Armed Seneca Valley Virus Oncolytic Therapy Compositions and Methods Thereof

Abstract

Provided herein are armed Seneca Valley viruses that have been modified to carry a therapeutic payload, i.e., to encode an agent for treating cancer. These armed Seneca Valley viruses are oncolytic and express a cancer therapeutic agent. Also provided herein are compositions and methods that use armed Seneca Valley viruses to treat cancer in a subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 138,999, filed January 19, 2021, the disclosure of which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. This ASCII copy, created on January 19, 2022, is named 115029_000049_SL.txt and is 142,398 bytes in size.

[0003] Technical Field The disclosed invention relates to compositions and methods for treating cancer. More particularly, the disclosed invention relates to the field of treating cancer in a subject using oncolytic viruses, particularly Seneca Valley Virus, engineered to encode therapeutic agents useful in the treatment of cancer. [Background technology]

[0004] Cancer is the second leading cause of death in the United States. One in four people die from cancer, and more than one million new cases are diagnosed each year. The disease begins with the unregulated proliferation and growth of abnormally altered cells. However, the definition does not end with a single disease description, but rather a description of hundreds of different diseases. No two cancers are the same, nor are they clonal. The mutations induced and acquired during cellular transformation may be similar, but they are never identical. This challenge adds to the complexity and heterogeneity of the pathologies patients develop. Current cancer treatments, including chemotherapy and radiation therapy, work best when combined with immunomodulatory agents to create and strengthen an antitumor microenvironment. Many malignant tumors may be resistant to treatment by these traditional methods.

[0005] Oncolytic viruses have great potential as anticancer drugs. The picornavirus Seneca Valley Virus (SVV) is a single-stranded (+)RNA virus that has been investigated as an oncolytic therapy. SVV has been shown to be able to target and promote the regression of many intractable malignancies, including small cell carcinoma, non-small cell lung cancer, and pediatric solid tumors. Often, such oncolytic viruses are combined with another compound useful in cancer treatment. Such combination therapy requires that different compounds are administered by different routes.

[0006] Thus, there is a need for improved therapeutic approaches that combine oncolytic viruses, particularly SVV, with other agents useful in the treatment of cancer. Summary of the Invention

[0007] Provided herein is an armed Seneca Valley virus engineered to express an agent useful for the treatment of cancer. In certain embodiments, the agent is a binding fragment of an anti-PD-1 antibody, CXCL9, a TGFβ receptor decoy, an IL-2 mutant, or a nitroreductase. In certain embodiments, the present disclosure provides an armed Seneca Valley virus comprising a Seneca Valley virus or an oncolytic fragment thereof and a nucleic acid encoding a therapeutic protein of interest, such as an interleukin, a chemokine, or a nanobody that acts as a checkpoint inhibitor. In certain embodiments, the armed Seneca Valley virus comprises a Seneca Valley virus or an oncolytic fragment thereof into which a nucleic acid encoding a therapeutic protein of interest is inserted.

[0008] An armed Seneca Valley virus can comprise a sequence of a Seneca Valley virus or an oncolytic fragment thereof having inserted therein a nucleic acid that is at least 85%, 95%, 99%, or 100% identical to the nucleic acid of SEQ ID NO: 1, 3, 5, 7, 9, 11, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 51. Alternatively, an armed Seneca Valley virus can comprise a sequence of a Seneca Valley virus or an oncolytic fragment thereof having inserted therein a nucleic acid that encodes a protein that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52.

[0009] Alternatively, the armed Seneca Valley virus comprises a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to nucleotides 1-7762 of SEQ ID NO:13; nucleotides 1-7783 of SEQ ID NO:14; nucleotides 1-7759 of SEQ ID NO:15; nucleotides 1-7984 of SEQ ID NO:16; nucleotides 1-8140 of SEQ ID NO:17; or nucleotides 1-7738 of SEQ ID NO:18.

[0010] The present disclosure also provides a vector, such as a plasmid, that comprises an armed Seneca Valley Virus. In certain embodiments, the plasmid comprises SEQ ID NO: 13-18 or 53-64, or a fragment thereof.

[0011] Also provided herein are improved methods, compositions, kits, and pharmaceutical compositions for treating cancer using armed Seneca Valley Virus engineered to express an agent useful in the treatment of cancer.

[0012] In particular, the cancer includes triple-negative breast cancer, small cell lung cancer, non-small cell lung cancer, non-small cell squamous cell carcinoma, adenocarcinoma, glioblastoma, skin cancer, hepatocellular carcinoma, colon cancer, cervical cancer, ovarian cancer, endometrial cancer, neuroendocrine cancer, pancreatic cancer, thyroid cancer, renal cancer, bone cancer, esophageal cancer, or soft tissue cancer. The cancer may also be a neuroblastoma, melanoma, neuroendocrine carcinoma, or small cell lung cancer (SCLC) tumor.

[0013] One embodiment of the invention is a method of treating cancer in a subject in need thereof comprising administering to the subject an effective amount of armed Seneca Valley virus, a virus genetically engineered to express an agent useful for treating cancer.

[0014] Another embodiment of the present invention is a method of improving the success of oncolytic cancer virus therapy comprising administering an effective amount of an armed Seneca Valley virus, where the virus has been genetically engineered to express an agent useful for treating cancer.

[0015] Also provided herein is a pharmaceutical composition for treating cancer in a subject, the pharmaceutical composition comprising an armed SVV and a pharma- ceutical acceptable carrier, wherein the armed SVV is genetically engineered to express an agent useful for treating cancer.

[0016] Further provided herein is a kit for treating cancer in a subject, comprising an armed Seneca Valley virus, wherein the armed SVV has been modified to express an agent useful for treating cancer.

[0017] Further provided herein is an armed Seneca Valley Virus (SVV) for use in the manufacture of a medicament for the treatment of cancer, wherein the armed SVV encodes an agent useful for the treatment of cancer.

[0018] Another embodiment of the invention is an armed SVV. Yet another embodiment is a method of producing an armed SVV. The armed SVV is modified to carry a nucleic acid encoding an agent useful in the treatment of cancer.

[0019] Other features and advantages of the present invention will become apparent from the detailed description and examples that follow. [Brief description of the drawings]

[0020] The Summary, and the following Detailed Description, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, exemplary embodiments of the invention are shown in the drawings. However, the invention is not limited to the particular methods and compositions disclosed, nor is the invention limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings. Additionally, the drawings are not necessarily drawn to scale. In the drawings:

[0021] [Figure 1] FIG. 1 shows a map of plasmid pNTX-11 VHH aPDL-1 (SEQ ID NO: 13). [Diagram 2] FIG. 2 is a schematic diagram showing the method for making plasmid pNTX-11 VHH aPDL-1 (SEQ ID NO: 13). [Diagram 3] FIG. 3 shows a map of plasmid pNTX-11 IL2 quad mutant (SEQ ID NO: 14). [Figure 4] FIG. 4 is a schematic diagram showing the method for making the plasmid pNTX-11 IL2 quad mutant (SEQ ID NO: 14). [Diagram 5] FIG. 5 shows a map of plasmid pNTX-11 CXCL9 (SEQ ID NO: 15). [Figure 6] FIG. 6 is a schematic diagram showing the method for producing plasmid pNTX-11 CXCL9 (SEQ ID NO: 15). [Figure 7] FIG. 7 shows a map of plasmid pNTX-11+TGFbDNRII (SEQ ID NO: 16). [Figure 8]FIG. 8 is a schematic diagram showing the method for producing plasmid pNTX-11+TGFbDNRII (SEQ ID NO: 16). [Figure 9] Figure 9 shows a map of plasmid pNTX-11 nfsa mut 22 (SEQ ID NO: 17). [Figure 10] FIG. 10 is a schematic diagram showing the method for constructing plasmid pNTX-11 nfsa mut 22 (SEQ ID NO: 17). [Figure 11] FIG. 11 shows a map of plasmid pNTX-11 Neoleukin 2-15 (SEQ ID NO: 18). [Figure 12] FIG. 12 is a schematic diagram showing the method for constructing plasmid pNTX-11 Neoleukin 2-15 (SEQ ID NO: 18). [Figure 13] FIG. 13 shows a map of plasmid pNTX-11 ova+covid epitope (SEQ ID NO: 19). [Figure 14] FIG. 14 is a schematic diagram showing the method for making plasmid pNTX-11 ova+covid epitope (SEQ ID NO: 19). [Figure 15] Figure 15A is a schematic diagram of the Seneca Valley Virus (SVV-001) genome. Specifically, Figure 15A shows the insertion site of the armed construct of the present invention between the nucleotide sequences encoding SVV protein 2A and SVV protein 2B. Figure 15B is a close-up schematic diagram of the insertion site including the restriction enzyme binding sites. Figure 15C shows the insertion strategy of the exogenous GFP coding sequence into the SVV-001 genome. A portion of the SVV-001 polyprotein is reproduced with long sequence gaps between the junctions indicated by dashes. Proteolytic events are indicated by arrows and ribosomal skips are indicated by diamond-tipped shapes. [Figure 16] Figures 16A and 16B show the rapid generation of armed SVV virus in cell lines. Figure 16A shows the results for SVV-GFP (SVV engineered to express GFP) and Figure 16B shows the results for SVV-mCherry (SVV engineered to express mCherry). [Figure 17] Figure 17 shows RT-PCR data of armed SVV engineered to express IL-2, CXCL9, and IL-2 / 15. The RT-PCR data demonstrate that armed SVV express therapeutic transgenes. [Figure 18] FIG. 18 shows transfection of linear DNA in PerC-T7 pol cells. [Figure 19] 19A and 19B show bioassays for IL-2 and IL2-15. [Figure 20] Figures 20A and 20B show the functional activity of SVV-IL2 and SVV-IL2 / 15 (Neoleukin2-15). Figure 20A shows the activity for SVV-IL2 and SVV-IL2 / 15 (Neoleukin2-15). Figure 20B shows the activity for the IL-2 standard positive control. [Figure 21] Figures 21A and 21B show SVV-CXCL9 Elisa data. Figure 21A shows the human CXCL standard curve. Figure 21B shows human CXCL9 level detection in amplified SVV-CXCL9 supernatant. [Figure 22] Figure 22A shows the nucleic acid sequence of plasmid pSVV-aCTLA4 VHH (SEQ ID NO: 53) containing armed SVV encoding the anti-CLA nanobody of SEQ ID NO: 30. Figure 22B shows a map of plasmid pSVV-aCTLA4 VHH. [Diagram 23] Figure 23A shows the nucleic acid sequence of plasmid pSVV-CD3 VHH (SEQ ID NO: 54) containing armed SVV encoding the anti-CD3 nanobody of SEQ ID NO: 32. Figure 23B shows a map of plasmid pSVV-CD3 VHH. [Figure 24] Figure 24A shows the nucleic acid sequence of plasmid pSVV-new aPDL1 VHH v.2 (SEQ ID NO: 55) containing armed SVV encoding the anti-PDL1 nanobody of SEQ ID NO: 34. Figure 24B shows a map of plasmid pSVV-new aPDL1 VHH v.2. [Diagram 25]Figure 25A shows the nucleic acid sequence of plasmid pSVV-new aPDL1-GSS-aCTLA4 (SEQ ID NO: 56) containing armed SVV encoding anti-PDL1 and anti-CTLA4 nanobodies of SEQ ID NO: 36. [Figure 26] Figure 26A shows the nucleic acid sequence of plasmid pSVV-aCD3-GSS-aCTLA4 (SEQ ID NO: 57) containing armed SVV encoding anti-CD3 and anti-CTLA4 nanobodies of SEQ ID NO: 38. Figure 26B shows a map of plasmid pSVV-aCD3-GSS-aCTLA4. [Figure 27] Figure 27A shows the nucleic acid sequence of plasmid pSVV-new aPDL1-GSS-aCD3 (SEQ ID NO: 58) containing armed SVV encoding anti-CD3 and anti-PDL1 nanobodies of SEQ ID NO: 40. Figure 27B shows a map of plasmid pSVV-new aPDL1-GSS-aCD3. [Figure 28] Figure 28A shows the nucleic acid sequence of plasmid pSVV-hIL-2 v.3 no signal seq (SEQ ID NO: 59) containing armed SVV encoding IL-2 of SEQ ID NO: 42. Figure 28B shows a map of plasmid pSVV-hIL-2 v.3 no signal seq. [Figure 29] Figure 29A shows the nucleic acid sequence of plasmid pSVV-hIL-2 v.2 (SEQ ID NO:60) containing armed SVV encoding IL-2 of SEQ ID NO:44. Figure 29B shows a map of plasmid pSVV-hIL-2 v.2. [Diagram 30] Figure 30A shows the nucleic acid sequence of plasmid pSVV-TGFbR DN v.2 no signal seq+met+pro (SEQ ID NO: 61) containing armed SVV encoding the TGF-beta RII decoy of SEQ ID NO: 46. Figure 30B shows a map of pSVV-TGFbR DN v.2 no signal seq+met+pro. [Diagram 31]Figure 31A shows the nucleic acid sequence of plasmid pSVV-TGFbR DN delta v.3 ser+met (SEQ ID NO: 62) containing armed SVV encoding the TGF-βRII decoy of SEQ ID NO: 48. Figure 31B shows a map of plasmid pSVV-TGFbR DN delta v.3 ser+met. [Diagram 32] Figure 32A shows the nucleic acid sequence of the plasmid pSVV-FCY2+3 mutant (SEQ ID NO:63) encoding the cytosine deaminase of SEQ ID NO:50. Figure 32B shows a mutation map of the plasmid pSVV-FCY2+3. [Diagram 33] Figure 33A shows the nucleic acid sequence of plasmid pSVV-nfsa mut 22-78, which encodes the Nfsa mutant of SEQ ID NO: 52. Figure 33B shows a map of plasmid pSVV-nfsa mut 22-78. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, which is defined in the appended claims. Other aspects of the invention will be apparent to those skilled in the art from a consideration of the detailed description of the invention provided herein.

[0023] The present invention relates to armed Seneca Valley Viruses (SVVs) that encode agents useful in the treatment of cancer. The present invention also relates to methods for producing such armed SVVs.

[0024] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to carry out the testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terms are used.

[0025] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0026] As used herein, the articles "a" and "an" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0027] As used herein, when referring to a measurable value such as an amount, duration, and the like, the term "about" is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the stated value, as appropriate variations for carrying out the disclosed methods.

[0028] The term "biological" or "biological sample" refers to a sample obtained from an organism or components of an organism (e.g., cells). The sample may be either a biological tissue or a fluid. Often the sample is a "clinical sample" that is a sample derived from a patient. Such samples include, but are not limited to, bone marrow, cardiac tissue, sputum, blood, lymph, blood cells (e.g., white blood cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, pleural fluid, or cells thereof. Biological samples also include sections of tissues, such as frozen sections taken for histological purposes.

[0029] As used herein, the terms "comprising," "including," "containing," and "characterized by" are interchangeable, inclusive, and readily modifiable and do not exclude additional, implied elements or method steps. Any recitation of the term "comprising" herein, particularly in the description of a component of a composition or the description of an element of a device, is understood to encompass compositions and methods consisting essentially of, and consisting of, the recited components or elements.

[0030] As used herein, the term "consisting of" excludes any element, step, or ingredient not specified in the claim element.

[0031] The term "Seneca Valley Virus" or "SVV" as used herein includes wild-type SVV or SVV derivatives. Exemplary suitable SSV strains include SVV-001, NTX-010, and the SVV strain with ATCC Patent Deposit No. PTA-5343. As used herein, the term "derivative" provides that a derivative of a virus may have a difference in nucleic acid or amino acid sequence with respect to the template viral nucleic acid or amino acid sequence. As used herein, the term SVV also includes fragments of SVV that maintain the lytic activity of SVV. For example, an SVV derivative can refer to an SVV that has a different nucleic acid or amino acid sequence with respect to the wild-type SVV nucleic acid or amino acid sequence of ATCC Patent Deposit No. PTA-5343. In other embodiments, the SVV derivative can be ONCR-788. In some embodiments, the SVV derivative includes an SVV mutant, an SVV variant, or a modified SVV (e.g., a genetically engineered SVV). In some embodiments, modified SVV derivatives are modified to recognize different cell receptors or to evade the immune system, while still being able to invade, replicate and kill target cells (i.e., cancer cells).Generally, SVV or SVV derivatives can be derived from existing virus stocks that are passaged to produce more virus.SVV or SVV derivatives can also be derived from plasmids.

[0032] As used herein, the phrase "armed Seneca Valley Virus" or "armed SVV" encompasses "Seneca Valley Virus" or "SVV" as defined above that has been modified to express an agent useful in the treatment of cancer. Armed SVV encodes an agent useful in the treatment of cancer. Armed SVV has been engineered to express a therapeutic gene.

[0033] As used herein, "higher" refers to an expression level that is at least 10% or more, such as 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or more higher than the reference value, and / or 1.1-fold, 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold, 2.0-fold or more higher than the reference value, and any whole or partial increment therebetween. A higher expression level than the reference value disclosed herein refers to an expression level (mRNA or protein) that is higher than the normal or control level from the expression (mRNA or protein) measured in a healthy subject, or from the expression (mRNA or protein) as defined or used in the art.

[0034] As used herein, "lower" refers to an expression level that is at least 10% or more lower than a reference value, such as 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or more lower, and / or 1.1-fold, 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold, 2.0-fold or more lower, and any whole or partial increment therebetween. An expression level lower than a reference value disclosed herein refers to an expression level (mRNA or protein) that is lower than a normal or control level from expression (mRNA or protein) measured in a healthy subject, or expression (mRNA or protein) as defined or used in the art.

[0035] As used herein, the terms "control" or "reference" can be used interchangeably and refer to a value used as a standard for comparison.

[0036] As used herein, "combination therapy" means that a first agent is administered in combination with another agent. "In combination with" or "in combination with" means administering one therapeutic modality in addition to another therapeutic modality. Thus, "in combination with" refers to administering one therapeutic modality before, during, or after delivery of the other therapeutic modality to an individual. Such a combination is considered a single therapeutic regimen or part of a regimen. As used herein, combination therapy can include therapeutic regimens that each include administration of an oncolytic virus and another anticancer agent to treat the same hyperproliferative disease or condition, such as the same tumor or cancer. Combination therapy can also include using armed SVV in combination with unarmed SVV.

