Nucleic acids encoding tgf-beta inhibitor and IL-12 and uses thereof

Abstract

The present disclosure provides nucleic acids encoding a transforming growth factor inhibitor and IL-12. Also provided herein are nucleic acids encoding the chemokine receptor CXCR3. Additionally described herein are oncolytic viruses comprising the nucleic acids described herein. The use of the compositions described herein in the treatment of cancer is also described.

Description

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 417,487, filed Oct. 19, 2022, and U.S. Provisional Application No. 63 / 471,811, filed Jun. 8, 2023, both of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application includes a sequence listing that has been electronically submitted in ST.26 xml format and is hereby incorporated by reference in its entirety. The xml copy created on Sep. 28, 2023 is named 199249-720601_SL.xml and is 110,350 bytes in size.

[0005] Background

[0006] Cancer is a major source of disease, especially in developed countries. Transforming growth factor β (“TGF-β”, “TGF-beta” or “TGF-b”) is highly overexpressed in many cancer cell types and provides a means of evading the host immune response. Accordingly, there is a need for compositions and methods for targeting and modulating TGF-b activity in the microenvironment.

[0007] Brief Description

[0008] Compositions are described herein, where the composition comprises: a vector, where the vector comprises: an exogenous nucleic acid comprising a sequence encoding a cytokine or a functional variant thereof; an exogenous nucleic acid comprising a sequence encoding a chemokine receptor or a functional variant thereof; and a first promoter region, where the first promoter region is upstream of the sequence encoding the chemokine receptor and provides for expression of the chemokine receptor prior to expression of the cytokine.

[0009] Nucleic acids are described herein, where the nucleic acid comprises sequences encoding at least two polypeptides, where the at least two polypeptides comprise: interleukin-12 (IL-12) or a functional variant thereof; and an inhibitor of transforming growth factor β (TGF-β) activity.

[0010] Nucleic acids are described herein, where the nucleic acid comprises: a first region encoding a first polypeptide comprising a sequence having at least 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:12 or SEQ ID NO:15; and a second region encoding a second polypeptide comprising a sequence having at least 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:40 or SEQ ID NO:41.

[0011] This text describes nucleic acids, wherein the nucleic acid molecule comprises: an insert containing, in 5' to 3' order at the A52R locus: a first promoter region, wherein the promoter comprises the A52R promoter; an insert encoding a first region of human CXCR3; an insert containing, in 5' to 3' order at the TK locus: a second promoter region, wherein the promoter comprises P135; a second region encoding human IL-12; a third promoter region, wherein the promoter comprises P7.5; and a third region encoding a TGFβ variant.

[0012] This text describes nucleic acids, wherein the nucleic acid molecule comprises: an insert containing the sequence listed in SEQ ID NO:88 at the A52R locus; an insert containing the sequence listed in SEQ ID NO:85 at the TK locus.

[0013] This text describes a pharmaceutical composition, wherein the pharmaceutical composition comprises: a nucleic acid as described herein or a vector as described herein; and a pharmaceutically acceptable excipient.

[0014] This text describes a method for treating cancer, comprising administering to a subject having cancer a pharmaceutical composition as described herein in an amount sufficient to treat cancer.

[0015] This text describes a method for activating an anti-tumor immune response, comprising administering to a subject having cancer a pharmaceutical composition as described herein.

[0016] This text describes a method for reducing the incidence of tumor cell growth, comprising: administering to tumor cells a pharmaceutical composition as described herein in an effective amount sufficient to reduce the incidence of tumor cell growth. Brief Description of the Drawings

[0018] The novel features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description which illustrates illustrative embodiments utilizing the principles of the present disclosure and the accompanying drawings, in which:

[0019] Figure 1A and Figure 1B shows the interaction of TGF-β receptor subunits I (RI) and II (RII) with native and modified TGF-β ligands. Figure 1A Illustrates the binding of unmodified TGF-β to RII and RI to produce a receptor complex. Figure 1B Shows the binding of modified TGF-β as described herein to RII, without the ability to bind to RI, thereby preventing the formation of a receptor complex.

[0020] Figure 2A diagram showing an expression construct having a P7.5 promoter driving the expression of an IL-12 polypeptide and a P28 promoter driving the expression of a TGFbf polypeptide, wherein the IL-12 polypeptide comprises IL-12β and α subunits linked by a 22-residue glycine-rich linker, and the TGFbf polypeptide comprises a signal peptide of interleukin 2 (IL-2sig) fused to a modified TGF-β variant, the modified TGF-β variant having cysteines 8 and 17 mutated to valine and arginine, respectively.

[0021] Figure 3A A line graph showing the number of days post-treatment on the x-axis and the average volume of induced Renca cell tumors on the y-axis in mice treated with buffer control, TK-control, or viruses expressing murine IL-12, TGFbf, or both IL-12 and TGFbf.

[0022] Figure 3B A line graph showing the number of days post-treatment on the x-axis and the average volume of induced B16 cell tumors on the y-axis in mice treated with buffer control, TK-control, or viruses expressing murine IL-12, TGFbf, or both IL-12 and TGFbf.

[0023] Figure 4A A line graph showing the probability of survival on the y-axis over time in days on the x-axis in mice with induced Renca cell tumors treated with PBS (1); TK-control (2); or viruses expressing murine IL-12 (3), both murine IL-12 and TGFbf1 (4), or TGFbf1 (5).

[0024] Figure 4B A line graph showing the probability of survival on the y-axis over time in days on the x-axis in mice with induced B16 cell tumors treated with PBS (1); TK-control (2); or viruses expressing murine IL-12 (3), both murine IL-12 and TGFbf1 (4), or TGFbf1 (5).

[0025] Figure 5 A diagram showing an expression construct having an A52R promoter driving the expression of CXCR3.

[0026] Figure 6 A diagram showing an expression construct having a P135 promoter driving the expression of a human IL-12 polypeptide and a P7.5 promoter driving the expression of a TGF-β variant TGF-bv2, the human IL-12 polypeptide comprising IL-12β and α subunits linked by a 22-residue glycine-rich linker.

[0027] Figure 7A figure showing inserts of sequences that express CXCR3 at the A52R locus and sequences that express IL-12 and a TGF-β inhibitor at the tyrosine kinase locus.

[0028] Figure 8A One-dimensional histograms of FACS analysis of Hela cells infected with vaccinia virus modified to express CXCR3, IL-12, and a TGF-β inhibitor (TGFBi / IL-12 / CXCR3); infected with a TK-control virus (CTRL); or uninfected (-), showing increased detection of CXCR3 expressed in Hela cells infected with the modified virus.

[0029] Figure 8B - Figure 8E A bar graph showing the migration of the indicated populations of peripheral blood mononuclear cells (PBMC) towards the CXCR3 ligand CXCL11 after infection with vaccinia virus modified to express CXCR3, IL-12, and a TGF-β inhibitor (TGFBi / IL-12 / CXCR3); infection with a TK-control virus (CTRL); or uninfection (-). Figure 8B Illustrates the migration of CD4 cells. Figure 8C Illustrates the migration of CD8 cells. Figure 8D Illustrates the migration of monocytes. Figure 8E Illustrates the migration of B cells.

[0030] Figure 8F A bar graph depicting the quantitative ELISA detection of IL-12 in the Hela supernatant of cells infected with vaccinia virus modified to express CXCR3, IL-12, and a TGF-β inhibitor; infected with a TK-control virus (CTRL); or uninfected (-), showing expression detectable only in the supernatant of Hela cells infected with the modified virus.

[0031] Figure 8G A photograph of a Western blot from Hela lysates infected with vaccinia virus modified to express CXCR3, IL-12, and a TGF-β inhibitor; infected with a TK-control virus (CTRL); or uninfected (-), showing a 12 kDa band corresponding to the TFG-β inhibitor, which is detectable only in lysates from cells infected with the modified virus.

[0032] Figure 9AShows the trace of fluorescence incorporation in cell generations of CD8 T cells infected with vaccinia virus modified to express CXCR3, IL-12, and TGF-β inhibitor (TGFBi / IL-12 / CXCR3); infected with TK-control virus (CTRL); or uninfected (-) and treated with 0 ng / ml, 10 ng / ml, or 50 ng / ml TGF-β1, as indicated by the peaks, showing that infection with the modified virus inhibits the inhibition by TGF-β.

[0033] Figure 9B Provides a FACS analysis plot of CD8 cells screened for CD44 and granzyme B (GZMB) after infection with vaccinia virus modified to express CXCR3, IL-12, and TGF-β inhibitor (TGFBi / IL-12 / CXCR3); infected with TK-control virus (CTRL); or uninfected (-), showing an increase in the expression of GZMB in cells infected with the modified virus.

[0034] Figure 10A Is a bar graph showing the PFU / ml detected in human lung adenocarcinoma (A549) cells, human cervical cancer (Hela) cells, and human foreskin fibroblasts (HFF) after infection with vaccinia virus modified to express CXCR3, IL-12, and TGF-β inhibitor, showing an increase in the virus in Hela cells.

[0035] Figure 10B Is a graph showing the copies of viral genome detected per mg of tumor in RENCA tumors after infection with vaccinia virus modified to express CXCR3, IL-12, and TGF-β inhibitor compared to the unmodified virus, showing an increase in virus count in tumors infected with the modified virus.

[0036] Figure 11A and Figure 11B Is the graph of tumor size within 51 days in EMT6 ( Figure 11A ) and MC38 ( Figure 11B ) tumor models infected with vaccinia virus modified to express CXCR3, IL-12, and TGF-β inhibitor (TGFBi / IL-12 / CXCR3); infected with TK-control virus (CTRL); or uninfected (-), showing almost complete inhibition of tumor growth in tumors infected with the modified virus.

[0037] Figure 11C and Figure 11D Is the graph of tumor size within 51 days in EMT6 infected with vaccinia virus modified to express CXCR3, IL-12, and TGF-β inhibitor (TGFBi / IL-12 / CXCR3); infected with TK-control virus (CTRL); or uninfected (-)Figure 11C ) and MC38( Figure 11D ) Graph of the probability of survival in a tumor model, showing an increased probability of survival in tumors infected with the modified virus.

[0038] Figure 12A Photographs of RENCA and MC38 tumor samples prepared after treatment with CD3, CD8, and nuclear staining after infection with vaccinia virus modified to express CXCR3, IL-12, and TGF-β inhibitor (TGFBi / IL-12 / CXCR3); infection with TK-control virus (CTRL); or no infection (-), showing increased infiltration of CD3 and CD8 cells into the tumor after infection with the modified virus.

[0039] Figure 12B - Figure 12E Bar graph of the total counts of CD3+ and CD8+ T cells in RENCA and MC38 tumor samples after infection with vaccinia virus modified to express CXCR3, IL-12, and TGF-β inhibitor (TGFBi / IL-12 / CXCR3); infection with TK-control virus (CTRL); or no infection (-). Figure 12B Shows increased CD3+ cells in RENCA tumors infected with the modified virus. Figure 12C Shows increased CD8+ cells in RENCA tumors infected with the modified virus. Figure 12D Shows increased CD3+ cells in MC38 tumors infected with the modified virus. Figure 12E Shows increased CD8+ cells in MC38 tumors infected with the modified virus.

[0040] Figure 13A Heat map showing the relative expression levels of type II interferon gamma (INFG)-related genes in cells treated with vaccinia virus modified to express CXCR3, IL-12, and TGF-β inhibitor and control cells compared to the overall average, showing increased expression of INFG-related genes in cells treated with the modified virus.

[0041] Figure 13B Heat map showing the relative expression levels of TGF-β1-related genes in cells treated with vaccinia virus modified to express CXCR3, IL-12, and TGF-β inhibitor and control cells compared to the overall average, showing increased expression of TGF-β1-related genes in cells treated with the modified virus.

[0042] Details

[0043] TGF-β1 (TGFB1)-mediated immune resistance is one of the major mechanisms of immunosuppression used across multiple tumor types. The immune resistance conferred by TGFB1 can be mediated by its pleiotropic effects on the vasculature, fibrogenesis, and regulatory / effector immune cells within the tumor microenvironment. Blocking TGFB1 with a TGF-β inhibitor (TGFBi) can improve the response to immunotherapy. In addition, IL-12 is a cytokine that can promote type 1 inflammatory responses, M1 macrophage skewing, and effector CD8 T cell responses through IFNg induction. Combining TGFB1 blockade with IL-12 can enhance the therapeutic benefit by simultaneously reducing immunosuppression and enhancing the anti-tumor immune response. In addition, CXCR3 expression from a viral backbone can enhance systemic viral delivery to tumors rich in CXCR3 ligands.

[0044] Current TGF-β inhibitors pose challenges in terms of specificity and delivery. For example, small molecule receptor kinase inhibitors (SMRKIs) that target receptors are prone to poor specificity and selectivity. Antibodies and receptor traps have been reported to be difficult to penetrate into dense tissues such as tumors. As a solution to these problems, the TGF-β variants provided herein offer specific receptor targeting and improved tissue penetration. The small size of the polypeptide allows for increased tissue penetration. In addition, as described herein, delivery can be further enhanced by providing nucleic acids encoding TGF-β inhibitors in a delivery vehicle. In addition, as described herein, delivery of a combination of TGF-β inhibitor and IL-12 shows greater overall response and survival.

[0045] The present disclosure provides compositions and methods for vaccinia-based immunotherapy that combine enhanced systemic viral delivery to tumors rich in CXCR3 ligands with locally expressed IL-12 and TGFBi within the tumor microenvironment. In some embodiments, the methods include the treatment of cancer. The compositions described herein can comprise one or more nucleic acids encoding polypeptides as described herein. The nucleic acids provided herein can comprise DNA, RNA, nucleic acid analogs, or any combination thereof. In short, the present disclosure describes (1) compositions for expressing TGFb inhibitors and IL-12, (2) combinations of nucleic acids, (3) compositions for expressing chemokine receptors, (4) vectors for expressing modified nucleic acids, (5) modified oncolytic viruses, (6) conditions being treated, and (7) the dosage, form, and method of administration of the compositions described herein.

[0046] Definitions

[0047] The terms used herein are for the purpose of describing particular situations only and are not intended to be limiting. Unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" as used herein include plural forms. In addition, to the extent that the terms "contain", "containing", "include", "including", "having", "has", "with" or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising".

[0048] The term "about" or "approximately" may mean within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measuring system. Where a particular value is described in this application and the claims, unless otherwise stated, the term "about" should be assumed to mean an acceptable error range for the particular value, such as ±10% of the value modified by the term "about".

[0049] As used herein, the term "heterologous nucleic acid sequence" or "exogenous nucleic acid sequence" or "transgene" associated with a particular virus may refer to a nucleic acid sequence derived from a source other than the designated virus.

[0050] As used herein, the term "mutation" may refer to a deletion, insertion of a heterologous nucleic acid, inversion or substitution, including an open reading frame that eliminates a mutation as commonly understood in the art.

[0051] As used herein, the term "gene" may refer to a nucleic acid segment (also referred to as a "coding sequence" or "coding region") that encodes a separate protein or RNA, optionally together with associated regulatory regions such as promoters, operators, terminators, etc., which may be located upstream or downstream of the coding sequence.

[0052] As used herein, a "promoter" can be a control sequence that is a region of a nucleic acid sequence that controls transcription initiation and transcription rate. In certain embodiments, a promoter can contain genetic elements at which regulatory proteins and molecules can bind, such as RNA polymerase and other transcription factors. The terms "operably positioned," "operably linked," "under control," and "under transcriptional control" can mean that the promoter is in the correct functional position and / or orientation relative to a nucleic acid sequence to control the initiation and / or expression of transcription of that sequence. In certain embodiments, a promoter can be used in combination with an "enhancer" or can be used without an "enhancer," where an "enhancer" refers to a cis-acting regulatory sequence that participates in the transcriptional activation of a nucleic acid sequence.

[0053] As used herein, the term "homology" can be a calculation of the "homology" or "percent homology" between two or more nucleic acid or amino acid sequences, which can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced into the sequence of the first sequence). The nucleic acids at the corresponding positions can then be compared, and the percent identity between the two sequences can be a function of the number of positions that are the same in both sequences (i.e., % homology = number of identical positions / total number of positions × 100). For example, if a position in the first sequence can be occupied by the same nucleic acid as the corresponding position in the second sequence, the molecules are identical at that position. The percent homology between two sequences can be a function of the number of positions that are the same in both sequences, where the number of gaps introduced for optimal alignment of the two sequences and the length of each gap are taken into account. In some embodiments, the length of the sequences aligned for comparison purposes can be at least about: 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference sequence. A search can determine the homology between two sequences. The homology can be between the entire lengths of the two sequences or between portions of the entire lengths of the two sequences. The two sequences can be genes, nucleic acid sequences, protein sequences, peptide sequences, amino acid sequences, or fragments thereof. The actual comparison of the two sequences can be done by well-known methods, e.g., using a mathematical algorithm. When using the BLAST and Gapped BLAST programs, any relevant parameters of the corresponding programs (e.g., NBLAST) can be used. For example, the parameters for sequence comparison can be set to score = 100, word length = 12, or can be varied (e.g., W = 5 or W = 20). Other examples include the algorithms of Myers and Miller, CABIOS (1989), ADVANCE, ADAM, BLAT, and FASTA.

[0054] The term "subject" can refer to an animal, including but not limited to a primate (e.g., human), cow, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms "subject" and "patient" are used interchangeably herein when referring to mammalian subjects such as human subjects.

[0055] The terms "treat", "treating", and "treatment" can be intended to include alleviating or eliminating a disorder, disease, or condition, or one or more symptoms associated with the disorder, disease, or condition; or alleviating or eradicating the cause itself of the disorder, disease, or condition.

[0056] The term "therapeutically effective amount" can refer to an amount of a compound that, when administered, is sufficient to prevent the development of one or more symptoms of the disorder, disease, or condition being treated or to alleviate it to some extent.

[0057] As used herein, the term "oncolytic" can refer to killing cancer cells or tumor cells by an agent, such as an oncolytic poxvirus, such as oncolytic vaccinia virus, e.g., the killing is by directly lysing the cells, such as by stimulating an immune response against the cells, apoptosis, expression of toxic proteins, autophagy, and termination of protein synthesis, inducing anti-tumor immunity, or any combination thereof. The direct lysis of cancer cells or tumor cells infected by an agent such as oncolytic vaccinia virus can be the result of viral replication within the cells. In certain instances, the term "oncolytic" can refer to killing cancer cells or tumor cells without lysing the cells.

[0058] As used herein, the term "oncolytic virus" can refer to a virus that preferentially infects and kills tumor cells. In some embodiments, the oncolytic virus can include but is not limited to: (i) a virus that naturally preferentially replicates in cancer cells and is generally non-pathogenic in humans due to increased sensitivity to innate antiviral signaling or dependence on oncogenic signaling pathways; and (ii) a virus that has been genetically engineered for use. In some embodiments, the oncolytic virus can be a measles virus, poliovirus, poxvirus, vaccinia virus, adenovirus, adeno-associated virus, herpes simplex virus, vesicular stomatitis virus, reovirus, Newcastle disease virus, Seneca virus, lentivirus, Mengo virus, or myxoma virus. In certain embodiments, the oncolytic virus can be a poxvirus. In certain embodiments, the oncolytic virus can be vaccinia virus.

[0059] As used herein, the term "modified oncolytic virus" can refer to an oncolytic virus that contains modifications to its components, such as but not limited to modifications in the native genome ("backbone") of the virus, such as mutations or deletions of viral genes, introduction of exogenous nucleic acids, chemical modifications of viral nucleic acids or viral proteins, and introduction of exogenous proteins or modified viral proteins into the viral capsid. Generally, oncolytic viruses can be modified (also referred to as "engineered") to obtain improved therapeutic effects against tumor cells. In some embodiments, the modified oncolytic virus can be a modified poxvirus. In some embodiments, the modified oncolytic virus can be a modified poxvirus. In some embodiments, the modified oncolytic virus can be a modified vaccinia virus.

[0060] The terms "systemic delivery" and "systemic administration," which are used interchangeably herein, can in some cases refer to the route of administering a drug, oncolytic virus, or other substance into the circulatory system. Systemic administration can include oral administration, intraperitoneal administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, intraarterial administration, or any combination thereof.

[0061] TGF-β activity inhibitor

[0062] The cytokine TGF-β, in each of its isoforms TGF-β1, TGF-β2, and TGF-β3, is a potent inhibitor of immunity. It has been reported that TGF-β inhibits the proliferation of cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, and dendritic cells (DCs). The cytokine also stimulates the proliferation and activation of regulatory T cells (Tregs).

[0063] Provided herein are TGF-β activity inhibitors for treating cancer and related symptoms. TGF-β signaling activity affects the progression of many diseases, including cancer. TGF-β binds to TGF-βRII and then recruits TGF-βRI to assemble the receptor TGF-βRI and TGF-βRII into a signaling tetramer. Blocking the binding or assembly with the receptor blocks the TGF-β signaling cascade. Provided herein are inhibitors that prevent the recruitment of TGF-βRI.

[0064] Provided herein are compositions comprising a nucleic acid encoding a TGF-β activity inhibitor. In some embodiments, the TGF-β inhibitor is capable of binding to TGF-β receptor II and antagonizing the expression of TGF-β ( Figure 1B)。Without being bound by theory, in some embodiments, the TGF-β inhibitor specifically targets the cognate receptor and antagonizes the receptor. In additional embodiments, the TGF-β inhibitor has fewer side effects associated with off-target activity. Nucleic acids encoding the TGF-β inhibitors described herein can provide increased tissue penetration. In some embodiments described herein, the TGF-β inhibitor binds to TGFβ receptor II. In some embodiments, the TGF-β inhibitor does not bind to TGFβ receptor I.

[0065] Native TGF-β is a dimer of two identical 112-residue peptides linked by disulfide bonds. In some embodiments, the nucleic acids described herein encode a human TGF-β monomer or any one of the three isoforms TGF-β1, TGF-β2, or TGF-β3 described as SEQ ID NO: 1-3 in Table 1, or a functional variant thereof. In some embodiments, the nucleic acids described herein encode engineered small monomers of TGF-β1, TGF-β2, and TGF-β3 described as SEQ ID NO: 4, 5, and 9, or functional variants thereof. In some embodiments, the engineered small monomer of TGF-β2 described herein includes a deletion of the α3 helix at residues 52-71 and a substitution of Cys-77 with serine, wherein the amino acid numbering is based on SEQ ID NO: 2. In some embodiments, the engineered small monomer of TGF-β2 includes a modification to include the cystine knot region of the protein related to Dan and Cerberus (PRDC). In some embodiments, the engineered small monomer of TGF-β2 lacks the α-3 heel helix of native TGF-β. Exemplary sequences for inclusion in the compositions described herein are listed in Table 1, SEQ ID NO: 4-9.

[0066] Exemplary variants of the TGF-β2 small monomer are described by SEQ ID NO: 6, 7, and 8. Nucleic acids encoding the small monomer variants are also provided herein, which contain substitutions that allow for increased solubility and higher binding affinity.

[0067] Table 1. Sequences of TGF-β Isoforms, Monomers, and Small Monomers

[0068]

[0069] In some embodiments, the nucleic acids described herein encode TGF-β inhibitors that comprise an antibody or a functional fragment thereof. In some embodiments, the nucleic acids described herein encode an antibody or a functional fragment thereof that binds to TGF-β. Non-limiting examples of antibodies that bind to TGF-β include 2G7, 1D11, GC1008, LY2382770, and TbetaM1. In some embodiments, the nucleic acids described herein encode an antibody or a functional fragment thereof that binds to a TGF-β receptor. In some embodiments, the antibody or a functional fragment thereof binds to TGF-β receptor I, TGF-β receptor II, or TGF-β receptor III.

