to about 10 15 pfu / kg body weight (e.g., about 10 2 pfu / kg body weight to about 10 13The pfu / kg body weight may be used. Oncolytic viruses are administered in single doses or multiple doses (e.g., two, three, four, six, or more doses). Multiple doses may be administered simultaneously or consecutively (e.g., over several days or weeks).

[0088] In some embodiments, the provided method involves administering to a subject one or more therapeutic agents, for example, one or more agents for treating cancer, such as breast cancer, brain cancer, pancreatic cancer, prostate cancer, skin cancer, bladder cancer, liver cancer, ovarian cancer, kidney cancer, endometrial cancer, colorectal cancer, gastric cancer, skin cancer (such as malignant melanoma), head and neck cancer (such as HNSCC), or thyroid cancer.

[0089] Administration of oncolytic viruses may be accompanied by administration of other anticancer agents or therapeutic procedures (such as surgical resection of the tumor). Any suitable anticancer agent may be administered in combination with the oncolytic viruses disclosed herein. Exemplary anticancer agents include, but are not limited to, chemotherapeutic agents, e.g., mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, antisurvivators, bioresponse modifiers, antihormonal agents (e.g., antiandrogens), CDK inhibitors, and anti-angiogenic agents. Other anticancer treatments include radiotherapy and other antibodies that specifically target cancer cells (e.g., biologics).

[0090] Non-exclusive examples of alkylating agents include nitrogen mustards (such as mechloretamine, cyclophosphamide, melphalan, uracil mustard, or chlorambucil), alkyl sulfonates (such as busulfan), and nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).

[0091] Non-exclusive examples of antimetabolites include folate analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.

[0092] Non-exclusive examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (such as L-asparaginase).

[0093] Other non-exclusive examples of drugs include platinum coordination complexes (such as cis-diamine-dichloroplatinum II, also known as cisplatin), substituted ureas (such as hydroxyureas), methylhydrazine derivatives (such as procarbazine), and corticosteroid inhibitors (such as mitotane and aminoglutethimide).

[0094] Non-exclusive examples of hormones and antagonists include corticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (such as diethylstilbestrol and ethinylestradiol), antiestrogens (such as tamoxifen), and androgens (such as testosterone propionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatin, Cisplatin, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mitramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, e.g., Docetaxel), Verban, Vincristine, and VP-16. Some newer drugs include gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin, and Calcitriol.

[0095] Non-exclusive examples of immunomodulatory agents that can be used include AS-101 (Wyeth-Ayerst Labs.), bropyrimin (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte-macrophage colony-stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immunoglobulin (Cutter Biological), IMREG (Imreg of New Orleans, La.), SK&F106528, and TNF (tumor necrosis factor; Genentech).

[0096] Another common treatment for some types of cancer is surgical intervention, such as surgical removal of the cancer or a portion thereof. Another example of intervention is radiation therapy, such as the administration of radioactive materials or energy (external beam radiation therapy, for example) to the tumor site to help eradicate or shrink the tumor before surgical removal.

[0097] CDK (cyclin-dependent kinase) inhibitors are drugs that inhibit the function of CDK. Non-exclusive examples of CDK inhibitors for use in the provided method include AG-024322, AT7519, AZD5438, flavopiridol, indisulam, P1446A-05, PD-0332991, and P276-00 (see, for example, Lapenna et al., Nature Reviews, 8:547-566, 2009). Other CDK inhibitors include LY2835219, palbociclib, LEE011 (Novartis), the pan-CDK inhibitor AT7519, sericiclib, CYC065, butyrolactone I, himenialdisine, SU9516, CINK4, PD0183812, or fascapricin.

[0098] In some cases, CDK inhibitors are broad-spectrum inhibitors (such as flavopyridol, oromoucin, roscovitine, kaempaulon, SNS-032, AT7519, AG-024322, (S)-roscovitine, or R547). In other cases, CDK inhibitors are specific inhibitors (such as fascapricin, luvidine, pluvaranol A, NU2058, BML-259, SU9516, PD0332991, or P-276-00).

[0099] For example, the additional therapeutic agent may include one or more immunomodulators, e.g., a PD-1 antagonist, a PD-L1 antagonist, a CTLA4 antagonist, or a T-cell agonist (such as a 4-1BB agonist, an OX40 agonist, or a glucocorticoid-induced tumor necrosis factor (TNF) receptor (GITR) agonist), or a combination thereof. For example, the anticancer agent may include a T-cell agonist, e.g., a 4-1BB agonist, an OX40 agonist, or a GITR agonist (such as an immune checkpoint protein, e.g., a monoclonal antibody (mAb) specific to one of the above proteins, a ligand for one of these proteins, or an aptamer for one of these proteins). For example, additional therapeutic agents may include antibodies that specifically bind to and antagonize PD-1 or PD-L1, such as atezolizumab, MPDL3280A, BNS-936558 (nivolumab), pembrolizumab, pizilizumab, CT011, AMP-224, AMP-514, MEDI-0680, BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, MSB-0010718C, MGA-271, indoximod, epacadostat, BMS-986016, MEDI-4736, MEDI-4737, MK-4166, BMS-663513, PF-05082566 (PF-2566), lirirumab, or durvalumab. In one example, the additional therapeutic agent includes a 4-1BB agonist, e.g., an antibody, e.g., PF-05082566 (utomirumab), or BMS-663513 (urerumab), or a ligand (e.g., 4-1BBL or SA-4-1BBL). In another example, the additional therapeutic agent includes an OX40 agonist, e.g., an mAb (e.g., PF-04518600, MEDI6469, MEDI0562, MEDI6383, MOXR0916, BMS986178, or GSK3174998), or a ligand (e.g., OX40L).In one example, the additional therapeutic agent includes an agonist GITR, e.g., mAb, e.g., DTA-1, TRX518, MK-4166, MK-1248, AMG228, INCAGN01876, GWN323 (Novartis), CK-302 (Checkpoint Therapeutics), or BMS-986156. In another example, the additional therapeutic agent includes a GITR agonist, e.g., GITR ligand (GITRL), e.g., native GITRL, or a polyvalent GITR ligand fusion protein, e.g., MEDI1873. In yet another example, the additional therapeutic agent includes an anti-CTLA4 (e.g., ipilimumab). For example, additional therapeutic agents may include anti-EGFR (e.g., cetuximab), anti-VEGF (e.g., bevacizumab), alemtuzumab, gemtuzumab, rituximab, panitumumab, pertuzumab, trastuzumab, and / or other therapeutic monoclonal antibodies.

[0100] The selection of drugs and dosages can be determined based on the specific disease being treated. Drugs or combinations of compositions can be administered simultaneously (e.g., as a mixture), individually but simultaneously (e.g., via separate intravenous routes), or sequentially (e.g., administering one drug first, followed by the second drug). Therefore, the term "combination" is used to refer to the simultaneous, concurrent, or sequential administration of two or more drugs or compositions.

[0101] VI. Exemplary Embodiments Embodiment 1. Substitution of cysteine ​​to serine or cysteine ​​to arginine in the amino acid residue corresponding to residue 77 of human TGF-β2 as described in Sequence ID No. 2; Deletion of the α3 helix corresponding to amino acid residues 52-71 of human TGF-β2 as described in Sequence ID No. 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 25 of Sequence ID No. 2; Substitution of arginine to lysine at the amino acid residue corresponding to residue 26 of SEQ ID NO: 2; Substitution of leucine to arginine at the amino acid residue corresponding to residue 51 in SEQ ID NO: 2; Substitution of alanine to lysine at the amino acid residue corresponding to residue 74 in Sequence ID No. 2; Substitution of leucine to valine at the amino acid residue corresponding to residue 89 in SEQ ID NO: 2; Substitution of isoleucine to valine at the amino acid residue corresponding to residue 92 in Sequence ID No. 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 94 in Sequence ID No. 2; Substitution of threonine to lysine in the amino acid residue corresponding to residue 95 of SEQ ID NO: 2; and Substitution of isoleucine to valine at the amino acid residue corresponding to residue 98 in SEQ ID NO: 2 An oncolytic virus that encodes a human recombinant transforming growth factor (TGF)-β2 monomer, including [the specified component].