[0037] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, with no limit on the maximum number of amino acids that a protein or peptide sequence may contain. A polypeptide includes a peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, and longer chains, of which there are many varieties, commonly referred to in the art as proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0038] The term "RNA" as used herein is defined as ribonucleic acid.

[0039] The term "treatment" as used in the context of the present invention is meant to include therapeutic as well as preventive or suppressive treatment of a disease or disorder. As used herein, the term "treatment" and related terms such as "treat" and "treating" refer to the alleviation of the progression, severity and / or duration of a disease state or at least one symptom thereof. Thus, the term "treatment" refers to any regimen that can benefit a subject. Treatment may be for an existing condition or may be preventative (prophylactic treatment). Treatment may include a curative, palliative, or preventive effect. References herein to "therapeutic" and "prophylactic" treatments are considered in their broadest context. The term "therapeutic" does not necessarily mean that a subject is treated until completely recovered. Similarly, "prophylactic" does not necessarily mean that a subject will not eventually suffer from a disease state. Thus, for example, the term treatment includes administering an agent before or after the onset of a disease or disorder, thereby preventing or eliminating all signs of the disease or disorder. As another example, administering an agent after clinical manifestation of a disease to combat the symptoms of a disease includes "treatment" of the disease.

[0040] As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, either analogs of RNA or DNA made from nucleotide analogs, and, where applicable to the described embodiment, single-stranded (sense or antisense) and double-stranded polynucleotides. ESTs, chromosomes, cDNAs, mRNAs, and rRNAs are representative examples of molecules that can be referred to as nucleic acids. In the present specification, when a sequenced nucleic acid is provided as a DNA sequence, it should be understood that an RNA sequence can also be used.

[0041] Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids may be DNA, both genomic and cDNA, RNA, or hybrids, where the nucleic acids may contain combinations of deoxyribo- and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. Nucleic acids may be obtained by chemical synthesis or recombinant methods. As used herein, "operably linked" means that the expression of a gene is under the control of a spatially linked promoter. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variations in this distance can be accommodated without impairing the function of the promoter.

[0042] As used herein, "substantially identical" means that the first and second amino acid sequences are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 50 It can mean at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a region of 0, 600, 700, 800, 900, 1000, 1100 or more amino acids. In addition, substantially the same means that the first nucleic acid sequence and the second nucleic acid sequence are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 60 It can mean at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a region of 0, 700, 800, 900, 1000, 1100 or more nucleotides.

[0043] As used herein, "coding sequence" or "encoding nucleic acid" refers to a nucleic acid (RNA or DNA molecule) that comprises a nucleotide sequence that codes for a protein. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered.

[0044] As used herein, "complement" or "complementary" refers to Watson-Crick (eg, AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0045] As used herein, "consensus" or "consensus sequence" refers to a synthetic nucleic acid sequence, or corresponding polypeptide sequence, constructed based on analysis of the alignment of multiple subtypes of a particular antigen. This sequence can be used to induce broad immunity against multiple subtypes, serotypes, or strains of a particular antigen. Synthetic antigens, such as fusion proteins, can be engineered to generate consensus sequences (or consensus antigens).

[0046] As used herein, "fragment" refers to a nucleic acid sequence or a portion thereof that encodes an oncolytic capable armed SVV and encodes a protein capable of treating cancer.

[0047] "Identical" or "identity" as used herein in the context of two or more nucleic acid or polypeptide sequences means that the sequences have a certain percentage of residues that are identical over a specified region. This percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions where identical residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different length or the alignment produces one or more sticky ends and the specified region of comparison contains only a single sequence, the residues of the single sequence are included in the denominator but not in the numerator of the calculation. Identity can be performed manually or using computer sequence algorithms such as BLAST and BLAST 2.0.

[0048] "Variant," as used herein with respect to a nucleic acid, means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of the referenced nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to the referenced nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0049] A variant can be further defined as a peptide or polypeptide that differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. Representative examples of "biological activity" include the ability to bind to a specific antibody, the ability to stimulate an immune response, and the like. A variant can also refer to a protein having an amino acid sequence substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are typically recognized in the art as involving minor changes. These minor changes can be identified, in part, by considering the hydrophilicity index of the amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydrophobicity index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophobicity indexes can be substituted while retaining protein function. In one embodiment, amino acids with hydrophobicity indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that will result in a protein retaining biological function. By considering the hydrophilicity of the amino acids that make up a peptide, the maximum local average hydrophilicity of the peptide can be calculated. Substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, such as immunogenicity, as understood in the art. Substitutions can be made with amino acids that have hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. Consistent with that observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, particularly their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0050] A variant may be a nucleic acid sequence that is substantially identical over the entire length of the entire gene sequence or a fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the gene sequence or a fragment thereof. A variant may be an amino acid sequence that is substantially identical over the entire length of the amino acid sequence or a fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the amino acid sequence or a fragment thereof.

[0051] As used herein, "vector" refers to a nucleic acid sequence that comprises a replication origin.Vector can be a virus vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome.Vector can be a DNA or RNA vector.Vector can be an autonomously replicating extrachromosomal vector, and is preferably a DNA plasmid.

[0052] As used herein, the term "pharmaceutical composition" refers to a mixture of at least one compound useful within the present invention with other chemical components, such as carriers, stabilizers, diluents, adjuvants, dispersants, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering the compound exist in the art, including, but not limited to, intratumoral administration, intravenous administration, intrapleural administration, oral administration, aerosol administration, parenteral administration, ophthalmic administration, pulmonary administration, and topical administration.

[0053] "Pharmaceutically acceptable carrier" includes pharma- ceutically acceptable salts, pharma-ceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, involved in carrying or transporting the compound(s) of the present invention (e.g., armed SVV) in or to a subject so that it performs its intended function. Typically, such compounds are carried or transported from one organ or part of the body to another organ or part of the body. Each salt or carrier must be "acceptable" in the sense of being compatible with the other components of the formulation and not harmful to the subject. Examples of substances which may function as pharma- ceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; glycols such as propylene glycol; glycerin, sorbitol, mannitol, polyethylene Polyols such as glycols; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; diluents; granulating agents; lubricants; binders; disintegrants; wetting agents; emulsifiers; colorants; release agents; coating agents; sweeteners; flavorings; fragrances; preservatives; antioxidants; plasticizers; gelling agents; thickeners; hardeners; coagulants; suspending agents; surfactants; humectants; carriers; stabilizers; and other non-toxic compatible substances used in pharmaceutical preparations, or any combination thereof. As used herein, "pharmaceutical acceptable carriers" also include any coating agent, antibacterial agent, antifungal agent, absorption retardant, etc., that is compatible with the activity of the compound and physiologically acceptable to the subject. Supplementary active compounds can also be incorporated into the composition.

[0054] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of viral particles or infectious units produced from a vector of the invention that is necessary to prevent a particular disease state or that reduces and / or ameliorates the severity of the disease state or at least one symptom thereof or a condition associated therewith.

[0055] As used herein, the phrase "cancer resistant to checkpoint inhibitor monotherapy" refers to a cancer that may be resistant to checkpoint inhibitor monotherapy at the start of treatment or that becomes resistant to checkpoint inhibitor monotherapy during treatment. This phrase also includes cancers that have been treated with checkpoint inhibitors but have not responded (i.e., are resistant to cancer treatment). This phrase also includes cancers that have been treated with checkpoint inhibitors and initially responded to treatment, but then the tumor recurs (relapsed / resistant). For purposes of this definition, checkpoint inhibitor monotherapy refers to a cancer that has been treated with a checkpoint inhibitor as the only anti-cancer agent. An example of such a cancer is a cold tumor, which is a cancer that is not recognized by the immune system or does not provoke a strong immune system response. Cold tumors are resistant to checkpoint inhibitors and / or checkpoint blockade.

[0056] As used herein, a "subject" or "patient" can be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is a human.

[0057] Ranges: Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity, and should not be construed as a flexible limitation of the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and individual numerical values ​​within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0058] Armed SVV The present disclosure provides armed Seneca Valley Virus modified to encode a therapeutic payload, i.e., an agent for treating cancer. Without being bound by theory, it is contemplated that when such armed SVV is used to treat cancer, the success of cancer treatment is improved compared to treatment using unarmed Seneca Valley Virus (i.e., SVV that does not carry a therapeutic payload).

[0059] In certain embodiments, the disclosure provides Seneca Valley Viruses modified to encode binding fragments of anti-PD-1 antibodies, CXCL9, TGFβ receptor decoys, IL-2 mutants, or nitroreductase.

[0060] Additional embodiments of the present disclosure include: armed SVV encoding an anti-CTLA4 nanobody; armed SVV encoding an anti-CD3 nanobody; armed SVV encoding an anti-PDL1 nanobody; armed SVV encoding both an anti-CTLA4 + anti-PDL1 nanobody; armed SVV encoding both an anti-CTLA4 + anti-CD3 nanobody; armed SVV encoding both an anti-CD3 + anti-PDL1 nanobody; armed SVV encoding IL-2 (versions 2 and 3); armed SVV encoding TGF-beta dominant negative RII decoy-no SS v.2; armed SVV encoding TGF-beta dominant negative RII decoy; armed SVV encoding cytosine deaminase; and armed SVV encoding Nfsa mut 22-78.

[0061] In one embodiment, the disclosure provides an armed Seneca Valley Virus comprising a nucleic acid encoding an IL-2 quadruple mutant, such as the IL-2 quadruple mutant T3A / F42A / Y45A / L72G(C125A). In another embodiment, the disclosure provides an armed SVV comprising Neoleukin2-15.

[0062] In certain embodiments, the present disclosure relates to an armed Seneca Valley virus, the armed Seneca Valley virus comprising a Seneca Valley virus or an oncolytic fragment thereof and a nucleic acid encoding a therapeutic protein of interest. In certain embodiments, the protein of interest is an interleukin, a chemokine, or a nanobody that acts as a checkpoint inhibitor. For example, the protein of interest can be an anti-PD-L1 nanobody, IL-2, CXCL9, IL-15, IL-2 / IL-15, TGF-β decoy, NfsA. In other embodiments, the protein of interest comprises an interleukin, a chemokine, or a nanobody that acts as a checkpoint inhibitor. In some embodiments, the therapeutic protein of interest comprises an anti-PD-L1 nanobody, IL-2 or a variant thereof, CXCL9, IL-15, IL-2 / IL-15 (Neoleukin2-15), TGF-β decoy or a variant thereof, NfsA or a variant thereof, an anti-CTLA4 nanobody, an anti-CD3 nanobody, an anti-CTLA-4+anti-PDLI-1 nanobody, an anti-CLTA4+anti-PLD-1 nanobody, or a cytosine deaminase.

[0063] In certain embodiments, the armed Seneca Valley Virus comprises a Seneca Valley Virus or a fragment thereof into which a nucleic acid encoding a therapeutic protein of interest has been inserted.

[0064] In certain embodiments, an armed Seneca Valley Virus is generated by inserting a nucleic acid sequence encoding a therapeutic protein into the genome of the Seneca Valley Virus or an oncolytic fragment thereof, between the coding sequences for proteins 2A and 2B.

[0065] The TGF-β decoy receptor is derived from the TGF-β receptor that binds to TGF-β (e.g., TGF-β1, TGF-β2 and / or TGF-β3) and lacks the amino acid sequence encoding the transmembrane domain. Expression of the TGF-β decoy reduces the immunosuppressive environment in the tumor microenvironment and enhances T cell responses.

[0066] Neoleukin2-15 is an improved IL-2 variant lacking the binding sites for IL-2Rα (also known as CD25) or IL-15Rα (also known as CD215). The molecule is highly stable and binds to human and mouse IL-2Rβγc with higher affinity than the native cytokine. The molecule is highly stable and binds to human and mouse IL-2Rβγc with higher affinity than the native cytokine.

[0067] In a specific embodiment, the SVV is armed with an IL-2 mutein capable of binding only to the IL-2 receptors CD122 and CD132 (beta and gamma chains) and lacking the CD25 binding domain.

[0068] Certain embodiments of the present invention relate to armed Seneca Valley viruses generated by inserting a nucleic acid of SEQ ID NO: 1, 3, 5, 7, 9, 11, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 51 into a Seneca Valley virus, wherein the resulting armed Seneca Valley virus is oncolytic and wherein a therapeutic protein encoded by SEQ ID NO: 1, 3, 5, 7, 9, 11, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 51 is expressed when the Seneca Valley virus is administered to a cancer cell.

[0069] Certain embodiments of the invention include a nucleic acid sequence that is 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%, or at least 99% identical to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 51. The present invention relates to an armed Seneca Valley virus generated by inserting a nucleic acid that is at least 99% identical to the Seneca Valley virus, wherein the resulting armed Seneca Valley virus is oncolytic and a therapeutic protein encoded by SEQ ID NO: 1, 3, 5, 7, 9, 11, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 or 51 is expressed when the Seneca Valley virus is administered to a cancer cell.

[0070] Another embodiment of the present invention is an armed Seneca Valley virus comprising a sequence of a Seneca Valley virus or an oncolytic fragment thereof modified to express a protein of SEQ ID NO: 2, 4, 6, 8, 10, 12, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52, wherein the armed Seneca Valley virus is oncolytic and the therapeutic protein is expressed when the Seneca Valley virus is administered to a cancer cell.

[0071] Yet another embodiment of the present invention is an armed Seneca Valley virus comprising a sequence of a Seneca Valley virus or an oncolytic fragment thereof that has been modified to express a protein that is 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%, or at least 99% identical to a protein sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52, wherein the armed Seneca Valley virus is oncolytic and the therapeutic protein is expressed when the Seneca Valley virus is administered to a cancer cell.

[0072] An alternative embodiment of the present invention includes an armed Seneca Valley virus that has been modified to include a nucleic acid sequence encoding a protein of SEQ ID NO: 2, 4, 6, 8, 10, 12, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52, or an oncolytic fragment thereof, wherein the armed Seneca Valley virus is oncolytic and the therapeutic protein is expressed when the Seneca Valley virus is administered to a cancer cell.

[0073] Yet another embodiment of the present invention is an armed Seneca Valley virus comprising a sequence of a Seneca Valley virus or an oncolytic fragment thereof that has been modified to contain a nucleic acid sequence encoding a protein that is 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%, or at least 99% identical to a protein sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52, wherein the armed Seneca Valley virus is oncolytic and the therapeutic protein is expressed when the Seneca Valley virus is administered to a cancer cell.

[0074] In one embodiment, the armed Seneca Valley virus comprises nucleotides 1-7762 of SEQ ID NO: 13. In another embodiment, the armed Seneca Valley virus comprises a nucleotide sequence that is 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%, or at least 99% identical to nucleotides 1-7762 of SEQ ID NO: 13.

[0075] In another embodiment, the armed Seneca Valley virus comprises nucleotides 1-7783 of SEQ ID NO: 14. In another embodiment, the armed Seneca Valley virus comprises a nucleotide sequence that is 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%, or at least 99% identical to nucleotides 1-7783 of SEQ ID NO: 14.

[0076] In an alternative embodiment, the armed Seneca Valley virus comprises nucleotides 1-7759 of SEQ ID NO: 15. In another embodiment, the armed Seneca Valley virus comprises a nucleotide sequence that is 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%, or at least 99% identical to nucleotides 1-7759 of SEQ ID NO: 15.

[0077] In yet another embodiment, the armed Seneca Valley virus comprises nucleotides 1-7984 of SEQ ID NO: 16. In another embodiment, the armed Seneca Valley virus comprises a nucleotide sequence that is 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%, or at least 99% identical to nucleotides 1-7984 of SEQ ID NO: 16.

[0078] In yet another embodiment, the armed Seneca Valley virus comprises nucleotides 1-8140 of SEQ ID NO: 17. In another embodiment, the armed Seneca Valley virus comprises a nucleotide sequence that is 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%, or at least 99% identical to nucleotides 1-8140 of SEQ ID NO: 17.

[0079] In yet additional embodiments, the armed Seneca Valley virus comprises nucleotides 1-7738 of SEQ ID NO: 18. In another embodiment, the armed Seneca Valley virus comprises a nucleotide sequence that is 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%, or at least 99% identical to nucleotides 1-7738 of SEQ ID NO: 18.

[0080] Another embodiment of the invention is an armed Seneca Valley Virus generated by inserting a nucleic acid sequence encoding a therapeutic protein into the genome of the Seneca Valley Virus between the coding sequences for proteins 2A and 2B. The nucleic acid sequence encoding the therapeutic protein may be a nucleic acid of SEQ ID NO: 1, 3, 5, 7, 9, 11, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 51; a nucleic acid that is at least 85%, 95% or 99% identical to a nucleic acid of SEQ ID NO: 1, 3, 5, 7, 9, 11, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 51; or a nucleic acid encoding a protein that is at least 85%, at least 90%, at least 95% or 99% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52. In certain embodiments, the armed Seneca Valley Virus is generated by arming SVV-001.

[0081] In other embodiments, the armed Seneca Valley virus comprises a sequence of a Seneca Valley virus or an oncolytic fragment thereof having nucleotides 3508-3885 of SEQ ID NO:13, nucleotides 3505-3906 of SEQ ID NO:14, nucleotides 3508-3882 of SEQ ID NO:15, nucleotides 3508-4107 of SEQ ID NO:16, nucleotides 3508-4263 of SEQ ID NO:17, or nucleotides 3508-3861 of SEQ ID NO:18 inserted therein. In yet another embodiment, the armed Seneca Valley Virus comprises a sequence of a Seneca Valley Virus or an oncolytic fragment thereof having inserted therein a nucleic acid that is at least 85%, 95%, or 99% identical to nucleotides 3508-3885 of SEQ ID NO: 13, nucleotides 3505-3906 of SEQ ID NO: 14, nucleotides 3508-3882 of SEQ ID NO: 15, nucleotides 3508-4107 of SEQ ID NO: 16, nucleotides 3508-4263 of SEQ ID NO: 17, or nucleotides 3508-3861 of SEQ ID NO: 18. The nucleic acid may be inserted between the coding sequences for proteins 2A and 2B in the SVV or SVV derivative.