[0070] In some embodiments, the nucleic acids described herein encode TGF-β inhibitors that comprise a receptor trap. In some embodiments, the encoded receptor trap blocks the entire receptor-binding interface. In some embodiments, the encoded receptor trap is an affinity-optimized soluble variant of the extracellular binding domain. In some embodiments, the target ligand preferentially binds to the receptor trap. In some embodiments, the encoded receptor trap prevents ligand binding to the receptor. In some embodiments, the encoded TGF-β inhibitor controls signal transduction with the receptor.

[0071] In some embodiments, the nucleic acids described herein encode TGF-β inhibitors that comprise a peptide. In non-exclusive embodiments, the peptide comprises P144 or P17. In selected embodiments, the nucleic acids described herein encode TGF-β inhibitors that comprise a sequence as described in Table 2. In some embodiments, the nucleic acids described herein encode a TGF-β inhibitor peptide as set forth in SEQ ID NO:10 or 11. In some embodiments, the nucleic acids described herein encode a TGF-β inhibitor that binds to TGF-β. In additional embodiments, the TGF-β-TGF-β inhibitor complex does not bind to a TGF-β receptor. The peptide optionally binds to an isotype of TGF-β.

[0072] Table 2. Inhibitor peptide sequences.

[0073] SEQ ID NO: Peptide Amino acid sequence 10 P17 KRIWFIPRSSWYERA 11 P144 TSLDASIIWAMMQN

[0074] In some embodiments, the nucleic acids described herein encode TGF-β inhibitors that comprise a dominant-negative receptor. In some embodiments, the dominant-negative receptor includes a truncated TGF-β receptor I, receptor II, or receptor III. In some embodiments, the nucleic acids described herein encode a dominant-negative receptor that comprises a truncated TGF-β receptor II. In some embodiments, the dominant-negative receptor is soluble. In some embodiments, the encoded dominant-negative receptor is a form of TGF-β receptor I, receptor II, or receptor III that lacks a transmembrane region.

[0075] In some embodiments, the nucleic acids described herein encode TGF-β inhibitors that include dominant negative inhibitors. In some embodiments, the encoded dominant negative inhibitor binds to TGF-β receptor I, receptor II, or receptor III. In some embodiments, the encoded dominant negative inhibitor binds to TGF-β receptor II. In some embodiments, the encoded dominant negative inhibitor optionally includes variants of TGF-β1, TGF-β2, or TGF-β3. Non-exclusive embodiments encoded by the nucleic acids described herein bind to the ligand-binding domain of TGF-β receptor II.

[0076] The present disclosure provides compositions that include nucleic acids encoding a TGF-β2 variant (TGFbv1). In some embodiments, the nucleic acid sequence encodes the peptide described by SEQ ID NO:7. In some embodiments, the encoded TGFbv1 includes at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO:7.

[0077] The present disclosure provides compositions that include nucleic acids encoding a TGF-β2 variant (TGFbv2). In some embodiments, the nucleic acid sequence encodes the peptide described by SEQ ID NO:8. In some embodiments, the encoded TGFbv2 includes at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO:8.

[0078] TGF-β can inhibit the proliferation of stimulated immune cells such as T cells to levels below baseline. Compositions that include the TGF-β inhibitors described herein can counteract the inhibition by TGF-β and can allow stimulated immune cells to proliferate at levels approaching baseline. In some embodiments, contacting TGF-β-inhibited cells with a TGF-β inhibitor as described herein can provide about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the proliferation at baseline in uninhibited cells.

[0079] TGF-β inhibitors can modulate the expression in related gene families. In some embodiments, modulation includes inducing the expression of genes. In some embodiments, contacting cells with a TGF-β inhibitor as described herein induces the expression of one or more interferon gamma (IFNG)-related genes compared to the median. In some embodiments, the expression of one or more IFNG-related genes is increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 120%, about 140%, about 160%, about 180%, about 200%, about 250%, about 300%, about 350%, or about 400% compared to the median across the expression in treated and untreated cells. The IFNG-related genes are selected from the group consisting of: CXCL11, XCR1, STAT1, IDO1, IL12B, IFNG, CIITA, H2-EB1, H2-AB1, TBX21, CXCR3, CD2, LTB, CXCL16, B2M, VCAM1, TAP1, IFIT2, TAP2, IL2RG, STAT2, CD274, and IRF1.

[0080] In some embodiments, contacting cells with a TGF-β inhibitor as described herein reduces the expression of TGF-B1-related genes compared to the median. In some embodiments, the expression of TGF-B1-related genes is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the median across the expression in treated and untreated cells. The TGF-B1-related genes are selected from the group consisting of: ILz1B, LPL, SLP1, FBN1, LCN2, CXCL5, OGN, PLOD2, TNFAIP6, CAN, ABCG1, ACKR3, and COL15A1.

[0081] Granzyme B

[0082] Granzyme B (GZMB) is found in the granules of immune cells such as natural killer cells (NK cells) and cytotoxic T cells. It can be secreted by these cells to mediate apoptosis of target cells. GZMB can also be produced by non-cytotoxic cells such as basophils and mast cells. It can help induce inflammation and extracellular matrix degradation. Activated immune cells can show increased GZMB expression. Compositions containing a TGF-β inhibitor as described herein can prevent the inhibition of TGF-β and allow stimulated immune cells to express GZMB.

[0083] Contacting immune cells with a modified virus as described herein can induce expression of granzyme B (GZMB) that is higher than that of untreated cells. In some embodiments, cells contacted with a TGF-β inhibitor contain about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000% more GZMB than untreated cells.

[0084] IL-12

[0085] Compositions are provided herein that contain a nucleic acid encoding IL-12 or a functional variant thereof. In some embodiments, the nucleic acids described herein encode at least two polypeptides. In some embodiments, the nucleic acid encodes a first polypeptide that comprises interleukin 12 or a functional variant thereof. In some embodiments, the nucleic acid encodes IL-12. In additional embodiments, IL-12 comprises subunit β (IL-12b) and subunit α (IL-12a). In some embodiments, the nucleic acid encodes the murine IL-12 (mIL-12) sequence as described in SEQ ID NO:12. In some cases, the encoded mIL-12 comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO:12. In some embodiments, the nucleic acid encodes the human IL-12 (hIL-12) sequence as described in SEQ ID NO:15. In some cases, the encoded hIL-12 comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO:15.

[0086] The present disclosure provides compositions comprising nucleic acids encoding murine interleukin-12 subunit alpha (IL-12a) (UniProtKB accession ID 43431.1). In some embodiments, the nucleic acid sequence encodes a peptide described by SEQ ID NO:13. In some cases, the encoded IL-12a comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% sequence identity to SEQ ID NO:13.

[0087] The present disclosure provides compositions comprising nucleic acids encoding murine interleukin-12 subunit beta (IL-12b) (UniProtKB accession ID P43432.1). In some embodiments, the nucleic acid sequence encodes a peptide described by SEQ ID NO:14. In some cases, the encoded IL-12b comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% sequence identity to SEQ ID NO:14.

[0088] The present disclosure provides compositions comprising nucleic acids encoding human interleukin-12 subunit alpha (hIL-12a) (UniProtKB accession ID P060595). In some embodiments, the nucleic acid sequence encodes a peptide described by SEQ ID NO:16. In some cases, the encoded hIL-12a comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% sequence identity to SEQ ID NO:16.

[0089] The present disclosure provides compositions comprising nucleic acids encoding the human IL-12 subunit beta (hIL-12b) (UniProtKB accession ID P29460). In some embodiments, the nucleic acid sequence encodes a peptide described by SEQ ID NO:17. In some cases, the encoded IL-12b comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% sequence identity to SEQ ID NO:17.

[0090] Exemplary amino acid sequences of the IL-12 regions for inclusion in the compositions described herein are listed in Table 3.

[0091] Table 3. IL-12 Amino Acid Sequences.

[0092]

[0093]

[0094]

[0095] Linkers and Signal Transduction Domains

[0096] The present disclosure provides compositions comprising nucleic acids encoding linkers. In some embodiments, the nucleic acids encoding the linkers are located between the various encoded biological functional units described herein. In some embodiments, the encoded linkers are flexible or rigid. In additional embodiments, the encoded linkers are cleavable linkers. In additional embodiments, the encoded cleavable linkers comprise disulfide bonds. In additional embodiments, the encoded cleavable linkers comprise protease-sensitive domains. A non-limiting list of exemplary linkers encoded by the nucleic acids included in the compositions described herein is listed in Table 4. In some embodiments, the compositions described herein comprise nucleic acids encoding a linker having the sequence described by SEQ ID NO:18. In some embodiments, the nucleic acids encode a linker having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO:18.

[0097] Table 4. Linkers.

[0098] SEQ ID NO: Linker type Amino acid sequence 18 Flexible GGGGSGGGGSGGGGSGGGGSAS 19 Flexible <![CDATA[(GGGGS) 1-3 > 20 Flexible <![CDATA[(Gly) 6-8 > 21 Rigid <![CDATA[(EAAAK) 1-3 > 22 Rigid <![CDATA[A(EAAAK)4ALEA(EAAAK)4A]]> 23 Rigid PAPAP 24 Cleavable S - S 25 Cleavable VSQTSKLTR↓AETVFPDV 26 Cleavable PLG↓LWA 27 Cleavable RVL↓AEA 28 Cleavable EDVVCC↓SMSY 29 Cleavable GGIEGR↓GS 30 Cleavable TRHRQPR↓GWE 31 Cleavable AGNRVRR↓SVG 32 Cleavable RRRRRRR↓R↓R 33 Cleavable GFLG↓ 34 Dipeptide LE 35 Cleavable GGGGSSPLGLWAGGGGS

[0099] Subscripts in the sequences indicate repeats.

[0100] The arrows in the sequence indicate the positions of the cleavage sites.

[0101] The present disclosure provides compositions comprising nucleic acids encoding signal transduction domains for controlling cell functions. The activity of interleukins (such as but not limited to IL-2 and IL-15) depends on the processing of signal peptides. The present disclosure provides compositions comprising nucleic acids encoding at least one signal peptide. Table 5 shows non-limiting examples of signal peptides encoded by the nucleic acids in the compositions described herein.

[0102] Table 5. Signal sequences.

[0103]

[0104] The present disclosure provides compositions comprising nucleic acids encoding the murine IL-2 signal sequence (mIL-2sig) (UniProtKB accession ID P04351.1). In some embodiments, the nucleic acid sequence encodes mIL-2sig corresponding to SEQ ID NO:36 or a functional variant thereof. In some embodiments, the encoded mIL-2sig has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO:36.

[0105] The present disclosure provides compositions comprising nucleic acids encoding the human IgE signal sequence (hIgEsig). In some embodiments, the nucleic acid sequence encodes hIgEsig corresponding to SEQ ID NO:37 or a functional variant thereof. In some embodiments, the encoded hIgEsig has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO:37.

[0106] Combined nucleic acid sequences

[0107] The present disclosure provides compositions comprising nucleic acids encoding combinations of technical features. In some embodiments, the nucleic acid encodes IL-12 or a functional variant thereof, a signal peptide, and a TGF-β variant polypeptide. In some embodiments, the nucleic acid encodes IL-12, the murine IL-2 signal peptide, and a TGF-β variant polypeptide or a functional variant thereof. In some embodiments, the nucleic acid encodes IL-12, the human IgE signal peptide, and a TGF-β variant polypeptide or a functional variant thereof.

[0108] In alternative embodiments, two nucleic acids are provided, wherein the first nucleic acid encodes a first polypeptide comprising interleukin 12 or a functional variant thereof, and the second nucleic acid encodes a second polypeptide comprising a TGF-β fusion (TGFbf) polypeptide comprising a signal peptide and a TGF-β variant. In some embodiments, the TGFbf polypeptide comprises the sequences set forth in SEQ ID NO:36 and SEQ ID NO:7 (TGFbf1). In some embodiments, the TGFbf polypeptide comprises the sequences set forth in SEQ ID NO:37 and SEQ ID NO:8 (TGFbf2).

[0109] Compositions are provided herein comprising nucleic acid sequences encoding polypeptides. In some embodiments, the nucleic acid encodes a polypeptide comprising TGFbf1 as described by SEQ ID NO:40 and shown in Table 6. In some embodiments, the encoded TGFbf comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% sequence identity to SEQ ID NO:40. In some embodiments, the nucleic acid encodes a polypeptide comprising TGFbf2 as described by SEQ ID NO:41 and shown in Table 6. In some embodiments, the encoded TGFbf comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% sequence identity to SEQ ID NO:41.

[0110] Table 6. TGF-β fusion amino acid sequences.

[0111]

[0112] Chemokine receptor

[0113] Chemokines are chemotactic cytokines that regulate cell trafficking and positioning by activating seven-transmembrane chemokine receptors. In some cases, chemokines are classified into four subfamilies based on the position of the first two N-terminal cysteine residues, including CC, CXC, CX3C, and XC subfamilies. Differential expression of chemokine receptors on leukocytes optionally leads to the selective recruitment of specific cell types under specific conditions, providing an appropriate and effective immune response suitable for infecting pathogens or foreign injuries. In addition to their crucial role in the coordinated migration of immune cells to the site of inflammation, in many cases, chemokines also play important roles in the development of lymphoid tissues, the maturation of immune cells, and the generation and delivery of adaptive immune responses.

[0114] Tumors are increasingly recognized as complex microenvironments composed of many different cell types that coexist and communicate with each other in complex signaling networks. Chemokines are important coordinators of cell migration and cell-cell interactions and thus have a great impact on tumor development. In the tumor microenvironment, tumor-associated host cells and cancer cells release a series of different chemokines, leading to the recruitment and activation of different cell types, which mediate the balance between anti-tumor and pro-tumor responses. In addition to their primary role as chemoattractants, chemokines are also involved in many other tumor-related processes in many cases, including tumor cell growth, angiogenesis, and metastasis.

[0115] Tumor cells have been shown to acquire the ability to produce growth-promoting chemokines. For example, melanoma has been found to express many chemokines, including CXCL1, CXCL2, CXCL3, CXCL8, CCL2, and CCL5, which are associated with tumor growth and progression. Increased CCL2 levels can be found in neuroblastoma cell lines and primary tumor cells isolated from human patients. Immunostaining studies have also shown elevated expression levels of CXCL12 in a variety of cancers, including breast cancer, carcinoid tumors, cervical cancer, colorectal cancer, endometrial cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, and gastric cancer.

[0116] Chemokine receptors are cytokine receptors that interact with chemokines and are found on the surface of certain cells. Twenty different chemokine receptors have been identified in humans. Each has a 7-transmembrane structure and is coupled to a G protein for intracellular signal transduction, making them members of the large protein family of G protein-coupled receptors. After interacting with their specific chemokine ligands, chemokine receptors trigger intracellular calcium (Ca 2+)Ion flow (calcium signaling). This causes a cellular response, including the initiation of a process called chemotaxis, which transports the cell to a desired location within an organism. Generally, as used herein, the term "chemokine receptor" can refer to a membrane-associated protein that selectively binds a chemokine ligand and induces chemotaxis toward the chemokine ligand.

[0117] It should be understood that the chemokine receptors disclosed herein in some cases refer not only to naturally occurring chemokine receptors identified in humans but also include chemokine receptors from other sources, such as but not limited to: (1) naturally occurring chemokine receptors identified in animals such as pigs, dogs, cows, sheep; and (2) non-naturally occurring chemokine receptors, such as mutant proteins, chimeric receptors, designed proteins that have binding affinity for a certain type of chemokine. In some instances, a fragment of a naturally occurring chemokine receptor is also considered a chemokine receptor if the function of binding and responding to the corresponding chemokine and directing chemotaxis of the cell remains in the fragment. As provided herein, in some embodiments, when a virus hijacks the gene expression machinery of a host cell, the virus containing the exogenous nucleic acid encoding a chemokine receptor forces the virus-infected cell to express the chemokine receptor.

[0118] In some embodiments, provided herein is a modified oncolytic virus containing an exogenous nucleic acid, also referred to herein as a transgene, encoding a chemokine receptor. In some cases, the exogenous nucleic acid is a therapeutic transgene. In some cases, the modified oncolytic virus contains an exogenous nucleic acid encoding a cytokine receptor whose cognate cytokine is expressed in the tumor microenvironment (e.g., IL15-R has the cognate cytokine IL15 expressed in the tumor microenvironment). In some cases, the modified oncolytic virus encodes a chemokine receptor whose cognate chemokine may be expressed on the tumor (e.g., CXCR4 has the cognate chemokine CXCL12 expressed on the tumor; CCR2 has the target CCL2 expressed on the tumor), and is systemically delivered as a naked virus. After the modified oncolytic virus enters the bloodstream, through systemic delivery, the virus infects lymphocytes, such as B cells, and redirects the infected B cells to the tumor, resulting in a significant increase in the viral load in the tumor. In certain embodiments, the increased viral load in the tumor is achieved shortly after systemic delivery. The ability to deliver the modified oncolytic viruses disclosed herein systemically provides an advantage over traditional intratumoral delivery methods of oncolytic viruses. While intratumoral delivery helps treat tumors that are easily accessible, in some cases, treating inaccessible or metastatic cancers is crucial, which are purportedly the main cause of death from the disease. In such cases, oncolytic viruses that rely on intratumoral delivery are ineffective because they would require systemic spread after being administered to distant sites. However, due to the development of an immune response to viral infection, such spread is generally transient and ineffective, at least in part.

[0119] Chemokine receptors are divided into different families. Non-limiting examples of chemokine receptors described herein include CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors, which correspond to four different subfamilies of chemokines to which they bind. Among the CXC chemokine receptors, CXCR1 and CXCR2 are closely related, and CXCR1 binds to CXCL8 and CXCL6, and CXCR2 binds to CXCL1 and CXCL7; CXCR3 binds to CXCL9, CXCL10, and CXCL11; CXCR4 binds to CXCL12 (or SDF-1); CXCR5 binds to CXCL13; CXCR6 binds to CXCL16. Among the CC chemokine receptors, the ligands of CCR1 include CCL4, CCL5, CCL6, CCL14, CCL15, CCL16, CCL23; the ligands of CCR2 include CCL2, CCL8, and CCL16; the ligands of CCR3 include CCL11, CCL26, CCL7, CCL13, CCL15, CCL24, CCL5, CCL28, and CCL18; the ligands of CCR4 include CCL3, CCL5, CCL17, and CCL22; the ligands of CCR5 include CCL3, CCL4, CCL5, CCL8, CCL11, CCL13, CCL14, and CCL16; the ligand of CCR6 includes CCL20; the ligands of CCR7 include CCL19 and CCL21; the ligands of CCR8 include CCL1, CCL16; the ligand of CCR9 includes CCL25; the ligands of CCR10 include CCL27, CCL28; the ligands of CCR11 include CCL19, CCL21, CCL25. The CX3C chemokine receptor CX3CR1 has the ligand CXCL1. The XC chemokine receptor XCR1 binds to both XCL1 and XCL2.

[0120] Non-limiting embodiments of the present disclosure provide an oncolytic virus comprising an exogenous nucleic acid encoding a chemokine receptor. In some embodiments, the chemokine receptor is a CXC chemokine receptor, a CC chemokine receptor, a CX3C chemokine receptor, an XC chemokine receptor, or any combination thereof. In some embodiments, the chemokine receptor is CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, or any combination thereof.

[0121] In certain embodiments, the modified oncolytic virus comprises a nucleic acid that exogenously expresses CXCR4. In certain embodiments, the modified oncolytic virus comprises a nucleic acid that exogenously expresses CCR2. Certain embodiments disclose a modified oncolytic virus, the modified oncolytic vaccinia virus comprising an exogenous nucleic acid encoding both CXCR4 and CCR2, and both chemokines are expressed from the same virus. In some cases, CXCL12 and / or CCL2, which are typically expressed in the tumor microenvironment, attract lymphocytes expressing CXCR4 and / or CCR2 or other migratory cells infected by the modified oncolytic virus, thereby enhancing the tumor-targeted delivery of the modified oncolytic virus. The nucleic acid and amino acid sequences of the selected chemokine receptors are listed in Table 7.

[0122] Table 7. Chemokine Receptor Sequences.

[0123]

[0124]

[0125]

[0126] In the compositions provided herein, the oncolytic virus gene can be mutated or replaced with a nucleic acid encoding a chemokine receptor listed in Table 7. In some embodiments, the chemokine receptor is murine CXCR3 as described by SEQ ID NO:42. In some embodiments, the encoded murine CXCR3 comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% sequence identity to SEQ ID NO:42. In some embodiments, the chemokine receptor is human CXCR3 as described by SEQ ID NO:43. In some embodiments, the encoded human CXCR3 comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% sequence identity to SEQ ID NO:43.

[0127] In some embodiments, provided herein are modified oncolytic viruses that comprise an exogenous nucleic acid encoding a membrane-associated protein that degrades hyaluronic acid, such as hyaluronidase. In some embodiments, provided herein are modified oncolytic viruses that comprise an exogenous nucleic acid, also referred to herein as a transgene, encoding a chemokine receptor. In some cases, the exogenous nucleic acid is a therapeutic transgene.

[0128] Promoter

[0129] Provided herein are compositions that comprise a nucleic acid, wherein the nucleic acid encodes at least one promoter region. A promoter region, or promoter, or promoter element, or regulatory region refers to a nucleic acid sequence to which a protein binds to initiate transcription. Promoters are typically located 5' or upstream of the DNA coding region that they control. In some embodiments, the nucleic acids described herein comprise one promoter. In some embodiments, one promoter drives the transcription of all polypeptides encoded on the nucleic acid. In some embodiments, the nucleic acids described herein comprise separate promoters for each polypeptide encoded on the nucleic acid. In some embodiments, the nucleic acid comprises two promoters, each driving the transcription of one of two polypeptides encoded on the nucleic acid.

[0130] The timing of expression can be regulated by modulating the structure of the promoter that drives gene expression. The number and affinity of transcription factor binding sites determine the relative timing of expression between different promoter regions. A promoter with more transcription factor binding sites and / or higher binding affinity can drive expression earlier than a promoter with fewer or lower affinity binding sites.

[0131] The application of the relative timing of protein expression can be utilized to express a particular factor from the modified viruses described herein earlier or later during the course of infection. Early promoters have repeated transcription factor binding sites. Late promoters have fewer binding sites than early promoters. In some embodiments, an early promoter is used to express a receptor. Expression early in infection allows for expression and processing by the cell before the cellular processes are disrupted. In some embodiments, a late promoter is used to express one or more cytokines.

[0132] In some embodiments, provided herein are promoters that include P7.5, P28, P135, the TK promoter, the A52R promoter, the 454 promoter, PB8, LEO, PF11, F7L, H5R, mH5, H1L, A1L, J3R, E4L, I1L, I3L, I4L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, L4R, the T7 promoter, the 28 kDa promoter, the short synthetic promoter (SSP), or any functional variant or combination thereof. In some embodiments, the promoter includes an early promoter. In some embodiments, the early promoter includes A52R, PB8, mH5, I4L, LEO, PF11, I3L, P7.5, the TK promoter, F7L, H5R, the short synthetic promoter (SSP), or any variant or combination thereof. In some embodiments, the promoter includes a late promoter. In some embodiments, the late promoter includes SSP, P7.5, P28, P135, the TK promoter, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, L4R, the 28 kDa promoter, or any functional variant or combination thereof. The sequences of the selected promoters are listed in Table 8.

[0133] Table 8. Promoter Nucleic Acid Sequences

[0134]

[0135] The compositions provided herein may comprise the P7.5 promoter and the P28 promoter. In some embodiments, the P7.5 promoter drives the expression of the region encoding the IL-12 polypeptide. In some embodiments, the P28 promoter drives the transcription of the TGFbf polypeptide. Schematic representations of the promoter and gene expression constructs are shown in Figure 2 In some embodiments, the P7.5 promoter comprises the nucleic acid sequence set forth in SEQ ID NO:57. In some embodiments, the P28 promoter comprises the nucleic acid sequence set forth in SEQ ID NO:58.