[0102] Embodiment 2. Substitution of leucine to arginine at the amino acid residue corresponding to residue 51 of SEQ ID NO: 2; Substitution of alanine to lysine at the amino acid residue corresponding to residue 74 in Sequence ID No. 2; Substitutions of cysteine ​​to serine or cysteine ​​to arginine in the amino acid residue corresponding to residue 77 of human TGF-β2 as described in Sequence ID No. 2; and Deletion of the α3 helix corresponding to amino acid residues 52-71 of human TGF-β2 as described in Sequence ID No. 2 An oncolytic virus that encodes a human recombinant transforming growth factor (TGF)-β monomer, including [specific component].

[0103] Embodiment 3. The oncolytic virus according to Embodiment 2, wherein the TGF-β monomer is a human TGF-β2 monomer.

[0104] Embodiment 4. The oncolytic virus according to Embodiment 3, wherein the human TGF-β2 monomer further comprises at least one amino acid substitution that enhances the monomer's affinity for TGF-β receptor II (TβRII).

[0105] Embodiment 5. The at least one amino acid substitution that enhances the affinity of the monomer to TβRII, Substitution of lysine to arginine at the amino acid residue corresponding to residue 25 of Sequence ID No. 2; Substitution of arginine to lysine at the amino acid residue corresponding to residue 26 of SEQ ID NO: 2; Substitution of leucine to valine at the amino acid residue corresponding to residue 89 in SEQ ID NO: 2; Substitution of isoleucine to valine at the amino acid residue corresponding to residue 92 in Sequence ID No. 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 94 in Sequence ID No. 2; Substitution of threonine to lysine in the amino acid residue corresponding to residue 95 of SEQ ID NO: 2; and / or Substitution of isoleucine to valine at the amino acid residue corresponding to residue 98 in SEQ ID NO: 2 The oncolytic virus according to Embodiment 4, including the following.

[0106] Embodiment 6. The human TGF-β2 monomer is Substitution of cysteine ​​to serine at the amino acid residue corresponding to residue 77 of human TGF-β2 as described in Sequence ID No. 2; Deletion of the α3 helix corresponding to amino acid residues 52-71 of human TGF-β2 as described in Sequence ID No. 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 25 of Sequence ID No. 2; Substitution of arginine to lysine at the amino acid residue corresponding to residue 26 of SEQ ID NO: 2; Substitution of leucine to arginine at the amino acid residue corresponding to residue 51 in SEQ ID NO: 2; Substitution of alanine to lysine at the amino acid residue corresponding to residue 74 in Sequence ID No. 2; Substitution of leucine to valine at the amino acid residue corresponding to residue 89 in SEQ ID NO: 2; Substitution of isoleucine to valine at the amino acid residue corresponding to residue 92 in Sequence ID No. 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 94 in Sequence ID No. 2; Substitution of threonine to lysine in the amino acid residue corresponding to residue 95 of SEQ ID NO: 2; and Substitution of isoleucine to valine at the amino acid residue corresponding to residue 98 in SEQ ID NO: 2 A tumor-lytic virus according to any one of Embodiments 1 to 5, including the above.

[0107] Embodiment 7. The oncolytic virus according to any one of Embodiments 1 to 6, wherein the amino acid sequence of the human TGF-β2 monomer includes or consists of SEQ ID NO: 7.

[0108] Embodiment 8. The oncolytic virus according to any one of Embodiments 1 to 5, wherein the human TGF-β2 monomer comprises, or further comprises, at least one amino acid substitution that reduces aggregation and / or improves folding of the monomer.

[0109] Embodiment 9. The human TGF-β2 monomer is Substitution of cysteine ​​to valine at the amino acid residue corresponding to residue 7 in SEQ ID NO: 2; Substitution of cysteine ​​to alanine at the amino acid residue corresponding to residue 16 of SEQ ID NO: 2; Substitution of cysteine ​​to arginine at the amino acid residue corresponding to residue 77 of SEQ ID NO: 2; and / or Substitution of valine to arginine at the amino acid residue corresponding to residue 79 in SEQ ID NO: 2 The oncolytic virus according to Embodiment 8, including the following:

[0110] Embodiment 10. The human TGF-β2 monomer is Deletion of the α3 helix corresponding to amino acid residues 52-71 of human TGF-β2 as described in Sequence ID No. 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 25 of Sequence ID No. 2; Substitution of arginine to lysine at the amino acid residue corresponding to residue 26 of SEQ ID NO: 2; Substitution of leucine to arginine at the amino acid residue corresponding to residue 51 in SEQ ID NO: 2; Substitution of alanine to lysine at the amino acid residue corresponding to residue 74 in Sequence ID No. 2; Substitution of cysteine ​​to arginine at the amino acid residue corresponding to residue 77 in SEQ ID NO: 2; Substitution of valine to arginine at the amino acid residue corresponding to residue 79 in SEQ ID NO: 2; Substitution of leucine to valine at the amino acid residue corresponding to residue 89 in SEQ ID NO: 2; Substitution of isoleucine to valine at the amino acid residue corresponding to residue 92 in Sequence ID No. 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 94 in Sequence ID No. 2; Substitution of threonine to lysine in the amino acid residue corresponding to residue 95 of SEQ ID NO: 2; and Substitution of isoleucine to valine at the amino acid residue corresponding to residue 98 in SEQ ID NO: 2 The oncolytic virus according to Embodiment 9, including the following:

[0111] Embodiment 11. The oncolytic virus according to Embodiment 10, wherein the amino acid sequence of the human TGF-β2 monomer includes or consists of SEQ ID NO: 9.

[0112] Embodiment 12. The human TGF-β2 monomer is Substitution of cysteine ​​to serine at the amino acid residue corresponding to residue 77 in Sequence ID No. 2; Deletion of the α3 helix corresponding to amino acid residues 52-71 of human TGF-β2 as described in Sequence ID No. 2; Substitution of cysteine ​​to valine at the amino acid residue corresponding to residue 7 in SEQ ID NO: 2; Substitution of cysteine ​​to alanine at the amino acid residue corresponding to residue 16 of SEQ ID NO: 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 25 of Sequence ID No. 2; Substitution of leucine to arginine at the amino acid residue corresponding to residue 51 in SEQ ID NO: 2; Substitution of alanine to lysine in the amino acid residue corresponding to residue 74 of SEQ ID NO: 2; and Substitution of lysine to arginine at the amino acid residue corresponding to residue 94 in SEQ ID NO: 2 The oncolytic virus according to Embodiment 9, including the following:

[0113] Embodiment 13. The oncolytic virus according to Embodiment 12, wherein the amino acid sequence of the human TGF-β2 monomer includes or consists of SEQ ID NO: 10.

[0114] Embodiment 14. The human TGF-β2 monomer is Deletion of the α3 helix corresponding to amino acid residues 52-71 of human TGF-β2 as described in Sequence ID No. 2; Substitution of cysteine ​​to valine at the amino acid residue corresponding to residue 7 in SEQ ID NO: 2; Substitution of cysteine ​​to alanine at the amino acid residue corresponding to residue 16 of SEQ ID NO: 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 25 of Sequence ID No. 2; Substitution of arginine to lysine at the amino acid residue corresponding to residue 26 of SEQ ID NO: 2; Substitution of leucine to arginine at the amino acid residue corresponding to residue 51 in SEQ ID NO: 2; Substitution of alanine to lysine at the amino acid residue corresponding to residue 74 in Sequence ID No. 2; Substitution of cysteine ​​to arginine at the amino acid residue corresponding to residue 77 in SEQ ID NO: 2; Substitution of valine to arginine at the amino acid residue corresponding to residue 79 in SEQ ID NO: 2; Substitution of leucine to valine at the amino acid residue corresponding to residue 89 in SEQ ID NO: 2; Substitution of isoleucine to valine at the amino acid residue corresponding to residue 92 in Sequence ID No. 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 94 in Sequence ID No. 2; Substitution of threonine to lysine in the amino acid residue corresponding to residue 95 of SEQ ID NO: 2; and Substitution of isoleucine to valine at the amino acid residue corresponding to residue 98 in SEQ ID NO: 2 The oncolytic virus according to Embodiment 9, including the following:

[0115] Embodiment 15. The oncolytic virus according to Embodiment 13, wherein the amino acid sequence of the human TGF-β2 monomer includes or consists of SEQ ID NO: 12.