[0082] Vector carrying an armed SSV The present disclosure also provides a vector that comprises the armed SVV construct of the present disclosure.The vector can have a nucleic acid sequence that comprises a replication origin.The vector can be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome.The vector can be either an autonomously replicating extrachromosomal vector or a vector that is integrated into the host genome.

[0083] In one embodiment, the vector comprises a nucleic acid sequence that is 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%, or at least 99% identical to a nucleic acid sequence of SEQ ID NO: 13-18 or 53-64.

[0084] In certain embodiments, the vector is a plasmid that forms a nucleic acid sequence of any one of SEQ ID NOs: 13-18 or 53-64, or a nucleic acid substantially identical to a nucleic acid sequence of SEQ ID NOs: 13-18 or 53-64.

[0085] In one embodiment, the plasmid comprises nucleotides 1-7762 of SEQ ID NO: 13. In another embodiment, the plasmid comprises a nucleotide sequence that is 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%, or at least 99% identical to nucleotides 1-7762 of SEQ ID NO: 13.

[0086] In another embodiment, the plasmid comprises nucleotides 1-7783 of SEQ ID NO: 14. In another embodiment, the plasmid comprises a nucleotide sequence that is 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%, or at least 99% identical to nucleotides 1-7783 of SEQ ID NO: 14.

[0087] In another embodiment, the plasmid comprises nucleotides 1-7759 of SEQ ID NO: 15. In another embodiment, the plasmid comprises a nucleotide sequence that is 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%, or at least 99% identical to nucleotides 1-7759 of SEQ ID NO: 15.

[0088] In another embodiment, the plasmid comprises nucleotides 1-7984 of SEQ ID NO: 16. In another embodiment, the plasmid comprises a nucleotide sequence that is 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%, or at least 99% identical to nucleotides 1-7984 of SEQ ID NO: 16.

[0089] In another embodiment, the plasmid comprises nucleotides 1-8140 of SEQ ID NO: 17. In another embodiment, the plasmid comprises a nucleotide sequence that is 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%, or at least 99% identical to nucleotides 1-8140 of SEQ ID NO: 17.

[0090] In a further embodiment, the plasmid comprises nucleotides 1-7738 of SEQ ID NO: 18. In another embodiment, the plasmid comprises a nucleotide sequence that is 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%, or at least 99% identical to nucleotides 1-7738 of SEQ ID NO: 18.

[0091] One or more of the vectors may be expression constructs, which are generally plasmids used to introduce specific genes into target cells. Once the expression vector enters a cell, the protein encoded by the gene is produced by the ribosomal complex of the cell's transcription and translation machinery. Plasmids are often engineered to contain regulatory sequences that act as enhancer and promoter regions and direct efficient transcription of the gene carried on the expression vector. The vectors of the present invention express large amounts of stable messenger RNA and therefore proteins. The vectors may have expression signals such as strong promoters, strong stop codons, adjustment of the distance between the promoter and the cloned gene, insertion of transcription termination sequences and PTIS (portable translation initiation sequences).

[0092] How to arm the SVV The present disclosure also provides a method for arming Seneca Valley Virus to express a therapeutic protein. The method includes providing a Seneca Valley Virus nucleic acid sequence, such as the nucleic acid sequence of SVV-001, NTX-010, and the SVV strain having ATCC Patent Deposit Number PTA-5343, and inserting a therapeutic protein at an appropriate location, whereby the resulting armed SSV virus is oncolytic and expresses a therapeutic protein. In a specific embodiment, the therapeutic protein is inserted into the Seneca Valley Virus between the nucleic acid sequence encoding the 2A peptide and the nucleic acid sequence encoding the 2B peptide of SVV. A schematic diagram of this insertion site is shown in Figures 15A-C.

[0093] In certain embodiments, the methods of the invention can be used to arm SVV to contain a nucleic acid encoding a therapeutic protein up to 800 base pairs in length. In one embodiment of the method, the Seneca Valley Virus is NTX-010 or SVV-001.

[0094] In one embodiment, the disclosure provides a method for generating an armed SVV construct, comprising the steps of: (1) constructing an armed SVV plasmid; (2) linearizing the armed SVV plasmid to define a 3' end; (3) generating RNA transcripts with authentic 5' and 3' ends by an in vitro transcription reaction using T7 polymerase; (4) transfecting the RNA into a target cell; and (5) isolating the armed SVV virus.

[0095] In another embodiment, the present disclosure provides a method for generating an armed SVV construct, comprising the steps of: (1) cloning a T7 polymerase-optimized mammalian expression plasmid into a target cell line; (2) linearizing the armed SVV plasmid; (3) transfecting the plasmid into T7-pol cells; and (4) isolating the armed SVV virus.

[0096] In an alternative embodiment, the method includes: constructing a plasmid containing Seneca Valley Virus or an oncolytic fragment thereof and a nucleic acid encoding a therapeutic protein of interest; linearizing the plasmid to define a 3' end; generating an RNA transcript having authentic 5' and 3' ends by an in vitro transcription reaction using T7 polymerase; transfecting the RNA transcript into a target cell; and isolating the armed SVV virus.

[0097] In yet another embodiment, the method includes the steps of: cloning a T7 polymerase-optimized mammalian expression plasmid into a target cell; providing a linearized armed SVV plasmid comprising a Seneca Valley Virus or an oncolytic fragment thereof and a nucleic acid encoding a therapeutic protein of interest; transfecting the armed SVV plasmid into a T7-pol target cell; and isolating the armed Seneca Valley Virus.

[0098] In certain embodiments, armed SVV constructs can be generated using the methods set forth in the Examples below.

[0099] In certain embodiments of the invention, the method comprises inserting into a Seneca Valley Virus a nucleic acid of SEQ ID NO: 1, 3, 5, 7, 9, 11, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 51. In other embodiments, the method comprises inserting into a Seneca Valley Virus or oncolytic fragment thereof a nucleic acid that is 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%, or at least 99% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 29, 31, 33, 37, 39, 41, 43, 45, 47, 49, or 51. Alternatively, the method includes inserting into the Seneca Valley Virus a nucleic acid encoding a protein of SEQ ID NO: 2, 4, 6, 8, 10, 12, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52. The method may also include inserting a nucleic acid encoding a protein that is 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%, or at least 99% identical to the protein sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 30, 32, 36, 38, 40, 42, 44, 48, 50, or 52. The method produces armed SSVs that are oncolytic and produce a functional therapeutic protein or a functional fragment thereof, ie, the therapeutic protein or fragment thereof maintains therapeutic function.

[0100] Immunogenic SVV construct The disclosure also provides Seneca Valley Virus modified to include screening proteins such as, for example, ovalbumin and COVID epitopes. Thus, one embodiment is a Seneca Valley Virus comprising nucleic acids 1-7891 of SEQ ID NO: 19. Another embodiment of the invention is a Seneca Valley Virus comprising a nucleic acid sequence that is 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%, or at least 99% identical to nucleotides 1-7891 of SEQ ID NO: 19.

[0101] Yet another embodiment is an SVV construct generated by inserting a nucleic acid sequence into the genome of Seneca Valley Virus between the coding sequences for proteins 2A and 2B, wherein the nucleic acid sequence comprises nucleotides 3508-4014 of SEQ ID NO:19, or a nucleic acid sequence that is 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%, or at least 99% identical to nucleotides 3508-4014 of SEQ ID NO:19.

[0102] In certain embodiments, the SVV constructs can be used to evaluate the in vivo immunogenicity of Seneca Valley Virus and / or armed Seneca Valley Virus. Specifically, the SVV constructs can be used to test the immune stimulatory properties of SVV.

[0103] How armed SVV can treat cancer The present disclosure provides methods, compositions, kits, and pharmaceutical compositions for treating cancer that utilize armed Seneca Valley viruses, whereby the armed Seneca Valley viruses encode agents useful for treating cancer. In particular, the present disclosure provides methods, compositions, kits, and pharmaceutical compositions for treating cancer that utilize armed Seneca Valley viruses as described herein.

[0104] The cancer treatment provided herein includes the treatment of solid tumors or the treatment of metastasis. Metastasis is a form of cancer in which transformed or malignant cells migrate and spread the cancer from one site to another. Such cancers include cancers of the skin, breast, brain, cervix, testis, and the like. More specifically, the cancer may include, but is not limited to, the following organs or systems: cardiac, pulmonary, gastrointestinal, genitourinary, liver, bone, nervous system, gynecological, blood, skin, and adrenal gland. More specifically, the methods herein can be used to treat gliomas (schwannoma, glioblastoma, astrocytoma), neuroblastoma, pheochromocytoma, paraganglioma, meningioma, adrenal cortical carcinoma, kidney cancer, various types of vascular cancer, osteoblastic bone cancer, prostate cancer, ovarian cancer, uterine leiomyoma, salivary gland cancer, choroid plexus cancer, breast cancer, pancreatic cancer, colon cancer, megakaryoblastic leukemia. Skin cancers include malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, lenticular nevus, lipoma, hemangioma, dermatofibroma, keloid, rhabdomyosarcoma, medulloblastoma, and psoriasis.

[0105] In some embodiments, the cancer treated by the methods disclosed herein comprises triple-negative breast cancer, small cell lung cancer, non-small cell lung cancer, non-small cell squamous cell carcinoma, adenocarcinoma, glioblastoma, skin cancer, hepatocellular carcinoma, colon cancer, cervical cancer, ovarian cancer, endometrial cancer, neuroendocrine cancer, pancreatic cancer, thyroid cancer, renal cancer, bone cancer, esophageal cancer, or soft tissue cancer.

[0106] In other embodiments, the cancer is neuroblastoma or melanoma. In yet other embodiments, the cancer is a neuroendocrine cancer or a small cell lung cancer (SCLC) tumor.

[0107] Combination therapy The compositions and methods for treating cancer in a subject using armed SVV described herein may be combined with at least one additional compound useful in the treatment of cancer. The additional compound may include commercially available compounds known to treat, prevent, or alleviate symptoms of cancer and / or metastasis.

[0108] In one embodiment, the pharmaceutical composition disclosed herein comprises armed SVV and a pharmaceutically acceptable carrier. The composition may also comprise additional SVV. The pharmaceutical composition can be used in combination with a therapeutic agent, such as an antitumor agent, including but not limited to chemotherapeutic agents, anti-cell proliferation agents, or any combination thereof. For example, any conventional chemotherapeutic agent of the following non-limiting exemplary classes: alkylating agents; nitrosoureas; antimetabolites; antitumor antibiotics; plant alkyloids; taxanes; hormonal agents; and miscellaneous agents are included in the present invention. In another embodiment, the pharmaceutical composition disclosed herein can be used in combination with radiation therapy.

[0109] Most alkylating agents are cell cycle non-specific. In certain embodiments, they stop tumor growth by crosslinking the guanine bases of DNA double helix strands. Non-limiting examples include busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, mechlorethamine hydrochloride, melphalan, procarbazine, thiotepa, and uracil mustard.

[0110] Antimetabolites block the incorporation of bases into DNA during the synthesis (S) phase of the cell cycle, preventing normal development and division. Non-limiting examples of antimetabolites include agents such as 5-fluorouracil, 6-mercaptopurine, capecitabine, cytosine arabinoside, floxuridine, fludarabine, gemcitabine, methotrexate, and thioguanine.

[0111] Antitumor antibiotics generally block cell division by inhibiting enzymes required for cell division or by altering the membranes that surround the cells. This class includes anthracyclines, such as doxorubicin, which act to block cell division by disrupting the structure of DNA and halting its function. These drugs are non-cell phase specific. Non-limiting examples of antitumor antibiotics include aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carubicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mitoxantrone, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin.

[0112] Plant alkaloids inhibit or stop mitosis or inhibit enzymes that prevent cells from producing proteins necessary for cell growth. Commonly used plant alkaloids include vinblastine, vincristine, vindesine, vinorelbine, etc. However, the present invention should not be construed as being limited to only these plant alkaloids.

[0113] Taxanes act on cellular structures called microtubules, which are important for cellular function. In normal cell growth, microtubules form when cell division begins, but disassemble or are destroyed when cell division stops. Taxanes inhibit the disassembly of microtubules, so cancer cells become clogged with microtubules and are unable to grow and divide. Non-limiting exemplary taxanes include paclitaxel and docetaxel.

[0114] Hormonal and hormone-like drugs are used in certain types of cancer, such as leukemia, lymphoma, and multiple myeloma. These drugs are often combined with other types of chemotherapy drugs to enhance their effectiveness. Sex hormones alter the action or production of female and male hormones and are used to slow the growth of cancers such as breast, prostate, and endometrial cancers. Blocking the production (aromatase inhibitors) or action (tamoxifen) of these hormones is often used as an adjunct to treatment. Other tumors are also hormone-dependent. Tamoxifen is a non-limiting example of a hormonal drug that blocks the activity of estrogen, which promotes the growth of breast cancer cells.

[0115] The various agents include chemotherapeutic agents such as bleomycin, hydroxyurea, L-asparaginase, and procarbazine.

[0116] Other examples of chemotherapeutic agents include, but are not limited to, the following and their pharma- ceutically acceptable salts, acids, and derivatives: MEK inhibitors, such as, but not limited to, refametinib, selumetinib, trametinib, or cobimetinib; nitrosamines, such as chlorambucil, chromafazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; Gen mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU; calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testosterone. Androgens such as lactones; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; eflornithine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenobarbital Namet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; polysaccharide-K (PSK); razoxane; schizofuran; spirogermanium; tenuazonic acid; triazicon; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOLO, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitors RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; and capecitabine.

[0117] Anti-cell proliferation agent can be further defined as apoptosis inducer or cytotoxic agent.Apoptosis inducer can be granzyme, Bcl-2 family member, cytochrome C, caspase, or combination thereof.Exemplary granzymes include granzyme A, granzyme B, granzyme C, granzyme D, granzyme E, granzyme F, granzyme G, granzyme H, granzyme I, granzyme J, granzyme K, granzyme L, granzyme M, granzyme N, or combination thereof.In other specific embodiments, Bcl-2 family member is, for example, Bax, Bak, Bcl-Xs, Bad, Bid, Bik, Hrk, Bok, or combination thereof.

[0118] In further embodiments, the caspase is caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, or a combination thereof. In certain embodiments, the cytotoxic agent is TNF-α, gelonin, prodigiosin, ribosome inhibitor protein (RIP), Pseudomonas aeruginosa exotoxin, Clostridium difficile toxin B, Helicobacter pylori VacA, Yersinia enterocolitica YopT, violacein, diethylenetriaminepentaacetic acid, irofulven, diphtheria toxin, mitogillin, ricin, botulinum toxin, cholera toxin, saporin 6, or a combination thereof.

[0119] Immunotherapeutic agents include, but are not limited to, interleukin-2 or other cytokines, monoclonal antibodies that bind to PD-1, and programmed cell death protein 1 (PD-1) signaling inhibitors such as ipilimumab. Immunotherapeutic agents can also inhibit cytotoxic T-lymphocyte-associated antigen A-4 (CTLA-4) signaling and can be associated with cancer vaccines and dendritic cell-based therapy.

[0120] In one embodiment, a subject suffering from cancer is administered at least one anti-cancer therapeutic agent selected from the group consisting of: a checkpoint inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a cytokine, a growth factor, a photosensitizer, a toxin, a siRNA molecule, a signal transduction modulator, an anti-cancer antibiotic, an anti-cancer antibody, an angiogenesis inhibitor, a chemotherapeutic compound, an anti-metastatic compound, an immunotherapeutic compound, a CAR therapy, a dendritic cell-based therapy, a cancer vaccine, an oncolytic virus, an engineered anti-cancer virus or virus derivative, and any combination thereof.

[0121] In certain embodiments, the subject is administered a checkpoint inhibitor. The checkpoint inhibitor can be a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a combination thereof. In certain embodiments, the checkpoint inhibitor is an anti-PD1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. In certain embodiments, the checkpoint inhibitor blocks one or more of the following checkpoint proteins on the cancer cells: PD-1, PD-L1, CTLA-4, B7-1, B7-2. In other embodiments, the checkpoint inhibitor blocks one or more of the following checkpoint proteins: LAG-3; TIM-3; TIGIT; VISTA, B7-H3, BTLA, and Siglec-15. See Qin, S. et al. Mol Cancer 18, 155 (2019); Gaynor et al. Semin Cancer Biol. 2020 Jul 2; S1044-579X(20)30152-8. The checkpoint inhibitor may be, for example, an antibody, such as a monoclonal antibody.

[0122] Additional exemplary suitable checkpoint inhibitors include, but are not limited to, ipilimumab (Yervoy®), pembrolizumab (Keytruda®), nivolumab (Opdivo®), and atezolizumab (Tecentriq®). In one embodiment, the checkpoint inhibitor is an anti-PD-1 antibody.

[0123] In one embodiment, at least one anti-cancer therapeutic agent is administered prior to, concurrently with, or following administration of armed SVV.

[0124] In one embodiment, the subject is administered an IFN-I inhibitor. IFN-I inhibitor as used herein includes any agent known in the art to partially or completely, temporarily or permanently inhibit, suppress or reduce IFN type I pathway. In some embodiments, the inhibitory effect of IFN-I inhibitor can be reversible. In other embodiments, the inhibition of IFN-I is reversed.

[0125] Inhibitors include siRNAs, ribozymes, antisense molecules, aptamers, peptidomimetics, small molecules, mTOR inhibitors, histone deacetylase (HDAC) inhibitors, Janus kinase (JAK) inhibitors, IFN inhibitors, IFN antibodies, IFN-α receptor 1 antibodies, IFN-α receptor 2 antibodies, viral peptides, and combinations thereof. Viral peptides include, but are not limited to, the NS1 protein from influenza virus, the NS2B3 protease complex from dengue virus, and the like.