[0136] The compositions provided herein may comprise the P135 promoter and the P7.5 promoter. In some embodiments, the P135 promoter drives the expression of the region encoding the IL-12 polypeptide. In some embodiments, the P7.5 promoter drives the expression of the region encoding the TGF-β polypeptide. In some embodiments, the P7.5 promoter comprises the nucleic acid sequence set forth in SEQ ID NO:57. In some embodiments, the P135 promoter comprises the sequence set forth in SEQ ID NO:56.

[0137] The compositions provided herein may comprise the P7.5 promoter (SEQ ID NO:57), the P135 promoter (SEQ ID NO:56), and the A52R promoter. In some embodiments, the P135 promoter drives the expression of a region encoding an IL-12 polypeptide. In some embodiments, the P7.5 promoter drives the expression of a region encoding a TGF-β polypeptide. In some embodiments, the A52R promoter drives the expression of a region encoding the CXCR3 receptor. In some embodiments, the expression of the CXCR3 receptor occurs prior to the expression of the IL-12 polypeptide or the TGF-β polypeptide.

[0138] The present invention provides a composition comprising an expression construct that, in a 5' to 3' order, comprises an IL-12β subunit, a linker, an IL-12α subunit, an IL-12 signal sequence, and a TGF-β variant. In some embodiments, the expression construct in a 5' to 3' order comprises hIL-12b (SEQ ID NO:17), a flexible linker (SEQ ID NO:18), hIL-12a (SEQ ID NO:16), a human IgEsig sequence (SEQ ID NO:37), and a TGF-β2 variant sequence (TGFbv2) (SEQ ID NO:8). In some embodiments, the expression construct in a 5' to 3' order comprises mIL-12b (SEQ ID NO:14), a flexible linker (SEQ IDNO:18), mIL-12a (SEQ ID NO:13), a murine IL-2sig (SEQ ID NO:36), and a TGF-β2 variant sequence (TGFbv1) (SEQ ID NO:7). In some embodiments, the expression construct in a 5' to 3' order comprises mIL-12 (SEQ ID NO:12) and TGFbf1 (SEQ ID NO:40). In some embodiments, the expression construct in a 5' to 3' order comprises hIL-12 (SEQID NO:15) and TGFbf2 (SEQ ID NO:41). In some embodiments, the exogenous nucleic acid described herein is incorporated into a viral genome.

[0139] The present disclosure provides compositions comprising an expression construct that, in 5' to 3' order, comprises an IL-12 signal sequence, a TGF-β variant, an IL-12β subunit, a linker, and an IL-12α subunit. In some embodiments, the expression construct, in 5' to 3' order, comprises a human IgE sig sequence (SEQ ID NO:37), a TGF-β2 variant sequence (TGFbv2) (SEQ ID NO:8), hIL-12b (SEQ ID NO:17), a flexible linker (SEQ ID NO:18), and hIL-12a (SEQ ID NO:16). In some embodiments, the expression construct, in 5' to 3' order, comprises a murine IL-2sig (SEQ ID NO:36), a TGF-β2 variant sequence (TGFbv1) (SEQ ID NO:7), mIL-12b (SEQ ID NO:14), a flexible linker (SEQ ID NO:18), and mIL-12a (SEQ ID NO:13). In some embodiments, the expression construct, in 5' to 3' order, comprises TGFbf1 (SEQ ID NO:40) and mIL-12 (SEQ ID NO:12). In some embodiments, the expression construct, in 5' to 3' order, comprises TGFbf2 (SEQ ID NO:41) and hIL-12 (SEQ ID NO:15).

[0140] vector

[0141] Vectors can be used to deliver foreign nucleic acids into cells for replication or expression. In some embodiments, the vector is a particle that comprises a plasmid, a viral vector, a cosmid, or an artificial chromosome. Vectors typically carry foreign nucleic acids inserted into the "backbone" nucleic acid of the vector. In some embodiments, the foreign nucleic acid is DNA or RNA. In some embodiments, the vector comprises at least one promoter sequence that drives the expression of the foreign nucleic acid.

[0142] oncolytic virus

[0143] Oncolytic viruses can preferentially infect and kill cancer cells. Oncolysis of the infected cancer cells can enable the spread of the virus to surrounding tissues to continue and can also stimulate the host anti-tumor immune response. Compositions comprising oncolytic viruses are provided herein, wherein the oncolytic viruses comprise modified nucleic acids as described herein. As used herein, oncolytic viruses kill cancer cells or tumor cells by mechanisms such as directly lysing the cells, by stimulating an immune response against the cells, apoptosis, expression of toxic proteins, autophagy and termination of protein synthesis, induction of anti-tumor immunity, or any combination thereof. In some embodiments, the oncolytic viruses as described herein replicate intracellularly. In some embodiments, the oncolytic viruses as described herein replicate in tumor cells, immune cells, somatic cells, hematopoietic cells, or another type of cell. Exemplary oncolytic viruses for inclusion in the compositions described herein include, but are not limited to, poxviruses, vaccinia virus, adeno-associated virus, adenovirus, reovirus, lentivirus, herpes simplex virus, vesicular stomatitis virus, Mengo virus, myxoma virus, Newcastle disease virus, measles virus, or poliovirus. These oncolytic viruses have a tendency to specifically target cancer cells and cause significant cell death and tumor regression upon viral replication. In some embodiments, the oncolytic virus is vaccinia virus. Exemplary vaccinia viruses include, but are not limited to, the following strains for modification by inclusion of the constructs described herein: Vaccinia virus Western Reserve (ATCC VR-1354), Vaccinia virus Ankara (ATCC VR-1508), Vaccinia virus Ankara (ATCC VR-1566), Wyeth Vaccinia virus strain (ATCC VR-1536), or Wyeth Vaccinia virus (ATCC VR-325). Additionally, in some embodiments, the recombinant vaccinia virus is a modified form of a wild-type or attenuated vaccinia virus strain. Non-limiting examples of vaccinia virus strains include the Western Reserve strain of vaccinia virus, Copenhagen strain, IHD strain, Wyeth (NYCBOH) strain, Tian Tan strain, Lister strain, USSR strain, Ankara strain, NYVAC strain, Ankara (MVA) strain, Paris strain, Bern strain, Temple of Heaven strain, Dairen strain, EM-63 strain, Evans strain, King strain, Patwadangar strain, or Tash Kent strain. The modified parental vaccinia virus strain as set forth herein optionally comprises one or more mutations relative to its parental strain, for example, but not limited to, one or more of the following: a deletion in TK (also referred to herein as "TK-") and a deletion in A52R (also referred to herein as "A52R-"). The vaccinia virus is optionally recombinant or selected to have low toxicity and accumulate in the target tissue.In some embodiments, the modifications in the viral backbone / viral genome are modifications that render vaccinia virus non-replicating or with poor replicative ability. Non-limiting examples of such modifications include mutations in the following viral genes: A1, A2, VH1, A33, and I7. In some embodiments, the viral backbone mutations are selected from the group consisting of: complete or partial deletion of the A52R gene; complete or partial deletion of the TK gene; complete or partial deletion of the B15R gene; complete or partial deletion of the K7R gene; complete or partial deletion of the B14R gene; complete or partial deletion of the N1L gene; complete or partial deletion of the K1L gene; complete or partial deletion of the M2L gene; complete or partial deletion of the A49R gene; complete or partial deletion of the VH1 gene; complete or partial deletion of the A33 gene; complete or partial deletion of the A1 gene; complete or partial deletion of the A2 gene; complete or partial deletion of the I7 gene; and complete or partial deletion of the A46R gene. As used herein, reference to a viral gene is made by reference to the protein encoded by the gene (e.g., the A33 gene means the gene encoding the A33 protein). In some embodiments, the viral backbone mutations, including any combination of substitutions, insertions, and deletions, result in a sequence having less than 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, or less sequence homology to the wild-type sequence of the viral gene or the viral protein encoded by the gene. In some embodiments, the viral genes and the proteins encoded thereby are selected from the group consisting of: B15R, K7R, B14R, N1L, K1L, M2L, A49R, VH1, A33, A1, A2, I7, and A46R. In some embodiments, the viral backbone contains 1, 2, 3, 4, 5, or more mutations in the amino acid sequence of a viral protein (e.g., a viral antigen). In some instances, the viral antigens are selected from the group consisting of: B15R, K7R, B14R, N1L, K1L, M2L, A49R, VH1, A33, A1, A2, I7, and A46R. In some embodiments, the present disclosure provides a recombinant vaccinia virus comprising one or more mutations in the viral genome (viral backbone) such that the mutation increases the T-cell arm of the immune response. The mutation can be an addition, deletion, or substitution of one or more nucleic acids in the viral genome (wild-type or attenuated natural strain of vaccinia virus). In a non-limiting example, the mutation is a complete or partial deletion of a gene known to inhibit cytokines involved in the Th1 immune response. As a non-limiting example, the mutation is a deletion of the nucleic acid encoding B8R (interferon gamma (IFN-γ) binding protein); C12L (interleukin-18 (IL-18) binding protein). In another non-limiting example, the mutation is a complete or partial deletion of a gene in innate immune signaling.As a non-limiting example, the mutation is a deletion of a nucleic acid encoding B18R (type I interferon (IFN) binding protein); A52R (nuclear factor κB (NF-κB) inhibitor protein); E3L (protein kinase (PKR) inhibitor); C4, C16 (STING pathway inhibitor).

[0144] The oncolytic viruses described herein contain one or more additional insertions or partial insertions of exogenous nucleic acids encoding one or more proteins. In some embodiments, the one or more proteins include a chemokine receptor or a functional variant thereof, a TGF-β inhibitor or a functional variant thereof, or interleukin-12 or a functional variant thereof. In some embodiments, the one or more proteins include a TGF-β inhibitor or a functional variant thereof and interleukin-12 or a functional variant thereof. Exemplary chemokine receptors included are, but are not limited to, wild-type and / or mutant CXCR3, CXCR4, CCR2, or CCL2. The vaccinia viruses of the present disclosure also contain one or more additional deletions or partial deletions of one or more genes from A52R, B15R, K7R, A46R, N1L, E3L, K1L, M2L, C16, N2R, B8R, B18R, VH1, and their functional domains or fragments or variants or any combination thereof. In some cases, the vaccinia viruses provided herein contain a complete or partial deletion of at least one of the following: the A52R or TK viral gene, and an insert of exogenous nucleic acid encoding one or more proteins (e.g., one or more immunomodulatory proteins).

[0145] In some embodiments, the oncolytic virus is a modified oncolytic virus that has one or more modifications that result in a greater therapeutic effect against tumor cells as compared to other identical viruses that do not contain the modification. In some non-limiting examples, the greater therapeutic effect includes each or any combination of the following: enhanced viral immune evasion, enhanced systemic delivery of the virus to tumors, enhanced intratumoral and intertumoral spread of the virus, and enhanced tumor-specific replication of the virus, or release of immunomodulators and anti-tumor agents into the extracellular matrix. In some cases, the modified oncolytic viruses of the present disclosure are used as platform vectors for systemic delivery.

[0146] The oncolytic viruses described herein contain the exogenous nucleic acids described herein. In some embodiments, the oncolytic viruses provided herein contain a complete or partial deletion of the TK gene and an insertion of a region encoding at least one of a transforming growth factor-β inhibitor and a cytokine such as IL-12. Exemplary sequences for integration were previously described herein.

[0147] In some embodiments, the oncolytic viruses provided herein contain a complete or partial deletion of the A52R gene and an insertion of a region encoding a chemokine receptor. In some embodiments, the chemokine receptor comprises CXCR3. In some embodiments, the region encoding the chemokine receptor comprises a sequence selected from Table 7. In some embodiments, the promoter driving chemokine receptor expression is an early promoter, a late promoter, a strong early promoter, a weak early promoter, a strong late promoter, a weak late promoter, or any combination thereof. In some embodiments, the A52R promoter drives the expression of the chemokine receptor. In some embodiments, the A52R promoter drives the expression of the region encoding CXCR3.

[0148] In some embodiments of the present disclosure, there are provided modified oncolytic viruses that contain modifications that enhance systemic delivery of the virus to tumors. Generally, oncolytic viruses are (a) administered systemically, (b) topically inoculated onto tumors, or (c) directly injected into tumors (“intratumoral delivery”). In some embodiments, systemic delivery of oncolytic viruses provides the opportunity to simultaneously treat primary tumors and any apparent or undiagnosed metastatic deposits. Thus, this delivery method is a very attractive option for treating patients with advanced / metastatic disease or patients with diseases that are difficult to access such as patients with pancreatic cancer or brain cancer, in whom access is difficult, for example, due to physiological barriers such as the blood-brain barrier. However, there are obstacles to the successful systemic delivery of many oncolytic viruses. For example, in some cases, as described above, host defenses limit the ability of most oncolytic viruses to infect tumors after systemic administration. Non-specific uptake by blood cells, complement, antibodies, and antiviral cytokines, as well as other tissues such as the lung, liver, and spleen, tissue-resident macrophages, and the poor escape of additional virus from the vascular compartment, are major obstacles to the systemic delivery of oncolytic viruses. In some embodiments of the present disclosure, the disclosed oncolytic viruses contain modifications that promote the persistence of the virus in the circulatory system, at least as mentioned above, by enhancing immune evasion. Alternatively, in certain cases, enhanced tumor-targeted delivery of the virus is desirable because it not only increases the therapeutic efficacy against cancer but also alleviates safety concerns surrounding virus-mediated tumor therapy because non-tumor infection is limited, avoiding unwanted side effects of viral infection. Certain embodiments herein relate to oncolytic viruses that contain modifications that promote tumor-targeted delivery of the virus.

[0149] In some embodiments of the present disclosure, there are provided modified oncolytic viruses that comprise modifications that enhance the intratumoral and intertumoral spread of the virus. Enhanced spread of the oncolytic virus within and between tumors is an effective way to enhance the therapeutic efficacy by increasing the number of cancer cells infected by the virus. In some embodiments, there are provided modified oncolytic viruses that comprise exogenous nucleic acids. In some embodiments, there are provided modified oncolytic viruses that comprise modifications to the viral genome. In some embodiments, there are provided modified oncolytic viruses that comprise exogenous nucleic acids as well as modifications in the viral genome.

[0150] In some embodiments, the oncolytic virus includes, but is not limited to: (i) a virus that naturally preferentially replicates in cancer cells and is generally non-pathogenic in humans due to increased sensitivity to innate antiviral signal transduction or dependence on oncogenic signal transduction pathways; and (ii) a virus that has been genetically engineered for use. In some embodiments, the oncolytic virus is a measles virus, a poliovirus, a poxvirus, a vaccinia virus, an adenovirus, an adeno-associated virus, a herpes simplex virus, a vesicular stomatitis virus, a reovirus, a Newcastle disease virus, a Seneca virus, a retrovirus, a Mengo virus, or a myxoma virus. In certain embodiments, the oncolytic virus is a poxvirus. In certain embodiments, the oncolytic virus is a vaccinia virus.

[0151] In some embodiments, modified oncolytic viruses are employed. Generally, such viruses comprise modifications to their components, such as, but not limited to, modifications in the native genome (“backbone”) of the virus, such as mutations or deletions of viral genes, introduction of exogenous nucleic acids, chemical modifications of viral nucleic acids or viral proteins, and introduction of exogenous proteins or modified viral proteins into the viral capsid.

[0152] In some embodiments, the modified oncolytic virus comprises a mutation or deletion of the TK gene and further comprises an exogenous nucleic acid encoding a TGF-β inhibitor. In some embodiments, the modified oncolytic virus comprises a mutation or deletion of the TK gene and further comprises an exogenous nucleic acid encoding a TGF-β inhibitor and an exogenous nucleic acid encoding a cytokine (e.g., IL-12). In some embodiments, the exogenous nucleic acid encoding IL-12 is inserted 5’ to the exogenous nucleic acid encoding a TGF-β inhibitor. In some embodiments, the exogenous nucleic acid encoding a TGF-β inhibitor is inserted 5’ to the exogenous nucleic acid encoding IL-12.

[0153] In some embodiments, the modified oncolytic virus comprises a mutation or deletion of the A52R gene and further comprises an exogenous nucleic acid encoding the CXCR3 receptor. In some embodiments, the modified oncolytic virus comprises a mutation or deletion of the A52R gene, wherein the A52R promoter is maintained, and further comprises an exogenous nucleic acid encoding the CXCR3 receptor.

[0154] In some embodiments, in a modified oncolytic virus, such as in an oncolytic vaccinia virus, the viral TK gene is replaced with a TK gene from herpes simplex virus (HSV-TK). The HSV TK optionally serves as a replacement for the missing TK and has multiple advantages. For example, in some embodiments, the HSV TK serves as an additional therapeutic prodrug convertase for converting ganciclovir (GCV) to its cytotoxic metabolite in tumors. In addition to the increased therapeutic effect, this modification also serves as a suicide gene, e.g., effectively killing vaccinia-expressing cells by addition of GCV, thereby halting the virus in the case of adverse events or uncontrolled replication. Thus, in some cases, the modified oncolytic virus of the present disclosure serves as a safety switch. In additional examples, a mutant form of HSV TK is used to allow for PET imaging of labeled substrates with a substantially increased sensitivity. Thus, in some cases, a modified oncolytic virus containing HSV TK for PET imaging can serve as a reporter of viral in vivo replication to determine early treatment activity after treatment.

[0155] In some cases, the modified oncolytic virus comprises the full-length viral backbone gene or viral backbone protein described above, or a truncated form thereof, or a functional domain thereof, or a fragment thereof, or a variant thereof. In various examples, as described above, the modified oncolytic virus comprises a mutation or deletion of one or more viral backbone genes or viral backbone proteins. Mutations of viral backbone genes and viral backbone proteins include insertions, deletions, substitutions, or modifications of nucleic acids in the nucleic acid sequence and amino acids in the protein sequence. In some examples, the deletion includes a complete or partial deletion of the viral backbone gene or protein.

[0156] In some embodiments, compared to oncolytic viruses that are otherwise identical to other aspects that do not contain the modification, the modification of the oncolytic virus results in an increase in the efficacy of systemic delivery of the virus to tumors by at least about 1.1-fold, 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 2.2-fold, 2.5-fold, 2.8-fold, 3-fold, 3.2-fold, 3.5-fold, 3.8-fold, 4-fold, 4.2-fold, 4.5-fold, 4.8-fold, 5-fold, 5.2-fold, 5.5-fold, 5.8-fold, 6-fold, 6.2-fold, 6.5-fold, 6.8-fold, 7-fold, 7.2-fold, 7.5-fold, 7.8-fold, 8-fold, 8.2-fold, 8.5-fold, 8.8-fold, 9-fold, 9.2-fold, 9.5-fold, 9.8-fold, 10-fold, 12-fold, 14-fold, 15-fold, 16-fold, 18-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 150-fold, 200-fold, 250-fold, 500-fold, 800-fold, 1000-fold, 2500-fold, 5000-fold, 10 4 -fold, 2.5×10 4 -fold, 5×10 4 -fold, 7.5×10 4 -fold, 2.5×10 5 -fold, 5×10 5 -fold, 7.5×10 5 -fold, 10 6 -fold, 2.5×10 6 -fold, 5×10 6 -fold, 7.5×10 6 -fold, 10 7 -fold, 2.5×10 7 -fold, 5×10 7 -fold, 7.5×10 7 -fold, 10 8 -fold, 2.5×10 8 -fold, 5×10 8 -fold, 7.5×10 8 -fold, 10 9 -fold, 2.5×10 9 -fold, 5×10 9 -fold, 7.5×10 9 -fold, 10 10times or even more. In certain embodiments, the efficacy of systemic delivery of a tumor-targeting virus is measured by quantifying the virus that infects tumor cells and optionally comparing it to the virus that infects non-tumor cells in the body. For example, in some cases, the virus is quantified by staining virus particles in tissue sections or, in the case of leukemia, lymphoma, or myeloma, by staining blood smears. In some cases, such quantification is performed by reporter molecules (e.g., luciferase and fluorescent proteins) engineered to be expressed by the virus. In some cases, such quantification is performed by quantifying the viral genome in the tumor. Without limitation, the systemic delivery of a tumor-targeting virus can also be measured by quantifying certain downstream effects of viral infection in tumor cells (such as cytokines in response to viral infection or lymphocyte accumulation). In some embodiments, the oncolytic virus comprises an exogenous nucleic acid encoding CXCR3, CXCR4, CCR2, or any combination thereof. In some embodiments, the presence of the exogenous nucleic acid results in an approximately 5-fold to 10-fold increase in the efficacy of systemic delivery of the tumor-targeting virus compared to an oncolytic virus that is otherwise identical but does not contain the exogenous nucleic acid.

[0157] In some embodiments, provided herein are modified oncolytic viruses that comprise an exogenous nucleic acid encoding a chemokine receptor, and the forced expression of the chemokine receptor by the modified oncolytic virus results in an enhanced immune response against the infected tumor. After infecting the tumor, the modified oncolytic virus replicates in the tumor cells and causes the expression of the chemokine receptor on the surface of the tumor cells. These membrane receptors act as decoy receptors, binding and sequestering immunosuppressive chemokines (e.g., CXCL12 and / or CCL2) within the tumor. Thus, compared to other identical viruses that do not contain a nucleic acid encoding a chemokine receptor, the immunosuppressive microenvironment in the tumor is altered, resulting in enhanced immunotherapeutic activity of the modified oncolytic virus. In some embodiments, the increase in immunotherapeutic activity is at least about 1.1-fold, 1.1-fold, 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 2.2-fold, 2.5-fold, 2.8-fold, 3-fold, 3.2-fold, 3.5-fold, 3.8-fold, 4-fold, 4.2-fold, 4.5-fold, 4.8-fold, 5-fold, 5.2-fold, 5.5-fold, 5.8-fold, 6-fold, 6.2-fold, 6.5-fold, 6.8-fold, 7-fold, 7.2-fold, 7.5-fold, 7.8-fold, 8-fold, 8.2-fold, 8.5-fold, 8.8-fold, 9-fold, 9.2-fold, 9.5-fold, 9.8-fold, 10-fold, 12-fold, 14-fold, 15-fold, 16-fold, 18-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 150-fold, 200-fold, 250-fold, 500-fold, 800-fold, 1000-fold, 2500-fold, 5000-fold, 10 4 times, 2.5×104 -fold, 5 × 10 4 -fold, 7.5 × 10 4 -fold, 2.5 × 10 5 -fold, 5 × 10 5 -fold, 10 6 -fold or even higher fold. Without limitation, the increased immunotherapeutic activity is reflected by increased B cell accumulation in the tumor, increased T cell responses to tumor-associated immunogens, or both. B cell accumulation is measured, for example, by quantifying B cells in the tumor, and T cell immunocompetence is measured, for example, by interferon-γ (interferon-gamma) secretion in an ELISPOT assay.