[0116] Embodiment 16. An oncolytic virus encoding a human recombinant transforming growth factor (TGF)-β2 monomer, wherein the amino acid sequence of the TGF-β2 monomer includes or consists of SEQ ID NO: 11.

[0117] Embodiment 17. The oncolytic virus according to Embodiment 1, wherein the TGF-β monomer is a human TGF-β1 monomer.

[0118] Embodiment 18. The human TGF-β1 monomer is Substitution of isoleucine to arginine at the amino acid residue corresponding to residue 52 in SEQ ID NO: 1; Substitution of alanine to lysine at the amino acid residue corresponding to residue 74 of Sequence ID No. 1; Substitution of alanine to serine at the amino acid residue corresponding to residue 75 of SEQ ID NO: 1; or Substitutions of isoleucine to arginine at the amino acid residue corresponding to residue 52 of SEQ ID NO: 1, substitution of alanine to lysine at the amino acid residue corresponding to residue 74, and substitution of alanine to serine at the amino acid residue corresponding to residue 75. The oncolytic virus according to Embodiment 17, further comprising the above.

[0119] Embodiment 19. The oncolytic virus according to Embodiment 17 or Embodiment 18, wherein the amino acid sequence of the human TGF-β1 monomer includes or consists of SEQ ID NO: 4.

[0120] Embodiment 20. The oncolytic virus according to Embodiment 2, wherein the TGF-β monomer is a human TGF-β3 monomer.

[0121] Embodiment 21. The human TGF-β3 monomer is Substitution of leucine to glutamic acid in the amino acid residue corresponding to residue 51 of SEQ ID NO: 3; Substitution of alanine to glutamic acid at the amino acid residue corresponding to residue 72 in SEQ ID NO: 3; Substitution of alanine to aspartic acid in the amino acid residue corresponding to residue 74 of SEQ ID NO: 3; or Substitutions of leucine to glutamate in the amino acid residue corresponding to residue 51 of SEQ ID NO: 3, substitution of alanine to glutamate in the amino acid residue corresponding to residue 72, and substitution of alanine to aspartate in the amino acid residue corresponding to residue 74. The oncolytic virus according to Embodiment 20, further comprising the above.

[0122] Embodiment 22. The oncolytic virus according to Embodiment 20 or Embodiment 21, wherein the amino acid sequence of the human TGF-β3 monomer includes or consists of SEQ ID NO: 6.

[0123] Embodiment 23. The oncolytic virus according to any one of Embodiments 1 to 22, wherein the TGF-β monomer further comprises a signal sequence.

[0124] Embodiment 24. The oncolytic virus according to Embodiment 23, wherein the signal sequence is an IL-2 signal sequence containing the amino acid sequence of SEQ ID NO: 8.

[0125] Embodiment 25. An oncolytic virus according to any one of Embodiments 1 to 24, which is a vaccinia virus, herpes simplex virus, or adenovirus.

[0126] Embodiment 26. A vaccinia virus, which is an oncolytic virus according to any one of Embodiments 1 to 25.

[0127] Embodiment 27. The oncolytic virus according to Embodiment 26, wherein the vaccinia virus comprises a modification of the gene encoding thymidine kinase (TK) and a modification of the gene encoding viral growth factor (VGF).

[0128] Embodiment 28. The oncolytic virus according to Embodiment 27, wherein the modification of the gene encoding the TK includes a complete or partial deletion of the gene.

[0129] Embodiment 29. The oncolytic virus according to Embodiment 27 or Embodiment 28, wherein at least a portion of the TK gene is replaced with a nucleic acid encoding the TGF-β monomer.

[0130] Embodiment 30. The oncolytic virus according to any one of Embodiments 27 to 29, wherein the modification of the gene encoding VGF includes a complete or partial deletion of the gene.

[0131] Embodiment 31. A composition comprising an oncolytic virus according to any one of Embodiments 1 to 30 and a pharmaceutically acceptable carrier.

[0132] 3. Embodiment 2. A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of an oncolytic virus described in any one of Embodiments 1 to 30 or a composition described in Embodiment 31.

[0133] Embodiment 33. A method for inhibiting tumor growth or metastasis in a subject having cancer, comprising administering to the subject a therapeutically effective amount of an oncolytic virus described in any one of Embodiments 1 to 30 or a composition described in Embodiment 31.

[0134] Embodiment 34. The method according to Embodiment 32 or Embodiment 33, wherein the cancer is melanoma, head and neck cancer, or pancreatic cancer.

[0135] The following examples are provided to illustrate certain features and / or embodiments. These examples should not be construed as limiting the disclosure to the specific features or embodiments described. [Examples]

[0136] (Example 1) Recombinant TGF-β minimonomer Recombinant human TGF-β proteins that cannot form dimers were designed as described in WO2018 / 094173 (which is incorporated herein by reference in its entirety). Specifically, recombinant human TGF-β1, human TGF-β2, and human TGF-β3 proteins were created with deletions of α-helix 3 and several adjacent residues (corresponding to residues 52-71 of each protein), and a cysteine-to-serine substitution at residue 77. The deletion length was chosen to leave a sufficient number of residues between the last residue of β-chain 4 (Gly-48) and the first residue of β-chain 5 (Cys-77 / Ser-77) to form an unrestricted loop that bridges β-chains 4 and 5. In addition, either two (TGF-β2) or three (TGF-β1 and -β3) loop-forming residues were substituted to increase the total net charge at pH 7.0 for the full-length TGF-β1, -β2, and -β3 monomers from -0.9, +1.1, and +4.4 to -3.1, +3.9, and +6.1, respectively. TGF-β proteins with these modifications are referred to herein as “minimonomers” and are denoted as mmTGF-β1, mmTGF-β2, and mmTGF-β3. An additional TGF-β2 minimonomer with seven amino acid substitutions to enhance its affinity for the TGF-β2 receptor (TβRII) was designed; this minimonomer is referred to herein as “mmTGF-β2-7M” or “dnTGFβ2 mm It is called "[name]". Table 1 provides a description of wild-type TGF-β and the variant TGF-β minimonomers disclosed herein. The protein sequences are listed (and shown below) in SEQ ID NOs: 1-7. The positions of single amino acid substitutions and deletions are relative to the corresponding wild-type TGF-β proteins.

[0137] [Table 1]

[0138] Sequence ID 1 - TGF-β1 ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS Sequence ID 2 - TGF-β2 ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSKVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS Sequence ID 3 - TGF-β3 ALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS Sequence ID 4 - mmTGF-β1 ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYRASKSPSCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS Sequence ID 5 - mmTGF-β2 ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYRASKSPSCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS Sequence ID 6 - mmTGF-β3 ALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYEESDSPSCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS Sequence ID 7 - mmTGF-β2-7M ALDAAYCFRNVQDNCCLRPLYIDFRKDLGWKWIHEPKGYNANFCAGACPYRASKSPSCVSQDLEPLTIVYYVGRKPKVEQLSNMIVKSCKCS

[0139] In some embodiments, each of the above sequences includes an N-terminal methionine (M) residue.

[0140] In some embodiments described herein, the TGF-β monomer includes an N-terminal signal sequence such as the IL-2 signal sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 8).