[0126] The mTOR pathway and its inhibition are known to be involved in various diseases, including cancer. Rapamycin is a natural product produced by the bacterium Streptomyces hygroscopicus, and can inhibit mTOR by associating with its intracellular receptor FK-506-binding protein 12 (FKBP12). The FKBP12-rapamycin complex directly binds to the FKBP12-rapamycin-binding domain of mTOR. mTOR functions as a catalytic subunit of two distinct molecular complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 is composed of mTOR regulatory associated protein (Raptor) and mammalian LST8 / G protein β subunit-like protein (mLST8 / GβL). This complex functions as a nutrient / energy / redox sensor and is responsible for controlling protein synthesis. mTORC1 activity is stimulated by insulin, growth factors, serum, phosphatidic acid, amino acids (especially leucine), and oxidative stress (Hay and Sonenberg, Genes Dev. 18(16):1926-1945, 2004; Wullschleger et al., Cell 124(3):471-484). In contrast, mTORC1 is known to be inhibited by low nutrient levels, growth factor deprivation, reductive stress, caffeine, rapamycin, farnesylthiosalicylic acid, and curcumin (Beevers et al., Int. J. Cancer 119(4):757-764, 2006; McMahon et al., Mol. Endocrinol. 19(1):175-183). Components of mTORC2 are rapamycin-insensitive companion of mTOR (Rictor), GβL, mammalian stress-activated protein kinase-interacting protein 1 and mTOR.mTORC2 has been shown to function as a key regulator of the cytoskeleton through stimulation of F-actin stress fibers, paxillin, RhoA, Rac1, Cdc42 and protein kinase C alpha (Sarbassov et al., Curr. Biol. 14(14): 1296-302, 2004; Sarbassov et al., Science 307(5712): 1098-101, 2005). Unlike mTORC1, mTORC2 is insensitive to rapamycin.

[0127] Many mTOR inhibitors are known in the art and have strong immunosuppressive and antitumor activity. mTOR inhibitors, such as rapamycin or rapamycin analogs or derivatives, are known to exhibit immunosuppressive and antiproliferative effects. Other mTOR inhibitors include everolimus, tacrolimus, zotarolimus (ABT-578), pimecrolimus, biolimus, FK-506, PP242 (2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol), Ku-0063794 (rel-5-[2-[(2R,6S)-2,6-dimethyl-4-morpholinyl]-4-(4-morpholinyl)pyrido[2,3-d]pyrimidin-7-yl]-2-methyl-1H-pyridin-5-ol), and 1H-methyl-1H-pyridin-5-ol (1H-methyl-1H-pyridin-5-ol). toxobenzenemethanol), PI-103 (3-(4-(4-morpholinyl)pyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl)phenol), PKI-179 (N-[4-[4-(4-morpholinyl)-6-(3-oxa-8-azabicyclo[3.2.1]oct-8-yl)-1,3,5-triazin-2-yl]phenyl]-N'-4-pyridinylurea hydrochloride), AZD8055 (5-[2,4-bis[(3S)-3-methyl-4-morpholinyl]pyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol), 1-{4-[1-(1,4-dioxa-spiro[4.5]dec-8-yl)-4-(8-oxa-3-aza-bicyclo[3.2.1]oct-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl]-phenyl}-3-methyl-urea; WYE-23 (4-{6-[4-(3-cyclopropyl-ureido)-phenyl]-4-morpholin- 4-yl-pyrazolo[3,4-d]pyrimidin-1-yl}-piperidine-1-carboxylic acid methyl ester), WYE-28 (4-(6-{4-[3-(4-hydroxymethyl-phenyl)-ureido]-phenyl}-4-morpholin-4-yl-pyrazolo[3,4-d]pyrimidin-1-yl)-piperidine-1-carboxylic acid methyl ester), WYE-354 (4-[6-[4-[(methoxycarbonyl)amino]phenyl]-4-(4-morpholinyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1-piperidine carboxylic acid methyl ester), C20-methallylrapamycin and C16-(S)-butylsulfonamidorapamycin, C16-(S)-3-methylindolerapamycin (C16-iRap), C16-(S)-7-methylindolerapamycin (AP21967 / C16-AiRap), CCI-779 (temsirolimus), RAD001 (40-O-(2-hydroxyethyl)-rapamycin), AP-23575, AP-23675, AP-23573, 20-thiarapamycin, 15-deoxo-19-sulfoxylrapamycin, WYE-592, ILS-920, 7-epi-rapamycin, 7-thiomethyl-rapamycin Synth, (3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,2-1,22,23,24,25,26,27,32,33,34,34a-Hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(-1S,3R,4R)-3 -Methoxy-4-tetrazol-1-yl)cyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1,4]oxaazacyclohentriacontin-1,5,11,28,29(4H,6H,31H)-pentone) (U.S. Patent No. 6,015,No. 815), A-94507, deforolimus, AP-23675, AP-23841, zotarolimus, CCI779 / temsirolimus, RAD-001 / everolimus, 7-epi-trimethoxy-rapamycin, 2-desmethyl-rapamycin, 42-O-(2-hydroxy)ethyl-rapamycin, ridaforolimus, ABI-009, MK8669, TOP216, TAFA93, TORISEL™ (prodrug), CERTICA N™, Ku-0063794, PP30, Torin1, Torin2, ECO371, AP23102, AP23573, AP23464, AP23841; 40-(2-hydroxyethyl)rapamycin, 40-[3-hydroxy(hydroxymethyl)methylpropanoate]-rapamycin (also called CC1779), 32-deoxorapamycin, and 16-pentynyloxy-32(S)-dihydrorapamycin. Non-rapamycin analog mTOR inhibitor compounds include, but are not limited to, LY294002, wortmannin, quercetin, myricentin, staurosporine, and ATP-competitive inhibitors. Other examples of suitable mTOR inhibitors are described in U.S. Pat. No. 6,329,386, U.S. Patent Application Publication No. 2003 / 129215, and U.S. Patent Application Publication No. 2002 / 123505.

[0128] In some embodiments, the mTOR inhibitor inhibits at least one of mTORC1 and mTORC2. In other embodiments, the mTOR inhibitor is Torin1 or Torin2.

[0129] Many HDAC inhibitors are known and used in the art. The most common HDAC inhibitors bind to the zinc-containing catalytic domain of HDAC. These HDAC inhibitors can be classified into several groups, named according to their chemical structure and the chemical moiety that binds to zinc ion. Some examples include, but are not limited to, hydroxamic acid or hydroxamate (such as trichostatin A (TSA) or vorinostat / SAHA (FDA approved)), aminobenzamide entinostat (MS-275), tacedinaline (CI994), mocetinostat (MGCD0103), cyclic peptides (apicidin, romidepsin (FDA approved)), cyclic tetrapeptides or epoxyketones (such as trapoxin B), depsipeptides, benzamides, electrophilic ketones, carboxylic aliphatic acid compounds (such as butyrate, phenylbutyrate, valproate, valproic acid, etc.). Other HDAC inhibitors include, but are not limited to, belinostat (PXD101), LAQ824, and panobinostat (LBH589). Examples of HDCA inhibitors in clinical trials include panobinostat (LBH-589), belinostat (PXD101), entinostat (MS275), mocetinostat (MGCD01030), divinostat (ITF2357), practinostat (SB939), chidamide (CS055 / HBI-8000), xinostat (JNJ-26481585), abexinostat (PCI-24781), CHR-3996, and AR-Z2. In one embodiment, the HDAC inhibitor is trichostatin A.

[0130] JAK inhibitors (also called JAK / SAT inhibitors) inhibit the activity of one or more enzymes of the Janus kinase family (e.g., JAK1, JAK2, JAK3, and / or TYK2), thereby inhibiting the JAK-STAT signaling pathway. Various JAK inhibitors are known and used in the art for the treatment of inflammatory diseases or cancer. Non-limiting examples of JAK inhibitors include FDA-approved compounds including ruxolitinib (Jakafi / Jakavi), tofacitinib (Jakvinus, formerly known as tasocitinib and CP-690550), oclacitinib (Apoquel), baricitinib (Olumient, LY3009104), and decernotinib (VX-509). Other JAK inhibitors are also in clinical trials and / or are used as experimental drugs. For example, these include filgotinib (G-146034, GLPG-0634), celduratinib (PRT062070), gandotinib (LY-2784544), lestaurtinib (CEP-701), momelotinib (GS-0387, CYT-387), pacritinib (SB1518), PF-04965842, upadacitinib (ABT-494), peficitinib (ASP015K, JNJ-54781532), fedratinib (SAR302503), cucurbitacin I, CHZ868, ABT-494, dimethyl fumarate (DMF, Tecfidera), GLPG0634, and CEP-33779. In one embodiment, the JAK / STAT inhibitor is staurosporine (STS; antibiotic AM-2282), an inhibitor of protein kinase C (PKC).

[0131] In one embodiment, the subject is further administered at least one IFN-I inhibitor selected from the group consisting of HDAC inhibitors, JAK / STAT inhibitors, IFN inhibitors, IFN antibodies, IFN-α receptor 1 antibodies, IFN-α receptor 2 antibodies, and viral peptides, and combinations thereof. In another embodiment, the at least one IFN-I inhibitor is administered before, simultaneously with, or after administration of armed SVV. In some embodiments, the at least one IFN-I inhibitor is removed once armed SVV has replicated in tumor cells, prior to adding an anti-cancer therapeutic agent (e.g., a checkpoint inhibitor).

[0132] In one embodiment, the anti-cancer therapeutic agent is administered before, simultaneously with, or after administration of at least one IFN-I inhibitor. In one embodiment, the anti-cancer therapeutic agent is administered after administration of at least one IFN-I inhibitor. In another embodiment, the anti-cancer therapeutic agent is administered subsequent to administration of at least one IFN-I inhibitor and armed SVV.

[0133] In one embodiment, armed SVV treatment is preceded by administration of an IFN-I inhibitor. In one embodiment, once armed SVV replication and cancer cell death are confirmed, administration of the IFN-I inhibitor is terminated. For example, cancer cells can be treated with an IFN-I inhibitor (e.g., (5-(tetradecyloxy)-2-furoic acid), acetyl-CoA carboxylase inhibitor: TOFA) 24 hours prior to armed SVV treatment, and both treatments can be continued for several weeks until robust armed SVV replication is observed and markers of cell death are detected. Then, IFN-I inhibitor treatment can be terminated and anti-cancer therapeutics can be initiated. Armed SVV replication generates various nucleic acids and cellular debris, which triggers the activation of influx of immune cells (e.g., T cells, NK cells, APCs, etc.) to inhibit, reduce, and / or eliminate / kill cancer cells. This immune response process is further enhanced by cessation of IFN-I inhibition.

[0134] Pharmaceutical Compositions In certain embodiments, the present invention relates to a pharmaceutical composition comprising an armed SVV, whereby the armed SVV encodes an agent for treating cancer. In certain embodiments, the pharmaceutical composition may be supplemented with one or more agents disclosed above.

[0135] Provided herein is a pharmaceutical composition for treating cancer in a subject in need thereof.The pharmaceutical composition comprises an armed SVV and a pharma- ceutical acceptable carrier; whereby, the armed SVV encodes an agent for treating cancer.

[0136] Also provided herein is a pharmaceutical composition for treating cancer in a subject in need thereof.The pharmaceutical composition comprises an armed SVV and a pharma- ceutical acceptable carrier; whereby the armed SVV encodes an agent for treating cancer.

[0137] Such pharmaceutical compositions are in a form suitable for administration to a subject, or the pharmaceutical compositions may further comprise one or more pharma- ceutically acceptable carriers, one or more additional components, or any combination thereof.Various components of the pharmaceutical compositions may be present in the form of physiologically acceptable salts, such as in combination with physiologically acceptable cations or anions, as is well known in the art.

[0138] In the embodiments provided herein, pharmaceutical compositions useful for practicing the methods of the invention may be administered to deliver a dose between 1 ng / kg / day and 100 mg / kg / day. In another embodiment, pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose between 1 ng / kg / day and 500 mg / kg / day. The relative amounts of the active ingredient, pharma- ceutically acceptable carrier, and any additional ingredients in the pharmaceutical compositions of the invention will vary depending on the identity, size, and condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the compositions may contain 0.1%-100% (w / w) active ingredient.

[0139] Pharmaceutical compositions useful in the methods of the invention may be suitably developed for inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ocular, intrathecal, intravenous, or other routes of administration. Other formulations are contemplated, such as projected nanoparticles, liposomal formulations, resealed erythrocytes containing the active ingredient, immunological formulations, and the like. The route of administration will be readily apparent to one of skill in the art and will depend on any number of factors, including the type and severity of the disease being treated, the species and age of the domestic animal or human patient being treated, and the like.

[0140] The pharmaceutical composition formulation described herein can be prepared by any method known or developed in the art of pharmacology.In general, such preparation method includes combining active ingredient with carrier or one or more other auxiliary ingredients, and then, if necessary or desired, shaping or packaging the product into desired single or multiple dose units.In some embodiments, SVV can be formulated as natural capsid, modified capsid, naked RNA, or encapsulated in protective coat.

[0141] The amount of active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject, or equal to a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage. The unit dosage form may be a single daily dose or multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0142] The description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for ethical administration to humans, but those skilled in the art will understand that such compositions are generally suitable for administration to any type of animal. Modifications to make pharmaceutical compositions suitable for administration to humans suitable for administration to various animals are well understood, and an ordinary skilled veterinary pharmacologist can design and implement such modifications with no more than routine experimentation, if any. Subjects to which the pharmaceutical compositions of the present invention are intended to be administered include, but are not limited to, mammals, including humans and other primates, commercially relevant mammals such as cows, pigs, horses, sheep, cats, and dogs. In one embodiment, the subject is a human, or a non-human mammal, such as, but not limited to, horses, sheep, cows, pigs, dogs, cats, and mice. In one embodiment, the subject is a human.

[0143] In one embodiment, the composition is formulated with one or more pharma- ceutically acceptable excipients or carriers. In one aspect, a pharmaceutical composition for treating cancer in a subject is disclosed. The pharmaceutical composition comprises an armed SVV and a pharma- ceutically acceptable carrier. Useful pharma- ceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, and other pharma- ceutically acceptable salt solutions, such as phosphates and salts of organic acids. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating agent such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, polyalcohols such as mannitol or sorbitol, in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0144] The preparations can be used in admixture with conventional excipients, i.e., pharma- ceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration known in the art. Pharmaceutical preparations can be sterilized and, if desired, can be mixed with auxiliary substances, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic buffers, colorants, flavors and / or aromatic substances, etc. These can also be combined with other active agents, such as other analgesics, if necessary.

[0145] The composition may contain about 0.005%-2.0% preservative by total weight of the composition. Preservatives are used to prevent spoilage when exposed to environmental contaminants. Examples of preservatives useful according to the present invention include, but are not limited to, those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea, and combinations thereof. A particularly preferred preservative is a combination of about 0.5%-2.0% benzyl alcohol and 0.05%-0.5% sorbic acid.

[0146] The composition may include antioxidants and chelating agents that inhibit degradation of the compound. Preferred antioxidants for some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid in the preferred range of about 0.01% to 0.3% by weight, more preferably BHT in the range of 0.03% to 0.1% by weight, based on the total weight of the composition. Preferably, the chelating agent is present in an amount of 0.01% to 0.5% by weight, based on the total weight of the composition. Particularly preferred chelating agents include edetate (e.g., disodium edetate) and citric acid in the range of about 0.01% to 0.20% by weight, more preferably 0.02% to 0.10% by weight, based on the total weight of the composition. Chelating agents are useful for chelating metal ions in the composition that may adversely affect the shelf life of the formulation. BHT and disodium edetate are particularly preferred antioxidants and chelating agents, respectively, for some compounds, although other suitable and equivalent antioxidants and chelating agents may be substituted, as known to those skilled in the art.

[0147] The pharmaceutical compositions disclosed herein can be used in combination with additional therapeutic agents, such as antitumor agents, including but not limited to chemotherapy agents, anti-cell proliferation agents, or any combination thereof.For example, any conventional chemotherapy agent of the following non-limiting exemplary classes is included in the present invention: alkylating agents; nitrosoureas; antimetabolites; antitumor antibiotics; plant alkyloids; taxanes; hormonal agents; and various agents.In another embodiment, the pharmaceutical compositions disclosed herein can be used in combination with radiation therapy.

[0148] Administration Armed SVV can be administered by any suitable administration route known in the art.In certain embodiments, armed SVV is used in combination with additional compounds useful for treating cancer.In other embodiments, armed SVV is used in combination with (unarmed) SVV.

[0149] In one embodiment of the invention, the armed SVV and an additional compound useful for treating cancer are administered simultaneously. In another embodiment, the additional compound is administered before the armed SVV is administered. In another embodiment, the additional inhibitor is administered after administration of the armed SVV.

[0150] The administration regimen may affect what constitutes an effective amount. For example, the therapeutic formulation may be administered to the patient subject before or after a surgical intervention related to cancer, or immediately after the patient is diagnosed with cancer. In addition, several divided doses and staggered doses may be administered daily or sequentially, or the dose may be continuously infused or bolus injected. Furthermore, the dose of the therapeutic formulation may be proportionally increased or decreased according to the exigencies of the therapeutic or prophylactic situation.

[0151] Generally, the SVV or armed SVV is 10 7 From 1×10 11 The exact amount administered will depend on a variety of factors, including the age, weight, and sex of the patient, and the size and severity of the tumor being treated.

[0152] SVV or armed SVV is usually administered in a therapeutically effective dose. A therapeutically effective dose refers to an amount of virus that results in an improvement in symptoms or a prolongation of survival in a patient. The toxicity and therapeutic effect of the virus can be determined by standard procedures in cell cultures or experimental animals, e.g., LD 50 (the dose that is lethal to 50% of a population of animals or cells; for viruses, the dose is in vp / kg), and ED 50 (dose, vp / kg, therapeutically effective in 50% of a population of animals or cells), or TC 10 (therapeutic concentration or dose that allows for 50% inhibition of tumor cells, which can be related to PFU), or EC 50 (the concentration effective in 50% of a population of animals or cells, vp / cell). The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 and ED 50 or EC 50 The viral dose can be expressed as the ratio between the ED 50 or EC 50 It is preferred that the dosage be within a circulating concentration range that includes the dosage form employed and the route of administration utilized.

