Seneca valley virus mutants

Abstract

Provided are nucleic acid molecules encoding mutant Seneca Valley Virus (SVV) that evade neutralizing antibodies with a binding specificity to SVV, as well as nanoparticles containing rapamycin and SVV or mutant SVV. The mutant SVV comprise one or more mutations in one or more SVV epitopes that prevent binding of the mutant SVV to the neutralizing antibodies. The one or more mutations of the mutant SVV may be in one or more SVV epitopes. The epitopes may be inVP1, VP2, and / or VP3 subunits of SVV. Also provided are methods of producing and using the mutant SVV for treating cancer. Also provided are methods of treating a subject comprising administering to the subject the nanoparticles comprising SVV, or mutant SVV, and rapamycin. Also provided are methods of treating a subject comprising administering to the subject SVV or mutant SVV, and cyclophosphamide.

Description

SENECA VALLEY VIRUS MUTANTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001]

[0001] This application claims priority to and the benefit of United States Provisional Patent Application No. 63 / 602,053 filed November 22, 2023, the disclosure of which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing XML which has been submitted electronically as an XML formatted sequence listing and is hereby incorporated by reference in its entirety. Said XML formatted sequence listing, created on November 20, 2024, is named 115029000085_Sequence_Listing20.xml and is 46,454 bytes in size.TECHNICAL FIELD

[0003] Disclosed herein are Seneca Valley Virus (SVV) mutants and methods for treating cancer with the SVV mutants. More particularly, the disclosed inventions relate to SVV mutants that evade SVV neutralizing antibodies and to methods for treating cancer using the SVV mutants that permit oncolytic therapies with multiple administrations of the SVV mutants.BACKGROUND

[0004] Cancer is the second most common cause of death in the United States. One out of every four individuals dies from it, and more than one million new cancer diagnoses are made every year. The disease begins with the uncontrolled proliferation and growth of abnormal, transformed cells. However, the definition does not end with a description of one disease but of hundreds of different diseases. No two cancers are the same, nor are they clonal. The mutations driving cell changes and cell transformation may be similar, but they are often not identical. This conundrum adds to the complexity and heterogeneity of the pathologies that patients develop. Current cancer therapies, including chemotherapeutics and radiation, are most effective when combined with immunomodulatory agents to create and enhance the antitumor microenvironment. Many malignancies may be resistant to treatment via these traditional methods.

[0005] Oncolytic viruses show enormous potential as anti-cancer agents. The picomavirus Seneca Valley virus (SVV) is a single stranded (+) RNA virus that has been investigated as an oncolytic therapy. However, a single administration of SVV can generate neutralizing antibodies, making repeat administrations of SVV less efficacious.

[0006] Accordingly, there is a need for improved SVV and methods that allow for repeat administrations of SVV to the same patient, where the SVV evades neutralizing antibodies.SUMMARY

[0007] In addressing these needs, provided herein are nucleic acid molecules encoding mutant Seneca Valley Virus (SVV) that evade neutralizing antibodies with a binding specificity to SVV. Also provided are the mutant SVV and methods of producing and using the mutant SVV for treating cancer.

[0008] The mutant SVV may comprise one or more mutations in one or more SVV epitopes that prevent binding of the mutant SVV to neutralizing antibodies with a binding specificity to SVV. The one or more mutations of the mutant SVV may be in one or more SVV epitopes in VP1, VP2, and / or VP3 subunits of SVV. The one or more SVV epitopes may be selected from the epitopes listed in Table 1.

[0009] The mutant SVV may comprise one or more point mutations listed in any one of Tables 1, 2, and / or 3. The mutant SVV may comprise one or more mutations selected from E22A, D155A, H158M, and E226A mutations in VP1 subunit, K148S and T227V mutations in VP2 subunit, and E57A, P62S. and 1183V mutations in the VP3 subunit.

[0010] Also provided are expression vectors comprising the nucleic acid molecules encoding the mutant SVV. The expression vectors may be plasmids, viral vectors, or bacmids.

[0011] Also provided are pharmaceutical compositions comprising the mutant SVV.

[0012] Provided herein are also methods of producing the mutant SVV. The methods of producing the mutant SVV may comprise expressing the nucleic acid molecules encoding the mutant SVV.