[0141] (Example 2) Metabolic modulation of the tumor microenvironment using oncolytic viruses The immune system faces numerous barriers within the tumor microenvironment, the tumor's unique metabolic landscape being central to these challenges (Scharping and Delgoffe, Vaccines (Basel) 4(4):46, 2016). As cancer cells proliferate, they deplete the local environment of nutrients and oxygen, leading to the accumulation of toxic byproducts such as lactic acid. Therefore, infiltrating immune cells must withstand both immune and metabolic suppression within the tumor microenvironment (Najjar et al., JCI Insight 4(5):e124989, 2019). Metabolic support is considered crucial for curative immunotherapy of cancer. T cells infiltrating tumors can do so even at a significant metabolic disadvantage, suppressing their ability to take up glucose and losing functional mitochondrial mass (Scharping et al., Immunity 45(2):374-388, 2016). In addition, several immunotherapy modalities can be improved through metabolic means, including mitochondrial reprogramming of adoptive cell therapy (Scharping et al., Immunity 45(2):374-388, 2016), as well as pharmacological (Scharping et al., Cancer Immunol Res 5(1):9-16, 2016) and immunotherapeutic (Menk et al., J Exp Med 215(4):1091-1100, 2018) metabolic enhancement of checkpoint blockade. Oncolytic virus immunotherapy can be improved by genetically encoding metabolic modulators rather than immunostimulants (such as GM-CSF). Genes encoding leptin, an adipokin that specifically enhances leptin expression in the tumor microenvironment, have been inserted into oncolytic vaccinia viruses (Rivadeneira et al., Immunity 51(3):548-560, 2019). Tumor-infiltrating T cells express high levels of leptin receptors, and therefore receive metabolic reprogramming signals when treated with this virus.Indeed, leptin was found to act on novel tumor-infiltrating T cells in mouse melanoma and pancreatic cancer models, enhancing mitochondrial activity and promoting robust anti-tumor immunity and long-term memory. Furthermore, during the course of these studies, the complete characterization of immunoinfiltrates induced by oncolytic viruses was described for the first time by scRNA-seq. These studies revealed that while oncolytic viruses induce robust remodeling of the immunological environment in cancer, these novel T cell exogenes still experience immunological suppression via TGF-β signaling, as well as metabolic suppression through exposure to hypoxia and sustained antigenic stimulation.

[0142] The examples disclosed herein describe how oncolytic viruses can be used to deliver metabolic modulation to the tumor microenvironment. Oncolytic viruses can “transduce” into tumor cells they infect, and the drugs themselves can be used to deliver gene therapy to tumors. The oncolytic form of the Western Reserve strain of vaccinia virus (lacking thymidine kinase and viral growth factor genes) (McCart et al., Cancer Res 61(24):8751-8757, 2001) was used for these studies. Vaccinia virus is an effective oncolytic virus because it encodes its own polymerase, replicates in the cytoplasm, replicates well under hypoxic conditions, and its large genome means it can be well engineered to express multiple transgenes (Yang et al., J Cancer Res Clin Oncol 144(12):2433-2440, 2018).

[0143] We used scRNAseq to confirm the direct consequences of oncolytic virus infection (Rivadeneira et al., Immunity 51(3):548-560, 2019). Specifically, we profiled CD45+ infiltrates 7 days after infection with oncolytic VV (oVV) or PBS control (Figures 1A and 1B). This analysis revealed significant remodeling of the tumor microenvironment and showed that CD45+ infiltrates were dominated by clonally distinct novel effector / memory T cells (Figure 1C, and Rivadeneira et al., Immunity 51(3):548-560, 2019). However, the novel infiltrates were ultimately ineffective, as tumors treated with this virus eventually escaped and grew unrestrained. Therefore, oVV can stimulate tumors and promote novel infiltrates, but other inhibitory mechanisms exist during its action. A deeper analysis of the 7-day infiltrates revealed that these T cells were effector / memory-like, but also contained transcriptome signatures consistent with TGF-β signaling (Figure 1D). Furthermore, metabolic analysis revealed that these T cells still succumbed to insufficient metabolism (Figure 1E) (Rivadeneira et al., Immunity 51(3):548-560, 2019).

[0144] Therefore, while oncolytic viruses can stimulate tumor inflammation and T cell infiltration, their novel infiltrations, primarily T cell-driven, encounter metabolic and immunological barriers that prevent a complete response. In the following examples, oncolytic viruses are used to express gene constructs that act to mitigate these barriers.

[0145] (Example 3) Manipulated targeting of TGF-β signaling within the tumor microenvironment using delivery of novel TGF-β inhibitors via oncolytic viruses. TGF-β represents a potent immunosuppressive signal within cancer cells and acts to inhibit antitumor immunity through multiple pathways (Ungefroren, Expert Opin Ther Targets 23(8):679-693, 2019; Derynck and Budi, Sci Signal 12(570):eaav5183, 2019). The signature of TGF-β signaling was very clear even in oVV-induced infiltrates, so we have taken steps to create drugs that can target the TGF-β-enhanced suppressive environment. However, despite its attractive profile as a target, safe and effective TGF-β inhibitors remain elusive (Connolly et al., Int J Biol Sci 8(7):964-978, 2012; Akhurst, Cold Spring Harb Perspect Biol 9(10):a022301, 2017). As a pluripotent cytokine, systemically inhibiting its activity carries some risk, and kinase inhibitors targeting receptor activity have significant off-target effects (Connolly et al., Int J Biol Sci 8(7):964-978, 2012; Akhurst, Cold Spring Harb Perspect Biol 9(10):a022301, 2017). Potent genetically encoded inhibitors are thought to be well-received when encoded by oncolytic viruses, as the drugs are expressed only by infected tumor cells and therefore confined to the tumor microenvironment.

[0146] With this objective in mind, we evaluated the antitumor activity of a genetically encoded TGF-β inhibitor (Kim et al., J Biol Chem 292(17):7173-7188, 2017). This inhibitor is an engineered TGF-β2 molecule that lacks cysteine, which is crucial for disulfide-mediated dimerization (and therefore exists as a monomer), but also interferes with the recruitment of TGF-β receptor I (TβRI) by replacing the "heel" helix with an inverted loop (see Example 1). Thus, this "mini-monomer" binds strongly to TGF-β receptor II (TβRII) as a monomer in a one-to-one ratio, interfering with the recruitment of TβRI. In this way, it acts as a dominant-negative (Figure 2A) (Kim et al., J Biol Chem 292(17):7173-7188, 2017). In fact, the minimonmer binds to the receptor with high affinity and lacks its own signaling activity (Figure 2B). In addition, it potently inhibits TGF-β1, TGF-β2, and TGF-β3 signaling in reporter cell lines (Figure 2B). An engineered construct containing the IL-2 signaling peptide (SEQ ID NO: 8) was cloned into the TK locus of oncolytic vaccinia virus. Viral infection of tumor cells induced the expression of minimonmeric TGF-β (Figure 3A), and the supernatant from these tumor cells suppressed TGF-β signaling (Figure 3B). When used as a treatment for B16 melanoma, VV-dnTgfb2 mm This induced a stronger antitumor response (Figure 3C).

[0147] (Example 4) Oncolytic virus-coding TGF-β inhibitor (VV-dnTgfb2 mm Immunological and environmental consequences of ) Targeting TGF-β as a pleomorphic cytokine can influence many factors within the tumor microenvironment (Derynck and Budi, Sci Signal 12(570):eaav5183, 2019). Therefore, several orthogonal approaches were used to understand the immunological consequences of viral delivery TGF-β inhibition. For these studies, melanoma and pancreatic cancer cell lines were used as immunologically active models compared to inactive tumors. Mice with B16 or clone 24 (cell lines derived from melanoma generated in the Pten / Braf mouse model; Najjar et al., JCI Insight 4(5):e124989, 2019) melanoma, or Panc02 pancreatic tumors were subjected to control oncolytic virus or dnTGFβ2 mm 2.5 × 10 of any of the viruses that express 6 PFU is injected into the tumor.

[0148] The "geography" of the tumor microenvironment is investigated using highly multiplexed imaging techniques utilizing oligotagged antibodies (co-detection by indexing, or CODEX) and nucleotide addition imaging, which involves repeatedly imaging dozens of antibodies within a single section (Goltsev et al., Cell 174(4):968-981, 2018). This technique is used to examine the location and status of multiple immune and stromal subsets. Anti-vaccinia and anti-phosphoSmad3 antibodies also enable the identification of infected cells and the evaluation of their effects on in situ TGF-β signaling.

[0149] scRNAseq is used to determine the cellular and transcriptional changes induced by this viral manipulation. Whole tumor homogenates or CD45+ enriched fractions are used and compared to control oncolytic virus and control injections using a 10× Genomics Chromium controller (Rivadeneira et al., Immunity 51(3):548-560, 2019).

[0150] These findings were confirmed using highly multiplexed flow cytometry, and further queries were conducted on VV-dnTgfb2. mm The quality of tumor infiltrates after therapy will be immunologically profiled and functionally evaluated.