[0158] In some embodiments, provided herein are modified oncolytic viruses that comprise exogenous nucleic acids encoding chemokine receptors, and the forced expression of the chemokine receptors by the modified oncolytic viruses results in increased replication of the virus in tumor cells as compared to other identical viruses that do not comprise nucleic acids encoding chemokine receptors. In some embodiments, the modified oncolytic virus comprises nucleic acids that ectopically express CXCR3. In some embodiments, the modified oncolytic virus comprises nucleic acids that express exogenous CCR2, which increases tumor-specific replication of the virus. In some embodiments, the modified oncolytic virus comprises nucleic acids that express exogenous CCR5, which increases tumor-specific replication of the virus. In some embodiments, the increase in tumor-specific replication is at least about 1.1-fold, 1.1-fold, 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 2.2-fold, 2.5-fold, 2.8-fold, 3-fold, 3.2-fold, 3.5-fold, 3.8-fold, 4-fold, 4.2-fold, 4.5-fold, 4.8-fold, 5-fold, 5.2-fold, 5.5-fold, 5.8-fold, 6-fold, 6.2-fold, 6.5-fold, 6.8-fold, 7-fold, 7.2-fold, 7.5-fold, 7.8-fold, 8-fold, 8.2-fold, 8.5-fold, 8.8-fold, 9-fold, 9.2-fold, 9.5-fold, 9.8-fold, 10-fold, 12-fold, 14-fold, 15-fold, 16-fold, 18-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 150-fold, 200-fold, 250-fold, 500-fold, 800-fold, 1000-fold, 2500-fold, 5000-fold, 10 4 -fold, 2.5 × 10 4 -fold, 5 × 10 4 -fold, 7.5 × 10 4 -fold, 2.5 × 10 5 -fold, 5 × 10 5 -fold, 10 6Folds or even higher folds. Exemplary methods for measuring the increased viral delivery and spread in tumors include, but are not limited to, fluorescence or bioluminescence-based reporter gene expression imaging, quantitative PCR for detecting the tumor concentration of viral genomes, or plaque determination of plaque-forming units, or immunohistochemistry of viral proteins.

[0159] In some embodiments, the modified oncolytic virus comprises an exogenous nucleic acid encoding a chemokine receptor that is a chimeric protein. At least a portion of its extracellular domain is from a chemokine receptor that promotes tumor-targeted delivery of the virus, and at least a portion of its intracellular domain is from a chemokine receptor that promotes tumor-specific replication, inhibits immunosuppressive activity, or confers some other beneficial effect, or vice versa. For example, the modified oncolytic virus comprises a nucleic acid encoding a protein having an intracellular GTPase domain of CCR5 or CXCR3 and an extracellular chemokine-binding domain of CXCR4 or CCR2. In some cases, combining domains with different functions achieves further improvement in the therapeutic performance of the modified oncolytic virus. In one embodiment of the present disclosure, the modified oncolytic virus comprises an exogenous nucleic acid encoding at least one chemokine receptor. In some cases, the modified oncolytic virus comprises an exogenous nucleic acid encoding two or more different chemokine receptors that are expressed simultaneously by the virus. Exemplary chemokine receptors expressed simultaneously from the modified oncolytic viruses described herein include CXCR4 and CCR2. In a modified oncolytic virus expressing more than one chemokine receptor, a combinatorial or synergistic effect on tumor cells is achieved in the therapeutic application of the oncolytic virus.

[0160] Condition being treated

[0161] Methods for treating cancer are provided herein, including administering the compositions described herein. In some embodiments, the treatment methods are for hyperproliferative diseases. In some embodiments, the hyperproliferative disease is cancer. In some embodiments, the hyperproliferative disease includes tumors. Treatments involving delivery of modified oncolytic viruses such as the oncolytic vaccinia virus described herein are envisioned. In some embodiments, the cancer is melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostatic intraepithelial neoplasia, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, intestinal type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma.

[0162] In some embodiments, the compositions described herein are administered to cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gingiva, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. Additionally, the cancer is optionally of the following histological types, but is not limited to these: neoplasm, malignant; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; trichoblastoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; oxyphilic carcinoma; oxyphilic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous gland carcinoma; ceraceous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; theca cell tumor, malignant; granulosa cell tumor, malignant; arrhenoblastoma, malignant; sertoli cell carcinoma; Leydig cell tumor, malignant; lipoid cell tumor, malignant; paraganglioma, malignant; extra-adrenal paraganglioma, malignant; pheochromocytoma; angiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; parosteal osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma multiforme; oligodendroglioma;Oligodendroglioblastoma; primitive neuroectoderm; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocyte; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myelosarcoma; or hairy cell leukemia. In some cases, a modified oncolytic virus of the present disclosure, such as a modified oncolytic vaccinia virus that facilitates systemic delivery, is used to treat metastatic solid cancers. In some cases, a modified oncolytic virus of the present disclosure, such as a modified oncolytic vaccinia virus that facilitates systemic delivery, is used to treat solid cancers that are inaccessible or difficult to access for the purpose of intratumoral delivery of therapeutic agents. In some embodiments, the compositions described herein are used to treat cancers associated with increased free fatty acid expression.;

[0163] The present disclosure also contemplates methods for inhibiting or preventing local invasion or metastasis or both of any type of primary cancer. In exemplary embodiments, the primary cancer is melanoma, non-small cell lung cancer, small cell lung cancer, lung cancer, liver cancer, retinoblastoma, astrocytoma, glioblastoma, gingival cancer, tongue cancer, leukemia, neuroblastoma, head cancer, neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, lymphoma, brain cancer, colon cancer or bladder cancer. In certain embodiments, the primary cancer is lung cancer. For example, the lung cancer is non-small cell lung cancer. Additionally, the present disclosure is optionally used to prevent cancer or treat pre-cancer or premalignant cells, including metaplasia, dysplasia and hyperplasia. It can also be used to inhibit unwanted but benign cells, such as squamous metaplasia, dysplasia, benign prostatic hyperplasia cells, hyperplastic lesions, and the like. In some embodiments, progression to cancer or a more severe form of cancer is halted, disrupted or delayed by methods of the present disclosure involving modified oncolytic viruses discussed herein.

[0164] In addition, the modified oncolytic viruses disclosed herein are administered to treat tumors having a high bioavailability of free fatty acids in the tumor microenvironment. In some cases, free fatty acids released by adipocytes in tumors of obese patients feed the modified oncolytic viruses in the tumor and enhance their replication and the formation of the viruses in the EEV form. This benefit can also be achieved in non-obese patients, particularly those suffering from peritoneal cancer. For example, several types of peritoneal cancer are targets for therapies using the modified oncolytic viruses of the present disclosure because these cancers tend to grow in the omental wall and are fed by adipocytes, and as described above, free fatty acids released by adipocytes in the tumor feed the modified oncolytic viruses in the tumor and enhance their replication. The modified oncolytic viruses disclosed herein form extracellular enveloped viruses (EEVs) with increased titers in tumors having a high bioavailability of free fatty acids.

[0165] The present disclosure provides methods of treating a subject by administering one or more modified oncolytic viruses as disclosed herein. "Individual" or "subject", as used interchangeably herein, refers to a human or non-human subject. Non-limiting examples of non-human subjects include non-human primates, dogs, cats, mice, rats, guinea pigs, rabbits, pigs, poultry, horses, cows, goats, sheep, cetaceans, etc. In some embodiments, the subject is human.

[0166] Methods of producing a toxic effect in cancer cells are provided, including administering a therapeutically effective amount of a modified virus, such as the oncolytic vaccinia virus as described above, or a pharmaceutical composition containing the same, to the cancer cells. The present disclosure also provides methods of inhibiting at least one of the growth and proliferation of a second cancer cell, the method including administering a modified oncolytic virus as described above to a first cancer cell such that the first cancer cell is infected by the virus. Thus, in some embodiments of the methods disclosed herein, it is contemplated that not every cancer cell or tumor cell will be infected after administering a therapeutically effective amount of the oncolytic vaccinia virus as described herein or a pharmaceutical composition containing the oncolytic vaccinia virus, and the growth of the uninfected cells is inhibited in the absence of direct infection.

[0167] In some instances, to use the methods and compositions of the present disclosure to induce oncolysis, kill cells, inhibit growth, inhibit metastasis, reduce tumor size, and otherwise reverse or reduce the malignant phenotype of tumor cells, cancer cells or tumors are contacted with a therapeutically effective dose of an exemplary oncolytic vaccinia virus as described herein or a pharmaceutical composition comprising the oncolytic vaccinia virus. In certain embodiments, an effective amount of a modified oncolytic virus of the present disclosure, such as the oncolytic vaccinia virus or its pharmaceutical composition as described herein, may comprise an amount sufficient to induce oncolysis, disrupt or lyse cancer cells, or inhibit or reduce the growth or size of cancer cells. For example, a reduction in the growth of cancer cells is manifested as cell death, or a slowdown in the replication rate or a decrease in the growth rate of a tumor containing the cells, or an increase in the survival period of a subject containing cancer cells.

[0168] In some embodiments, compared to an untreated tumor, the growth of the tumor is inhibited by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% using a modified virus as described herein.

[0169] In some embodiments, compared to an untreated tumor, the size of the tumor is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% using a modified virus as described herein.

[0170] Methods for treating a subject having cancer or a tumor are provided herein, the methods comprising administering to the subject an effective amount of a modified virus as described above. An effective amount in such methods includes an amount that slows the growth rate or spread of cancer, or increases the survival period of the subject. The present disclosure provides methods for slowing tumor growth, the methods comprising administering to a tumor an effective amount of a modified oncolytic virus as described above. In certain embodiments, an effective amount of a modified virus or its pharmaceutical composition includes an amount sufficient to induce a slowdown, inhibition, or reduction in tumor growth or size, and includes eradicating the tumor. For example, a slowdown in tumor growth is manifested as a decrease in the growth rate or an increase in the survival period of a subject containing the tumor. In certain embodiments, an effective amount of a modified virus or its pharmaceutical composition includes an amount sufficient to activate an anti-tumor response. In some embodiments, activating an anti-tumor response includes activating T cells. In certain embodiments, an effective amount of a modified virus or its pharmaceutical composition includes an amount sufficient to reduce the incidence of tumor growth. In some embodiments, reducing the incidence of tumor growth includes inhibiting metastasis, preventing primary tumor growth, inhibiting the growth of existing tumors, or any combination thereof.

[0171] The present disclosure provides methods for determining the infectivity or anti-tumor activity or tumor-specific viral replication of an oncolytic vaccinia virus as described herein, the method comprising: (i) administering to a subject a therapeutically effective amount of an oncolytic vaccinia virus or pharmaceutical composition according to the present disclosure, which also expresses a luciferase reporter gene, either alone or in combination with an additional therapy; (ii) collecting a first biological sample from the subject immediately after administering the virus and determining the level of the luciferase reporter gene in the first biological sample; (iii) collecting a second biological sample from the subject after administration in step (ii) and (iii) detecting the level of the luciferase reporter gene in the second biological sample, wherein if the level of luciferase in step (iii) is higher than in step (ii), it is determined that the oncolytic vaccinia virus is infectious, exhibits anti-tumor activity, and exhibits tumor-specific viral replication. The second biological sample is collected about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 15 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 1 month to about 2 months after administration in step (i). In some embodiments, the methods described above further comprise detecting in steps (i) and (iii) the levels of one or more assay cytokines (e.g., IL-2, IL-7, IL-8, IL-10, IFN-γ, GM-CSF, TNF-α, IL-6, IL-4, IL-5, and IL-13) in plasma samples collected from the subject after administering a therapeutically effective amount of the modified oncolytic virus of the present disclosure, such as an oncolytic vaccinia virus as described herein or a pharmaceutical composition comprising the oncolytic vaccinia virus. In some embodiments of the present disclosure, the modified oncolytic virus as described herein has a higher increase in luciferase bioluminescence between steps (ii) and (iv) compared to a virus that is otherwise identical but does not contain the modification in the modified oncolytic virus. Other exemplary techniques for detecting and monitoring viral load after administration of a modified oncolytic virus include real-time quantitative PCR.

[0172] The present disclosure provides methods for monitoring pharmacokinetics following administration of a therapeutically effective amount of a modified oncolytic virus according to the present disclosure (such as an oncolytic vaccinia virus or a pharmaceutical composition containing vaccinia virus described herein). Exemplary methods for monitoring pharmacokinetics include the steps of: (i) administering to a subject a therapeutically effective amount of an oncolytic vaccinia virus or a pharmaceutical composition containing the oncolytic vaccinia virus, alone or in combination with another therapy; (ii) collecting a biological sample from the subject at one or more time points selected from about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 120 minutes, about 180 minutes, and about 240 minutes, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 15 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 1 month to about 2 months after the administration in step (i), and (iii) detecting the amount of viral genome (or a reporter gene inserted within the viral genome, such as luciferase) in the biological sample collected at the above time points. In some cases, the viral genome copies / mL in the sample collected at the 15-minute time point is the highest, and further, the sample collected at the 240-minute time point does not contain a detectable amount of viral genome. Thus, in some cases, a viral peak is observed at about 15 minutes after administration, and most of the virus is cleared from the subject's system after about 240 minutes (or 4 hours). In some cases, a first viral peak is observed at about 15 minutes after administration, and a second viral peak is observed in biological samples collected at subsequent time points (e.g., at about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes). In an exemplary embodiment, the biological sample is blood, and the amount of viral genome / mL is determined by quantitative PCR or other suitable techniques. In some instances, a first viral peak is observed at about 15 minutes after administration of the modified oncolytic virus according to the present disclosure (such as an oncolytic vaccinia virus as described herein), and a second viral peak is observed at about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 15 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 1 month to about 2 months after administration.

[0173] In some cases, the tumor-selective replication of a modified virus (such as an oncolytic vaccinia virus) is measured by using a reporter gene (such as the luciferase gene). In some embodiments, the luciferase gene is inserted into the genome of the virus, and tumor cells are infected with the virus. The bioluminescence in the infected tumor cells is measured to monitor tumor-selective replication. Some examples show that the luciferase reporter molecule bioluminescence in the modified oncolytic viruses of the present disclosure is increased compared to an oncolytic vaccinia virus that is otherwise identical but does not contain the modification in the modified oncolytic virus.

[0174] Methods for delivering a modified virus as described herein are provided. The modified viruses provided herein are capable of increased replication in tumor cells compared to normal cells. In some embodiments, the modified virus produces about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 12-fold, about 14-fold, about 16-fold, about 18-fold, about 20-fold, about 30-fold, about 40-fold, or about 50-fold more copies / mg in tumor cells compared to production in normal cells.

[0175] Methods for contacting a tumor with a modified virus as described herein are provided. The modified viruses provided herein can increase the infiltration of CD3+CD8+ T cells into the tumor compared to an untreated tumor. In some embodiments, contacting a tumor with a modified virus as described herein results in a about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% increase in CD3+CD8+ T cells in the tumor compared to an untreated tumor. In some embodiments, contacting a tumor with a modified virus as described herein results in a about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold increase in CD3+CD8+ T cells in the tumor compared to an untreated tumor.

[0176] Dose

[0177] In some embodiments, the amount of the modified oncolytic virus described herein administered to a subject is between about 10 3 and 10 12 infectious virus particles or plaque forming units (PFU), or between about 10 5 PFU and 10 10 PFU, or between about 10 5 PFU and 10 8 PFU, or between about 10 8 PFU and 10 10 PFU. In some embodiments, the amount of the modified oncolytic virus of the present disclosure administered to a subject is between about 10 3and 10 12 between virus particles or plaque forming units (PFU), or at about 10 5 PFU and 10 10 PFU, or at about 10 5 PFU and 10 8 PFU, or at about 10 8 PFU and 10 10 PFU. In some embodiments, the modified oncolytic virus of the present disclosure is administered at a dose comprising about 10 3 PFU / dose to about 10 4 PFU / dose, about 10 4 PFU / dose to about 10 5 PFU / dose, about 10 5 PFU / dose to about 10 6 PFU / dose, about 10 7 PFU / dose to about 10 8 PFU / dose, about 10 9 PFU / dose to about 10 10 PFU / dose, about 10 10 PFU / dose to about 10 11 PFU / dose, about 10 11 PFU / dose to about 10 12 PFU / dose, about 10 12 PFU / dose to about 10 13 PFU / dose, about 10 13 PFU / dose to about 10 14 PFU / dose, or about 10 14 PFU / dose to about 10 15 PFU / dose. In some embodiments, the modified oncolytic virus of the present disclosure is administered at a dose comprising about 2×10 3 PFU / dose, 3×10 3 PFU / dose, 4×10 3 PFU / dose, 5×10 3 PFU / dose, 6×10 3 PFU / dose, 7×10 3 PFU / dose, 8×10 3 PFU / dose, 9×10 3 PFU / dose, about 10 4 PFU / dose, about 2×10 4 PFU / dose, about 3×10 4 PFU / dose, about 4×10 4 PFU / dose, about 5×10 4 PFU / dose, about 6×10 4 PFU / dose, about 7×104 PFU / dose, approximately 8×10 4 PFU / dose, approximately 9×10 4 PFU / dose, approximately 10 5 PFU / dose, 2×10 5 PFU / dose, 3×10 5 PFU / dose, 4×10 5 PFU / dose, 5×10 5 PFU / dose, 6×10 5 PFU / dose, 7×10 5 PFU / dose, 8×10 5 PFU / dose, 9×10 5 PFU / dose, approximately 10 6 PFU / dose, approximately 2×10 6 PFU / dose, approximately 3×10 6 PFU / dose, approximately 4×10 6 PFU / dose, approximately 5×10 6 PFU / dose, approximately 6×10 6 PFU / dose, approximately 7×10 6 PFU / dose, approximately 8×10 6 PFU / dose, approximately 9×10 6 PFU / dose, approximately 10 7 PFU / dose, approximately 2×10 7 PFU / dose, approximately 3×10 7 PFU / dose, approximately 4×10 7 PFU / dose, approximately 5×10 7 PFU / dose, approximately 6×10 7 PFU / dose, approximately 7×10 7 PFU / dose, approximately 8×10 7 PFU / dose, approximately 9×10 7 PFU / dose, approximately 10 8 PFU / dose, approximately 2×10 8 PFU / dose, approximately 3×10 8 PFU / dose, approximately 4×10 8 PFU / dose, approximately 5×10 8 PFU / dose, approximately 6×10 8 PFU / dose, approximately 7×10 8 PFU / dose, approximately 8×10 8 PFU / dose, approximately 9×10 8 PFU / dose, approximately 10 9 PFU / dose, approximately 2×10 9 PFU / dose, approximately 3×10 9 PFU / dose, approximately 4×10 9PFU / dose, about 5×10 9 PFU / dose, about 6×10 9 PFU / dose, about 7×10 9 PFU / dose, about 8×10 9 PFU / dose, about 9×10 9 PFU / dose, about 10 10 PFU / dose, about 2×10 10 PFU / dose, about 3×10 10 PFU / dose, about 4×10 10 PFU / dose, about 5×10 10 PFU / dose, about 6×10 10 PFU / dose, about 7×10 10 PFU / dose, about 8×10 10 PFU / dose, about 9×10 10 PFU / dose, about 10 10 PFU / dose, about 2×10 10 PFU / dose, about 3×10 10 PFU / dose, about 4×10 10 PFU / dose, about 5×10 10 PFU / dose, about 6×10 10 PFU / dose, about 7×10 10 PFU / dose, about 8×10 10 PFU / dose, about 9×10 10 PFU / dose, about 10 11 PFU / dose, about 2×10 11 PFU / dose, about 3×10 11 PFU / dose, about 4×10 11 PFU / dose, about 5×10 11 PFU / dose, about 6×10 11 PFU / dose, about 7×10 11 PFU / dose, about 8×10 11 PFU / dose, about 9×10 11 PFU / dose, or about 10 12 PFU / dose, about 10 12 PFU / dose to about 10 13 PFU / dose, about 10 13 PFU / dose to about 10 14 PFU / dose, or about 10 14 PFU / dose to about 10 15 PFU / dose of the dose is administered. In some embodiments, the modified oncolytic virus of the present disclosure comprises 5×10 9Administration of the dose of PFU / dose. In some embodiments, the modified oncolytic virus of the present disclosure is administered at a dose comprising up to 5×10 9 PFU / dose.

[0178] In some embodiments, the modified oncolytic virus of the present disclosure is administered at a dose comprising from about 10 3 viral particles / dose to about 10 4 viral particles / dose, from about 10 4 viral particles / dose to about 10 5 viral particles / dose, from about 10 5 viral particles / dose to about 10 6 viral particles / dose, from about 10 7 viral particles / dose to about 10 8 viral particles / dose, from about 10 9 viral particles / dose to about 10 10 viral particles / dose, from about 10 10 viral particles / dose to about 10 11 viral particles / dose, from about 10 11 viral particles / dose to about 10 12 viral particles / dose, from about 10 12 viral particles / dose to about 10 13 viral particles / dose, from about 10 13 viral particles / dose to about 10 14 viral particles / dose or from about 10 14 viral particles / dose to about 10 15 viral particles / dose.

[0179] In some embodiments, the modified oncolytic virus of the present disclosure is administered at a dose comprising from about 10 3 PFU / kg to about 10 4 PFU / kg, from about 10 4 PFU / kg to about 10 5 PFU / kg, from about 10 5 PFU / kg to about 10 6 PFU / kg, from about 10 7 PFU / kg to about 10 8 PFU / kg, from about 10 9 PFU / kg to about 10 10 PFU / kg, from about 10 10 PFU / kg to about 10 11 PFU / kg, from about 10 11 PFU / kg to about 10 12 PFU / kg, from about 10 12PFU / kg to about 10 13 PFU / kg, about 10 13 PFU / kg to about 10 14 PFU / kg or about 10 14 PFU / kg to about 10 15 PFU / kg are administered. In some embodiments, the modified oncolytic virus of the present disclosure is in a dose comprising about 2×10 3 PFU / kg, 3×10 3 PFU / kg, 4×10 3 PFU / kg, 5×10 3 PFU / kg, 6×10 3 PFU / kg, 7×10 3 PFU / kg, 8×10 3 PFU / kg, 9×10 3 PFU / kg, about 10 4 PFU / kg, about 2×10 4 PFU / kg, about 3×10 4 PFU / kg, about 4×10 4 PFU / kg, about 5×10 4 PFU / kg, about 6×10 4 PFU / kg, about 7×10 4 PFU / kg, about 8×10 4 PFU / kg, about 9×10 4 PFU / kg, about 10 5 PFU / kg, 2×10 5 PFU / kg, 3×10 5 PFU / kg, 4×10 5 PFU / kg, 5×10 5 PFU / kg, 6×10 5 PFU / kg, 7×10 5 PFU / kg, 8×10 5 PFU / kg, 9×10 5 PFU / kg, about 10 6 PFU / kg, about 2×10 6 PFU / kg, about 3×10 6 PFU / kg, about 4×10 6 PFU / kg, about 5×10 6 PFU / kg, about 6×10 6 PFU / kg, about 7×10 6 PFU / kg, about 8×10 6 PFU / kg, about 9×10 6PFU / kg, approximately 10 7 PFU / kg, approximately 2×10 7 PFU / kg, approximately 3×10 7 PFU / kg, approximately 4×10 7 PFU / kg, approximately 5×10 7 PFU / kg, approximately 6×10 7 PFU / kg, approximately 7×10 7 PFU / kg, approximately 8×10 7 PFU / kg, approximately 9×10 7 PFU / kg, approximately 10 8 PFU / kg, approximately 2×10 8 PFU / kg, approximately 3×10 8 PFU / kg, approximately 4×10 8 PFU / kg, approximately 5×10 8 PFU / kg, approximately 6×10 8 PFU / kg, approximately 7×10 8 PFU / kg, approximately 8×10 8 PFU / kg, approximately 9×10 8 PFU / kg, approximately 10 9 PFU / kg, approximately 2×10 9 PFU / kg, approximately 3×10 9 PFU / kg, approximately 4×10 9 PFU / kg, approximately 5×10 9 PFU / kg, approximately 6×10 9 PFU / kg, approximately 7×10 9 PFU / kg, approximately 8×10 9 PFU / kg, approximately 9×10 9 PFU / kg, approximately 10 10 PFU / kg, approximately 2×10 10 PFU / kg, approximately 3×10 10 PFU / kg, approximately 4×10 10 PFU / kg, approximately 5×10 10 PFU / kg, approximately 6×10 10 PFU / kg, approximately 7×10 10 PFU / kg, approximately 8×10 10 PFU / kg, approximately 9×10 10 PFU / kg, approximately 10 10 PFU / kg, approximately 2×10 10 PFU / kg, approximately 3×10 10 PFU / kg, approximately 4×10 10PFU / kg, approximately 5×10 10 PFU / kg, approximately 6×10 10 PFU / kg, approximately 7×10 10 PFU / kg, approximately 8×10 10 PFU / kg, approximately 9×10 10 PFU / kg, approximately 10 11 PFU / kg, approximately 2×10 11 PFU / kg, approximately 3×10 11 PFU / kg, approximately 4×10 11 PFU / kg, approximately 5×10 11 PFU / kg, approximately 6×10 11 PFU / kg, approximately 7×10 11 PFU / kg, approximately 8×10 11 PFU / kg, approximately 9×10 11 PFU / kg or approximately 10 12 PFU / kg, approximately 10 12 PFU / kg to approximately 10 13 PFU / kg, approximately 10 13 PFU / kg to approximately 10 14 PFU / kg or approximately 10 14 PFU / kg to approximately 10 15 administered at a dose of PFU / kg. In some embodiments, the modified oncolytic virus of the present disclosure is administered at a dose comprising 5×10 9 administered at a dose of PFU / kg. In some embodiments, the modified oncolytic virus of the present disclosure is administered at a dose comprising up to 5×10 9 administered at a dose of PFU / kg.