[0013] Also provided are methods of treating subjects with cancer by administering to the subjects the mutant SVV. The methods of treating the subjects may compriseadministering the mutant SVV two, three, four, or more times to the same subject over the course of treatment. The mutant SVV may be administered intratumorally or systemically, such as intravenously.

[0014] Also provided are nanoparticles containing SVV, or mutant SVV, and rapamycin. Also provided are nanoparticles containing any one or more of VP1, VP2, VP3, and VP4 (VP1 through VP4 recombinant proteins) and rapamycin, or any one or more of mutant VP1, mutant VP2, mutant VP3, and mutant VP4 (VP1 through VP4 recombinant mutant proteins) and rapamycin.

[0015] Also provided are methods of producing and using the mutant SVV for treating cancer. Also provided are methods of treating a subject comprising administering to the subject the nanoparticles comprising SVV and rapamycin, or the nanoparticle containing mutant SVV and rapamycin. Also provided are methods of treating cancer in a subject comprising administering to the subject the nanoparticles containing any one or more of VP1, VP2. VP3, and VP4 (VP1 through VP4 recombinant proteins) and rapamycin, or any one or more of mutant VP1, mutant VP2, mutant VP3, and mutant VP4 (VP1 through VP4 recombinant mutant proteins) and rapamycin. Also provided are methods of treating cancer in a subject comprising administering to the subject SVV and cy clophosphamide, or mutant SVV and cyclophosphamide.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 A is a ribbon model of a portion of SVV capsid pentamer and shows the contact amino acid KI 71 that contacts TEM8. The sequence shown. QELNEE, is SEQ ID NO: 20. FIG. IB is a flow diagram depicting the workflow for the neutralization assay. FIG. 1C is a diagram depicting an exemplary layout of a 96-well plate for the neutralizing assay with human or murine serum.

[0017] FIG. 2 is a graph depicting change in luminescence (% LMU reduction) of different single mutant viral constructs after 24-hour incubation with PerC6 cell in the presence of the indicated sera.

[0018] FIG. 3 is a graph depicting change in luminescence (% LMU reduction) of different double and triple mutant viral constructs after 24-hour incubation with PerC6 cell in the presence of murine sera.

[0019] FIG. 4 is a graph depicting change in luminescence (% LMU reduction) of different double and triple mutant viral constructs after 24-hour incubation with PerC6 cell in the presence of human sera.

[0020] FIG. 5 is a diagram depicting polylactide nanoparticles that contain rapamycin and recombinant proteins VP1, VP2, VP3 and / or VP4.

[0021] FIG. 6A is a graph depicting changes in tumor volume (mm3) over time (days) in a syngeneic murine tumor model. FIG. 6B is a graph depicting neutralization titer (fold dilution) over time (days) in the syngeneic murine tumor model.DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

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

[0023] The present invention relates to compositions comprising the SVV mutants and methods of using the SVV mutants. The SVV mutants allow for repeat administrations of the SVV mutants to the same patient, where the SVV mutants evade neutralizing antibodies. SVV is useful in a variety of applications such as treating a cancer, reducing, or inhibiting cancer cell growth, and increasing the survival of a subject suffering from cancer. The disclosed methods in certain embodiments particularly rely upon the use of SVV mutants that have mutations in epitopes recognized by neutralizing antibodies.Definitions