[0151] (Example 5) VV-dnTgfb2 in clone 24 and Panc02 models mm Treatment results The therapeutic efficacy of TGF-β-targeted oncolytic viruses will be tested in injectable melanoma (clone 24) and pancreatic cancer (Panc02) models that respond partially to oVV but more completely to immunologically / metabolic engineered strains (Rivadeneira et al., Immunity 51(3):548-560, 2019). Mice with 3-4 mm tumors will be injected with VV-dnTgfb2 mm or 2.5 × 10⁶ of either of the control oncolytic viruses 6 A single intratumoral injection of PFU is administered. Tumor growth is tracked using digital calipers, and survival is calculated based on IACUC guidelines at the time of euthanasia (when the tumor reaches 15 mm in either direction).

[0152] In mice experiencing a complete response, the parental tumor (untreated with virus) is reinjected after maintaining the surviving mice for 3–4 weeks. Simultaneously, the parental tumor is injected into a second cohort of untreated mice. When the mice have immunological memory of the tumor, they either reject the tumor or resist tumor growth compared to the untreated mouse cohort.

[0153] Additional experiments involve multiple virus injections and use a tumor model in which mice have two identical tumors but only one is injected with the virus.

[0154] (Example 6) Additional modifications of TGF-β2 monomers for in vivo administration This example describes a study evaluating modifications that enhance the in vivo delivery of TGF-β monomers. Four variants of mmTGF-β2-7M were prepared and are listed in Table 2. The positions of single amino acid substitutions and deletions are compared to those of human TGF-β2 as described in Sequence ID No. 2.

[0155] [Table 2]

[0156] Sequence ID 9 - mmTGF-β2-7M2R ALDAAYCFRNVQDNCCLRPLYIDFRKDLGWKWIHEPKGYNANFCAGACPYRASKSPRCRSQDLEPLTIVYYVGRKPKVEQLSNMIVKSCKCS Sequence ID 10 - mmTGF-β2-2M-Del8-17 ALDAAYVFRNVQDNCALRPLYIDFRRDLGWKWIHEPKGYNANFCAGACPYRASKSPSCVSQDLEPLTILYYIGRTPKIEQLSNMIVKSCKCS Sequence ID 11 - mmTGF-β2-7M-PRDC KEVLASSQEALVVTERKYLKSDWCKLRPLYIDFRKDLGWKWIHEPKGYNANFCYGQCNSFYIPRHVKKEEDSFQSSAFCVSQDLEPLTIVYYVGRKPKVEQLSNMIVKSCRCMSV Sequence ID 12 - mmTGF-β2-7M2R-Del8-17 ALDAAYVFRNVQDNCALRPLYIDFRKDLGWKWIHEPKGYNANFCAGACPYRASKSPRCRSQDLEPLTIVYYVGRKPKVEQLSNMIVKSCKCS

[0157] In some embodiments, each of the above sequences includes an N-terminal methionine (M) residue.

[0158] Disappearance or reduction of aggregation tendency We first investigated modifications that eliminate or reduce the aggregation tendency of mmTGF-β2-7M. While it had previously been shown that the modified mmTGF-β2-7M monomer has a significantly lower aggregation tendency than wild-type TGF-β2, mmTGF-β2-7M nevertheless retains some aggregation tendency (Kim et al., J Biol Chem 292(17):7173-7188, 2017). This was recorded in two dimensions, either in the absence (Figure 5D, 5E) or in the presence (Figure 5F) of the unmodified washing agent 3-[(3-coramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). 1 H- 15 The amide skeleton detected by N NMR shift correlation (HSQC, heteronuclide single quantum correlation) spectroscopy 1 H- 15 This was also evident from the appearance of the N signal. In the absence of CHAPS, the skeletal amide signal fluctuated greatly in intensity, and was partially detectable, particularly at pH 7.2, where it is known that protein solubility is reduced compared to pH 4.6. This type of fluctuation in signal intensity is caused by the transient formation of higher-order aggregates. The formation of such aggregates occurs over the protein's rotational correlation time (τ). c This extends the NMR signal, thus broadening it and decreasing the signal intensity. It has been observed that increasing concentrations of CHAPS at either pH 4.6 or 7.2 result in improvements in the intensity of many signals, and therefore increased uniformity of these in the observed spectra (Figure 5F). The improvement in signal intensity is dependent on the concentration of CHAPS, with substantial improvements occurring up to a concentration of approximately 10 mM.

[0159] We hypothesized that the role of CHAPS in reducing aggregation of mmTGF-β2-7M may be due to the transient formation of aggregation through certain hydrophobic residues that remain in a molecular region often described as the base of the fingers, which were previously part of the dimer interface in wild-type TGF-β homodimers (Figure 4A). After testing several substitutions that were found to have little effect on aggregation formation, we found that the substitution of two residues in mmTGF-β2-7M with arginine—S57R and V59R (Figure 4B)—resulted in a significant reduction in the tendency to aggregate. This variant of mmTGF-β2-7M in which two residues are replaced with arginine is called mmTGF-β2-7M2R (SEQ ID NO: 9). Evidence of the reduced tendency to aggregate was the observation of a considerably more uniform NMR signal intensity for this variant, regardless of pH or whether undenatured CHAPS was added (Figures 5A-5C).

[0160] Modifications to improve folding TGF-β proteins are formed from monomers classified as possessing a cystine knot growth factor fold (CKGF) (Hinck et al., Cold Spring Harb Prospect Biol 8(12):a022103, 2016). This fold is present in all proteins of the TGF-β family, but is also found in many other signaling proteins and signaling protein antagonists in humans. These include the signaling proteins platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and nerve growth factor (NGF), as well as antagonists such as noggin, sclerostin, and Dan and Cerubus-related proteins (PRDC). TGF-β family proteins are unique among CKGF proteins in that they all possess an N-terminal prodomain. While the role of prodomains is still under investigation, it is well known that they play a regulatory role in many protein families (Hinck et al., Cold Spring Harb Prospect Biol 8(12):a022103, 2016). This regulation arises from the prodomain binding to the growth factor domain (GFD) with an affinity (from nanomolar to sub-nanomolar concentrations) sufficient to completely block the GFD's ability to bind to type I and type II receptors. Only some prodomains, such as those of TGF-β1, TGF-β2, and TGF-β3, bind to the GFD with very high affinity, maintaining them in an inactive (latent) state until they are activated, which is also necessary for proper GFD folding. The GFD of TGF-β, like that of other CKGF proteins, is characterized by a cystine knot, a structural motif stabilized by three disulfides (Schwarz, Biol Chem 398(12): 1295-1308, 2017). The three disulfides are spatially very close to each other, and therefore their formation is complex, with many possible alternative topological configurations existing in addition to the exact one.

[0161] This is related to mmTGF-β2-7M because it retains a cystine knot and one additional disulfide known as the 8-17 disulfide (corresponding to cysteine ​​at residues 7 and 16 compared to SEQ ID NO: 2). One method of producing the mmTGF-β2-7M protein is to express it in bacteria in the form of insoluble inclusion bodies and refold the protein to form the native pair of disulfides (Huang and Hinck, Methods Mol Biol 1344:63-92, 2016). Nevertheless, the overall folding yield is limited by the aggregates formed as a result of misfolding of its eight cysteine ​​residues and improper pairing. The mmTGF-β2-7M protein can also be produced by expressing the protein as a secreted protein in eukaryotic hosts. This allows the protein to pass through the endoplasmic reticulum (ER) and Golgi apparatus, and thus facilitate folding by endogenous disulfide exchange and glycosylation mechanisms, as well as by eukaryotic cell-specific chaperones to promote the folding of disulfide-rich proteins. However, attempts to use this method for the expression of mmTGF-β2-7M resulted in the formation of misfolded disulfide-binding aggregates. This likely occurred because mmTGF-β2-7M was so dramatically modified compared to wild-type TGF-β that it was predicted not to bind to and interact with its prodomain.