[0180] In some embodiments, the modified oncolytic virus of the present disclosure is administered at a dose comprising approximately 10 3 virus particles / kg to approximately 10 4 virus particles / kg, approximately 10 4 virus particles / kg to approximately 10 5 virus particles / kg, approximately 10 5 virus particles / kg to approximately 10 6 virus particles / kg, approximately 10 7 virus particles / kg to approximately 10 8 virus particles / kg, approximately 10 9 virus particles / kg to approximately 10 10 virus particles / kg, approximately 10 10 virus particles / kg to approximately 10 11 virus particles / kg, approximately 10 11from about 10 virus particles / kg to about 10 12 virus particles / kg, about 10 12 virus particles / kg to about 10 13 virus particles / kg, about 10 13 virus particles / kg to about 10 14 virus particles / kg or about 10 14 virus particles / kg to about 10 15 virus particles / kg and administered at a dose of about 10

[0181] In certain embodiments, the liquid dosage form of the oncolytic virus as described herein comprises from about 10 3 PFU / mL to about 10 4 PFU / mL, about 10 4 PFU / mL to about 10 5 PFU / mL, about 10 5 PFU / mL to about 10 6 PFU / mL, about 10 7 PFU / mL to about 10 8 PFU / mL, about 10 9 PFU / mL to about 10 10 PFU / mL, about 10 10 PFU / mL to about 10 11 PFU / mL, about 10 11 PFU / mL to about 10 12 PFU / mL, about 10 12 PFU / mL to about 10 13 PFU / mL, about 10 13 PFU / mL to about 10 14 PFU / mL or about 10 14 PFU / mL to about 10 15 PFU / mL of viral dose. In some embodiments, the modified oncolytic virus of the present disclosure is in a form comprising from about 2×10 3 PFU / mL, 3×10 3 PFU / mL, 4×10 3 PFU / mL, 5×10 3 PFU / mL, 6×10 3 PFU / mL, 7×10 3 PFU / mL, 8×10 3 PFU / mL, 9×10 3 PFU / mL, about 10 4 PFU / mL, about 2×10 4 PFU / mL, about 3×10 4PFU / mL, approximately 4×10 4 PFU / mL, approximately 5×10 4 PFU / mL, approximately 6×10 4 PFU / mL, approximately 7×10 4 PFU / mL, approximately 8×10 4 PFU / mL, approximately 9×10 4 PFU / mL, approximately 10 5 PFU / mL, 2×10 5 PFU / mL, 3×10 5 PFU / mL, 4×10 5 PFU / mL, 5×10 5 PFU / mL, 6×10 5 PFU / mL, 7×10 5 PFU / mL, 8×10 5 PFU / mL, 9×10 5 PFU / mL, approximately 10 6 PFU / mL, approximately 2×10 6 PFU / mL, approximately 3×10 6 PFU / mL, approximately 4×10 6 PFU / mL, approximately 5×10 6 PFU / mL, approximately 6×10 6 PFU / mL, approximately 7×10 6 PFU / mL, approximately 8×10 6 PFU / mL, approximately 9×10 6 PFU / mL, approximately 10 7 PFU / mL, approximately 2×10 7 PFU / mL, approximately 3×10 7 PFU / mL, approximately 4×10 7 PFU / mL, approximately 5×10 7 PFU / mL, approximately 6×10 7 PFU / mL, approximately 7×10 7 PFU / mL, approximately 8×10 7 PFU / mL, approximately 9×10 7 PFU / mL, approximately 10 8 PFU / mL, approximately 2×10 8 PFU / mL, approximately 3×10 8 PFU / mL, approximately 4×10 8 PFU / mL, approximately 5×10 8 PFU / mL, approximately 6×10 8 PFU / mL, approximately 7×10 8 PFU / mL, approximately 8×108 PFU / mL, approximately 9×10 8 PFU / mL, approximately 10 9 PFU / mL, approximately 2×10 9 PFU / mL, approximately 3×10 9 PFU / mL, approximately 4×10 9 PFU / mL, approximately 5×10 9 PFU / mL, approximately 6×10 9 PFU / mL, approximately 7×10 9 PFU / mL, approximately 8×10 9 PFU / mL, approximately 9×10 9 PFU / mL, approximately 10 10 PFU / mL, approximately 2×10 10 PFU / mL, approximately 3×10 10 PFU / mL, approximately 4×10 10 PFU / mL, approximately 5×10 10 PFU / mL, approximately 6×10 10 PFU / mL, approximately 7×10 10 PFU / mL, approximately 8×10 10 PFU / mL, approximately 9×10 10 PFU / mL, approximately 10 10 PFU / mL, approximately 2×10 10 PFU / mL, approximately 3×10 10 PFU / mL, approximately 4×10 10 PFU / mL, approximately 5×10 10 PFU / mL, approximately 6×10 10 PFU / mL, approximately 7×10 10 PFU / mL, approximately 8×10 10 PFU / mL, approximately 9×10 10 PFU / mL, approximately 10 11 PFU / mL, approximately 2×10 11 PFU / mL, approximately 3×10 11 PFU / mL, approximately 4×10 11 PFU / mL, approximately 5×10 11 PFU / mL, approximately 6×10 11 PFU / mL, approximately 7×10 11 PFU / mL, approximately 8×10 11 PFU / mL, approximately 9×10 11 PFU / mL or approximately 10 12 PFU / mL, approximately 10 12 PFU / mL to approximately 10 13PFU / mL, about 10 13 PFU / mL to about 10 14 PFU / mL or about 10 14 PFU / mL or about 10 15 PFU / mL. In some embodiments, the modified oncolytic virus of the present disclosure is administered at a dose comprising 5×10 9 PFU / mL. In some embodiments, the modified oncolytic virus of the present disclosure is administered at a dose comprising up to 5×10 9 PFU / mL.

[0182] In some cases, when administering the modified oncolytic virus by injection, the dose comprises about 10 3 viral particles per injection, 10 4 viral particles per injection, 10 5 viral particles per injection, 10 6 viral particles per injection, 10 7 viral particles per injection, 10 8 viral particles per injection, 10 9 viral particles per injection, 10 10 viral particles per injection, 10 11 viral particles per injection, 10 12 viral particles per injection, 2×10 12 viral particles per injection, 10 13 viral particles per injection, 10 14 viral particles per injection, or 10 15 viral particles per injection. In additional cases, when administering the modified oncolytic virus by injection, the dose comprises about 10 3 infectious viral particles per injection, 10 4 infectious viral particles per injection, 10 5 infectious viral particles per injection, 10 6 infectious viral particles per injection, 10 7 infectious viral particles per injection, 10 8 infectious viral particles per injection, 10 9 infectious viral particles per injection, 10 10 infectious viral particles per injection, 10 11 infectious viral particles per injection, 10 12 infectious viral particles per injection, 2×10 12 infectious viral particles per injection, 10 13 infectious viral particles per injection, 1014 infectious viral particles, or 10 per injection 15 infectious viral particles. In some embodiments, the virus is administered in an amount sufficient to induce oncolysis in at least about 20% of the cells in the tumor, at least about 30% of the cells in the tumor, at least about 40% of the cells in the tumor, at least about 50% of the cells in the tumor, at least about 60% of the cells in the tumor, at least about 70% of the cells in the tumor, at least about 80% of the cells in the tumor, or at least about 90% of the cells in the tumor. In certain embodiments, a single dose of the virus refers to the amount administered to a subject or tumor over a period of 1 hour, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours, or 24 hours. In certain embodiments, the dose is extended over time or by separate injections. In certain embodiments, more than one dose (e.g., 2, 3, 4, 5, 6, or more doses) of vaccinia virus is administered to a subject, for example, wherein the second treatment is performed within 1, 2, 3, 4, 5, 6, 7 days or weeks after the first treatment. In certain embodiments, more than one dose of the modified oncolytic virus is administered to a subject over a period of 1, 2, 3, 4, 5, 6, 7, or more days or weeks. In certain embodiments, the oncolytic virus or pharmaceutical composition as described herein is administered over a period of about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks, or about 52 weeks or longer. In certain cases, the frequency of administration of the oncolytic vaccinia virus or pharmaceutical composition as described herein is once daily, twice daily, once weekly, once every three weeks, once every four weeks (or once monthly), once every 8 weeks (or once every 2 months), once every 12 weeks (or once every 3 months), or once every 24 weeks (once every 6 months). In some embodiments of the methods disclosed herein, the oncolytic vaccinia virus or pharmaceutical composition is administered independently at an initial dose for a first period, at an intermediate dose for a second period, and at a high dose for a third period. In some embodiments, the initial dose is lower than the intermediate dose, and the intermediate dose is lower than the high dose. In some embodiments, the first period, the second period, and the third period are independently about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks, or about 52 weeks or longer.

[0183] In some instances, according to any of the treatment methods described herein, a subject is placed on a reduced carbohydrate diet, e.g., a ketogenic diet, before, concurrently with, and after administration of a modified oncolytic virus such as oncolytic vaccinia virus or a pharmaceutical composition comprising the modified oncolytic virus as described herein. In certain embodiments, the subject is placed on a diet that includes consuming less than 500 grams of carbohydrates per day, less than 450 grams of carbohydrates per day, less than 400 grams of carbohydrates per day, less than 350 grams of carbohydrates per day, less than 300 grams of carbohydrates per day, less than 250 grams of carbohydrates per day, less than 200 grams of carbohydrates per day, less than 150 grams of carbohydrates per day, less than 100 grams of carbohydrates per day, less than 90 grams of carbohydrates per day, less than 80 grams of carbohydrates per day, less than 70 grams of carbohydrates per day, less than 60 grams of carbohydrates per day, less than 50 grams of carbohydrates per day, less than 40 grams of carbohydrates per day, less than 30 grams of carbohydrates per day, less than 20 grams of carbohydrates per day, or less than 10 grams of carbohydrates per day.

[0184] Exemplary methods of delivering a modified oncolytic virus of the present disclosure, such as an oncolytic vaccinia virus as described herein, or a pharmaceutical composition comprising the modified oncolytic virus, to cancer cells or tumor cells are via intratumoral injection. However, alternative methods of administration are also used, for example, intravenous, via infusion, parenteral, intravenous, intradermal, intramuscular, transdermal, rectal, intraurethral, intravaginal, intranasal, intrathecal, or intraperitoneal. The route of administration varies with the location and nature of the tumor. In certain embodiments, the route of administration is intraodontal, transdermal, parenteral, intraperitoneal, intravenous, intramuscular, intranasal, subcutaneous, regional (e.g., near the tumor, particularly the blood vessels of the tumor or adjacent blood vessels), percutaneous, intrathecal, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, by lavage, or oral. The injectable dose of the oncolytic virus is administered as a bolus injection or a slow infusion. In certain embodiments, the modified oncolytic virus is administered to a patient from a source implanted within the patient. In certain embodiments, the administration of the modified oncolytic virus is carried out by continuous infusion over a selected period of time. In some cases, an oncolytic vaccinia virus as described herein, or a pharmaceutical composition comprising the oncolytic vaccinia virus, is administered in a therapeutically effective dose by infusion over a period of about 15 min, about 30 min, about 45 min, about 50 min, about 55 min, about 60 min, about 75 min, about 90 min, about 100 min, or about 120 min or longer. The oncolytic virus or pharmaceutical composition of the present disclosure is administered as a liquid dose, wherein the total volume administered is about 1 mL to about 5 mL, about 5 mL to 10 mL, about 15 mL to about 20 mL, about 25 mL to about 30 mL, about 30 mL to about 50 mL, about 50 mL to about 100 mL, about 100 mL to 150 mL, about 150 mL to about 200 mL, about 200 mL to about 250 mL, about 250 mL to about 300 mL, about 300 mL to about 350 mL, about 350 mL to about 400 mL, about 400 mL to about 450 mL, about 450 mL to 500 mL, about 500 mL to 750 mL, or about 750 mL to 1000 mL.

[0185] Preparation

[0186] A pharmaceutical composition containing a modified virus (such as an oncolytic vaccinia virus) as described herein is prepared as a solution, a dispersion in glycerol, liquid polyethylene glycol, and any combination thereof in oil, solid dosage forms, as an inhalable dosage form, as a nasal dosage form, as a liposomal formulation, a dosage form containing nanoparticles, a dosage form containing microparticles, a polymeric dosage form, or any combination thereof. In some embodiments, the pharmaceutical composition as described herein contains a stabilizer and a buffer. In some embodiments, the pharmaceutical composition as described herein may contain a solubilizer, such as sterile water, Tris buffer. In some embodiments, the pharmaceutical composition as described herein may contain excipients. Non-limiting examples of suitable excipients may include buffers, preservatives, stabilizers, binders, compacting agents, lubricants, chelating agents, dispersion enhancers, disintegrants, flavoring agents, sweetening agents, coloring agents.

[0187] In certain embodiments, the buffer includes phosphate buffered saline (PBS), Dulbecco's PBS (DPBS), TRIS buffered saline (TBS), Hank's balanced salt solution (HBSS), Earle's balanced salt solution (EBSS), standard citrate saline (SSC), HEPES buffered saline (HBS), or Gey's balanced salt solution.

[0188] In certain embodiments, the pharmaceutical composition of the present disclosure contains an effective amount of the modified virus disclosed herein, in combination with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" includes any carrier that does not interfere with the effectiveness of the biological activity of the active ingredient and / or is non-toxic to the patient to whom it is administered. Non-limiting examples of suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, and sterile solutions. Additional non-limiting examples of pharmaceutically compatible carriers include gels, bioabsorbable matrix materials, implantable elements containing modified oncolytic viruses, or any other suitable vehicle, delivery, or dispersion tool or material. Such carriers are formulated by conventional methods and administered to a subject in an effective amount.

[0189] Production method

[0190] The modified oncolytic viruses of the present disclosure are produced by methods known to those skilled in the art. In certain embodiments, the modified oncolytic viruses are propagated in suitable host cells (e.g., HeLa cells, 293 cells, or Vero cells), isolated from the host cells, and stored under conditions that promote the stability and integrity of the virus, such that the loss of infectivity over time is minimized. In certain exemplary methods, cell stacks, roller bottles, or perfusion bioreactors are used to propagate the modified oncolytic viruses in host cells. In some instances, downstream methods for purifying the modified oncolytic viruses include filtration (e.g., depth filtration, tangential flow filtration, or a combination thereof), ultracentrifugation, chromatographic capture, or any combination thereof. For example, the modified oncolytic viruses are stored by freezing or drying, such as by lyophilization. In certain embodiments, prior to administration, the stored modified oncolytic viruses are reconstituted (if stored dry) and diluted in a pharmaceutically acceptable carrier for administration.

[0191] Some embodiments provide that, compared to viruses modified in other aspects but not containing the modifications in the modified oncolytic viruses, the modified oncolytic viruses as described herein exhibit higher titers in HeLa cells and 293 cells. In certain cases, higher titers of the modified oncolytic viruses are observed in HeLa cells and 293 cells.

[0192] Kit

[0193] In an embodiment, the present disclosure provides a kit for administering the modified oncolytic viruses as described herein. In certain embodiments, the kit of the present disclosure contains the modified oncolytic viruses as described above or a pharmaceutical composition containing the modified oncolytic viruses. In certain embodiments, the kit of the present disclosure further contains one or more components, such as instructions for use, devices, and additional reagents, as well as components for performing the methods disclosed above, such as tubes, containers, and syringes. In certain embodiments, the kit of the present disclosure further contains one or more agents, for example, at least one of an anti-cancer agent, an immunomodulator, or any combination thereof, which is administered in combination with the modified virus.

[0194] In certain embodiments, the kit of the present disclosure contains one or more containers that contain the modified viruses disclosed herein. For example, and without limitation, the kit of the present disclosure contains one or more containers that contain the modified oncolytic viruses of the present disclosure.

[0195] In certain embodiments, the kit of the present disclosure includes instructions for use, a device for administering a modified oncolytic virus to a subject, or a device for administering an additional agent or compound to a subject. For example, and without limitation, the instructions for use include a description of the modified oncolytic virus and optionally other components included in the kit, as well as a method of administration, which includes methods for determining the appropriate condition of the subject, the appropriate dosage amount, and the appropriate method of administration of the modified virus. The instructions for use also optionally include guidelines for monitoring the subject during the duration of the treatment time.

[0196] In some embodiments, the kit of the present disclosure includes a device for administering a modified oncolytic virus to a subject. Any of a variety of devices known in the art for administering drugs and pharmaceutical compositions are included in the kits provided herein. For example, and without limitation, such devices include subcutaneous injection needles, intravenous injection needles, catheters, needleless injection devices, inhalers, and liquid dispensers such as eye droppers. In certain embodiments, for example, a modified oncolytic virus to be systemically delivered by intravenous injection, intratumoral injection, or intraperitoneal injection is included in a kit having a subcutaneous injection needle and syringe.

[0197] Although the preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the present disclosure. The appended claims are intended to define the scope of the present disclosure and thereby cover the methods and structures within the scope of these claims and their equivalents.

[0198] Exemplary embodiments

[0199] The present invention provides compositions, wherein the compositions comprise a vector, and the vector comprises: an exogenous nucleic acid comprising a sequence encoding a cytokine or a functional variant thereof; an exogenous nucleic acid comprising a sequence encoding a chemokine receptor or a functional variant thereof; and a first promoter region, wherein the first promoter region is upstream of the sequence encoding the chemokine receptor and provides expression of the chemokine receptor prior to expression of the cytokine. The present invention also provides compositions, wherein the encoded chemokine receptor comprises at least one of the following: a CXC receptor, a CC receptor, a CX3C receptor, an XC receptor, a functional fragment thereof, a functional variant thereof, or any combination thereof. The present invention also provides compositions, wherein the encoded chemokine receptor comprises at least one of the following: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, a functional fragment thereof, a functional variant thereof, or any combination thereof. The present invention also provides compositions, wherein the encoded chemokine receptor is CXCR3. The present invention also provides compositions, wherein the encoded chemokine receptor comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:42 or SEQ ID NO:43. The present invention also provides compositions, wherein the encoded chemokine receptor comprises the amino acid sequence set forth in SEQ ID NO:42 or SEQ ID NO:43. The present invention also provides compositions, wherein the first promoter region comprises an early promoter. The present invention also provides compositions, wherein the early promoter comprises any one of A52R, pB8, mH5, I4L, LEO, pF11, I3L, P7.5, TK promoter, F7L, H5R, short synthetic promoter (SSP), or any variant or combination thereof. The present invention also provides compositions, wherein the early promoter comprises the A52R promoter. The present invention also provides compositions, wherein the encoded cytokine comprises IL-12 or a functional variant thereof. The present invention also provides compositions, wherein the encoded IL-12 is murine IL-12 or human IL-12. The present invention also provides compositions, wherein the encoded IL-12 comprises an α subunit and a β subunit. The present invention also provides compositions, wherein the sequences encoding the IL-12α subunit and the IL-12β subunit further comprise a sequence encoding a linker. The present invention also provides compositions, wherein the encoded IL-12α subunit comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO:13 or SEQ ID NO:16. The present invention also provides compositions, wherein the encoded IL-12α subunit comprises the amino acid sequence set forth in any one of SEQ ID NO:13 or SEQ ID NO:16.The present invention also provides compositions, wherein the encoded IL-12β subunit comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NO:14 or SEQ ID NO:17. The present invention also provides compositions, wherein the IL-12β subunit comprises the amino acid sequence set forth in any one of SEQ ID NO:14 or SEQ ID NO:17. The present invention also provides compositions, wherein the encoded linker comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:18. The present invention also provides compositions, wherein the encoded linker comprises the amino acid sequence set forth in SEQ ID NO:18. The present invention also provides compositions, wherein the encoded IL-12 comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NO:12 or SEQ ID NO:15. The present invention also provides compositions, wherein the encoded IL-12 comprises the amino acid sequence set forth in any one of SEQ ID NO:12 or SEQ ID NO:15. The present invention also provides compositions, wherein the sequence encoding IL-12 comprises a nucleic acid sequence having at least 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NO:64 or SEQ ID NO:66. The present invention also provides compositions, wherein the sequence encoding IL-12 comprises the nucleic acid sequence set forth in any one of SEQ ID NO:64 or SEQ ID NO:66. The present invention also provides compositions further comprising an exogenous nucleic acid, the exogenous nucleic acid comprising a sequence encoding an inhibitor of transforming growth factor β (TGF-β) activity; wherein the first promoter region provides expression of the chemokine receptor prior to expression of the inhibitor of TGF-β activity. The present invention also provides compositions, wherein the encoded inhibitor of TGF-β activity comprises a TGF-β dominant negative, a TGF-β receptor dominant negative, a protein that binds to TGF-β or a protein that binds to a TGF-β receptor. The present invention also provides compositions, wherein the encoded protein that binds to a TGF-β receptor is a protein comprising a domain of TGF-β. The present invention also provides compositions, wherein the encoded protein comprising a domain of TGF-β comprises the amino acid sequence set forth in any one of SEQ ID NO:1-9. The present invention also provides compositions, wherein the encoded protein comprising a domain of TGF-β comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NO:7 or SEQ ID NO:8. The present invention also provides compositions, wherein the encoded protein comprising a domain of TGF-β comprises the amino acid sequence set forth in any one of SEQ ID NO:7 or SEQ ID NO:8.The present invention also provides a composition, wherein the sequence encoding an inhibitor of transforming growth factor β (TGF-β) activity comprises a nucleic acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 67. The present invention also provides a composition, wherein the sequence encoding an inhibitor of TGF-β activity comprises the nucleic acid sequence set forth in SEQ ID NO: 67. The present invention also provides a composition, wherein the protein that binds to the TGF-β receptor is a TGF-β fusion protein. The present invention also provides a composition, wherein the encoded inhibitor of TGF-β activity comprises a protein that binds to the TGF-β receptor, and wherein the protein that binds to the TGF-β receptor comprises a TGF-β fusion 1 (TGFbf1) protein. The present invention also provides a composition, wherein the TGFbf1 protein comprises a murine IL-2 signal peptide and a TGF-β variant 1 (TGFbv1). The present invention also provides a composition, wherein the murine IL-2 signal peptide comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 36. The present invention also provides a composition, wherein the murine IL-2 signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 36. The present invention also provides a composition, wherein the TGFbv1 comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 7. The present invention also provides a composition, wherein the TGFbv1 comprises the sequence set forth in SEQ ID NO: 7. The present invention also provides a composition, wherein the encoded inhibitor of TGF-β activity comprises a protein that binds to the TGF-β receptor, and wherein the protein that binds to the TGF-β receptor comprises a TGF-β fusion 2 (TGFbf2) protein. The present invention also provides a composition, wherein the TGFbf2 comprises a human IgE signal peptide and a TGF-β variant 2 (TGFbv2). The present invention also provides a composition, wherein the human IgE signal peptide comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 37. The present invention also provides a composition, wherein the human IgE signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 37. The present invention also provides a composition, wherein the TGFbv2 comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 8. The present invention also provides a composition, wherein the TGFbv2 comprises the sequence set forth in SEQ ID NO: 8. The present invention also provides a composition, wherein the sequence encoding the cytokine comprises a second promoter region that provides for cytokine expression, and wherein the sequence encoding the inhibitor of TGF-β activity comprises a third promoter region that provides for expression of the inhibitor of TGF-β activity. The present invention also provides a composition, wherein each of the second promoter region and the third promoter region comprises a late promoter.The present invention also provides a composition, wherein the late promoter comprises any one of SSP, P7.5, P28, P135, TK promoter, E / L, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, PH5, L4R, 28 kDa promoter, or any variant or combination thereof. The present invention also provides a composition, wherein the late promoter comprises a weak late promoter. The present invention also provides a composition, wherein the weak late promoter comprises the P135 promoter. The present invention also provides a composition, wherein the second promoter region comprises the P135 promoter. The present invention also provides a composition, wherein the late promoter comprises a strong late promoter. The present invention also provides a composition, wherein the strong late promoter comprises the P7.5 promoter. The present invention also provides a composition, wherein the third promoter region that provides for the expression of a TGF-β activity inhibitor comprises the P7.5 promoter. The present invention also provides a composition, wherein the exogenous nucleic acid encoding a cytokine or a functional variant thereof, the exogenous nucleic acid encoding a TGF-β activity inhibitor, and the exogenous nucleic acid encoding a chemokine receptor or a functional variant thereof are located on a single genome. The present invention also provides a composition, wherein the vector is an oncolytic virus, and wherein the oncolytic virus comprises at least one genomic modification. The present invention also provides a composition, wherein the at least one modification comprises a mutation or deletion of at least one gene selected from the group consisting of: thymidine kinase (TK), F13L, A36R, A34R, A33R, A52R, B5R, B8R, B18R, SPI-1, SPI-2, B15R, VGF, E3L, K3L, A41L, K7R, or N1L, a functional fragment thereof, or any combination thereof. The present invention also provides a composition, wherein the at least one modification comprises a deletion of genes A52R and TK. The present invention also provides a composition, wherein the oncolytic virus is a poxvirus, an adeno-associated virus, an adenovirus, a reovirus, a lentivirus, a herpes simplex virus, a vesicular stomatitis virus, a Mengo virus, a myxoma virus, a Newcastle disease virus, a measles virus, or a poliovirus. The present invention also provides a composition, wherein the poxvirus is vaccinia virus. The present invention also provides a composition, wherein the vaccinia virus is the Western Reserve strain. The present invention also provides a composition, wherein the at least one genomic modification results in an increase in the efficacy of systemic delivery of the virus to tumors by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 12-fold, about 14-fold, about 16-fold, about 18-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, or about 100-fold.