[0153] The SVV or armed SVV can be present in the composition in multiple doses as well as in a single dose, and may be present in at least, or at least about, 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 1×10 11 , or 1 × 10 12 pfu, approximately 1 x 10 5 ~1×1012 p.f.u., 1 × 10 6 ~1×10 10 p.f.u., or 1 × 10 7 ~1×10 10 During pfu, each includes but is not limited to:

[0154] The volume of the composition can be any volume and can be for single or multiple dose administration, including but not limited to about 0.01 mL to 100 mL, 0.1 mL to 100 mL, 1 mL to 100 mL, 10 mL to 100 mL, 0.01 mL to 10 mL, 0.1 mL to 10 mL, 1 mL to 10 mL, 0.02 mL to 20 mL, 0.05 mL to 5 mL, 0.5 mL to 50 mL, or 0.5 mL to 5 mL, respectively.

[0155] The infectivity of the SVV and / or armed SVV can be manifested, for example, by an increase in the titer or half-life of the oncolytic virus when exposed to a bodily fluid such as blood or serum. The infectivity can be increased by any amount, including, but not limited to, at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold.

[0156] Administration of the compositions of the present invention to a patient subject, preferably a mammal, more preferably a human, can be carried out using known procedures at a dosage and duration effective to treat the cancer of the subject. The effective amount of the therapeutic compound required to achieve a therapeutic effect can vary depending on the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and previous medical history of the patient being treated, and such factors as are well known in the medical art. Dosing regimens can be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily, or the dosage may be proportionally reduced depending on the exigencies of the treatment situation. A non-limiting example of an effective dose range of the therapeutic compound is about 0.01 to about 50 mg / kg body weight / day.

[0157] Armed SVV can be administered to a subject as frequently as several times a day, or can be administered once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every few months, or once a year or less. It is understood that the amount of compound administered per day can be administered, in non-limiting examples, every day, every other day, every third day, every fourth day, or every fifth day. For example, in the case of alternate day administration, a dose of 5 mg per day can be administered starting on Monday, followed by a first dose of 5 mg per day on Wednesday, followed by a second dose of 5 mg per day on Friday. The frequency of the dose is readily apparent to one skilled in the art and depends on any number of factors, including, but not limited to, the type and severity of the disease being treated, the species and age of the animal. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. A physician having ordinary skill in the art, such as a physician or veterinarian, can easily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start the dosage of the compound of the present invention used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0158] In certain embodiments, it is particularly advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to a physically separate unit suitable as a unitary dosage for the patient to be treated, each unit containing a predetermined amount of therapeutic compound calculated to produce a desired therapeutic effect in association with the required pharmaceutical vehicle.The dosage unit form of the present invention is determined by and directly depends on (a) the unique characteristics of the therapeutic compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations of the technology of compounding / formulating such therapeutic compound to treat cancer in patients.

[0159] Route of administration One of skill in the art will recognize that multiple routes of administration can be used, with certain routes providing more immediate and effective responses than others. In one embodiment, armed SVV is administered intratumorally.

[0160] Routes of administration of the disclosed compositions (including armed SVV) include inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, buccal, urethral, ​​vaginal (e.g., vaginal and perivaginal), nasal, and rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical administration. Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for intranasal or oral administration, dry powders or aerosolized formulations for inhalation, and compositions and dosage forms for intravesical administration. It should be understood that the formulations and compositions that may be useful in the present invention are not limited to the specific formulations and compositions described herein. In one embodiment, armed SVV therapy comprises a route of administration selected from the group consisting of inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ocular, intrahepatic artery, intrathoracic, intrathecal, intratumoral, intravenous, and any combination thereof.

[0161] kit The invention also includes kits that contain armed SVV, whereby the kits are used to treat cancer.

[0162] In further embodiments, a kit for treating or ameliorating cancer is provided, as described elsewhere herein, where the kit comprises: a) an armed SVV or a composition comprising armed SVV; and optionally b) an additional agent or therapy as described herein. The kit may further comprise instructions or a label for using the kit to treat or ameliorate cancer. The kit may also comprise an assay to confirm that the cancer is indeed refractory to a checkpoint inhibitor. In yet other embodiments, the invention extends to a kit assay for a given cancer, such as, but not limited to, small cell lung cancer or triple negative breast cancer, as described herein. Such a kit may comprise, for example, reagents for PCR or other nucleic acid hybridization techniques (microarrays), or reagents for immunologically-based detection techniques (e.g., ELISpot, ELISA).

[0163] Working Example The present invention will now be described with reference to the following examples, which are provided for illustrative purposes only, and the present invention should in no way be construed as being limited to these examples, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0164] Without further explanation, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. Accordingly, the following examples specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0165] Example 1: Construction of Armed Seneca Valley Virus Hales et al., 2008, described the genome sequence of SVV-001. The RNA genome of SVV-001 consists of 7280 nt, excluding the 39 poly(A) tail, with a 5'UTR of 666 nt and a shorter 3'UTR (71 nt). The deduced amino acid sequence of the open reading frame (ORF) revealed a large single ORF of 6543 bp that potentially encodes a polyprotein precursor of 2181 aa.

[0166] Poirer et al., 2012, describe the construction of pNTX-11, an SVV plasmid encoding GFP, in which the GFP cDNA is inserted between the SVV 2A and 2B coding sequences, as shown below. The method used by Poirer et al. is shown in Example 2.

[0167] The therapeutic cDNA is inserted into the SVV-001 clone GFP deletion pNTX-11, where the GFP cDNA is excised and replaced with the therapeutic gene at nt. 3508. Thus, all therapeutic gene insertions occur at nucleotide 3508 of GFP deletion pNTX-11 as shown. To facilitate cloning, a new transgene was synthesized incorporating SVV sequences from the Nhe I (nt 3199) to the Hind III (nt 4484) site, allowing for easy cloning into the GFP deletion pNTX-11 backbone. The SVV genome begins at nt. 1 of the GFP deletion pNTX-11 plasmid, and the SVV ORF ends at nt. 7281. The pGEM4Z plasmid sequence follows the SVV 3'UTR and (A)n sequences at nt. 7383-9885.

[0168] When generating armed SVV by the above procedure, a nucleic acid sequence encoding a drug useful for cancer therapy is used instead of the nucleic acid sequence of GFP. Specifically, the therapeutic cDNA is inserted into the SVV-001 clone pNTX-11 GFP, where the GFP cDNA is excised and replaced with the therapeutic gene at nt.3508. During translation of the SVV-therapeutic polyprotein, the ribosome first "skips" at the "TNPG↓P" motif of the SVV 2A protein, and then "skips" again at another "TNPG↓P" motif of the T2A protein. The two ribosome skipping events release the therapeutic payload protein flanked by one extra N-terminal proline and the C-terminal T2A cleavage product.

[0169] The parental plasmid for all armed SVV viruses described in this example is GFP-deleted pNTX-11, a 9,885 bp plasmid derived from pNTX-11, a 10,596 bp plasmid encoding GFP with the GFP cDNA deleted from nt. 3508-4218. The SVV genome of pNTX-11 is contained in the pGEM-4Z plasmid backbone (Promega). Other parental plasmids can also be used.

[0170] Plasmids carrying armed SVV and therapeutic proteins have been constructed (see SEQ ID NO: 15-18). These plasmids are: pNTX-11 CXCL9 (SEQ ID NO: 15); pNTX-11+TGFbDNRII (SEQ ID NO: 16); pNTX-11 nfsa mut 22 (SEQ ID NO: 17); and pNTX-11 Neoleukin 2-15 (SEQ ID NO: 18). Additionally, a plasmid carrying both chicken ovalbumin and Sars-Cov-2 (Covid virus) epitopes has been designed (pNTX-11 ova+covid epitopes (SEQ ID NO: 19)).

[0171] The armed SVV and protein locations in the plasmids are shown in Table 1. A detailed description of each plasmid follows. [Table 1]

[0172] Construction of armed SVV encoding anti-PDL-1 To construct an armed SVV encoding anti-PDL-1, the nucleotide sequence of an anti-PDL-1 encoding VHH nanobody (SEQ ID NO: 1) was inserted into GFP-deleted pNTX-11 using the procedures described herein. The plasmid map of the resulting plasmid, pNTX-11 VHH aPDL-1, is shown in FIG. 1. A schematic diagram of the generation of this plasmid (pNTX-11 VHH aPDL-1) is shown in FIG. 2. The resulting modified SVV expresses the anti-PDL1 protein (SEQ ID NO: 2). TIFF2024503703000002.tif54150

[0173] Referring to the resulting plasmid carrying the armed SVV construct (SEQ ID NO: 13), the SVV-anti-PD-L1 virus is nt. 1-7762. The anti-PD-L1 cDNA is nt. 3508-3885. The sequence used to generate the SVV armed virus is from SEQ ID NO: 1 of WO 2017 / 157334 A.

[0174] Construction of armed SVV encoding the IL-2 quadruple mutant Quadruple mutant IL-2 does not bind to CD25 (alpha receptor) but only to the IL-2 receptors CD122 and CD132 (beta and gamma chains). Removal of the CD25 binding domain reduces T suppressor development and decreases the potential for IL-2 toxicity via vascular leak syndrome.

[0175] To construct an armed SVV encoding the IL-2 quadruple mutant, the nucleotide sequence of the IL-2 quadruple mutant (T3A / F42A / Y45A / L72G) (SEQ ID NO: 3) was inserted into the GFP deleted pNTX-11 using the procedures described herein. The plasmid map of the resulting plasmid pNTX-11 IL2 quad mutant is shown in FIG. 3. A schematic diagram of the generation of this plasmid (pNTX-11 IL2 quad mutant) is shown in FIG. 4. The resulting modified SVV expresses the IL-2 quadruple mutant protein (SEQ ID NO: 4). TIFF2024503703000003.tif81143

[0176] Referring to the resulting plasmid (SEQ ID NO: 14) carrying the armed SVV construct, the sequence of the SVV IL-2 quad mutant protein virus is 1-7783. The IL-2 quad mutant protein cDNA sequence is 3508-3906 of the SVV virus. The IL-2 quad mutant protein used to generate the armed SVV is derived from Ast et al., 2010, EP3075745B1, and US9266938B2.

[0177] Construction of armed SVV (417bp) encoding CXCL9 CXCL9 is a chemokine thought to be involved in T cell trafficking. The encoded protein binds the CXC motif chemokine 3 and is a chemoattractant for lymphocytes but not neutrophils. CXCL9 is also known as MIG-1 (monokine induced by interferon-gamma). CXCL9 has 125 amino acids and a molecular weight of 14,019 Da.

[0178] To construct armed SVV encoding CXCL9, the nucleotide sequence of CXCL9 (SEQ ID NO: 5) was inserted into GFP-deleted pNTX-11 using the procedures described herein. The GFP gene was excised from pNTX-11 and CXCL9 cDNA was inserted into nt. 3508 of the SVV-001 clone. During translation of the SVV-CXCL9 polyprotein, the ribosome first skipped at the "TNPG↓" motif of the SVV 2A protein and then skipped again at another "TNPG↓P" motif of the T2A protein. The two ribosome skipping events release the CXCL9 protein flanked by one extra N-terminal proline and the C-terminal T2A cleavage product.

[0179] The plasmid map of the resulting plasmid, pNTX-11 CXCL9, is shown in Figure 5. A schematic diagram of the generation of this plasmid (pNTX-11 IL2 quad mutant) is shown in Figure 6. The resulting modified SVV expresses the CXCL9 protein (SEQ ID NO: 6). TIFF2024503703000004.tif59145

[0180] Referring to the resulting plasmid (SEQ ID NO: 15) carrying the armed SVV construct, the SVV CXCL9 viral sequence is 1-7759. The CXCL9 cDNA sequence is 3508-3882 of the SVV virus.

[0181] Construction of armed SVV encoding TGF-beta decoy The construction of this armed SVV used nucleic acids encoding the extracellular domains of human TβRI ECD1-128 (384bp) and TβRII ECD1-184 (486bp). The TGF-β decoy receptor is derived from the TGF-β receptor, which binds TGF-β (e.g., TGF-β1, TGF-β2, and / or TGF-β3) and lacks the amino acid sequence encoding the transmembrane domain. Expression of the TGF-β decoy reduces the immunosuppressive environment in the tumor microenvironment and enhances T cell responses.

[0182] To construct an armed SVV encoding TGF-beta decoy, the nucleotide sequence of TGF-beta decoy (SEQ ID NO: 7) was inserted into GFP-deleted pNTX-11 using the procedures described herein. The plasmid map of the resulting plasmid pNTX-11+TGFbDNRII is shown in FIG. 7. A schematic diagram of the generation of this plasmid (pNTX-11+TGFbDNRII) is shown in FIG. 8. The resulting modified SVV expresses the TGF-beta decoy protein (SEQ ID NO: 8). This construct has a myc tag added to the COOH-tail of the protein. This sequence was obtained from Addgene-plasmid-130888. The myc tag may be removed. TIFF2024503703000005.tif66144

[0183] Referring to the resulting plasmid (SEQ ID NO: 16) carrying the armed SVV construct, the sequence of the SVV TGF-beta decoy virus is 1-7984. The TGF-beta decoy cDNA sequence is 3508-4107 of the SVV virus.

[0184] Construction of armed SVV expressing NfsA Gene-directed enzyme prodrug therapy (GDEPT) is a developing strategy for cancer treatment in which exogenous genes encoding enzymes capable of converting non-toxic prodrugs into cytotoxic products are introduced into tumor cells. In principle, optimal therapeutic efficacy can be achieved by locally generating highly reactive cytotoxins in cancer cells, while systemic toxicity remains lower than with conventional chemotherapy. The nitroreductase NfsB from Escherichia coli can activate the prodrug CB1954 to a potent bifunctional alkylating agent. NfsA preferentially reduced the 2-NO2 group of CB1954, resulting in enhanced killing of bystander cells compared to Nfsb. Overall, the results suggest that NfsA may be superior to NfsB in GDEPT when used in combination with CB1954 or several other nitroaromatic prodrugs.

[0185] To construct an armed SVV encoding NfsA, the nucleotide sequence of NfsA (SEQ ID NO: 9) was inserted into GFP-deleted pNTX-11 using the procedures described herein. The plasmid map of the resulting plasmid, pNTX-11 nfsa mut 22, is shown in FIG. 9. A schematic diagram of the generation of this plasmid (pNTX-11 nfsa mut 22) is shown in FIG. 10. The resulting modified SVV expresses the Nfsa protein (SEQ ID NO: 10). TIFF2024503703000006.tif77142

[0186] Referring to the resulting plasmid (SEQ ID NO: 17), the SVV Nfsa viral sequence is 1-8140. The Nfsa cDNA sequence is 3508-4263 of the SVV virus.

[0187] The NsFA sequence used to construct this armed SVV is derived from sequence ID number: 32 of WO2012008860. Vass, S., Jarrom, D., Wilson, W. et al. E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954. Br J Cancer 100, 1903-1911 (2009). See also https: / / doi.org / 10.1038 / sj.bjc.6605094.

[0188] Construction of armed SVV expressing Neoleukin2-15 Neoleukin2-15 is an improved IL-2 variant lacking the binding sites for IL-2Rα (also known as CD25) or IL-15Rα (also known as CD215). The molecule is highly stable and binds human and mouse IL-2Rβγc with higher affinity and greater potency than the native cytokine.

[0189] To construct an armed SVV encoding Neoleukin 2-15, the nucleotide sequence of Neoleukin 2-15 (SEQ ID NO: 11) was inserted into GFP-deleted pNTX-11 using the procedures described herein. The plasmid map of the resulting plasmid, pNTX-11 sig seq Neoleukin, is shown in FIG. 11. A schematic diagram of the generation of this plasmid (pNTX-11 sig seq Neoleukin) is shown in FIG. 12. The resulting modified SVV expresses Neoleukin 2-15 (SEQ ID NO: 12). TIFF2024503703000007.tif46142

[0190] Referring to the resulting plasmid (SEQ ID NO: 18), the SVVNeoleukin2-15 viral sequence is 1-7738. The Neoleukin2-15 cDNA sequence is 3508-3861 of the SVV virus.

[0191] The sequence used to generate this construct is derived from Silva DA, Yu S, Ulge UY, et al. De novo design of potent and selective mimics of IL-2 and IL-15. Nature. 2019;565(7738):186-191.

[0192] Construction of SVV immunogen constructs We engineered a novel armed virus encoding multiple immunogenic epitopes derived from chicken ovalbumin, including SIINFEKYL and immunogenic COVID peptides: gly-pro-lys-lys-ser-thr-asn-leu. Immunogenic Covid mouse H2-D peptide derived from the spike (S) protein of SARS-CoV-2. d The -restricted CD8+ CTL epitope GPKKSTNL (aa 526-533) is described in Muraoka et al., 2020.

[0193] A map of the plasmid carrying this SVV construct (NTX-11 ova+covid epitope) is shown in Figure 13. Figure 14 is a schematic diagram of the generation of this plasmid. Referring to the resulting plasmid (SEQ ID NO: 19), the SVV ova+covid epitope viral sequence is 1-7891. The ova+covid epitope cDNA sequence is 3508-4014 of the SVV virus.

[0194] Example 2: Cloning of full-length SVV cDNA, insertion, and rescue of SVV-GFP The synthesis of the full SVV-001 genome and cloning into a bacterial plasmid has been described previously (Poirier et al.). The production of a GFP-expressing derivative of SVV-001 is described in http: / / vir.sgmjournals.org 2611, the disclosure of which is incorporated herein.

[0195] Briefly, three cDNA fragments representing the full-length SVV001 genome were amplified by triplicate PCR reactions using six sets of SVV001-specific primers (see Table 2 ). [Table 2] PCR was performed using Turbo Pfu polymerase (Stratagene). First, a fragment representing the 5' end of the SVV-001 genome was amplified with primers 59SVV-001-A and SVV0011029RT-RI, and the resulting fragment was digested with ApaI and EcoRI and gel purified. The gel-purified fragment was ligated to NdeApaT7SVV-001, an annealed oligonucleotide duplex containing the first 17 nt of SVV-001 with an engineered NdeI site at the 5' end, a T7 core promoter sequence in the middle, and an ApaI site at the 39 end, and cloned by three-way ligation into the NdeI and EcoRI sites of pGEM-3Z (Promega) to generate pNTX-03.