[0162] Therefore, we investigated modifications aimed at improving the folding of mmTGF-β2-7M. To improve folding, each of the four disulfides of mmTGF-β2-7M was removed at a rate of one disulfide at a time. To do this, the two cysteines forming each disulfide were replaced with valine-alanine pairs, and then the modified protein was expressed, refolded, and purified according to the previous procedure (Kim et al., J Biol Chem 292(17):7173-7188, 2017). To enhance the possibility of achieving a naturally folded protein, substitutions were made in the context of the engineered TGF-β2 monomer, but instead of the seven substitutions in the case of mmTGF-β2-7M, only two essential residues were changed to TGF-β1 residues. Because TGF-β2 is known to fold with much higher efficiency than TGF-β1, these variants were expected to still bind to TβRII with high affinity but with improved efficiency (Huang and Hinck, Methods Mol Biol 1344:63-92, 2016). The results showed that, in this background, the variant with cysteine ​​forming the 8-17 disulfide substituted with valine and alanine, designed as mmTGF-β2-2M-Del8-17 (SEQ ID NO: 10; Figure 4C), folded naturally (Figures 6A-6C), while the variant with three other disulfides, with 16-59, 45-90, and 49-92 removed, exhibited unnatural folding. In the absence of CHAPS, there were significantly significant fluctuations in NMR signal intensity suggesting aggregation, but these differences were attenuated upon addition of CHAPS (Figures 6A-6C). The fact that 8-17 disulfides can be removed without disrupting protein folding suggests that this can lead to a significant improvement in folding, regardless of whether the protein is produced in bacteria and refolded in vitro, or whether it is produced as a secreted protein in eukaryotic cells.

[0163] The third type of modification investigated also aimed to improve the folding of mmTGF-β2-7M. The chosen strategy was to take advantage of the fact that some CKGF proteins exist that are naturally produced as monomers and do not possess a prodomain for folding, or are prodomain-independent, such as the bone morphogenetic protein (BMP) antagonist PRDC. To take advantage of the potential improvement in PRDC folding while retaining high-affinity TβRII binding, a chimeric mmTGF-β2-7M:PRDC construct was created in which the finger 1-2 and 3-4 regions of mmTGF-β2-7M, which are the regions responsible for TβRII binding, were grafted onto the cystine knot region of PRDC. This construct, named mmTGF-β2-7M-PRDC (SEQ ID NO: 11; Figure 4D), was expressed in E. coli and refolded in a manner similar to that used for mmTGF-β27M (Kim et al., J Biol Chem 292(17):7173-7188, 2017), and then purified to homogeneity using high-resolution cation exchange chromatography. Through NMR analysis, this protein was found to be: 1 It was shown to fold naturally, as evidenced by the dispersion of amide signals well outside the random coil region corresponding to 7.9–8.5 ppm in the H dimension (Figures 7A–7C). This indicates that the cystine knot region of PRDC was well incorporated together with the finger region of mmTGF-β2-7M, and that the design was successful.

[0164] Binding properties of mmTGF-β2-7M variant A prerequisite for any designed mmTGF-β2-7M variant to be functional in cells and in vivo is that it binds to TβRII with high affinity. To evaluate the ability of the mmTGF-β2-7M variants described herein (mmTGF-β2-7M2R (SEQ ID NO: 9), mmTGF-β2-2M-Del8-17 (SEQ ID NO: 10), and mmTGF-β2-7M-PRDC (SEQ ID NO: 11)) to bind to TβRII, isothermal titration calorimetry (ITC) and unmodified gels were used. ITC binding experiments were performed by injecting increasing amounts of TβRII into mmTGF-β2-7M2R (SEQ ID NO: 9) or mmTGF-β2-2M-Del8-17 (SEQ ID NO: 10), with mmTGF-β2-7M (SEQ ID NO: 7) used as a reference control. These titrations yielded easily detectable isotherms with large negative enthalpy and nearly 1:1 bond stoichiometry (Figure 8). Fitting the integrated heat to the 1:1 bond model yielded dissociation constants (K) for the TβRII bond of 75.1 nM and 80.1 nM for mmTGF-β2-7M2R and mmTGF-β2-2M-Del8-17, respectively. D ) resulted (Figure 8). These K D The value determined for mmTGF-β2-7M (60.5 nM) was within the experimental error range, indicating that the substitutions introduced to reduce aggregation or improve folding did not have a detrimental effect on the protein's ability to bind to TβRII.

[0165] Alternatively, the binding of mmTGF-β2-7M-PRDC (SEQ ID NO: 11) was evaluated using an undenatured gel. Since the detection of the complex requires the two proteins to remain bound for a timescale equivalent to the electrophoresis timescale of approximately 1 hour, these showed high affinity binding, but K DThis does not provide quantitative measurement. In the undenatured gel, mmTGF-β2-7M, mmTGF-β2-7M2R, and mmTGF-β2-7M-PRDC all formed bands that moved approximately one-quarter of the gel length, while TβRII showed migration across almost the entire length of the gel (Figure 7D). This, along with previous findings that mmTGF-β2-7M, mmTGF-β2-7M2R, and mmTGF-β2-7M-PRDC do not enter the gel individually, suggests that all three of these proteins bind to TβRII with high affinity. This is consistent with ITC results for mmTGF-β2-7M and mmTGF-β2-7M2R variants and also indicates that this is true for mmTGF-β2-7M-PRDC.

[0166] Inhibitory properties of mmTGF-β2-7M variant For any designed mmTGF-β2-7M variant to be functional in vivo, it should inhibit TGF-β signaling in cells. To evaluate this for the mmTGF-β2-7M variants of this disclosure, mmTGF-β2-7M2R (SEQ ID NO: 9), mmTGF-β2-2M-Del8-17 (SEQ ID NO: 10), and mmTGF-β2-7M-PRDC (SEQ ID NO: 11), we used the HEK-293 TGF-β luciferase reporter cell line, in which cells were stably transfected with a TGF-β CAGA enhancer element fused to a luciferase reporter gene. To evaluate the inhibitory activity of this assay, cells were seeded in 96-well plates, and various concentrations of mmTGF-β2-7M2R, mmTGF-β2-2M-Del8-17, and mmTGF-β2-7M-PRDC were added, with mmTGF-β2-7M used as a control. After 30 minutes, TGF-β signaling was stimulated by adding TGF-β3 at a final concentration of 10 pM. After 12 hours, cells were lysed and luciferase activity was evaluated. The results showed that mmTGF-β2-7M2R, mmTGF-β2-2M-Del8-17, and mmTGF-β2-7M-PRDC each strongly inhibited TGF-β3-induced signaling, and fitted IC50 was obtained. 50The values ​​were 53 nM, 111 nM, and 283 nM, respectively. The values ​​for mmTGF-β2-7M2R and mmTGF-β2-2M-Del8-17 were both within twice the value measured for mmTGF-β2-7M, which indicates that these proteins are both approximately as effective as mmTGF-β2-7M (IC). 50 It was shown that it is 58nM. Furthermore, the IC of mmTGF-β2-7M-PRDC is more powerful. 50 The value is 283 nM, which is about one-fifth of that of mmTGF-β2-7M. This indicates that while mmTGF-β2-7M-PRDC is a functional TGF-β inhibitor, its potency may be slightly impaired by some small change in the orientation of the two finger regions.

[0167] summary The mmTGF-β2-7M variants disclosed herein have substitutions that reduce their aggregation tendency and increase their folding tendency. Each mmTGF-β2-7M variant has been shown to retain the ability to bind to TβRII with high affinity and to potently inhibit TGF-β3 signaling in cultured cells. Therefore, the mmTGF-β2-7M variants of this disclosure possess attributes that improve their ability to be administered in vivo and thus provide novel pathways for therapeutic interventions to attenuate the progression of TGF-β-mediated diseases.

[0168] (Example 7) Oncolytic vaccinia viruses expressing the mmTGF-β2 variant show superior efficacy in cancer resistance models. Vaccinia virus (VV) expressing the mmTGFβ2 variant mmTGF-β2-7M2R-Del8-17 (SEQ ID NO: 12) (also known as "variant 1" or "var1") was tested in two cancer resistance models. The first model was a MEER subclone (HPV+HNSCC) of the head and neck squamous cell carcinoma (HNSCC) cell line, which is resistant to oncolytic virus therapy. The second model was a melanoma cell line clone 24 (CL24 melanoma; Pten-deficient BrafV600E This condition does not respond to immunotherapy such as oncolytic viruses or anti-PD1.

[0169] In the first test, C57 / BL6J mice were given 1 × 10⁶ 5 Individual MEER cells were subcutaneously inoculated, and after 7 days, mice were given either control VV or mmTGF-β2-7M2R-Del8-17(VV). mmTGFβ(var1) 2.5 × 10¹⁶ of any VV modified to express (SEQ ID NO: 12) 5 PFU was injected intratumorally. As shown in Figure 10 (top row), the control virus had a slight curative effect, but VV mmTGFβ(var1) Half of the mice treated with the drug showed a complete response and demonstrated a long-lasting survival benefit.