[0200] The present invention provides nucleic acids, wherein the nucleic acids comprise sequences encoding at least two polypeptides, and the at least two polypeptides comprise: interleukin-12 (IL-12) or a functional variant thereof; and an inhibitor of transforming growth factor-β (TGF-β) activity. The present invention provides nucleic acids, wherein the nucleic acids comprise sequences encoding: a first polypeptide comprising interleukin-12 (IL-12) or a functional variant thereof; and a second polypeptide comprising an inhibitor of transforming growth factor-β (TGF-β) activity. The present invention also provides nucleic acids, wherein the nucleic acids comprise DNA or RNA. The present invention also provides nucleic acids, wherein IL-12 is murine IL-12 or human IL-12. The present invention also provides nucleic acids, wherein IL-12 or a functional variant thereof comprises an α subunit and a β subunit. The present invention also provides nucleic acids, wherein the α subunit and the β subunit are linked by a linker. The present invention also provides nucleic acids, wherein the first polypeptide comprising the IL-12 α subunit and the IL-12 β subunit further comprises a linker. The present invention also provides nucleic acids, wherein the IL-12 α subunit comprises a sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:13 or SEQ ID NO:16. The present invention also provides nucleic acids, wherein the IL-12 α subunit comprises the sequence listed in SEQ ID NO:13 or SEQ ID NO:16. The present invention also provides nucleic acids, wherein the IL-12 β subunit comprises a sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:14 or SEQ ID NO:17. The present invention also provides nucleic acids, wherein the IL-12 β subunit comprises the sequence listed in SEQ ID NO:14 or SEQ ID NO:17. The present invention also provides nucleic acids, wherein the linker comprises a sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:18. The present invention also provides nucleic acids, wherein the linker comprises the sequence listed in SEQ ID NO:18. The present invention also provides nucleic acids, wherein the encoded IL-12 comprises a sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:12 or SEQ ID NO:15. The present invention also provides nucleic acids, wherein the encoded IL-12 comprises the sequence listed in SEQ ID NO:12 or SEQ ID NO:15. The present invention also provides nucleic acids, wherein the nucleic acid encoding IL-12 comprises a sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:64 or SEQ ID NO:66. The present invention also provides nucleic acids, wherein the nucleic acid encoding IL-12 comprises the sequence listed in SEQ ID NO:64 or SEQ ID NO:66. The present invention also provides nucleic acids, wherein the inhibitor of TGF-β activity comprises a TGF-β dominant negative, a TGF-β receptor dominant negative, a protein that binds to TGF-β or a protein that binds to a TGF-β receptor.The present invention also provides nucleic acids, wherein the TGF-β activity inhibitor comprises a protein that binds to TGF-β, and wherein the protein that binds to TGF-β is an antibody or a functional variant thereof. The present invention also provides nucleic acids, wherein the TGF-β activity inhibitor comprises a protein that binds to the TGF-β receptor, and wherein the protein that binds to the TGF-β receptor is an antibody or a functional variant thereof. The present invention also provides nucleic acids, wherein the TGF-β activity inhibitor comprises a protein that binds to the TGF-β receptor, and wherein the protein that binds to the TGF-β receptor is a protein comprising a domain of TGF-β. The present invention also provides nucleic acids, wherein the protein comprising a domain of TGF-β comprises a sequence listed in any one of SEQ ID NO: 1-9. The present invention also provides nucleic acids, wherein the protein comprises at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 7 or SEQ ID NO: 8. The present invention also provides nucleic acids, wherein the protein comprising a domain of TGF-β comprises the sequence listed in SEQ ID NO: 7 or SEQ ID NO: 8. The present invention also provides nucleic acids, wherein the nucleic acid encoding the TGF-β activity inhibitor comprises at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 67. The present invention also provides nucleic acids, wherein the nucleic acid encoding the TGF-β activity inhibitor comprises the sequence listed in SEQ ID NO: 67. The present invention also provides nucleic acids, wherein the protein that binds to the TGF-β receptor comprises a TGF-β fusion 1 (TGFbf1) protein. The present invention also provides nucleic acids, wherein TGFbf1 comprises a murine IL-2 signal peptide and a TGF-β variant 1 (TGFbv1). The present invention also provides nucleic acids, wherein the murine IL-2 signal peptide comprises a sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 36. The present invention also provides nucleic acids, wherein the murine IL-2 signal peptide comprises the sequence listed in SEQ ID NO: 36. The present invention also provides nucleic acids, wherein TGFbv1 comprises a sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 7. The present invention also provides nucleic acids, wherein TGFbv1 comprises the sequence listed in SEQ ID NO: 7. The present invention also provides nucleic acids, wherein the protein that binds to the TGF-β receptor comprises a TGF-β fusion 2 (TGFbf2) protein. The present invention also provides nucleic acids, wherein TGFbf2 comprises a human IgE signal peptide and a TGF-β variant 2 (TGFbv2). The present invention also provides nucleic acids, wherein the human IgE signal peptide comprises a sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 37. The present invention also provides nucleic acids, wherein the human IgE signal peptide comprises the sequence listed in SEQ ID NO: 37.The present invention also provides nucleic acids, wherein TGFbv2 comprises a sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:8. The present invention also provides nucleic acids, wherein TGFbv2 comprises the sequence set forth in SEQ ID NO:8. The present invention also provides nucleic acids, wherein the protein that binds to the TGF-β receptor binds to the TGF-β receptor II but not to the TGF-β receptor I. The present invention also provides nucleic acids that further comprise at least one promoter region. The present invention also provides nucleic acids, wherein the at least one promoter region drives the expression of at least two polypeptides. The present invention also provides nucleic acids, wherein the at least one promoter region comprises a first promoter region and a second promoter region, wherein the first promoter region drives the expression of a polypeptide comprising IL-12, and the second promoter region drives the expression of an inhibitor of TGF-β activity. The present invention also provides nucleic acids, wherein the first promoter region and the second promoter region each comprise any one of SSP, P7.5, P28, P135, TK promoter, E / L, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, PH5, L4R, 28kDa promoter or any variant or combination thereof. The present invention also provides nucleic acids, wherein the first promoter region comprises the P7.5 promoter. The present invention also provides nucleic acids, wherein the first promoter region comprises the P135 promoter. The present invention also provides nucleic acids, wherein the second promoter region comprises the P28 promoter. The present invention also provides nucleic acids, wherein the second promoter region comprises the P7.5 promoter. The present invention also provides nucleic acids that further comprise a sequence encoding a chemokine receptor or a functional variant thereof. The present invention also provides nucleic acids, wherein the chemokine receptor comprises at least one of the following: CXC receptor, CC receptor, CX3C receptor, XC receptor, a functional fragment thereof, a functional variant thereof or any combination thereof. The present invention also provides nucleic acids, wherein the chemokine receptor comprises at least one of the following: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, a functional fragment thereof or a functional variant thereof, or any combination thereof. The present invention also provides nucleic acids, wherein the chemokine receptor is CXCR3. The present invention also provides nucleic acids, wherein the chemokine receptor comprises at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:42 or SEQ ID NO:43. The present invention also provides nucleic acids, wherein the chemokine receptor comprises the sequence set forth in SEQ ID NO:42 or SEQ ID NO:43.The present invention also provides a nucleic acid, wherein the sequence encoding the chemokine receptor further comprises a third promoter region, and the third promoter region provides expression of the chemokine receptor prior to expression of the IL-12 and TGF-β activity inhibitors. The present invention also provides a nucleic acid, wherein the third promoter region comprises any one of A52R, pB8, mH5, I4L, LEO, pF11, I3L, P7.5, TK promoter, F7L, H5R, short synthetic promoter (SSP) or any variant or combination thereof. The present invention also provides a nucleic acid, wherein the third promoter comprises the A52R promoter.

[0201] The present disclosure provides nucleic acids, wherein the nucleic acids comprise: a first region encoding a first polypeptide, the first polypeptide comprising a sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:12 or SEQ ID NO:15; and a second region encoding a second polypeptide, the second polypeptide comprising a sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:40 or SEQ ID NO:41. The present disclosure also provides nucleic acids, wherein the nucleic acids comprise DNA or RNA. The present disclosure also provides nucleic acids further comprising at least one promoter region. The present disclosure also provides nucleic acids, wherein the at least one promoter region drives the expression of at least two polypeptides. The present disclosure also provides nucleic acids comprising a first promoter region and a second promoter region, wherein the first promoter region drives the expression of the first polypeptide, and the second promoter region drives the expression of the second polypeptide. The present disclosure also provides nucleic acids, wherein the first promoter region and the second promoter region each comprise any one of SSP, P7.5, P28, P135, TK promoter, E / L, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, PH5, L4R, 28kDa promoter or any variant or combination thereof. The present disclosure also provides nucleic acids, wherein the first promoter region comprises the P7.5 promoter. The present disclosure also provides nucleic acids, wherein the first promoter region comprises the P135 promoter. The present disclosure also provides nucleic acids, wherein the second promoter region comprises the P28 promoter. The present disclosure also provides nucleic acids, wherein the second promoter region comprises the P7.5 promoter. The present disclosure also provides nucleic acids, wherein: the first region encodes a polypeptide comprising the sequence listed in SEQ ID NO:12 or SEQ ID NO:15; and the second region encodes a polypeptide comprising the sequence listed in SEQ ID NO:40 or SEQ ID NO:41. The present disclosure also provides nucleic acids, wherein the nucleic acids comprise: a first region having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:64 or SEQ ID NO:66; and a second region having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:65 or SEQ ID NO:67. The present disclosure also provides nucleic acids, wherein the nucleic acids comprise DNA and in 5' to 3' order comprise the following sequences: a first region encoding IL-12 or a functional variant thereof and a second region encoding an inhibitor of TGF-β activity. The present disclosure also provides nucleic acids, wherein the nucleic acids comprise DNA encoding the sequences listed in SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:13, SEQ ID NO:36 and SEQ ID NO:7 in 5' to 3' order.The present invention also provides nucleic acids, wherein the nucleic acids comprise DNA encoding the sequences of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:16, SEQ ID NO:37 and SEQ ID NO:8 in a 5' to 3' order. The present invention also provides nucleic acids, wherein the nucleic acids comprise sequences encoding SEQ ID NO:12 and SEQ ID NO:40 in a 5' to 3' order. The present invention also provides nucleic acids, wherein the nucleic acids comprise sequences encoding SEQ ID NO:15 and SEQ ID NO:41 in a 5' to 3' order. The present invention also provides nucleic acids further comprising a third region encoding a third polypeptide, wherein the third polypeptide comprises a sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:42 or SEQ ID NO:43. The present invention also provides nucleic acids, wherein the third region comprises SEQ ID NO:42 or SEQ ID NO:43. The present invention also provides nucleic acids further comprising a third region having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:69 or SEQ ID NO:86. The present invention also provides nucleic acids, wherein the third region comprises the sequences listed in SEQ ID NO:69 or SEQ ID NO:86. The present invention also provides nucleic acids, wherein the third region encoding the third polypeptide further comprises a third promoter region, which provides for the expression of the third polypeptide prior to the expression of the first and second polypeptides. The present invention also provides nucleic acids, wherein the third promoter comprises any one of A52R, pB8, mH5, I4L, LEO, pF11, I3L, P7.5, TK promoter, F7L, H5R, short synthetic promoter (SSP) or any variant or combination thereof. The present invention also provides nucleic acids, wherein the third promoter comprises the A52R promoter. The present invention also provides nucleic acids, wherein the nucleic acids are present in an oncolytic virus. The present invention also provides nucleic acids, wherein the oncolytic virus is a poxvirus, adeno-associated virus, adenovirus, reovirus, lentivirus, herpes simplex virus, vesicular stomatitis virus, Mengo virus, myxoma virus, Newcastle disease virus, measles virus or poliovirus. The present invention also provides nucleic acids, wherein the poxvirus is vaccinia virus. The present invention also provides nucleic acids, wherein the vaccinia virus is a modified strain of Western Reserve vaccinia virus (ATCC VR-1354), Ankara vaccinia virus (ATCC VR-1508), Ankara vaccinia virus (ATCC VR-1566), Wyeth vaccinia virus strain (ATCC VR-1536) or Wyeth vaccinia virus (ATCC VR-325). The present invention also provides nucleic acids, wherein the nucleic acids are inserted into the viral genome.The present invention also provides a nucleic acid comprising a mutation or deletion of at least one viral gene selected from the group consisting of: thymidine kinase (TK), F13L, A36R, A34R, A33R, A52R, B5R, B8R, B18R, SPI-1, SPI-2, B15R, VGF, E3L, K3L, A41L, K7R or N1L, functional fragments thereof, or any combination thereof.

[0202] The present invention provides a nucleic acid molecule, wherein the nucleic acid molecule comprises: an insert comprising, in 5' to 3' order, at the A52R locus: a first promoter region, wherein the promoter comprises the A52R promoter; an insert encoding a first region of human CXCR3; an insert comprising, in 5' to 3' order, at the TK locus: a second promoter region, wherein the promoter comprises P135; a second region encoding human IL-12; a third promoter region, wherein the promoter comprises P7.5; and a third region encoding a TGFβ variant. The present invention also provides a nucleic acid molecule, wherein the P135 promoter comprises the sequence set forth in SEQ ID NO:56, and the P7.5 promoter comprises the sequence set forth in SEQ ID NO:57. The present invention also provides a nucleic acid molecule, wherein the first region encoding human CXCR3 comprises the sequence set forth in SEQ ID NO:86; wherein the second region encoding human IL-12 comprises the sequence set forth in SEQ ID NO:66; and wherein the third region encoding a TGF-β inhibitor comprises the sequence set forth in SEQ ID NO:67. The present invention also provides a nucleic acid molecule, wherein the nucleic acid molecule comprises: an insert comprising the sequence set forth in SEQ ID NO:88 at the A52R locus; an insert comprising the sequence set forth in SEQ ID NO:85 at the TK locus.

[0203] The present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises: a nucleic acid as described herein or a vector as described herein; and a pharmaceutically acceptable excipient. The present invention also provides a pharmaceutical composition, wherein the composition is in a liquid dosage form. The present invention also provides a pharmaceutical composition, wherein the pharmaceutically acceptable excipient is buffered saline. The present invention also provides a pharmaceutical composition, wherein the buffered saline is phosphate buffered saline (PBS), Dulbecco's PBS (DPBS), TRIS buffered saline (TBS), Hank's balanced salt solution (HBSS), Earle's balanced salt solution (EBSS), standard citrate saline (SSC), HEPES buffered saline (HBS), or Gey's balanced salt solution. The present invention also provides a pharmaceutical composition, wherein the composition further comprises liposomes or nanoparticles. The present invention also provides a pharmaceutical composition, wherein the nucleic acid or vector is associated with the liposomes or nanoparticles.

[0204] The present invention provides methods for treating cancer, comprising administering to a subject having cancer a pharmaceutical composition as described herein in an amount sufficient to treat cancer. The present invention also provides methods wherein the cancer is a solid tumor or a hematological cancer. The present invention also provides methods wherein the cancer comprises melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostatic epithelial cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, intestinal type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm or sarcoma. The present invention also provides methods wherein the administration comprises intratumoral administration. The present invention also provides methods wherein the administration comprises systemic administration. The present invention also provides methods wherein the systemic administration comprises oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration or any combination thereof.

[0205] The present invention provides methods for activating an anti-tumor immune response, comprising administering to a subject having cancer a pharmaceutical composition as described herein. The present invention also provides methods wherein the cancer is a solid tumor, leukemia or lymphoma. The present invention also provides methods wherein the cancer comprises melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostatic epithelial cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, intestinal type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm or sarcoma. The present invention also provides methods wherein the administration step is intratumoral administration. The present invention also provides methods wherein the administration step is systemic administration. The present invention also provides methods wherein the systemic administration comprises oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration or any combination thereof.

[0206] The present disclosure provides methods for reducing the incidence of tumor cell growth, including: administering to the tumor cells a pharmaceutical composition as described herein in an effective amount sufficient to reduce the incidence of tumor cell growth. The present disclosure also provides methods, wherein the tumor cells are from solid tumors or blood cancers. The present disclosure also provides methods, wherein the tumor cells are from melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostatic epithelial cancer, hepatocellular carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, intestinal type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma. The present disclosure also provides methods, wherein the administering step is intratumoral administration. Examples

[0207] The following examples further illustrate the described embodiments and do not limit the scope of the disclosure.

[0208] Example 1: mIL12 and TGFbf1 Construct Design

[0209] A vaccinia virus containing the construct depicted in Figure 2 was constructed by replacing the open reading frame encoding viral thymidine kinase with a nucleic acid containing the sequences encoding murine IL-12 (murine IL-12) (SEQ ID NO:64) and TGF-β fusion (TGF-bf1) (SEQ ID NO:65) as shown in Table 9. Expression of murine IL-12 is driven by the viral promoter P7.5 (SEQ ID NO:57). Expression of TGF-bf1 is driven by the viral promoter P28 (SEQ ID NO:58). The murine IL-12 polypeptide comprises a covalent dimer consisting of murine interleukin-12 subunit β (mIL-12b) (UniProtKB accession ID P43432.I) (SEQ ID NO:14) linked via a 22-residue glycine-rich linker (SEQ ID NO:18) to murine interleukin-12 subunit α (mIL-12a) (UniProtKB accession ID 43431) residues 11-215 (SEQ ID NO:13). The TGFbf1 polypeptide comprises the signal peptide of murine interleukin-2 (IL-2sig) (UniProtKB accession ID P04351.1) (SEQ ID NO:36) fused to a TGF-β inhibitor (TGFbv1) (SEQID NO:7) in which cysteines 8 and 17 (original PDB numbering) are mutated to valine and alanine, respectively.

[0210] ​​Table 9. Nucleic acid sequences.

[0211]

[0212]

[0213] Example 2: Design of hIL12 and TGFbf2 constructs

[0214] The vaccinia virus containing the construct depicted in Figure 6 was constructed by replacing the open reading frame encoding viral thymidine kinase with nucleic acid encoding a fusion of human IL-12 (SEQ ID NO:66) and TGF-β (TGF-bf2) (SEQ ID NO:67) as shown in Table 10. Expression of hIL-12 was driven by the viral promoter P135 (SEQ ID NO:56). Expression of TGF-bf2 was driven by the viral promoter P7.5 (SEQ ID NO:57). The human IL-12 polypeptide comprises a covalent dimer consisting of human interleukin-12 subunit β (hIL-12b) (SEQ ID NO:17) linked via a 22-residue glycine-rich linker (SEQ ID NO:18) to human interleukin-12 subunit α (hIL-12a) (SEQ ID NO:16). The TGFbf2 polypeptide comprises the signal peptide of human IgE (SEQ ID NO:37) fused to a TGF-β inhibitor (TGFbv2) (SEQ ID NO:8) containing a cystine knot region of PRDC.

[0215] Table 10. Nucleic acid sequences.

[0216]

[0217]

[0218] Example 3: Measurement of tumor growth after treatment with TGF-β inhibitor and murine IL-12

[0219] Renca cells were implanted into the flanks of Balb / c mice. After 12 days, the mice were grouped such that each treatment group had an average volume of 58 mm 3 . The mice were treated with 1E7 PFU virus via IT injection.

[0220] B16F10 cells were mixed 1:1 with Matrigel and implanted into the flanks of C57 / Black 6 mice. After 5 days, the mice were grouped such that each treatment group had an average volume of 93 mm 3 . The mice were treated with 1E7 PFU virus via IT injection.

[0221] The tumor volume was measured twice a week, and the mice were weighed once a week. Signs of morbidity and mortality in the mice were monitored and none were observed (data not shown).

[0222] Mice with induced tumors were divided into treatment groups including PBS, TK-75, TK-75 expressing IL12, TK-75 expressing a TGF-β inhibitor, and TK-75 expressing both IL-12 and a TGF-β inhibitor. The mean tumor volume in each treatment group was measured and is shown for the Renca tumor group in Figure 3A and for the B16 tumor group in Figure 3B .

[0223] Mice with induced Renca tumors treated with a virus expressing only a TGF-β inhibitor showed a mean tumor volume of approximately 225 mm 3 at 17 days post-treatment. Groups treated with a combination of IL-12 and a TGF-β inhibitor or a virus expressing IL-12 alone showed a mean tumor volume of less than 50 mm 3 at 17 days post-treatment. In contrast, control mice treated with PBS had a mean tumor volume of approximately 450 mm 3 .

[0224] Mice with induced B16 tumors treated with a virus expressing only a TGF-β inhibitor had a mean tumor volume of approximately 600 mm 3 at 17 days post-treatment. Mice treated with a combination of IL-12 and a TGF-β inhibitor or a virus expressing IL-12 alone had a mean tumor volume of less than 100 mm 3 at 17 days post-treatment. In contrast, control mice treated with PBS had a mean tumor volume of approximately 1200 mm 3 .

[0225] Treatment with a modified virus expressing both a TGF-β inhibitor and IL-12 inhibited tumor growth in mice more than treatment with a virus expressing only a TGF-β inhibitor.