[0196] A fragment representing the 3' end of the viral genome was then amplified by PCR using primers SVV-0016056 and SVV-0017309NsiB. The reverse primer, SVV0017309NsiB, was used to introduce a 30 nt long poly(A) tail and an NsiI recognition sequence at the 3' end for cloning into the PstI site of the pGEM-3Z plasmid. The resulting PCR product was digested with BamHI and gel purified.

[0197] A fragment covering the interior of the viral genome was amplified with primers SVV-001911L and SVV0016157R. The resulting PCR product was digested with EcoRI and BamHI and gel purified. The two gel-purified fragments, corresponding to the middle and 3' end of the SVV genome, were cloned by three-way ligation into the EcoRI and SmaI sites of pGEM-4Z to generate pNTX-02.

[0198] To generate full-length SVV-001 cDNA, pNTX-02 was digested with EcoRI and NsiI, and the resulting 7.3-kb fragment was gel purified and cloned into the EcoRI and PstI sites of pNTX-03. pNTX-04 was further modified at both the 5′ and 3′ ends to facilitate in vitro transcription and virus rescue after RNA transfection into PER.C6 cells.

[0199] First, a SwaI restriction enzyme site was inserted immediately downstream of the poly(A) tail to liberate the 3' end of the SVV-001 cDNA from the plasmid backbone prior to in vitro transcription and to provide a blunt end for termination. The site was inserted by PCR using primer pair SVV-0016056 and SVV0013SwaRev and pNTX-04 as template. The reverse primer SVV0013SwaRev contained 58 nt representing the 3' end of the SVV-001 genome and recognition sequences for SwaI and SphI restriction enzyme sites. The resulting PCR fragment was digested with BamHI and SphI and used to replace the corresponding fragment in pNTX04 to create pNTX-06. Next, the four extra nucleotides present between the T7 promoter transcription start site in pNTX-06 and the 59 end of the SVV-001 cDNA were removed using an annealed oligonucleotide duplex approach. The double-stranded oligonucleotide was engineered to contain a KpnI recognition site, a T7 core promoter sequence, and the first 17 nt of SVV-001 with an ApaI site at the 3' end. The annealed oligonucleotide was used to replace the corresponding portion of pNTX-06 with the KpnI and ApaI sites, generating pNTX-07. Finally, the 2 bp deletion observed in the 3D polymerase coding region of pNTX-07 was repaired by replacing the BamHI and SphI fragment with the corresponding fragment amplified from SVV-001 cDNA by PCR, generating pNTX-09.

[0200] Insertion of the GFP coding sequence into the full-length SVV001 plasmid Overlap extension PCR was used to insert the GFP coding sequence fused to the F2A protein between the SVV-001 2A- and 2B-coding regions. Six primers were designed, each with overlapping sequences that amplified three PCR fragments. The first PCR fragment (PCR ab) was amplified using forward primer NI-03, which bound upstream of the 2A sequence, and reverse primer NI-04, which bound 18 bp of 2A sequence, 3 bp of 2B sequence, and 15 bp of 5'GFP sequence. The second PCR fragment (PCR cd) with the GFP coding sequence was amplified using forward primer NI-05, which bound 9 bp of 2A sequence, 3 bp of 2B sequence, and 29 bp of GFP 5' sequence, and reverse primer (NI-06), which bound 21 bp of GFP 3' sequence, and 48 bp of F2A sequence. The third PCR fragment (PCR ef) was amplified using forward primer NI-07, containing 46 bp of F2A sequence and 24 bp of 2B sequence, and reverse primer NI-08, which binds 615 bp downstream of the SVV-001 2A sequence. The PCR fragments ab and cd were fused by amplification with primers NI-03 and NI-06 to generate the PCR ad fragment. Finally, the PCR ad and ef fragments were fused by amplification with primers NI-03 and NI-08 to generate the PCR af fragment. The PCR af fragment was digested with NheI and HindIII and inserted into the corresponding sites of pNTX-09 to generate the SVV full-length plasmid, pNTX-11, containing the GFP coding sequence fused to F2A.

[0201] In vitro transcription and infectivity of RNA The infectivity of the in vitro transcribed RNA was tested by first digesting pNTX-09 with SwaI to liberate the 3' end of the SVV-001 sequence from the plasmid backbone. The linearized plasmid was subjected to in vitro transcription using T7 RNA polymerase (Promega). To evaluate the transfection of the in vitro transcribed RNA in SVV-permissive cells, PER.C6 cells were plated in 6-well tissue culture dishes. The next day, the cells were transfected with the in vitro transcribed RNA (1.5 mg) using Lipofectamine reagent (Invitrogen) according to the supplier's recommendations. CPE due to virus production was observed by 36 hours post-transfection. The transfected cells were subjected to three freeze-thaw cycles and further infected with PER.C6 cells to confirm the presence of virus in the lysates.

[0202] result The cDNA encoding the full-length genome of wild-type SVV-001 was cloned into the pGEM-4Z expression vector. To generate a recombinant reporter virus expressing GFP, a fusion protein of GFP and F2A protein was subsequently cloned into the SVV-001 2A (S2A) protein in pNTX-09, resulting in pNTX-11, as depicted in Figure 15A-C.

[0203] The F2A sequence was chosen to prevent undesired recombination events between the duplicated sequences, rather than a repeat of the S2A sequence. During translation of the SVV-GFP polypeptide, the ribosome skips at the TNPGQP of the S2A sequence, continues in frame to produce a GFP-F2A fusion protein with one additional N-terminal proline from SVV-001 2B (S2B), and skips a second time at the F2A SNPGQP sequence, continues in frame a second time to translate the remainder of the SVV-001 polyprotein. pNTX-11 was digested with SwaI and used as a template for in vitro transcription. RNA transcripts were transfected into ten 15 cm dishes of PER.C6 cells. GFP-expressing plaques were observed and purified. Plaques were purified and amplified SVV-GFP was used to infect SCLC H446 cells. Cytopathic effects (CPE) typical of wild-type SVV-001 infection were observed, as well as bright green fluorescence. Individual infected cells were sufficiently bright to be easily detected by flow cytometry at over 4 logs and by fluorescence scanning as plaques of various sizes. Proteins were extracted from H446 cells infected with SVV001 or SVV-GFP and Western blotted for GFP, the F2A epitope, and glyceraldehyde-3-phosphate dehydrogenase. Strong signals for both GFP and F2A were detected at 30 kDa, corresponding to the GFP-F2A fusion protein. The approach described above can also be applied to integrate therapeutic transgenes: the successful generation of SVV-GFP indicates that transgenes of up to 800 bp can be accommodated.

[0204] Example 3: Development of a mammalian T7 polymerase cell line (PerC.6-T7#2) Current systems for generating armed SVV constructs require the following steps: (1) constructing an armed SVV plasmid; (2) linearizing the armed SVV plasmid to define the 3' end; (3) generating RNA transcripts with authentic 5' and 3' ends by an in vitro transcription reaction using T7 polymerase; (4) transfecting the RNA into target cells; and (5) isolating armed SVV virus.

[0205] A new system for rapid generation of armed SVV constructs has been devised. The new system relies on a mammalian T7 polymerase cell line (PerC.6-T7#2). The new system requires the following steps: (1) develop a T7 polymerase plasmid optimized for expression in mammalian cells; (2) clone the T7 polymerase-optimized mammalian expression plasmid into a target cell line and select the optimal clone (PerC.6-T7#2); (2) linearize the armed SVV plasmid; (3) transfect the plasmid into T7-pol cells; and (4) isolate the armed SVV virus.

[0206] Armed SVV constructs were designed and armed SVV viruses were generated using both the current and new systems, and armed SVV viruses from both systems performed comparably.

[0207] Figures 16A and 16B show the rapid generation of armed SVV virus in cell lines using the new system (PerC.6-T7#2). Figure 16A shows the results for SVV-GFP (SVV engineered to express GFP) and Figure 16B shows the results for SVV-mCherry (SVV engineered to express mCherry). This data shows that it is possible to generate a variety of SVV constructs using the current system.

[0208] Using the current system, armed SVV constructs expressing IL-2, CXCL9, and IL-2 / 15 were generated. Figure 17 shows RT-PCR data of armed SVV generated to express IL-2, CXCL9, and IL-2 / 15. The RT-PCR data shows that armed SVV express therapeutic transgenes.

[0209] Furthermore, this new system was used to design an armed SVV construct carrying GFP. Figure 18 shows the transfection of linearized DNA in PerC-T7 pol cells. As can be seen from Figure 18, it is possible to produce armed SVV constructs using PerC6-T7 pol cells.

[0210] Example 4: Testing armed SVV with IL-2 and IL-2 / -15 Interleukin-2 (IL-2), reported in 1976 as a "T-cell growth factor," is a small 15.5 kDa monomer secreted by a variety of cell types, including CD4+ and CD8+ T cells, natural killer (NK) cells, and activated dendritic cells. IL-2 exerts pleiotropic effects on the immune system. IL-2 plays a key role in the generation, maintenance, and proliferation of CD4+ regulatory T cells, promotes the cytotoxic activity of NK and CD8+ cells, and controls homeostasis through the elimination of harmful autoreactive T cells by activation-induced cell death. IL-2 can signal through the intermediate affinity dimeric CD122 / CD132 IL-2R or the high affinity IL-2 receptor, which is composed of the trimer CD25 / CD122 / CD132.

[0211] Interleukin-15 (IL-15) is a cytokine structurally similar to interleukin-2 (IL-2). Like IL-2, IL-15 binds to and signals through a complex consisting of the IL-2 / IL-15 receptor beta chain (CD122) and the common gamma chain (CD132). IL-15 is constitutively expressed in many cell types and tissues. This cytokine induces the proliferation of natural killer cells, cells of the innate immune system whose main role is to kill virus-infected cells. IL-15 is a pluripotent cytokine and plays an important role in innate and adaptive immunity. IL-15 has been shown to enhance antitumor immunity of CD8+ T cells.

[0212] Armed SVV constructs engineered to express IL-2 and IL-2 / -15 were generated and tested for activity. The constructs were generated using the general procedures described in Example 1 above.

[0213] IL-2 bioassay cells are engineered to express luc2 in response to IL-2 signals. When IL-2 binds to the IL-2 bioassay cells, the receptor transmits an intracellular signal that results in luminescence. The bioluminescent signal is detected and quantified. In the absence of IL-2, no signaling occurs downstream of the IL-2R and no luminescent signal is generated. The IL-2 receptor (IL-2R) is composed of three subunits: IL-2Rα (CD25), IL-2Rβ (CD122) and IL-2Rγ (CD132). IL-15 transmits signals through IL-2Rβ (CD122) and IL-2Rγ (CD132). Thus, the IL-2 bioassay cells are capable of detecting cytokine binding and signaling through the IL-2 or IL-15 receptors. These bioassays are shown in Figures 19A and 19B.

[0214] These bioassays were used to evaluate the activity of the armed SVV constructs. The results of this study are shown in Figures 20A and 20B. Figure 20A shows the activity for SVV-IL2 and SVV-IL2 / 15 (Neoleukin2-15). Figure 20B shows the activity for the IL-2 standard positive control. SVV-IL2 / 15 (Neoleukin2-15) protein activity is in the supernatant and pellet. SVV-IL-2 protein activity is only in the pellet. This study demonstrated that SVV can be transfected to express IL-2 and IL-2 / 15 (Neoleukin2-15).

[0215] Example 5: Testing armed SVV with CXCL9 Chemokine (C-X-C motif) ligand 9 (CXCL9), also known as monokine induced by gamma interferon (MIG), is a small cytokine belonging to the C-X-C chemokine family. CXCL9 is one of the chemokines that play a role in inducing chemotaxis, promoting differentiation and proliferation of leukocytes, and causing tissue migration.

[0216] CXCL9 / CXCR3 receptors regulate immune cell migration, differentiation and activation. Antitumor immune responses occur through the recruitment of immune cells, such as cytotoxic lymphocytes (CTLs), natural killer (NK) cells, NKT cells and macrophages. CXCL9 mainly mediates lymphocyte infiltration into tumor sites and suppresses tumor growth.

[0217] PerC.6-T7#2 cells were transfected with the plasmid pSVV-CXCL9, and the supernatant (S) and pellet lysate (PS) were used to infect fresh PerC.6 cells. Supernatant samples were collected on days 1, 2, and 3 (D1–3) and tested using the CXCL9 ELISA.

[0218] The results of this study are shown in Figures 21A and 21B. Figures 21A and 21B show the SVV-CXCL9 Elisa data. Figure 21A shows the human CXCL standard curve. Figure 21B shows human CXCL9 level detection in the amplified SVV-CXCL9 supernatant. As with Example 3 above, this example confirms that SVV is armed to carry a therapeutic protein that is functionally expressed in cells after transfection.

[0219] Example 6: Construction of additional armed SVV constructs Additional SVV constructs were generated using the general procedure shown in Example 1. Specifically, the following SVV armed constructs were designed: armed SVV encoding anti-CTLA4 nanobody; armed SVV encoding anti-CD3 nanobody; armed SVV encoding anti-PDL1 nanobody; armed SVV encoding both anti-CTLA4+anti-PDL1 nanobody; armed SVV encoding both anti-CTLA4+anti-CD3 nanobody; armed SVV encoding both anti-CD3+anti-PDL1 nanobody; armed SVV encoding IL-2 (versions 2 and 3); armed SVV encoding TGF-beta dominant negative RII decoy-no SS v.2; armed SVV encoding TGF-beta dominant negative RII decoy-delta SerMet v.3; armed SVV encoding cytosine deaminase (FCY2+3); and armed SVV encoding Nfsa mut 22-78.

[0220] The protein and nucleic acid sequences of these inserts are shown in Table 2 below. [Table 3] TIFF2024503703000010.tif204149TIFF2024503703000011.tif208148TIFF2024503703000012.tif205148

[0221] The sequences of the plasmids harboring these constructs (SEQ ID NOs: 53-64) and maps of these plasmids are shown in Figures 22A-33B.

[0222] Exemplary embodiments Provided herein are exemplary embodiments of the disclosed technology. These embodiments are illustrative only and are not intended to limit the scope of the disclosure or the appended claims.

[0223] Further embodiment 1. A modified Seneca Valley virus, said modified Seneca Valley virus comprising a sequence of a Seneca Valley virus or an oncolytic fragment thereof inserted with a nucleic acid that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to nucleotides 1-7891 of SEQ ID NO:19.

[0224] Further embodiment 2. An armed Seneca Valley virus, wherein the armed Seneca Valley virus comprises a sequence of a Seneca Valley virus or an oncolytic fragment thereof having a nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 11 inserted therein.

[0225] Further embodiment 3. An armed Seneca Valley virus, wherein the armed Seneca Valley virus comprises a sequence of a Seneca Valley virus or an oncolytic fragment thereof having inserted therein a nucleic acid encoding an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12.

[0226] Further embodiment 4. An armed Seneca Valley virus, wherein the armed Seneca Valley virus comprises a sequence of a Seneca Valley virus or an oncolytic fragment thereof inserted with a nucleic acid encoding a protein that is at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12.

[0227] Further embodiment 5. An armed Seneca Valley virus, said armed Seneca Valley virus comprising a sequence of a Seneca Valley virus or an oncolytic fragment thereof having nucleotides 3508-3885 of SEQ ID NO: 13, nucleotides 3505-3906 of SEQ ID NO: 14, nucleotides 3508-3882 of SEQ ID NO: 15, nucleotides 3508-4107 of SEQ ID NO: 15, nucleotides 3508-4107 of SEQ ID NO: 16, nucleotides 3508-4263 of SEQ ID NO: 17, or nucleotides 3508-3861 of SEQ ID NO: 18 inserted therein.

[0228] Further embodiment 6. An armed Seneca Valley virus, said armed Seneca Valley virus comprising a sequence of a Seneca Valley virus or an oncolytic fragment thereof into which nucleotides 3508-3885 of SEQ ID NO: 13, nucleotides 3505-3906 of SEQ ID NO: 14, nucleotides 3508-3882 of SEQ ID NO: 15, nucleotides 3508-4107 of SEQ ID NO: 15, nucleotides 3508-4107 of SEQ ID NO: 16, nucleotides 3508-4263 of SEQ ID NO: 17, or nucleotides 3508-3861 of SEQ ID NO: 18 have been inserted.

[0229] Further embodiment 7. An armed Seneca Valley virus, the armed Seneca Valley virus comprising a nucleotide sequence at least 85%, at least 90%, at least 95%, or at least 99% identical to nucleotides 1-7762 of SEQ ID NO:13; a nucleotide sequence at least 85%, at least 90%, at least 95%, or at least 99% identical to nucleotides 1-7783 of SEQ ID NO:14; or a nucleotide sequence at least 85%, at least 90%, at least 95%, or at least 99% identical to nucleotides 1-7759 of SEQ ID NO:15. a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to nucleotides 1-7984 of SEQ ID NO:16; a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to nucleotides 1-8140 of SEQ ID NO:17; or a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 99% identical to nucleotides 1-7738 of SEQ ID NO:18.

[0230] Further embodiment 8. An armed Seneca Valley virus produced by inserting a nucleic acid sequence encoding a therapeutic protein into the genome of the Seneca Valley virus between the coding sequences for proteins 2A and 2B, wherein the nucleic acid encoding the therapeutic protein comprises a nucleic acid of SEQ ID NO: 1, 3, 5, 7, 9, or 11; a nucleic acid that is at least 85%, 95%, or 99% identical to the nucleic acid of SEQ ID NO: 1, 3, 5, 7, 9, or 11; a nucleic acid encoding an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12; or a nucleic acid encoding a protein that is at least 85%, at least 90%, at least 95% or 99% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12.

[0231] Further embodiment 9. The armed Seneca Valley virus of further embodiment 8, wherein said Seneca Valley virus is SVV-001.

[0232] Further embodiment 10. The armed Seneca Valley virus of further embodiments 7-9, wherein the armed Seneca Valley virus comprises nucleotides 1-7762 of SEQ ID NO:13; nucleotides 1-7783 of SEQ ID NO:14; nucleotides 1-7759 of SEQ ID NO:15; nucleotides 1-7984 of SEQ ID NO:16; nucleotides 1-8140 of SEQ ID NO:17; or nucleotides 1-7738 of SEQ ID NO:18.