[0170] In the second test, C57 / BL6J mice were given 1 × 10⁶ 5 Individual CL24 cells were subcutaneously inoculated, and after 7 days, mice were inoculated with either a VV control or a VV control. mmTGFβ(var1) 2.5 × 10 5 PFU was administered. As shown in Figure 10 (center column), targeting TGFβ resulted in a complete response in 40% of animals in this invasive melanoma model. Addition of anti-PD1 enhanced the tumor inhibitory effect in the CL24 model (Figure 10, bottom column) and resulted in a significant synergistic effect among the inhibition of VV, anti-PD1, and TGFβ.

[0171] In view of the many possible embodiments to which the principles of the subject matter of this disclosure can be applied, it should be recognized that these exemplary embodiments are merely examples of this disclosure and should not be construed as limiting the scope of this disclosure. Rather, the scope of this disclosure is defined by the following claims. Accordingly, the inventors claim all that is encompassed within the scope of these claims and the technical idea. In certain embodiments, for example, the following are provided: (Item 1) An oncolytic virus encoding a human recombinant transforming growth factor (TGF)-β2 monomer, wherein the human TGF-β2 monomer is Substitutions of cysteine ​​to serine or cysteine ​​to arginine in the amino acid residue corresponding to residue 77 of human TGF-β2 as described in Sequence ID No. 2; Deletion of the α3 helix corresponding to amino acid residues 52-71 of human TGF-β2 as described in Sequence ID No. 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 25 of Sequence ID No. 2; Substitution of arginine to lysine at the amino acid residue corresponding to residue 26 of SEQ ID NO: 2; Substitution of leucine to arginine at the amino acid residue corresponding to residue 51 in SEQ ID NO: 2; Substitution of alanine to lysine at the amino acid residue corresponding to residue 74 in Sequence ID No. 2; Substitution of leucine to valine at the amino acid residue corresponding to residue 89 in SEQ ID NO: 2; Substitution of isoleucine to valine at the amino acid residue corresponding to residue 92 in Sequence ID No. 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 94 in Sequence ID No. 2; Substitution of threonine to lysine in the amino acid residue corresponding to residue 95 of SEQ ID NO: 2; and Substitution of isoleucine to valine at the amino acid residue corresponding to residue 98 in SEQ ID NO: 2 Oncolytic viruses, including [specific virus name]. (Item 2) An oncolytic virus encoding a human recombinant transforming growth factor (TGF)-β monomer, wherein the human TGF-β2 monomer is Substitution of leucine to arginine at the amino acid residue corresponding to residue 51 in SEQ ID NO: 2; Substitution of alanine to lysine at the amino acid residue corresponding to residue 74 in Sequence ID No. 2; Substitutions of cysteine ​​to serine or cysteine ​​to arginine in the amino acid residue corresponding to residue 77 of human TGF-β2 as described in Sequence ID No. 2; and Deletion of the α3 helix corresponding to amino acid residues 52-71 of human TGF-β2 as described in Sequence ID No. 2 Oncolytic viruses, including [specific virus name]. (Item 3) The oncolytic virus described in item 2, wherein the TGF-β monomer is a human TGF-β2 monomer. (Item 4) The oncolytic virus according to item 2, wherein the human TGF-β2 monomer further comprises at least one amino acid substitution that enhances the affinity of the monomer for TGF-β receptor II (TβRII). (Item 5) The at least one amino acid substitution that enhances the affinity of the monomer to TβRII is Substitution of lysine to arginine at the amino acid residue corresponding to residue 25 of Sequence ID No. 2; Substitution of arginine to lysine at the amino acid residue corresponding to residue 26 of SEQ ID NO: 2; Substitution of leucine to valine at the amino acid residue corresponding to residue 89 in SEQ ID NO: 2; Substitution of isoleucine to valine at the amino acid residue corresponding to residue 92 in Sequence ID No. 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 94 in Sequence ID No. 2; Substitution of threonine to lysine in the amino acid residue corresponding to residue 95 of SEQ ID NO: 2; and / or Substitution of isoleucine to valine at the amino acid residue corresponding to residue 98 in SEQ ID NO: 2 Oncolytic viruses, including those listed in item 4. (Item 6) The aforementioned human TGF-β2 monomer, Substitution of cysteine ​​to serine at the amino acid residue corresponding to residue 77 of human TGF-β2 as described in Sequence ID No. 2; Deletion of the α3 helix corresponding to amino acid residues 52-71 of human TGF-β2 as described in Sequence ID No. 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 25 of Sequence ID No. 2; Substitution of arginine to lysine at the amino acid residue corresponding to residue 26 of SEQ ID NO: 2; Substitution of leucine to arginine at the amino acid residue corresponding to residue 51 in SEQ ID NO: 2; Substitution of alanine to lysine at the amino acid residue corresponding to residue 74 in Sequence ID No. 2; Substitution of leucine to valine at the amino acid residue corresponding to residue 89 in SEQ ID NO: 2; Substitution of isoleucine to valine at the amino acid residue corresponding to residue 92 in Sequence ID No. 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 94 in Sequence ID No. 2; Substitution of threonine to lysine in the amino acid residue corresponding to residue 95 of SEQ ID NO: 2; and Substitution of isoleucine to valine at the amino acid residue corresponding to residue 98 in SEQ ID NO: 2 Oncolytic viruses, including those listed in item 1. (Item 7) The oncolytic virus described in item 6, wherein the amino acid sequence of the human TGF-β2 monomer includes or consists of SEQ ID NO: 7. (Item 8) The oncolytic virus according to item 1, wherein the human TGF-β2 monomer comprises, or further comprises, at least one amino acid substitution that reduces aggregation and / or improves folding of the monomer. (Item 9) The aforementioned human TGF-β2 monomer, Substitution of cysteine ​​to valine at the amino acid residue corresponding to residue 7 in SEQ ID NO: 2; Substitution of cysteine ​​to alanine at the amino acid residue corresponding to residue 16 of SEQ ID NO: 2; Substitution of cysteine ​​to arginine at the amino acid residue corresponding to residue 77 of SEQ ID NO: 2; and / or Substitution of valine to arginine at the amino acid residue corresponding to residue 79 in SEQ ID NO: 2 Oncolytic viruses, including those listed in item 8. (Item 10) The aforementioned human TGF-β2 monomer, Deletion of the α3 helix corresponding to amino acid residues 52-71 of human TGF-β2 as described in Sequence ID No. 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 25 of Sequence ID No. 2; Substitution of arginine to lysine at the amino acid residue corresponding to residue 26 of SEQ ID NO: 2; Substitution of leucine to arginine at the amino acid residue corresponding to residue 51 in SEQ ID NO: 2; Substitution of alanine to lysine at the amino acid residue corresponding to residue 74 in Sequence ID No. 2; Substitution of cysteine ​​to arginine at the amino acid residue corresponding to residue 77 in SEQ ID NO: 2; Substitution of valine to arginine at the amino acid residue corresponding to residue 79 in SEQ ID NO: 2; Substitution of leucine to valine at the amino acid residue corresponding to residue 89 in SEQ ID NO: 2; Substitution of isoleucine to valine at the amino acid residue corresponding to residue 92 in Sequence ID No. 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 94 in Sequence ID No. 2; Substitution of threonine to lysine in the amino acid residue corresponding to residue 95 of SEQ ID NO: 2; and Substitution of isoleucine to valine at the amino acid residue corresponding to residue 98 in SEQ ID NO: 2 Oncolytic viruses, including those listed in item 9. (Item 11) The oncolytic virus described in item 10, wherein the amino acid sequence of the human TGF-β2 monomer includes or consists of SEQ ID NO: 9. (Item 12) The aforementioned human TGF-β2 monomer, Substitution of cysteine ​​to serine at the amino acid residue corresponding to residue 77 in Sequence ID No. 2; Deletion of the α3 helix corresponding to amino acid residues 52-71 of human TGF-β2 as described in Sequence ID No. 2; Substitution of cysteine ​​to valine at the amino acid residue corresponding to residue 7 in SEQ ID NO: 2; Substitution of cysteine ​​to alanine at the amino acid residue corresponding to residue 16 of SEQ ID NO: 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 25 of Sequence ID No. 2; Substitution of leucine to arginine at the amino acid residue corresponding to residue 51 in SEQ ID NO: 2; Substitution of alanine to lysine in the amino acid residue corresponding to residue 74 of SEQ ID NO: 2; and Substitution of lysine to arginine at the amino acid residue corresponding to residue 94 in SEQ ID NO: 2 Oncolytic viruses, including those listed in item 9. (Item 13) The oncolytic virus described in item 12, wherein the amino acid sequence of the human TGF-β2 monomer includes or consists of SEQ ID NO: 10. (Item 14) The aforementioned human TGF-β2 monomer, Deletion of the α3 helix corresponding to amino acid residues 52-71 of human TGF-β2 as described in Sequence ID No. 2; Substitution of cysteine ​​to valine at the amino acid residue corresponding to residue 7 in SEQ ID NO: 2; Substitution of cysteine ​​to alanine at the amino acid residue corresponding to residue 16 of SEQ ID NO: 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 25 of Sequence ID No. 2; Substitution of arginine to lysine at the amino acid residue corresponding to residue 26 of SEQ ID NO: 2; Substitution of leucine to arginine at the amino acid residue corresponding to residue 51 in SEQ ID NO: 2; Substitution of alanine to lysine at the amino acid residue corresponding to residue 74 in Sequence ID No. 2; Substitution of cysteine ​​to arginine at the amino acid residue corresponding to residue 77 in SEQ ID NO: 2; Substitution of valine to arginine at the amino acid residue corresponding to residue 79 in SEQ ID NO: 2; Substitution of leucine to valine at the amino acid residue corresponding to residue 89 in SEQ ID NO: 2; Substitution of isoleucine to valine at the amino acid residue corresponding to residue 92 in Sequence ID No. 2; Substitution of lysine to arginine at the amino acid residue corresponding to residue 94 in Sequence ID No. 2; Substitution of threonine to lysine in the amino acid residue corresponding to residue 95 of SEQ ID NO: 2; and Substitution of isoleucine to valine at the amino acid residue corresponding to residue 98 in SEQ ID NO: 2 Oncolytic viruses, including those listed in item 9. (Item 15) The oncolytic virus according to item 13, wherein the amino acid sequence of the human TGF-β2 monomer includes or consists of SEQ ID NO: 12. (Item 16) An oncolytic virus encoding a human recombinant transforming growth factor (TGF)-β2 monomer, wherein the amino acid sequence of the TGF-β2 monomer includes or consists of SEQ ID NO: 11. (Item 17) The oncolytic virus described in item 2, wherein the TGF-β monomer is a human TGF-β1 monomer. (Item 18) The aforementioned human TGF-β1 monomer, Substitution of isoleucine to arginine at the amino acid residue corresponding to residue 52 in SEQ ID NO: 1; Substitution of alanine to lysine at the amino acid residue corresponding to residue 74 of Sequence ID No. 1; Substitution of alanine to serine at the amino acid residue corresponding to residue 75 of SEQ ID NO: 1; or Substitutions of isoleucine to arginine at the amino acid residue corresponding to residue 52 of SEQ ID NO: 1, substitution of alanine to lysine at the amino acid residue corresponding to residue 74, and substitution of alanine to serine at the amino acid residue corresponding to residue 75. Furthermore, the oncolytic viruses listed in item 17, including the following. (Item 19) The oncolytic virus described in item 18, wherein the amino acid sequence of the human TGF-β1 monomer includes or consists of SEQ ID NO: 4. (Item 20) The oncolytic virus described in item 2, wherein the TGF-β monomer is a human TGF-β3 monomer. (Item 21) The aforementioned human TGF-β3 monomer, Substitution of leucine to glutamic acid in the amino acid residue corresponding to residue 51 of SEQ ID NO: 3; Substitution of alanine to glutamic acid at the amino acid residue corresponding to residue 72 in SEQ ID NO: 3; Substitution of alanine to aspartic acid in the amino acid residue corresponding to residue 74 of SEQ ID NO: 3; or Substitutions of leucine to glutamate in the amino acid residue corresponding to residue 51 of SEQ ID NO: 3, substitution of alanine to glutamate in the amino acid residue corresponding to residue 72, and substitution of alanine to aspartate in the amino acid residue corresponding to residue 74. Furthermore, the oncolytic viruses listed in item 20, including the following. (Item 22) The oncolytic virus described in item 21, wherein the amino acid sequence of the human TGF-β3 monomer includes or consists of SEQ ID NO: 6. (Item 23) The oncolytic virus described in item 1, wherein the TGF-β monomer further comprises a signal sequence. (Item 24) The oncolytic virus described in item 23, wherein the signal sequence is an IL-2 signal sequence containing the amino acid sequence of sequence number 8. (Item 25) A vaccinia virus, herpes simplex virus, or adenovirus, which is an oncolytic virus as described in item 1. (Item 26) Vaccinia virus, an oncolytic virus as described in item 1. (Item 27) The oncolytic virus described in item 26, wherein the vaccinia virus comprises modifications of the gene encoding thymidine kinase (TK) and modifications of the gene encoding viral growth factor (VGF). (Item 28) The oncolytic virus described in item 27, wherein the modification of the gene encoding the TK includes a complete or partial deletion of the gene. (Item 29) The oncolytic virus described in item 28, wherein at least a portion of the TK gene is replaced with a nucleic acid encoding the TGF-β monomer. (Item 30) The oncolytic virus described in item 27, wherein the modification of the gene encoding VGF includes a complete or partial deletion of the gene. (Item 31) A composition comprising the oncolytic virus described in item 1 and a pharmaceutically acceptable carrier. (Item 32) A method for treating cancer in a subject, comprising administering a therapeutically effective amount of the composition described in item 31 to the subject. (Item 33) A method for inhibiting tumor growth or metastasis in a subject having cancer, comprising administering a therapeutically effective amount of the composition described in item 31 to the subject. (Item 34) The method according to item 32, wherein the cancer is melanoma, head and neck cancer, or pancreatic cancer.