[0226] Example 4: Survival assay using a TGF-β inhibitor and mIL-12

[0227] Mice were induced to develop tumors with Renca and B16 cells and then treated with modified viruses as described in Example 2. Treatment groups included PBS, the TK-virus, the TK-virus expressing IL12, the TK-virus expressing a TGF-β inhibitor, and the TK-virus expressing both IL-12 and a TGF-β inhibitor. The survival probability for each treatment group was calculated and is shown for the Renca tumor group in Figure 4A and for the B16 tumor group inFigure 4B Shown in. In mice with Renca cell tumors, the treatment groups receiving viruses expressing IL-12 or IL-12 and TGF-β inhibitor showed a survival probability of approximately 75% after 56 days, while the other groups had zero surviving subjects on day 56. In mice with B16 tumors, compared to 0 surviving subjects in the other treatment groups, those mice treated with the modified virus expressing TGF-β inhibitor and IL-12 showed a survival rate of approximately 35%.

[0228] Example 5: Addition of murine CXCR3 expression system

[0229] Vaccinia virus was modified by replacing the gene encoding A52 with nucleic acids encoding murine CXCR3 and a fluorescent reporter. A seamless plasmid transfer vector was generated that sequentially contained the following: upstream recombination directing sequence A (SEQ ID NO:68), open reading frame encoding murine CXCR3 (SEQ ID NO:69), stop codon, SacI cloning site and short spacer (SEQ ID NO:70), loxP site (SEQ ID NO:71), spacer followed by a viral promoter driving GFP-pac reporter expression (SEQ ID NO:72), PacI cloning site and short spacer A (SEQ ID NO:73), loxP site (SEQ ID NO:71), and downstream recombination directing sequence A (SEQ ID NO:74). After recombination and treatment with cre recombinase, the viral genome contained the incorporated sequences listed in SEQ ID NO:75. The sequences are listed in Table 11. A schematic representation of the promoter and gene expression constructs is shown in Figure 5 Shown in.

[0230] Table 11. Murine CXCR3 recombinant sequences.

[0231]

[0232]

[0233]

[0234]

[0235] Example 6: Construction of murine IL-12 and TGF-β inhibitor expression systems

[0236] The modified vaccinia virus of Example 5 was further modified by replacing the gene encoding thymidine kinase (VACV094, J2R) with nucleic acids encoding single-chain murine IL-12, TGF-β2, and a fluorescent reporter. A seamless plasmid transfer vector was generated that sequentially contained the following: upstream recombination guidance sequence B (SEQ ID NO:76), an SbfI cloning site (SEQ ID NO:77) followed by the P135 promoter, an open reading frame encoding murine IL-12 (SEQ ID NO:64), a SalI cloning site followed by a spacer region (SEQ ID NO:78), the vaccinia virus promoter P7.5 (SEQ ID NO:79) followed by a KpnI cloning site, an open reading frame encoding a TGF-β inhibitor (TGF-β2) (SEQ ID NO:67), a SacI cloning site (SEQ ID NO:81), a loxP site (SEQ ID NO:71), a spacer region (SEQ ID NO:72) followed by a viral promoter driving GFP-pac reporter expression, a PacI cloning site and a short spacer region B (SEQ ID NO:82), a loxP site (SEQ ID NO:71), and a downstream recombination guidance sequence B (SEQ ID NO:83). After recombination and treatment with cre recombinase, the viral genome incorporated the sequences listed in SEQ ID NO:84. The selected sequences are listed in Table 12. A schematic representation of the promoter and gene expression constructs is shown in Figure 6 is shown in.

[0237] Table 12. Recombinant sequences for murine IL-12 and TGF-β inhibitor.

[0238]

[0239]

[0240]

[0241]

[0242] Example 7: Construction of a human IL-12 and TGF-β inhibitor expression system

[0243] Vaccinia virus was modified by replacing the gene encoding thymidine kinase (VACV094, J2R) with nucleic acids encoding single-stranded human IL-12, the TGF-β inhibitor TGFbf2, and a fluorescent reporter. A seamless plasmid transfer vector was generated that sequentially contained the following: upstream recombination guidance sequence B (SEQ ID NO:76), an SbfI cloning site (SEQ ID NO:77) followed by the P135 promoter, an open reading frame encoding human IL-12 (SEQ ID NO:66), a SalI cloning site followed by a spacer region (SEQ ID NO:78), the vaccinia virus promoter P7.5 (SEQ ID NO:79) followed by a KpnI cloning site, an open reading frame encoding the TGF-β inhibitor (TGFbf2) (SEQ ID NO:67), a sacI cloning site (SEQ ID NO:81), a loxP site (SEQ ID NO:71), a spacer region followed by a viral promoter driving GFP-pac reporter expression (SEQ ID NO:72), a PacI cloning site and a short spacer region B (SEQ ID NO:82), a loxP site (SEQ ID NO:71), and a downstream recombination guidance sequence B (SEQ ID NO:83). After recombination and treatment with cre recombinase, the viral genome contained the incorporated sequences listed in SEQ ID NO:85. The sequences are listed in Table 13.

[0244] Table 13. Recombinant sequences of human IL-12 and TGF-β inhibitor.

[0245]

[0246]

[0247]

[0248] Example 8: Addition of chemokine receptor sequences

[0249] The modified vaccinia virus described in Example 7 was further modified by replacing the gene encoding A52 (VACV178, A52R) with a nucleic acid encoding human CXCR3 and a fluorescent reporter. A non-gapped plasmid transfer vector was generated that sequentially contained the following: upstream recombination guide sequence A (SEQ ID NO:68), open reading frame encoding human CXCR3 isoform 1 (SEQ ID NO:86), a stop codon, a SacI cloning site and a short spacer (SEQ ID NO:70), loxP site (SEQ ID NO:71), a spacer followed by a viral promoter driving GFP-pac reporter expression (SEQ ID NO:72), PacI cloning site and short spacer A (SEQ ID NO:73), loxP site (SEQ ID NO:71) and downstream recombination guide sequence A (SEQ ID NO:74). Reporter gene-positive viruses were isolated and then treated with a reporter gene-free transfer vector that contained upstream recombination guide sequence A (SEQ ID NO:68), open reading frame encoding human CXCR3 isoform 1 (SEQ ID NO:86) and a second downstream recombination guide sequence (SEQ ID NO:87). After recombination, the viral genome incorporated the sequence listed in SEQ ID NO:88. The selected sequences are listed in Table 14.

[0250] Table 14. Human CXCR3 recombinant sequences.

[0251]

[0252]

[0253]

[0254] Example 9: Generation of Modified Oncolytic Vaccinia Viruses Expressing CXCR3, IL-12, and TGFB Inhibitors

[0255] Modified oncolytic vaccinia viruses expressing CXCR3 as described in SEQ ID NO:43, IL-12 as described in SEQ ID NO:15, and the TGFB1 antagonist small monomer construct as described in SEQ ID NO:41 as shown in Figure 7 were generated according to the methods described in Example 7 and Example 8. Expression and function of the transgenes in the modified viruses were confirmed as follows.

[0256] CXCR3

[0257] Figure 8AFluorescence-activated cell sorting (FACS) analysis of HeLa cells infected with the modified virus depicted in Figure 8B - Figure 8E confirmed the expression of CXCR3. The function of CXCR3 expressed was tested in peripheral blood mononuclear cells (PBMCs) (including CD4+ cells, CD8+ cells, monocytes, and B cells). The migration of subsets of PBMCs infected with the modified virus was compared to that of TK-virus-infected controls (CTRL) or uninfected cells (-) towards 100 ng / ml ITAC (CXCL11), a ligand of CXCR3. The counting of the migrated cells in each test group is shown in the bar graph in Figure 8B It was shown that the number of modified virus-infected CD4 cells that migrated was more than 4-fold higher compared to control cells. Figure 8C It was shown that the number of modified virus-infected CD8 cells that migrated was increased by approximately 2.5-fold compared to control cells. Figure 8D The migration of monocytes in infected cells was shown compared to no response in control cells. Figure 8E It was shown that the number of modified virus-infected B cells that migrated was increased by approximately 10-fold compared to control cells. The increased amount of migration towards the CXCR3 ligand indicates the expression of the active form of the receptor in cells infected with the modified virus.

[0258] IL-12

[0259] ELISA assays of the culture supernatants from Hela cells infected with the modified virus confirmed the expression of IL-12 with a conserved epitope. Figure 8F is a bar graph showing the quantitative ELISA results of the supernatants from Hela cells infected with the modified virus compared to TK-virus-infected controls (CTRL) or uninfected cells (-). Approximately 170,000 pg / ml of IL-12 was detected in the supernatants from modified virus-infected cells. No IL-12 was detected from untreated and negative control cells.

[0260] TGFBi

[0261] Western blot analysis was used on Hela cell culture lysates from cells infected with the modified virus, TK-virus-infected controls (CTRL), or uninfected cells (-). As shown in Figure 8G the lysates from modified virus-infected cells showed a 12 kDa protein corresponding to the TGFBi protein. No corresponding bands were shown in untreated and negative control cultures.

[0262] Example 10: Modified virus rescues CD8 T cells from TGF-β inhibition and induces more granzyme B

[0263] Hela cells were treated with medium (-) or infected with a TK-control virus (CTRL) at a multiplicity of infection (MOI) of 10 or a modified virus (TGFBi / IL-12 / CXCR3) for 24 hours. Virus-free supernatants were harvested from the infected cells and used for subsequent T cell experiments. CD8+ T cells treated with supernatants from Hela cells infected with the modified virus showed less inhibition when treated with TGF-β1 (TGFB1), indicating the expression of a functional TGFB inhibitor. CD8+ T cells were labeled with 2 mM CFSE and stimulated with anti-CD3 antibody and anti-CD28 antibody in the presence of supernatants from Hela cells infected with the modified virus or the TK-control virus (CTRL) or uninfected cells. The T cells were then exposed to 0 ng / ml, 10 ng / ml or 50 ng / ml TGFB1.

[0264] CD44 and GZMB expression of T cells was analyzed. T cells were stained with an antibody against surface-expressed CD44 and then fixed and permeabilized using the Cytofix / Cytoperm TM fixation / permeabilization kit (BD Biosciences, Franklin Lakes, NJ). Cells were stained with an antibody against cytoplasmic GZMB and analyzed using an Attune flow cytometer (ThermoFisher Scientific, Carlsbad, CA).

[0265] The initial cell population with incorporated stain appeared as a peak with the highest intensity in the trace. Subsequent generations of cells contained a diluted amount of stain and appeared as a peak with decreased fluorescence or shifted to the left. Cells sensitive to TGFB1 inhibition showed smaller generational peaks in the trace. Figure 9A The traces depicted in panel (b) show a decrease in generational peaks in TK-control virus (CTRL)-infected and uninfected (-) CD8 T cells after exposure to 10 ng / ml and 50 ng / ml TGFB1, indicating inhibited proliferation. Stimulated T cells treated with the modified virus showed less proliferation inhibition after contact with TGFB1 compared to the control.

[0266] Granzyme B (GZMB) induction in CD8 cells was also evaluated. FCS files generated by Attune were analyzed using FCS 7 software. Gating was for live T cells. Density plots were drawn with CD44 on the x-axis and GZMB on the y-axis. The percentage of GZMB was analyzed by gating GZMB+ T cells in the density plot. Figure 9BDot plots of each sample group are shown. The figure shows an increase in the level of CD44+ GZMB+ in CD8 T cells infected with the modified virus compared to untreated (-) and TK-virus-infected control cells.

[0267] Example 11: In Vitro Selectivity and Enhanced In Vivo Tumor-Specific Delivery of Modified Vaccinia Virus

[0268] Using an in vivo murine RENCA tumor model, the selectivity of the modified virus compared to the control virus was tested. The infectivity of the modified virus in cancer cell lines and non-cancer cell lines was tested as described in Example 9. At 48 hours post-infection, the viral loads of three cell types, human lung adenocarcinoma (A549) cells, human cervical cancer (Hela) cells, and human foreskin fibroblasts (HFF), were analyzed. The PFU / ml detected in each cell type is shown in the Figure 10A bar graph. At 48 hours post-infection, more than 10-fold higher PFU / ml of virus was detected in Hela cells compared to HFF cells. A549 cells showed approximately 4-fold higher viral loads compared to infected HFF cells. The modified virus showed preferential in vitro infectivity in tumor cells.

[0269] Renca tumors were implanted subcutaneously into BALB / c mice. Modified viruses with and without modification to express CXCR3 were administered intravenously to the mice at 1×10 7 PFU. The tumors were harvested 24 hours later, and the viral genomes were quantified by qPCR. The results are shown in the Figure 10B scatter plot. Approximately 10-fold more viral genomes were detected in the Renca tumors of mice treated with the virus expressing CXCR3 compared to the tumors of mice treated with the virus without CXCR3 modification. The results indicate that the modified virus increased in vivo infectivity compared to the unmodified virus.

[0270] Example 12: Modified Vaccinia Virus Reduces Tumor Burden and Increases Mouse Survival in Multiple Tumor Models

[0271] Using in vivo murine EMT-6 and MC38 tumor models, the therapeutic efficacy of the modified virus was tested by comparison with a control virus and a buffer negative control. Mice were inoculated with EMT6 and MC38 tumors.

[0272] BalbC mice were implanted with 1×10 5 EMT6 cells. Thirteen days after implantation, the tumors reached an average volume of 80.26 mm 3 (26.70 mm 3 -136.23 mm 3) Exclude mice with tumors smaller than 36.92 mm 3 and larger than 117.43 mm 3 and group the mice such that each treatment group (n = 10) has an average tumor volume of ~78 mm 3 Treat the mice with 1×10 4 PFU / dose, 1×10 5 PFU / dose, 1×10 6 PFU / dose or 1×10 7 PFU / dose of the modified vaccinia virus (TGFBi / IL-12 / CXCR3) as described in Example 9, buffer (-) or TK-control virus (CTRL). Treat the mice via IV injection (tail vein) on Day 0 and Day 3. Measure the tumor volume twice a week and weigh the mice once a week.

[0273] Implant 5×10 cells into C57Bl / 6 mice and allow tumors to form over 8 days to reach an average volume of 50.46 mm 3 (10.17 mm 3 - 84.79 mm 3 ) Exclude mice with tumors smaller than 31.99 mm 3 and group the mice such that each group (n = 10) has an average volume of 53.06 mm 3 Treat the mice with 2×1×10 5 PFU / dose, 2×1×10 6 PFU / dose or 1×1×10 7 PFU / dose of the modified vaccinia virus (TGFBi / IL-12 / CXCR3) as described in Example 9, buffer (-) or TK-control virus (CTRL). Treat the mice via IV injection (tail vein) on Day 0 and Day 3. Measure the tumor volume twice a week and weigh the mice once a week.

[0274] All buffer control (-) and control virus (CTRL)-treated mice in both tumor models were euthanized on Day 42. Eight out of 10 EMT6 mice treated with the modified virus showed complete inhibition of tumor growth by Day 54. MC38 mice treated with the modified virus showed complete inhibition of tumor growth for more than 39 days, with 9 out of 10 mice showing complete inhibition by Day 51. The probabilities of survival to Day 54 after treatment in EMT6 tumor mice and MC38 tumor mice are shown in Figure 11C and Figure 11D respectively. Asterisks indicate P-values: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; and ****P ≤ 0.0001. As Figure 11CAs shown, mice treated with the modified virus maintained a 70% survival probability until day 54. As Figure 11D shown, mice treated with the modified virus maintained a 90% survival probability until day 54. The results indicate that in multiple tumor models, treatment with the modified virus inhibited tumor growth and increased the survival probability.

[0275] Example 13: Modified virus treatment increases CD3+CD8+ T cell infiltration in tumors

[0276] Postmortem analysis was used to analyze the effect of the modified virus on the immune / stromal / endothelial environment of tumors and to determine the toxicity profile. Tumors from Example 11 (Renca) and Example 12 (MC38) were sectioned and processed by staining with nucleic acids, CD3, and CD8. Representative stained sections are shown in Figure 12A Compared to corresponding sections from untreated or control samples, tumors treated with the modified virus showed greater infiltration of CD3 and CD8 markers, indicating cytotoxic T cell infiltration.

[0277] Figure 12B - Figure 12E are bar graphs of the total counts of CD3+ and CD8+ T cells in RENCA and MC38 tumor samples treated with the modified virus or untreated or control samples. Figure 12B shows that the CD3+ T cells in RENCA tumors treated with the modified virus increased by approximately 3-fold compared to control cells. Figure 12C shows that the CD8+ T cells in RENCA tumors treated with the modified virus increased by approximately 3.5-fold compared to control cells. Figure 12D shows that the average number of CD3+ T cells increased by approximately 2-fold after treatment with the modified virus compared to control cells. Figure 12E shows that the average number of CD8+ T cells increased by approximately 2 - 3-fold after treatment with the modified virus compared to control cells.

[0278] Example 14: Modified virus induces type II interferon-G-related genes in tumors but eliminates the TGFB1-related gene signature

[0279] Intact RENCA tumors were harvested from the treated mice described in Example 11 and homogenized in Qiazol (Qiagen, Hilden, Germany) using a bead mill. RNA was extracted using the Rneasy kit (Qiagen). The extracted RNA was sequenced using next-generation sequencing methods. The raw RNA sequence data (FASTQ) files were analyzed for differential gene expression between the TK control virus (CTRL) and the modified virus using Rosalind software, and generated as shown in Figure 13A andFigure 13B The gene expression heat map shown in

[0280] Figure 13A is a heat map showing the relative expression levels of type II interferon - γ (INFG) - related genes specifically compared to the overall average. The expression levels of CXCL11, XCR1, STAT1, IDO1, IL12B, IFNG, CIITA, H2 - EB1, H2 - AB1, TBX21, CXCR3, CD2, LTB, CXCL16, B2M, VCAM1, TAP1, IFIT2, TAP2, IL2RG, STAT2, CD274, IRF1 were tested. Region 1 shows that compared to the overall average, control CD3+ and CD8+ cells generally showed lower expression levels, while region 2 shows that CD3+ and CD8+ cells treated with the modified virus showed higher expression levels. This indicates that contacting cells with the modified virus expressing TGF - β inhibitor activates the expression of IFNG - related genes.

[0281] Figure 13B is a heat map showing the relative expression levels of TGF - β1 - related genes compared to the overall average. The expression levels of ILz1B, LPL, SLP1, FBN1, LCN2, CXCL5, OGN, PLOD2, TNFAIP6, CAN, ABCG1, ACKR3, COL15A1 were tested. Region 3 shows that compared to the overall average, control cells generally showed higher expression levels, while region 4 shows that cells treated with the modified virus showed lower expression levels. This indicates that contacting cells with the modified virus expressing TGF - β inhibitor reduces the expression of TGF - β1 - related genes.

[0282] The foregoing description and drawings set forth many representative embodiments of the present. Of course, various modifications, additions, and alternative designs will become apparent to those skilled in the art without departing from the scope of the foregoing teachings, which scope is indicated by the appended claims rather than the foregoing description. All changes and variations that fall within the equivalent meaning and scope of the claims should be included within the scope of the claims.

Claims

1. A composition, wherein the composition comprises: a carrier, wherein the carrier comprises: an exogenous nucleic acid comprising a sequence encoding a cytokine or a functional variant thereof; an exogenous nucleic acid comprising a sequence encoding a chemokine receptor or a functional variant thereof; and a first promoter region, wherein the first promoter region is upstream of the sequence encoding the chemokine receptor and provides expression of the chemokine receptor before expression of the cytokine.

2. The composition according to claim 1, wherein the encoded chemokine receptor comprises at least one of the following: CXC receptor, CC receptor, CX3C receptor, XC receptor, a functional fragment thereof, a functional variant thereof, or any combination thereof.

3. The composition according to claim 1, wherein the encoded chemokine receptor comprises at least one of the following: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, a functional fragment thereof, or a functional variant thereof, or any combination thereof.

4. The composition according to claim 1, wherein the encoded chemokine receptor comprises CXCR3.

5. The composition according to claim 4, wherein the encoded chemokine receptor comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:42 or SEQ ID NO:

43.

6. The composition according to claim 4, wherein the encoded chemokine receptor comprises the amino acid sequence listed in SEQ ID NO:42 or SEQ ID NO:

43.

7. The composition according to claim 1, wherein the first promoter region comprises an early promoter.

8. The composition according to claim 7, wherein the early promoter comprises any one of A52R, pB8, mH5, I4L, LEO, pF11, I3L, P7.5, TK promoter, F7L, H5R, short synthetic promoter (SSP), or any variant or combination thereof.

9. The composition according to claim 7, wherein the early promoter comprises the A52R promoter.

10. The composition according to claim 1, wherein the encoded cytokine comprises IL-12 or a functional variant thereof.

11. The composition according to claim 10, wherein the encoded IL-12 is murine IL-12 or human IL-12.

12. The composition according to claim 10, wherein the encoded IL-12 comprises an α subunit and a β subunit.

13. The composition according to claim 12, wherein the sequences encoding the IL-12α subunit and the IL-12β subunit further comprise a sequence encoding a linker.

14. The composition according to claim 12, wherein the encoded IL-12α subunit comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO:13 or SEQ ID NO:

16.

15. The composition according to claim 12, wherein the encoded IL-12α subunit comprises the amino acid sequence listed in any one of SEQ ID NO:13 or SEQ ID NO:

16.

16. The composition according to claim 12, wherein the encoded IL-12β subunit comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO:14 or SEQ ID NO:

17.

17. The composition according to claim 12, wherein the IL-12β subunit comprises the amino acid sequence listed in any one of SEQ ID NO:14 or SEQ ID NO:

17.

18. The composition according to claim 13, wherein the encoded linker comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:

18.

19. The composition according to claim 13, wherein the encoded linker comprises the amino acid sequence listed in SEQ ID NO:

18.

20. The composition according to claim 10, wherein the encoded IL-12 comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO:12 or SEQ ID NO:

15.

21. The composition according to claim 10, wherein the encoded IL-12 comprises the amino acid sequence listed in any one of SEQ ID NO:12 or SEQ ID NO:

15.

22. The composition according to claim 10, wherein the sequence encoding the IL-12 comprises a nucleic acid sequence having at least 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO:64 or SEQ ID NO:

66.

23. The composition according to claim 10, wherein the sequence encoding the IL-12 comprises the nucleic acid sequence listed in any one of SEQ ID NO:64 or SEQ ID NO:

66.

24. The composition according to any one of claims 1 to 23, further comprising an exogenous nucleic acid, the exogenous nucleic acid comprising a sequence encoding an inhibitor of transforming growth factor β (TGF-β) activity; wherein the first promoter region provides expression of the chemokine receptor before expression of the TGF-β activity inhibitor.

25. The composition according to claim 24, wherein the encoded TGF-β activity inhibitor comprises a TGF-β dominant negative, a TGF-β receptor dominant negative, a protein that binds to TGF-β or a protein that binds to the TGF-β receptor.

26. The composition according to claim 25, wherein the encoded protein that binds to the TGF-β receptor is a protein comprising a domain of TGF-β.

27. The composition according to claim 26, wherein the encoded protein comprising a domain of TGF-β comprises an amino acid sequence listed in any one of SEQ ID NOs: 1-9.

28. The composition according to claim 26, wherein the encoded protein comprising a domain of TGF-β comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO: 7 or SEQ ID NO:

8.

29. The composition according to claim 26, wherein the encoded protein comprising a domain of TGF-β comprises an amino acid sequence listed in any one of SEQ ID NO: 7 or SEQ ID NO:

8.

30. The composition according to claim 24, wherein the sequence encoding the transforming growth factor β (TGF-β) inhibitor comprises a nucleic acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:

67.

31. The composition according to claim 24, wherein the sequence encoding the TGF-β inhibitor comprises the nucleic acid sequence listed in SEQ ID NO:

67.

32. The composition according to claim 25, wherein the encoded protein that binds to the TGF-β receptor is a TGF-β fusion protein.