[0233] Further embodiment 11. The armed Seneca Valley virus of any one of further embodiments 1-10, wherein said armed Seneca Valley virus is oncolytic, and wherein said armed Seneca Valley virus expresses a therapeutic agent or a functional fragment thereof capable of treating cancer.

[0234] Further embodiment 12. A vector comprising an armed Seneca Valley Virus according to any one of further embodiments 1 to 11.

[0235] Further embodiment 13. A plasmid comprising a nucleic acid sequence of SEQ ID NO: 13-18 or 53-64.

[0236] Further embodiment 14. A plasmid comprising a nucleic acid that is at least 85%, at least 90%, at least 95% or at least 99% identical to a nucleic acid sequence of SEQ ID NO: 13-18 or 53-64.

[0237] Further embodiment 15. A plasmid comprising a nucleic acid that is at least 85% or at least 90% identical to nucleotides 677-8050 of any one of the nucleic acid sequences of SEQ ID NOs: 13-18 or 53-64.

[0238] Further embodiment 16. A method for producing an armed Seneca Valley virus, comprising inserting into a Seneca Valley virus a nucleic acid encoding a nucleic acid of SEQ ID NO: 1, 3, 5, 7, 9, or 11, or an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12.

[0239] Further embodiment 17. A method of generating an armed Seneca Valley virus comprising inserting a nucleic acid into a Seneca Valley virus, wherein the nucleic acid is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to nucleotides 1-7762 of SEQ ID NO:13; nucleotides 1-7783 of SEQ ID NO:14; nucleotides 1-7759 of SEQ ID NO:15; nucleotides 1-7984 of SEQ ID NO:16; nucleotides 1-8140 of SEQ ID NO:17; or nucleotides 1-7738 of SEQ ID NO:18.

[0240] Further embodiment 18. The method of further embodiment 16 or 17, wherein the nucleic acid is inserted into the genome of the Seneca Valley Virus between the coding sequences for proteins 2A and 2B.

[0241] Further embodiment 19. The method of any one of further embodiments 16-18, wherein the Seneca Valley Virus is SVV-001.

[0242] Further embodiment 20. The method of any one of further embodiments 16-19, wherein the armed Seneca Valley virus is oncolytic and the armed Seneca Valley virus expresses a therapeutic agent or a functional fragment thereof capable of treating cancer.

[0243] Further embodiment 21. A method of treating cancer in a subject in need thereof, said method comprising administering to said subject an effective amount of an armed Seneca Valley virus according to any one of further embodiments 1-11.

[0244] Further embodiment 22. The method of further embodiment 21, wherein the subject is administered at least one anti-cancer therapeutic agent selected from the group consisting of a checkpoint inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a cytokine, a growth factor, a photosensitizer, a toxin, a siRNA molecule, a signal transduction modulator, an anti-cancer antibiotic, an anti-cancer antibody, an angiogenesis inhibitor, a chemotherapeutic compound, an anti-metastatic compound, an immunotherapeutic compound, CAR therapy, dendritic cell-based therapy, a cancer vaccine, an oncolytic virus, an IFN-I inhibitor, an engineered anti-cancer virus or virus derivative, and combinations thereof.

[0245] Further embodiment 23. The method of further embodiment 21 or 22, wherein at least one anti-cancer therapeutic agent is administered before, simultaneously with, or after the step of administering said armed Seneca Valley Virus.

[0246] Further embodiment 24. The method of any one of further embodiments 21-23, wherein the subject is further administered at least one additional IFN-I inhibitor selected from the group consisting of an HDAC inhibitor, a JAK / STAT inhibitor, an IFN inhibitor, an IFN antibody, an IFN-α receptor 1 antibody, an IFN-α receptor 2 antibody and a viral peptide, and combinations thereof.

[0247] Further embodiment 25. The method of further embodiment 24, wherein the HDAC inhibitor is trichostatin A.

[0248] Further embodiment 26 The method of further embodiment 24, wherein the JAK / STAT inhibitor is staurosporine.

[0249] Further embodiment 27. The method of any one of further embodiments 21-26, wherein the cancer comprises triple-negative breast cancer, small cell lung cancer, non-small cell lung cancer, non-small cell squamous cell carcinoma, adenocarcinoma, glioblastoma, skin cancer, hepatocellular carcinoma, colon cancer, cervical cancer, ovarian cancer, endometrial cancer, neuroendocrine cancer, pancreatic cancer, thyroid cancer, renal cancer, bone cancer, esophageal cancer, or soft tissue cancer.

[0250] Further embodiment 28. The method of any one of further embodiments 21 to 227, wherein the success of the cancer treatment is improved compared to treatment with a disarmed Seneca Valley Virus.

[0251] Further embodiment 29. A pharmaceutical composition for treating cancer in a subject in need thereof, said pharmaceutical composition comprising an armed Seneca Valley virus according to any one of further embodiments 1-9 and a pharma- ceutically acceptable carrier.

[0252] Further embodiment 30. The pharmaceutical composition of further embodiment 29, wherein the composition comprises a checkpoint inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a cytokine, a growth factor, a photosensitizer, a toxin, a siRNA molecule, a signal transduction modulator, an anti-cancer antibiotic, an anti-cancer antibody, an angiogenesis inhibitor, a chemotherapeutic compound, an anti-metastatic compound, an immunotherapeutic compound, CAR therapy, dendritic cell based therapy, a cancer vaccine, an oncolytic virus, an IFN-I inhibitor, an engineered anti-cancer virus or virus derivative, and combinations thereof.

[0253] Further embodiment 31. The pharmaceutical composition of further embodiment 29 or 30, wherein the cancer comprises triple negative breast cancer, small cell lung cancer, non-small cell lung cancer, non-small cell squamous cell carcinoma, adenocarcinoma, glioblastoma, skin cancer, hepatocellular carcinoma, colon cancer, cervical cancer, ovarian cancer, endometrial cancer, neuroendocrine cancer, pancreatic cancer, thyroid cancer, renal cancer, bone cancer, esophageal cancer, or soft tissue cancer.

[0254] Further embodiment 32. An armed Seneca Valley Virus according to any one of further embodiments 1 to 11 for use in the manufacture of a medicament for treating cancer.

[0255] Further embodiment 33. Use of an armed Seneca Valley Virus according to any one of further embodiments 1 to 11 for treating cancer.

[0256] Further embodiment 34. The use of further embodiment 32 or 33, wherein the cancer comprises triple negative breast cancer, small cell lung cancer, non-small cell lung cancer, non-small cell squamous cell carcinoma, adenocarcinoma, glioblastoma, skin cancer, hepatocellular carcinoma, colon cancer, cervical cancer, ovarian cancer, endometrial cancer, neuroendocrine cancer, pancreatic cancer, thyroid cancer, renal cancer, bone cancer, esophageal cancer, or soft tissue cancer.

[0257] Further embodiment 35. An armed Seneca Valley Virus according to any one of further embodiments 1-1, a vector according to further embodiment 10 or 11, or a plasmid according to any one of further embodiments 11-13, wherein the nucleic acid is RNA.

[0258] Further embodiment 36. A modified Seneca Valley Virus comprising nucleotides 1-7891 of SEQ ID NO: 19.

[0259] Further embodiment 37. A modified Seneca Valley virus, created by inserting a nucleic acid sequence into the genome of the Seneca Valley virus between the coding sequences for proteins 2A and 2B, said nucleic acid sequence comprising nucleotides 3508-4014 of SEQ ID NO: 19; or a nucleic acid sequence that is at least 85%, at least 95%, or at least 99% identical to nucleotides 3508-4014 of SEQ ID NO: 19.

[0260] Further embodiment 38. A vector or plasmid comprising nucleotides 1-7891 of SEQ ID NO: 19.

[0261] Alternative embodiment 1. A modified Seneca Valley virus, the modified Seneca Valley virus comprising a sequence of a Seneca Valley virus or an oncolytic fragment thereof inserted with a nucleic acid that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to nucleotides 1-7891 of SEQ ID NO:19.

[0262] Alternative embodiment 2. A vector or plasmid encoding the modified Seneca Valley virus of embodiment 1.

[0263] Alternative embodiment 3. A plasmid comprising SEQ ID NO:19, or nucleotides which are at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to nucleotides 1-7891 of SEQ ID NO:19.

[0264] Alternative embodiment 4: A modified Seneca Valley Virus, wherein said modified virus is oncolytic and expresses ova and Covid epitopes.

[0265] Alternative embodiment 4. A method for generating a modified Seneca Valley Virus, comprising inserting a nucleic acid into a Seneca Valley Virus, wherein the nucleic acid is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to nucleotides 1-7891 of SEQ ID NO:19.

[0266] Additional embodiment 1. An armed Seneca Valley virus, said armed Seneca Valley virus comprising a Seneca Valley virus or an oncolytic fragment thereof and a nucleic acid encoding a therapeutic protein of interest.

[0267] Additional embodiment 2. The armed Seneca Valley virus of additional embodiment 1, wherein said protein of interest comprises an interleukin, a chemokine, or a nanobody that acts as a checkpoint inhibitor.

[0268] Additional embodiment 3. The armed Seneca Valley virus of additional embodiment 2, wherein said therapeutic protein of interest comprises an anti-PD-L1 nanobody, IL-2 or a variant thereof, CXCL9, IL-15, IL-2 / IL-15 (Neoleukin2-15), TGF-β decoy or a variant thereof, NfsA or a variant thereof, an anti-CTLA4 nanobody, an anti-CD3 nanobody, an anti-CTLA-4 + anti-PDLI-1 nanobody, an anti-CLTA4 + anti-PLD-1 nanobody, or a cytosine deaminase.

[0269] Additional embodiment 4. The armed Seneca Valley virus of any one of additional embodiments 1-3, wherein said therapeutic protein of interest comprises IL-2, CXCL-9, or IL-2 / IL-15.

[0270] Additional embodiment 5. An armed Seneca Valley virus according to any one of additional embodiments 1-4, wherein the armed Seneca Valley virus comprises a Seneca Valley virus or an oncolytic fragment thereof having inserted therein a nucleic acid encoding a therapeutic protein of interest.

[0271] Additional embodiment 6. The armed Seneca Valley virus of additional embodiment 5, wherein the armed Seneca Valley virus comprises a sequence of a Seneca Valley virus or an oncolytic fragment thereof having inserted therein a nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 51.

[0272] Additional embodiment 7. The armed Seneca Valley virus of additional embodiment 5, wherein the armed Seneca Valley virus comprises a sequence of a Seneca Valley virus or an oncolytic fragment thereof having inserted therein a nucleic acid encoding an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52.

[0273] Additional embodiment 8. The armed Seneca Valley virus of additional embodiment 5, wherein the armed Seneca Valley virus comprises a sequence of a Seneca Valley virus or an oncolytic fragment thereof inserted with a nucleic acid encoding a protein that is at least 85%, at least 90%, at least 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52.

[0274] Additional embodiment 9. The armed Seneca Valley virus of additional embodiment 5, wherein the armed Seneca Valley virus comprises a sequence of the Seneca Valley virus or an oncolytic fragment thereof inserted with nucleotides 3508-3885 of SEQ ID NO: 13, nucleotides 3505-3906 of SEQ ID NO: 14, nucleotides 3508-3882 of SEQ ID NO: 15, nucleotides 3508-4107 of SEQ ID NO: 15, nucleotides 3508-4107 of SEQ ID NO: 16, nucleotides 3508-4263 of SEQ ID NO: 17, or nucleotides 3508-3861 of SEQ ID NO: 18.

[0275] Additional embodiment 10. The armed Seneca Valley virus of additional embodiment 5, wherein the armed Seneca Valley virus comprises a sequence of the Seneca Valley virus or an oncolytic fragment thereof inserted with nucleotides 3508-3885 of SEQ ID NO: 13, nucleotides 3505-3906 of SEQ ID NO: 14, nucleotides 3508-3882 of SEQ ID NO: 15, nucleotides 3508-4107 of SEQ ID NO: 15, nucleotides 3508-4107 of SEQ ID NO: 16, nucleotides 3508-4263 of SEQ ID NO: 17, or nucleotides 3508-3861 of SEQ ID NO: 18.

[0276] Additional embodiment 11. The armed Seneca Valley virus of additional embodiment 5, wherein the armed Seneca Valley virus comprises: (a) a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to nucleotides 1-7762 of SEQ ID NO:13; (b) a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to nucleotides 1-7783 of SEQ ID NO:14; (c) a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to nucleotides 1-7759 of SEQ ID NO:15. (d) a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to nucleotides 1-7984 of SEQ ID NO:16; (e) a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to nucleotides 1-8140 of SEQ ID NO:17; or (f) a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to nucleotides 1-7738 of SEQ ID NO:18.

[0277] Additional embodiment 12. The armed Seneca Valley virus of additional embodiment 5, wherein the armed Seneca Valley virus is produced by inserting a nucleic acid sequence encoding a therapeutic protein into the genome of the Seneca Valley virus between the coding sequences for proteins 2A and 2B, wherein the nucleic acid encoding the therapeutic protein is selected from the group consisting of: (a) a nucleic acid of SEQ ID NO: 1, 3, 5, 7, 9, 11, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 51; (b) a nucleic acid of SEQ ID NO: 1, 3, 5, 7, 9, 11, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 51; (c) a nucleic acid that encodes an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52; or (d) a nucleic acid that encodes a protein that is at least 85%, at least 90%, at least 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52.

[0278] Additional embodiment 13. The armed Seneca Valley virus of any one of additional embodiments 1-12, wherein the Seneca Valley virus is SVV-001.

[0279] Additional embodiment 14. The armed Seneca Valley virus of additional embodiment 13, wherein the armed Seneca Valley virus comprises: (a) nucleotides 1-7762 of SEQ ID NO:13; (b) nucleotides 1-7783 of SEQ ID NO:14; (c) nucleotides 1-7759 of SEQ ID NO:15; (d) nucleotides 1-7984 of SEQ ID NO:16; (e) nucleotides 1-8140 of SEQ ID NO:17; or (f) nucleotides 1-7738 of SEQ ID NO:18.

[0280] Additional embodiment 15. An armed Seneca Valley virus, wherein the armed Seneca Valley virus comprises a sequence of a Seneca Valley virus or an oncolytic fragment thereof having a nucleic acid inserted therein that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to nucleotides 1-7891 of SEQ ID NO: 19.

[0281] Additional embodiment 16. The armed Seneca Valley virus of any one of additional embodiments 1-15, wherein the armed Seneca Valley virus is oncolytic, and wherein the armed Seneca Valley virus expresses a therapeutic agent or a functional fragment thereof capable of treating cancer.

[0282] Additional embodiment 17. A vector comprising an armed Seneca Valley Virus according to any one of additional embodiments 1 to 16.

[0283] Additional embodiment 18. A plasmid comprising an armed Seneca Valley Virus according to any one of additional embodiments 1 to 16.

[0284] Additional embodiment 19. The plasmid of additional embodiment 18, wherein the plasmid comprises a nucleic acid sequence of SEQ ID NO: 13-18 or 53-64.

[0285] Additional embodiment 20. The plasmid of additional embodiment 18, wherein the plasmid comprises a nucleic acid that is at least 85%, at least 90%, at least 95% or at least 99% identical to a nucleic acid sequence of SEQ ID NO: 13-18 or 53-64.

[0286] Additional embodiment 21. The plasmid of additional embodiment 18, wherein the plasmid comprises a nucleic acid that is at least 85% or at least 90% identical to nucleotides 677-8050 of any one of the nucleic acid sequences of SEQ ID NOs: 13-18 or 53-64.

[0287] Further embodiment 22. A method of generating an armed Seneca Valley Virus, comprising inserting a nucleic acid encoding a therapeutic protein of interest into a Seneca Valley Virus or an oncolytic fragment thereof.

[0288] Further embodiment 23. The method of further embodiment 22, wherein the protein of interest comprises an interleukin, a chemokine, or a nanobody that acts as a checkpoint inhibitor.

[0289] Further embodiment 24. The method of further embodiment 23, wherein said therapeutic protein of interest comprises an anti-PD-L1 nanobody, IL-2, CXCL9, IL-15, IL-2 / IL-15, TGF-β decoy, or NfsA.

[0290] Additional embodiment 25. The method of any one of additional embodiments 22-24, wherein the therapeutic protein of interest comprises IL-2, CXCL-9, or IL-2 / IL-15.

[0291] Additional embodiment 26. The method of any one of additional embodiments 22-26, comprising constructing a plasmid comprising a Seneca Valley virus or an oncolytic fragment thereof and a nucleic acid encoding a therapeutic protein of interest, linearizing the plasmid to define a 3' end, generating an RNA transcript having authentic 5' and 3' ends by an in vitro transcription reaction using T7 polymerase, transfecting the RNA transcript into a target cell; and isolating the armed SVV virus.

[0292] Additional embodiment 27. A method according to any one of additional embodiments 22 to 27, comprising the steps of: cloning a T7 polymerase-optimized mammalian expression plasmid into a target cell; providing a linearized armed SVV plasmid comprising a Seneca Valley Virus or an oncolytic fragment thereof and a nucleic acid encoding a therapeutic protein of interest; transfecting the armed SVV plasmid into a T7-pol target cell; and isolating the armed Seneca Valley Virus.

[0293] Additional embodiment 28. The method of additional embodiment 27, further comprising constructing a plasmid comprising the Seneca Valley Virus or an oncolytic fragment thereof and a nucleic acid encoding a therapeutic protein of interest.

[0294] Additional embodiment 29. The method of additional embodiment 28, further comprising generating a linearized armed SVV plasmid.

[0295] Additional embodiment 30. The method of any one of additional embodiments 22 to 29, wherein the nucleic acid is inserted into the genome of the Seneca Valley Virus between the coding sequences for proteins 2A and 2B.

[0296] Additional embodiment 31. The method of any one of additional embodiments 22-30, wherein the Seneca Valley Virus is SVV-001.

[0297] Additional embodiment 32. The method of any one of additional embodiments 22-31, wherein the armed Seneca Valley virus is oncolytic, and the armed Seneca Valley virus expresses a therapeutic agent or a functional fragment thereof capable of treating cancer.