Claims

1. An oncolytic virus encoding a human recombinant transforming growth factor (TGF)-β2 monomer, wherein the amino acid sequence of the human recombinant TGF-β2 monomer includes or consists of SEQ ID NO:

9.

2. An oncolytic virus encoding a human recombinant transforming growth factor (TGF)-β2 monomer, wherein the amino acid sequence of the human recombinant TGF-β2 monomer includes or consists of SEQ ID NO:

10.

3. An oncolytic virus encoding a human recombinant transforming growth factor (TGF)-β2 monomer, wherein the amino acid sequence of the human recombinant TGF-β2 monomer includes or consists of SEQ ID NO:

12.

4. An oncolytic virus encoding a human recombinant transforming growth factor (TGF)-β2 monomer, wherein the amino acid sequence of the human recombinant TGF-β2 monomer includes or consists of SEQ ID NO:

11.

5. The oncolytic virus according to any one of claims 1 to 4, wherein the human recombinant TGF-β2 monomer further comprises a signal sequence.

6. The oncolytic virus according to claim 5, wherein the signal sequence is an IL-2 signal sequence comprising the amino acid sequence of SEQ ID NO:

8.

7. The oncolytic virus according to any one of claims 1 to 4, which is a vaccinia virus, herpes simplex virus, or adenovirus.

8. A vaccinia virus, the oncolytic virus according to any one of claims 1 to 4.

9. The oncolytic virus according to claim 8, wherein the vaccinia virus comprises modifications of a gene encoding thymidine kinase (TK) and a gene encoding viral growth factor (VGF).

10. The oncolytic virus according to claim 9, wherein the modification of the gene encoding TK includes a complete or partial deletion of the gene.

11. The oncolytic virus according to claim 10, wherein at least a portion of the TK gene is replaced with a nucleic acid encoding the human recombinant TGF-β2 monomer.

12. The oncolytic virus according to claim 9, wherein the modification of the gene encoding VGF includes a complete or partial deletion of the gene.

13. A composition comprising an oncolytic virus according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

14. The composition according to claim 13 for treating cancer in a subject.

15. The composition according to claim 13 for inhibiting tumor growth or tumor metastasis in a subject having cancer.

16. The composition according to claim 14, wherein the cancer is melanoma, head and neck cancer, or pancreatic cancer.

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