33. The composition according to claim 25, wherein the encoded TGF-β inhibitor comprises a protein that binds to the TGF-β receptor, and wherein the protein that binds to the TGF-β receptor comprises a TGF-β fusion 1 (TGFbf1) protein.

34. The composition according to claim 33, wherein the TGFbf1 protein comprises a murine IL-2 signal peptide and a TGF-β variant 1 (TGFbv1).

35. The composition according to claim 34, wherein the murine IL-2 signal peptide comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:

36.

36. The composition according to claim 34, wherein the murine IL-2 signal peptide comprises the amino acid sequence listed in SEQ ID NO:

36.

37. The composition according to claim 34, wherein the TGFbv1 comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:

7.

38. The composition according to claim 34, wherein the TGFbv1 comprises the sequence listed in SEQ ID NO:

7.

39. The composition according to claim 25, wherein the encoded TGF-β inhibitor comprises a protein that binds to the TGF-β receptor, and wherein the protein that binds to the TGF-β receptor comprises a TGF-β fusion 2 (TGFbf2) protein.

40. The composition according to claim 39, wherein the TGFbf2 comprises a human IgE signal peptide and TGF-β variant 2 (TGFbv2).

41. The composition according to claim 40, wherein the human IgE signal peptide comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:

37.

42. The composition according to claim 40, wherein the human IgE signal peptide comprises the amino acid sequence set forth in SEQ ID NO:

37.

43. The composition according to claim 40, wherein the TGFbv2 comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:

8.

44. The composition according to claim 40, wherein the TGFbv2 comprises the sequence set forth in SEQ ID NO:

8.

45. The composition according to claim 24, wherein the sequence encoding the cytokine comprises a second promoter region providing for the expression of the cytokine, and wherein the sequence encoding the TGF-β activity inhibitor comprises a third promoter region providing for the expression of the TGF-β activity inhibitor.

46. The composition according to claim 45, wherein each of the second promoter region and the third promoter region comprises a late promoter.

47. The composition according to claim 46, wherein the late promoter comprises any one of SSP, P7.5, P28, P135, TK promoter, E / L, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, PH5, L4R, 28kDa promoter or any variant or combination thereof.

48. The composition according to claim 46, wherein the late promoter comprises a weak late promoter.

49. The composition according to claim 48, wherein the weak late promoter comprises the P135 promoter.

50. The composition according to claim 46, wherein the second promoter region comprises the P135 promoter.

51. The composition according to claim 46, wherein the late promoter comprises a strong late promoter.

52. The composition according to claim 51, wherein the strong late promoter comprises the P7.5 promoter.

53. The composition according to claim 46, wherein the third promoter region comprises the P7.5 promoter.

54. The composition according to claim 24, wherein the exogenous nucleic acid encoding the cytokine or a functional variant thereof, the exogenous nucleic acid encoding the TGF-β activity inhibitor, and the exogenous nucleic acid encoding the chemokine receptor or a functional variant thereof are located on a single genome.

55. The composition according to any one of claims 1-54, wherein the vector is an oncolytic virus, and wherein the oncolytic virus comprises at least one genomic modification.

56. The composition according to claim 55, wherein the at least one modification comprises a mutation or deletion of at least one gene selected from the group consisting of thymidine kinase (TK)56, F13L, A36R, A34R, A33R, A52R, B5R, B8R, B18R, SPI-1, SPI-2, B15R, VGF, E3L, K3L, A41L, K7R or N1L, a functional fragment thereof or any combination thereof.

57. The composition according to claim 56, wherein the at least one modification comprises deletions of genes A52R and TK.

58. The composition according to claim 55, wherein the oncolytic virus is a poxvirus, an adeno-associated virus, an adenovirus, a reovirus, a lentivirus, a herpes simplex virus, a vesicular stomatitis virus, a Mengo virus, a myxoma virus, a Newcastle disease virus, a measles virus or a poliovirus.

59. The composition according to claim 58, wherein the poxvirus is vaccinia virus.

60. The composition according to claim 59, wherein the vaccinia virus is the Western Reserve strain.

61. The composition according to claim 55, wherein the at least one genomic modification results in an increase in the efficacy of systemic delivery of the virus to tumors by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 12-fold, about 14-fold, about 16-fold, about 18-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold or about 100-fold.

62. A nucleic acid, wherein the nucleic acid comprises a sequence encoding: a first polypeptide comprising interleukin-12 (IL-12) or a functional variant thereof; and a second polypeptide comprising an inhibitor of transforming growth factor β (TGF-β) activity.

63. The nucleic acid according to claim 62, wherein the nucleic acid comprises DNA or RNA.

64. The nucleic acid according to claim 62, wherein the IL-12 is murine IL-12 or human IL-12.

65. The nucleic acid according to claim 62, wherein the IL-12 comprises an α subunit and a β subunit.

66. The nucleic acid according to claim 65, wherein the first polypeptide comprising the IL-12α subunit and the IL-12β subunit further comprises a linker.

67. The nucleic acid according to claim 65, wherein the IL-12α subunit comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NO:13 or SEQ ID NO:

16.

68. The nucleic acid according to claim 65, wherein the IL-12α subunit comprises the sequence listed in any one of SEQ ID NO:13 or SEQ ID NO:

16.

69. The nucleic acid according to claim 65, wherein the IL-12β subunit comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO:14 or SEQ ID NO:

17.

70. The nucleic acid according to claim 65, wherein the IL-12β subunit comprises the amino acid sequence set forth in any one of SEQ ID NO:14 or SEQ ID NO:

17.

71. The nucleic acid according to claim 66, wherein the linker comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:

18.

72. The nucleic acid according to claim 66, wherein the linker comprises the amino acid sequence set forth in SEQ ID NO:

18.

73. The nucleic acid according to claim 62, wherein the encoded IL-12 comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO:12 or SEQ ID NO:

15.

74. The nucleic acid according to claim 62, wherein the encoded IL-12 comprises the amino acid sequence set forth in any one of SEQ ID NO:12 or SEQ ID NO:

15.

75. The nucleic acid according to claim 62, wherein the sequence encoding the IL-12 comprises a nucleic acid sequence having at least 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO:64 or SEQ ID NO:

66.

76. The nucleic acid according to claim 62, wherein the sequence encoding the IL-12 comprises the nucleic acid sequence set forth in any one of SEQ ID NO:64 or SEQ ID NO:

66.

77. The nucleic acid according to claim 62, wherein the TGF-β activity inhibitor comprises a TGF-β dominant negative, a TGF-β receptor dominant negative, a protein that binds to TGF-β or a protein that binds to a TGF-β receptor.

78. The nucleic acid according to claim 77, wherein the TGF-β activity inhibitor comprises a protein that binds to TGF-β, and wherein the protein that binds to TGF-β is an antibody or a functional variant thereof.

79. The nucleic acid according to claim 77, wherein the TGF-β activity inhibitor comprises a protein that binds to a TGF-β receptor, and wherein the protein that binds to the TGF-β receptor is an antibody or a functional variant thereof.

80. The nucleic acid according to claim 77, wherein the TGF-β activity inhibitor comprises a protein that binds to a TGF-β receptor, and wherein the protein that binds to the TGF-β receptor is a protein comprising a domain of TGF-β.

81. The nucleic acid according to claim 80, wherein the protein comprising a domain of TGF-β comprises the amino acid sequence set forth in any one of SEQ ID NO:1-9.

82. The nucleic acid according to claim 80, wherein the protein comprising the TGF-β-containing domain comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO:7 or SEQ ID NO:

8.

83. The nucleic acid according to claim 80, wherein the protein comprising the TGF-β-containing domain comprises the amino acid sequence set forth in any one of SEQ ID NO:7 or SEQ ID NO:

8.

84. The nucleic acid according to claim 62, wherein the sequence encoding the transforming growth factor β (TGF-β) activity inhibitor comprises a nucleic acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:

67.

85. The nucleic acid according to claim 62, wherein the sequence encoding the TGF-β activity inhibitor comprises the nucleic acid sequence set forth in SEQ ID NO:

67.

86. The nucleic acid according to claim 77, comprising the protein that binds to the TGF-β receptor, and wherein the protein that binds to the TGF-β receptor comprises a TGF-β fusion 1 (TGFbf1) protein.

87. The nucleic acid according to claim 86, wherein the TGFbf1 protein comprises a murine IL-2 signal peptide and a TGF-β variant 1 (TGFbv1).

88. The nucleic acid according to claim 87, wherein the murine IL-2 signal peptide comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:

36.

89. The nucleic acid according to claim 87, wherein the murine IL-2 signal peptide comprises the amino acid sequence set forth in SEQ ID NO:

36.

90. The nucleic acid according to claim 87, wherein the TGFbv1 comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:

7.

91. The nucleic acid according to claim 87, wherein the TGFbv1 comprises the sequence set forth in SEQ ID NO:

7.

92. The nucleic acid according to claim 77, comprising the protein that binds to the TGF-β receptor, and wherein the protein that binds to the TGF-β receptor comprises a TGF-β fusion 2 (TGFbf2) protein.

93. The nucleic acid according to claim 92, wherein the TGFbf2 comprises a human IgE signal peptide and a TGF-β variant 2 (TGFbv2).

94. The nucleic acid according to claim 93, wherein the human IgE signal peptide comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:

37.

95. The nucleic acid according to claim 93, wherein the human IgE signal peptide comprises the amino acid sequence set forth in SEQ ID NO:

37.

96. The nucleic acid according to claim 93, wherein the TGFbv2 comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:

8.

97. The nucleic acid according to claim 93, wherein the TGFbv2 comprises the sequence set forth in SEQ ID NO:

8.

98. The nucleic acid according to claim 77, wherein the protein that binds to the TGF-β receptor binds to TGF-β receptor II and does not bind to TGF-β receptor I.

99. The nucleic acid according to claim 62, further comprising at least one promoter region.

100. The nucleic acid according to claim 99, wherein the at least one promoter region drives the expression of the at least two polypeptides.

101. The nucleic acid according to claim 99, wherein the at least one promoter region comprises a first promoter region and a second promoter region, wherein the first promoter region drives the expression of a polypeptide comprising the IL-12, and the second promoter region drives the expression of the TGF-β activity inhibitor.

102. The nucleic acid according to claim 101, wherein each of the first promoter region and the second promoter region comprises any one of SSP, P7.5, P28, P135, TK promoter, E / L, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, PH5, L4R, 28kDa promoter or any variant or combination thereof.

103. The nucleic acid according to claim 102, wherein the first promoter region comprises the P7.5 promoter.

104. The nucleic acid according to claim 102, wherein the first promoter region comprises the P135 promoter.

105. The nucleic acid according to claim 102, wherein the second promoter region comprises the P28 promoter.

106. The nucleic acid according to claim 102, wherein the second promoter region comprises the P7.5 promoter.

107. The nucleic acid according to any one of claims 62-106, further comprising a sequence encoding a third polypeptide comprising a chemokine receptor or a functional variant thereof.

108. The nucleic acid according to claim 107, wherein the chemokine receptor comprises at least one of the following: CXC receptor, CC receptor, CX3C receptor, XC receptor, a functional fragment thereof, a functional variant thereof or any combination thereof.

109. The nucleic acid according to claim 107, wherein the chemokine receptor comprises at least one of the following: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, a functional fragment thereof or a functional variant thereof, or any combination thereof.

110. The nucleic acid according to claim 109, wherein the chemokine receptor comprises CXCR3.

111. The nucleic acid according to claim 110, wherein the chemokine receptor comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:42 or SEQ ID NO:

43.

112. The nucleic acid according to claim 110, wherein the chemokine receptor comprises the amino acid sequence set forth in SEQ ID NO:42 or SEQ ID NO:

43.

113. The nucleic acid according to claim 107, wherein the third polypeptide comprises a third promoter region that provides expression of the chemokine receptor prior to expression of the IL-12 and the TGF-β activity inhibitor.

114. The nucleic acid according to claim 113, wherein the third promoter region comprises any one of A52R, pB8, mH5, I4L, LEO, pF11, I3L, P7.5, TK promoter, F7L, H5R, short synthetic promoter (SSP) or any variant or combination thereof.

115. The nucleic acid according to claim 114, wherein the third promoter comprises the A52R promoter.

116. A nucleic acid, wherein the nucleic acid comprises: a first region encoding a first polypeptide, the first polypeptide comprising an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO:12 or SEQ ID NO:15; and a second region encoding a second polypeptide, the second polypeptide comprising an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO:40 or SEQ ID NO:

41.

117. The nucleic acid according to claim 116, wherein the nucleic acid comprises DNA or RNA.

118. The nucleic acid according to claim 116, further comprising at least one promoter region.

119. The nucleic acid according to claim 119, wherein the at least one promoter region drives the expression of the at least two polypeptides.

120. The nucleic acid according to claim 119, comprising a first promoter region and a second promoter region, wherein the first promoter region drives the expression of the first polypeptide and the second promoter region drives the expression of the second polypeptide.

121. The nucleic acid according to claim 120, wherein each of the first promoter region and the second promoter region comprises any one of SSP, P7.5, P28, P135, TK promoter, E / L, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, PH5, L4R, 28 kDa promoter or any variant or combination thereof.

122. The nucleic acid according to claim 120, wherein the first promoter region comprises the P7.5 promoter.

123. The nucleic acid according to claim 120, wherein the first promoter region comprises the P135 promoter.

124. The nucleic acid according to claim 120, wherein the second promoter region comprises the P28 promoter.

125. The nucleic acid according to claim 120, wherein the second promoter region comprises the P7.5 promoter.

126. The nucleic acid according to claim 116, wherein: the first polypeptide comprises the sequence listed in any one of SEQ ID NO:12 or SEQ ID NO:15; and the second polypeptide comprises the sequence listed in any one of SEQ ID NO:40 or SEQ ID NO:

41.

127. The nucleic acid according to claim 116, wherein: the first region has a nucleic acid sequence having at least 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO:64 or SEQ ID NO:66; and the second region has a nucleic acid sequence having at least 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO:65 or SEQ ID NO:

67.

128. The nucleic acid according to claim 117, wherein the nucleic acid comprises the DNA, and wherein the DNA contains, in 5' to 3' order, a sequence comprising: a first region encoding the IL-12 or a functional variant thereof, and a second region encoding an inhibitor of TGF-β activity.

129. The nucleic acid according to claim 117, wherein the nucleic acid comprises DNA, and wherein the DNA contains a sequence encoding, in 5' to 3' order, the amino acid sequences listed in SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:13, SEQ ID NO:36 and SEQ ID NO:

7.

130. The nucleic acid according to claim 117, wherein the nucleic acid comprises DNA, and wherein the DNA contains a sequence encoding, in 5' to 3' order, the amino acid sequences listed in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:16, SEQ ID NO:37 and SEQ ID NO:

8.

131. The nucleic acid according to claim 117, wherein the nucleic acid comprises DNA, and wherein the DNA contains a sequence encoding, in 5' to 3' order, the amino acid sequences listed in SEQ ID NO:12 and SEQ ID NO:

40.

132. The nucleic acid according to claim 117, wherein the nucleic acid comprises DNA, and wherein the DNA contains a sequence encoding, in 5' to 3' order, the amino acid sequences listed in SEQ ID NO:15 and SEQ ID NO:

41.

133. The nucleic acid according to any one of claims 116 - 132, wherein the nucleic acid further comprises a third region encoding a third polypeptide, and the third polypeptide comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO: 42 or SEQ ID NO:

43.

134. The nucleic acid according to claim 133, wherein the third polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NO: 42 or SEQ ID NO:

43.

135. The nucleic acid according to any one of claims 116 - 132, wherein the nucleic acid further comprises a third region, and the third region comprises a nucleic acid sequence having at least 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO: 69 or SEQ ID NO:

86.

136. The nucleic acid according to claim 135, wherein the third region comprises the nucleic acid sequence set forth in any one of SEQ ID NO: 69 or SEQ ID NO:

86.

137. The nucleic acid according to claim 133, wherein the third region encoding the third polypeptide further comprises a third promoter region, and the third promoter region provides expression of the third polypeptide before expression of the first polypeptide and the second polypeptide.

138. The nucleic acid according to claim 137, wherein the third promoter comprises any one of A52R, pB8, mH5, I4L, LEO, pF11, I3L, P7.5, TK promoter, F7L, H5R, short synthetic promoter (SSP) or any variant or combination thereof.

139. The nucleic acid according to claim 138, wherein the third promoter comprises the A52R promoter.

140. The nucleic acid according to any one of claims 116 to 139, wherein the nucleic acid is present in an oncolytic virus.

141. The nucleic acid according to claim 140, wherein the oncolytic virus is a poxvirus, adeno - associated virus, adenovirus, reovirus, lentivirus, herpes simplex virus, vesicular stomatitis virus, Mengo virus, myxoma virus, Newcastle disease virus, measles virus or poliovirus.

142. The nucleic acid according to claim 141, wherein the poxvirus is vaccinia virus.

143. The nucleic acid according to claim 142, wherein the vaccinia virus is a modified strain of Western Reserve vaccinia virus (ATCC VR - 1354), Ankara vaccinia virus (ATCC VR - 1508), Ankara vaccinia virus (ATCC VR - 1566), Wyeth vaccinia virus strain (ATCC VR - 1536) or Wyeth vaccinia virus (ATCC VR - 325).

144. The nucleic acid according to claim 140, wherein the nucleic acid is inserted into the viral genome.

145. The nucleic acid according to claim 140 further comprises a mutation or deletion of at least one viral gene selected from the group consisting of: thymidine kinase (TK), F13L, A36R, A34R, A33R, A52R, B5R, B8R, B18R, SPI-1, SPI-2, B15R, VGF, E3L, K3L, A41L, K7R or N1L, a functional fragment thereof or any combination thereof.

146. A nucleic acid molecule, wherein the nucleic acid molecule comprises: an insert comprising, in 5' to 3' order, at the A52R locus: a first promoter region, wherein the promoter comprises the A52R promoter; an insert encoding a first region of human CXCR3; an insert comprising, in 5' to 3' order, at the TK locus: a second promoter region, wherein the promoter comprises P135; a second region encoding human IL-12; a third promoter region, wherein the promoter comprises P7.5; and a third region encoding a TGFβ variant.

147. The nucleic acid molecule according to claim 146, wherein the P135 promoter comprises the nucleic acid sequence set forth in SEQ ID NO:56, and the P7.5 promoter comprises the nucleic acid sequence set forth in SEQ ID NO:

57.

148. The nucleic acid according to claim 146, wherein the first region encoding human CXCR3 comprises the nucleic acid sequence set forth in SEQ ID NO:86; wherein the second region encoding human IL-12 comprises the nucleic acid sequence set forth in SEQ ID NO:66; and wherein the third region encoding a TGF-β inhibitor comprises the nucleic acid sequence set forth in SEQ ID NO:

67.

149. A nucleic acid molecule, wherein the nucleic acid molecule comprises: an insert comprising the sequence set forth in SEQ ID NO:88 at the A52R locus; an insert comprising the sequence set forth in SEQ ID NO:85 at the TK locus.

150. A pharmaceutical composition, wherein the pharmaceutical composition comprises: the composition according to any one of claims 1 to 61 or the nucleic acid according to any one of claims 62 to 149; and a pharmaceutically acceptable excipient.

151. The pharmaceutical composition according to claim 150, wherein the composition is in a liquid dosage form.

152. The pharmaceutical composition according to claim 150, wherein the pharmaceutically acceptable excipient is buffered saline.

153. The pharmaceutical composition according to claim 152, wherein the buffered saline is phosphate buffered saline (PBS), Dulbecco's PBS (DPBS), TRIS buffered saline (TBS), Hank's balanced salt solution (HBSS), Earle's balanced salt solution (EBSS), standard citrate saline (SSC), HEPES buffered saline (HBS) or Gey's balanced salt solution.

154. The pharmaceutical composition according to claim 150, wherein the composition further comprises liposomes or nanoparticles.

155. The pharmaceutical composition according to claim 154, wherein the nucleic acid or vector is associated with the liposomes or nanoparticles.

156. A method for treating cancer, comprising administering to a subject having cancer the pharmaceutical composition according to any one of claims 150 to 155 in an amount sufficient to treat cancer.

157. The method according to claim 156, wherein the cancer is a solid tumor, leukemia or lymphoma.

158. The method according to claim 156, wherein the cancer includes melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostatic epithelial cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, intestinal type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm or sarcoma.

159. The method according to claim 156, wherein the administration comprises intratumoral administration.

160. The method according to claim 156, wherein the administration comprises systemic administration.

161. The method according to claim 160, wherein the systemic administration comprises oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration or any combination thereof.

162. A method for activating an anti-cancer immune response, comprising administering to a subject having cancer the pharmaceutical composition according to any one of claims 150 to 155.

163. The method according to claim 162, wherein the cancer is a solid tumor, leukemia or lymphoma.

164. The method according to claim 162, wherein the cancer includes melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostatic epithelial cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, intestinal type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm or sarcoma.

165. The method according to claim 162, wherein the administration step is intratumoral administration.

166. The method according to claim 162, wherein the administration step is systemic administration.

167. The method according to claim 166, wherein the systemic administration comprises oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration or any combination thereof.

168. The method according to claim 162, wherein the anti-cancer response is defined by an increased infiltration of CD3+CD8+ T cells into the tumor.

169. The method according to claim 168, wherein the increased infiltration of CD3+CD8+ T cells is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold higher in the tumor compared to an untreated tumor.

170. The method according to claim 168, wherein the increased infiltration of CD3+CD8+ T cells in the tumor is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% higher than in an untreated tumor.

171. The method according to claim 162, wherein the anti-tumor immune response comprises an increase in the expression of one or more genes associated with interferon-gamma (IFNG).

172. The method according to claim 171, wherein the one or more genes associated with IFNG are selected from the group consisting of CXCL11, XCR1, STAT1, IDO1, IL12B, IFNG, CIITA, H2-EB1, H2-AB1, TBX21, CXCR3, CD2, LTB, CXCL16, B2M, VCAM1, TAP1, IFIT2, TAP2, IL2RG, STAT2, CD274, and IRF1.

173. The method according to claim 171, wherein the increase in the expression of the one or more genes associated with IFNG is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 120%, about 140%, about 160%, about 180%, about 200%, about 250%, about 300%, about 350%, or about 400% compared to the median expression across treated and untreated cells.

174. The method according to claim 162, wherein the anti-cancer immune response comprises a decrease in the expression of one or more genes associated with TGFB1.

175. The method according to claim 174, wherein the one or more genes associated with TGFB1 are selected from the group consisting of ILz1B, LPL, SLP1, FBN1, LCN2, CXCL5, OGN, PLOD2, TNFAIP6, CAN, ABCG1, ACKR3, and COL15A1.

176. The method according to claim 174, wherein the decrease in the expression of the one or more genes associated with TGFB1 is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the median expression across treated and untreated cells.

177. The method according to claim 162, wherein the anti-cancer response comprises an increase in granzyme B expression.

178. The method according to claim 177, wherein the increase in granzyme B expression is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000% more than that of untreated cells.

179. A method for reducing the incidence of tumor cell growth, comprising: administering to the tumor cells a pharmaceutical composition according to any one of claims 150 to 155 in an effective amount sufficient to reduce the incidence of tumor cell growth.

180. The method according to claim 179, wherein the tumor cells are from a solid tumor, leukemia or lymphoma.

181. The method according to claim 179, wherein the tumor cells are from melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostatic intraepithelial neoplasia, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, intestinal type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm or sarcoma.

182. The method according to claim 179, wherein the administering step is intratumoral administration.

183. The method according to claim 179, wherein the administering step is systemic administration.

184. The method according to claim 183, wherein the systemic administration comprises oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration or any combination thereof.

185. The method according to claim 179, wherein the incidence of tumor cell growth is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% or about 100% compared to the incidence of untreated tumor cell growth.

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