[0298] Additional embodiment 33. The method of any one of additional embodiments 22-32, the method comprising inserting a nucleic acid encoding a nucleic acid of SEQ ID NO: 1, 3, 5, 7, 9, 11, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 51, or an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52 into a Seneca Valley virus or an oncolytic fragment thereof.

[0299] Additional embodiment 34. The method of any one of additional embodiments 22-32, wherein the method comprises inserting a nucleic acid into a Seneca Valley virus or an oncolytic fragment thereof, wherein the nucleic acid is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to nucleotides 1-7762 of SEQ ID NO: 13, nucleotides 1-7783 of SEQ ID NO: 14, nucleotides 1-7759 of SEQ ID NO: 15, nucleotides 1-7984 of SEQ ID NO: 16, nucleotides 1-8140 of SEQ ID NO: 17, or nucleotides 1-7738 of SEQ ID NO: 18.

[0300] Additional embodiment 35. A method of treating cancer in a subject in need thereof, said method comprising administering to said subject an effective amount of an armed Seneca Valley virus described in any one of additional embodiments 1-16.

[0301] Additional embodiment 36. The method of additional embodiment 35, wherein the subject is administered at least one anti-cancer therapeutic agent selected from the group consisting of a checkpoint inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a cytokine, a growth factor, a photosensitizer, a toxin, a siRNA molecule, a signal transduction modulator, an anti-cancer antibiotic, an anti-cancer antibody, an angiogenesis inhibitor, a chemotherapeutic compound, an anti-metastatic compound, an immunotherapeutic compound, CAR therapy, dendritic cell-based therapy, a cancer vaccine, an oncolytic virus, an IFN-I inhibitor, an engineered anti-cancer virus or virus derivative, and combinations thereof.

[0302] Additional embodiment 37. The method of additional embodiment 35 or 36, wherein at least one anti-cancer therapeutic agent is administered before, simultaneously with, or after the step of administering the armed Seneca Valley Virus.

[0303] Additional embodiment 38. The method of any one of additional embodiments 35-37, wherein the subject is further administered at least one additional IFN-I inhibitor selected from the group consisting of an HDAC inhibitor, a JAK / STAT inhibitor, an IFN inhibitor, an IFN antibody, an IFN-α receptor 1 antibody, an IFN-α receptor 2 antibody, and a viral peptide, and combinations thereof.

[0304] Additional embodiment 39. The method of any one of additional embodiments 35-38, wherein the HDAC inhibitor is trichostatin A.

[0305] Additional embodiment 40. The method of any one of additional embodiments 35-38, wherein the JAK / STAT inhibitor is staurosporine.

[0306] Additional embodiment 41. The method of any one of additional embodiments 35-40, wherein the cancer comprises triple-negative breast cancer, small cell lung cancer, non-small cell lung cancer, non-small cell squamous cell carcinoma, adenocarcinoma, glioblastoma, skin cancer, hepatocellular carcinoma, colon cancer, cervical cancer, ovarian cancer, endometrial cancer, neuroendocrine cancer, pancreatic cancer, thyroid cancer, renal cancer, bone cancer, esophageal cancer, or soft tissue cancer.

[0307] Additional embodiment 42. The method of any one of additional embodiments 35-41, wherein the success of the cancer treatment is improved compared to treatment with a disarmed Seneca Valley Virus.

[0308] Additional embodiment 43. A pharmaceutical composition for treating cancer in a subject in need thereof, comprising an armed Seneca Valley virus according to any one of additional embodiments 1-10 and a pharma- ceutically acceptable carrier.

[0309] Additional embodiment 44. The pharmaceutical composition of additional embodiment 43, wherein the composition comprises a checkpoint inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a cytokine, a growth factor, a photosensitizer, a toxin, a siRNA molecule, a signal transduction modulator, an anti-cancer antibiotic, an anti-cancer antibody, an angiogenesis inhibitor, a chemotherapeutic compound, an anti-metastatic compound, an immunotherapeutic compound, a CAR therapy, a dendritic cell based therapy, a cancer vaccine, an oncolytic virus, an IFN-I inhibitor, an engineered anti-cancer virus or virus derivative, and combinations thereof.

[0310] Further embodiment 45. The pharmaceutical composition of further embodiment 43, wherein the cancer comprises triple negative breast cancer, small cell lung cancer, non-small cell lung cancer, non-small cell squamous cell carcinoma, adenocarcinoma, glioblastoma, skin cancer, hepatocellular carcinoma, colon cancer, cervical cancer, ovarian cancer, endometrial cancer, neuroendocrine cancer, pancreatic cancer, thyroid cancer, renal cancer, bone cancer, esophageal cancer, or soft tissue cancer.

[0311] Additional embodiment 46. An armed Seneca Valley Virus according to any one of additional embodiments 1-16 for use in the manufacture of a medicament for treating cancer.

[0312] Further embodiment 47. The use of further embodiment 46, wherein the cancer comprises triple negative breast cancer, small cell lung cancer, non-small cell lung cancer, non-small cell squamous cell carcinoma, adenocarcinoma, glioblastoma, skin cancer, hepatocellular carcinoma, colon cancer, cervical cancer, ovarian cancer, endometrial cancer, neuroendocrine cancer, pancreatic cancer, thyroid cancer, renal cancer, bone cancer, esophageal cancer, or soft tissue cancer.

[0313] Further embodiment 48. The use of an armed Seneca Valley Virus according to any one of further embodiments 1 to 10 for treating cancer.

[0314] Further embodiment 49. The use of further embodiment 48, wherein the cancer comprises triple negative breast cancer, small cell lung cancer, non-small cell lung cancer, non-small cell squamous cell carcinoma, adenocarcinoma, glioblastoma, skin cancer, hepatocellular carcinoma, colon cancer, cervical cancer, ovarian cancer, endometrial cancer, neuroendocrine cancer, pancreatic cancer, thyroid cancer, renal cancer, bone cancer, esophageal cancer, or soft tissue cancer.

[0315] Additional embodiment 50. An armed Seneca Valley Virus comprising nucleotides 1-7891 of SEQ ID NO:19.

[0316] Additional embodiment 51. An armed Seneca Valley virus produced by inserting a nucleic acid sequence into the genome of the Seneca Valley virus between the coding sequences for proteins 2A and 2B, said nucleic acid sequence comprising a nucleic acid sequence comprising nucleotides 3508-4014 of SEQ ID NO: 19, or a nucleic acid sequence that is at least 85%, at least 95%, or at least 99% identical to nucleotides 3508-4014 of SEQ ID NO: 19.

[0317] Additional embodiment 52. A vector or plasmid comprising nucleotides 1-7891 of SEQ ID NO:19.

[0318] Although the present disclosure has been described in conjunction with its preferred specific embodiments, it should be understood that the foregoing description and the following examples are intended to illustrate, but not to limit, the scope of the present disclosure. It will be understood by those skilled in the art that various changes can be made and equivalents can be substituted without departing from the scope of the present disclosure, and that other aspects, advantages, and modifications will be apparent to those skilled in the art to which the present disclosure pertains. In addition to the embodiments described herein, the present disclosure contemplates and claims inventions resulting from combinations of features of the disclosure cited herein and features of the cited prior art documents that complement the features of the present disclosure. Similarly, it will be understood that any described material, feature, or article can be used in combination with any other material, feature, or article, and such combinations are considered within the scope of the present disclosure.

[0319] The disclosures of each patent, patent application, and publication cited or described in this specification are hereby incorporated by reference in their entirety for all purposes.

Claims

1. an armed Seneca Valley virus, the armed Seneca Valley virus comprising a Seneca Valley virus or an oncolytic fragment thereof and a nucleic acid encoding a therapeutic protein of interest; Said therapeutic protein of interest comprises an anti-CTLA4 nanobody, an anti-CTLA-4+anti-PD-L1 nanobody, an anti-PD-L1 nanobody, an anti-CD3 nanobody, IL-2 or a mutant thereof, CXCL9, IL-15, IL-2 / IL-15 (Neoleukin 2-15), TGF-β decoy or a mutant thereof, NfsA or a mutant thereof, or cytosine deaminase; Armed Seneca Valley Virus.

2. 2. The armed Seneca Valley virus of claim 1, wherein the armed Seneca Valley virus comprises a Seneca Valley virus or an oncolytic fragment thereof having inserted therein a nucleic acid encoding a therapeutic protein of interest.

3. 3. The armed Seneca Valley virus according to claim 1 or 2, wherein the armed Seneca Valley virus is (a) a sequence of Seneca Valley Virus or an oncolytic fragment thereof, wherein the nucleic acid sequence of SEQ ID NO: 29, 35, 33, 3, 5, 7, 9, 11, 31, 37, 39, 41, 43, 45, 47, 49, or 51 is inserted between the coding sequences for proteins 2A and 2B; (b) a sequence of Seneca Valley Virus or an oncolytic fragment thereof, wherein a nucleic acid encoding an amino acid sequence of SEQ ID NO: 30, 36, 34, 4, 6, 8, 10, 12, 32, 38, 40, 42, 44, 46, 48, 50, or 52 is inserted between the coding sequences for proteins 2A and 2B; (c) a sequence of the Seneca Valley virus or an oncolytic fragment thereof, in which a nucleic acid encoding a protein at least 85%, at least 90%, at least 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 30, 36, 34, 4, 6, 8, 10, 12, 32, 38, 40, 42, 44, 46, 48, 50, or 52 is inserted between the coding sequences for proteins 2A and 2B; or (d) a sequence of Seneca Valley virus or an oncolytic fragment thereof, in which nucleotides 3508-3885 of SEQ ID NO:13, nucleotides 3505-3906 of SEQ ID NO:14, nucleotides 3508-3882 of SEQ ID NO:15, nucleotides 3508-4107 of SEQ ID NO:15, nucleotides 3508-4107 of SEQ ID NO:16, nucleotides 3508-4263 of SEQ ID NO:17, nucleotides 3508-3861 of SEQ ID NO:18, or nucleotides 3508-4014 of SEQ ID NO:19 are inserted between the coding sequences for proteins 2A and 2B; Including, Armed Seneca Valley Virus.

4. 4. The armed Seneca Valley virus according to claim 3, comprising: (a) a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to nucleotides 1-7762 of SEQ ID NO:13; (b) a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to nucleotides 1-7783 of SEQ ID NO:14; (c) a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to nucleotides 1-7759 of SEQ ID NO:15; (d) a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to nucleotides 1-7984 of SEQ ID NO:16; (e) a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to nucleotides 1-8140 of SEQ ID NO:17; (f) a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to nucleotides 1-7738 of SEQ ID NO:18, or (g) a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to nucleotides 1-7891 of SEQ ID NO: 19; Including, Armed Seneca Valley Virus.

5. 3. The armed Seneca Valley virus of claim 2, wherein the armed Seneca Valley virus is produced by inserting a nucleic acid sequence encoding a therapeutic protein into the genome of the Seneca Valley virus between the coding sequences for proteins 2A and 2B, the nucleic acid encoding the therapeutic protein comprising: (a) a nucleic acid of SEQ ID NO: 29, 35, 33, 3, 5, 7, 9, 11, 31, 37, 39, 41, 43, 45, 47, 49, or 51; (b) a nucleic acid that is at least 85%, 95% or 99% identical to a nucleic acid of SEQ ID NO: 29, 35, 33, 3, 5, 7, 9, 11, 31, 37, 39, 41, 43, 45, 47, 49, or 51; (c) a nucleic acid encoding the amino acid sequence of SEQ ID NO: 30, 36, 34, 4, 6, 8, 10, 12, 32, 38, 40, 42, 44, 46, 48, 50, or 52; or (d) a nucleic acid encoding a protein that is at least 85%, at least 90%, at least 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 30, 36, 34, 4, 6, 8, 10, 12, 32, 38, 40, 42, 44, 46, 48, 50, or 52; Including, Armed Seneca Valley Virus.

6. 2. The armed Seneca Valley virus of claim 1, wherein the Seneca Valley virus is SVV-001.

7. 7. The armed Seneca Valley virus according to claim 6, comprising: (a) nucleotides 1-7762 of SEQ ID NO:13; (b) nucleotides 1-7783 of SEQ ID NO:14; (c) nucleotides 1-7759 of SEQ ID NO:15; (d) nucleotides 1-7984 of SEQ ID NO:16; (e) nucleotides 1-8140 of SEQ ID NO:17; (f) nucleotides 1-7738 of SEQ ID NO:18; or (g) nucleotides 1-7891 of SEQ ID NO: 19; Including, Armed Seneca Valley Virus.

8. A vector comprising the armed Seneca Valley virus of any one of claims 1 to 7.

9. A plasmid comprising the armed Seneca Valley virus of any one of claims 1 to 7.

10. 10. The plasmid of claim 9, wherein the plasmid comprises a nucleic acid sequence of SEQ ID NO: 13-18 or 53-64, or a nucleic acid that is at least 85%, at least 90%, at least 95%, or at least 99% identical to nucleotides 677-8050 of any one of the nucleic acid sequences of SEQ ID NO: 13-18 or 53-64.

11. A method for generating an armed Seneca Valley Virus, comprising inserting a nucleic acid encoding a therapeutic protein of interest into a Seneca Valley Virus or an oncolytic fragment thereof, wherein the protein of interest comprises a nanobody, an interleukin, or a chemokine that acts as a checkpoint inhibitor.

12. 12. The method of claim 11, wherein the therapeutic protein of interest comprises an anti-PD-L1 nanobody, IL-2, CXCL9, IL-15, IL-2 / IL-15, TGF-β decoy, or NfsA.

13. 13. The method of claim 12, further comprising: (a) constructing a plasmid containing the Seneca Valley Virus or an oncolytic fragment thereof and a nucleic acid encoding a therapeutic protein of interest; Linearizing the plasmid to define the 3' end; generating RNA transcripts having authentic 5' and 3' ends by an in vitro transcription reaction using T7 polymerase; transfecting the RNA transcripts into a target cell; and Isolating the armed SVV virus; Contains, or (b) cloning the T7 polymerase-optimized mammalian expression plasmid into a target cell; providing a linearized armed SVV plasmid comprising a Seneca Valley Virus or an oncolytic fragment thereof and a nucleic acid encoding a therapeutic protein of interest; transfecting the armed SVV plasmid into T7-pol target cells; and isolating the armed Seneca Valley virus; A method comprising:

14. The method of claim 13, wherein (b) further comprises constructing a plasmid containing the Seneca Valley Virus or an oncolytic fragment thereof and a nucleic acid encoding a therapeutic protein of interest, and / or further comprises generating a linearized armed SVV plasmid.

15. 12. The method of claim 11, wherein the nucleic acid is inserted into the genome of the Seneca Valley Virus between the coding sequences for proteins 2A and 2B.

16. The method of claim 11, wherein the Seneca Valley Virus is SVV-001.

17. The method of claim 11, wherein the armed Seneca Valley virus is oncolytic, the armed Seneca Valley virus expresses a therapeutic agent or functional fragment thereof capable of treating cancer, and the armed Seneca Valley virus is defined in any one of claims 1 to 3.

18. An armed Seneca Valley virus for use in the treatment of cancer in a subject in need thereof, said use comprising administering to the subject an effective amount of an armed Seneca Valley virus described in any one of claims 1 to 7.

19. 19. The armed Seneca Valley Virus for use according to claim 18, wherein the subject is selected from the group consisting of checkpoint inhibitors, PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, cytokines, growth factors, photosensitizers, toxins, siRNA molecules, signal transduction regulators, anti-cancer antibiotics, anti-cancer antibodies, angiogenesis inhibitors, chemotherapeutic compounds, anti-metastatic compounds, immunotherapeutic compounds, CAR therapy, dendritic cell-based therapy, cancer vaccines, oncolytic viruses, IFN-I inhibitors, artificial anti-cancer viruses or virus derivatives, and combinations thereof. and / or the at least one anti-cancer therapeutic agent is administered before, simultaneously with, or after the step of administering the armed Seneca Valley virus, and / or the subject is further administered at least one additional IFN-I inhibitor selected from the group consisting of an HDAC inhibitor, a JAK / STAT inhibitor, an IFN inhibitor, an IFN antibody, an IFN-α receptor 1 antibody, an IFN-α receptor 2 antibody, and a viral peptide, and combinations thereof.

20. 20. The armed Seneca Valley Virus of claim 19, wherein the HDAC inhibitor is trichostatin A or the JAK / STAT inhibitor is staurosporine, and the cancer comprises triple-negative breast cancer, small cell lung cancer, non-small cell lung cancer, non-small cell squamous cell carcinoma, adenocarcinoma, glioblastoma, skin cancer, hepatocellular carcinoma, colon cancer, cervical cancer, ovarian cancer, endometrial cancer, neuroendocrine cancer, pancreatic cancer, thyroid cancer, renal cancer, bone cancer, esophageal cancer, or soft tissue cancer.

21. 21. The armed Seneca Valley Virus of claim 20, wherein the success of the cancer treatment is improved compared to treatment with a non-armed Seneca Valley Virus.

22. A pharmaceutical composition comprising an armed Seneca Valley virus according to any one of claims 1 to 7 and a pharma- ceutically acceptable carrier.

23. 23. The pharmaceutical composition of claim 22, wherein the composition comprises a checkpoint inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a cytokine, a growth factor, a photosensitizer, a toxin, an siRNA molecule, a signal transduction modulator, an anti-cancer antibiotic, an anti-cancer antibody, an angiogenesis inhibitor, a chemotherapeutic compound, an anti-metastatic compound, an immunotherapeutic compound, a CAR therapy, a dendritic cell based therapy, a cancer vaccine, an oncolytic virus, an IFN-I inhibitor, an engineered anti-cancer virus or virus derivative, or a combination thereof, and / or the pharmaceutical composition is for treating cancer, including triple negative breast cancer, small cell lung cancer, non-small cell lung cancer, non-small cell squamous cell carcinoma, adenocarcinoma, glioblastoma, skin cancer, hepatocellular carcinoma, colon cancer, cervical cancer, ovarian cancer, endometrial cancer, neuroendocrine cancer, pancreatic cancer, thyroid cancer, kidney cancer, bone cancer, esophageal cancer, or soft tissue cancer.

24. 8. An armed Seneca Valley Virus according to any one of claims 1 to 7 for use in the manufacture of a medicament for treating cancer.

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