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be 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, a temporal duration, and the like, the term “about” is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, or ± 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0028] The term “biological” or “biological sample” refers to a sample obtained from an organism or from components (e g., cells) of an organism. The sample may be of any biological tissue or fluid. Frequently the sample will be a “clinical sample” which is a sample derived from a patient. Such samples include, but are not limited to, bone marrow, cardiac tissue, sputum, blood, lymphatic fluid, blood cells (e.g.. white cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom. Biological samples may 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 exchangeable, inclusive, open-ended and do not exclude additional, unrecited elements or method steps. Any recitation herein of the term “comprising,” particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those 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] As used herein the term “Seneca Valley Virus” or “SVV” encompasses wild type SVV. Exemplary suitable SVV strains include SVV-001 (SEQ ID NO: 1 and SEQ ID NO: 2, see Table 4 below), NTX-0I0, and the SVV strain having ATCC Patent Deposit Number PTA-5343. As used herein, the term “mutant” specifies a derivative of a virus having a nucleic acid and / or amino acid sequence difference in respect to a template viral nucleic acid and / or amino acid sequence. For instance, an SVV mutant (also referred to herein as “mutant SVV”) can refer to an SVV that has a nucleic acid and / or amino acid sequence different with respect to the wild-type SVV nucleic acid and / or amino acid sequence of ATCC Patent Deposit Number PTA-5343. In some embodiments, the SVVmutant encompasses an SVV variant or a modified SVV (e.g. genetically engineered SVV). In one embodiment, the SVV mutant is a SVV virus modified to comprise one or more point mutations in VP1, VP2, or VP3 subunits of SVV. The SVV mutant can also express a therapeutic protein. Examples of therapeutic proteins may be found in Inti. Publ. No. WO2022 / 159508, published on July 28, 2022, the disclosure of which is incorporated herein by reference in its entirety. Exemplary' suitable therapeutic proteins include (1) IL-2; (2) Anti-PD-Ll; (3) CXCL9; (4) TGF beta decoy; (5) Nitroreductase; (6) IL2-IL15 Fusion Protein; and (7) Ovalbumin epitope. In some embodiments, the SVV mutants are also modified to be capable of recognizing different cell receptors or to be capable of being undetected by the immune system while still being able to invade, replicate and kill the cell of interest (i.e. cancer cell). In other embodiments, mutant SVV is further modified to express an agent that is useful for treating cancer. In general, an SVV can be derived from a pre-existing stock of virus that is passaged to produce more viruses. SV V can also be derived from a plasmid.

[0032] As used herein, the term “evade’' in the context of SVV mutants evading neutralizing antibodies with binding specificity to SVV refers to the actions of replicating, packaging, and infecting host cells in the presence of the neutralizing antibodies with binding specificity to wild type SVV.

[0033] As used herein, the terms “control” and “reference” can be used interchangeably to refer to a value that is used as a standard of comparison.

[0034] As used herein, by “combination therapy” is meant that a first agent is administered in conjunction with another agent. “In combination with” or “In conjunction with” refers to administration of one treatment modality in addition to another treatment modality. As such, “in combination with” refers to administration of one treatment modality before, during, or after delivery of the other treatment modality to the individual. Such combinations are considered to be part of a single treatment regimen or regime. For purposes herein, a combination therapy can include a treatment regime that includes administration of an oncolytic virus (e.g., mutant SVV) and another anti-cancer agent, each for treating the same hyperproliferative disease or conditions, such as the same tumor or cancer.

[0035] As used herein, the terms “peptide.” “polypeptide.” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and nolimitation is placed on the maximum number of amino acids that may comprise a protein or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. ‘‘Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0036] As used herein, plaque forming units (PFU) refers to a measure of number of infectious virus particles. It is determined by plaque forming assay.

[0037] As used herein, viral genomes (VG) or viral particles (VP) refers to a measure of the number of viral genomes as determined by polymerase chain reaction.

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

[0039] The term “treatment” as used within the context of the present invention is meant to include therapeutic treatment as well as prophylactic, or suppressive measures for the disease or disorder. As used herein, the term “treatment” and associated terms such as “treat” and “treating” means the reduction of the progression, severity and / or duration of a disease condition or at least one symptom thereof. The term “treatment” therefore refers to any regimen that can benefit a subject. The treatment may be in respect of an existing condition or may be prophylactic (preventative treatment). Treatment may include curative, alleviative or prophylactic effects. References herein to “therapeutic” and “prophylactic” treatments are to be considered in their broadest context. The term “therapeutic” does not necessarily imply that a subject is treated until total recovery. Similarly, “prophylactic” does not necessarily mean that the subject will not eventually develop a disease condition. Thus, for example, the term treatment includes the administration of an agent prior to or following the onset of a disease or disorder thereby preventing or removing one or more signs of the disease or disorder. As another example, administration of the agent after clinical manifestation of the disease to combat the symptoms of the disease comprises “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, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides. ESTs, chromosomes, cDNAs, rnRNAs, and rRNAs are representative examples of molecules that may be referred to as nucleic acids.

[0041] As used herein, the term “pharmaceutical composition” refers to a mixture of at least one compound useful within the invention with other chemical components, such as carriers, stabilizers, diluents, adjuvants, dispersing agents, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to intra-tumoral, intravenous, intrapleural, oral, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

[0042] The language “pharmaceutically acceptable carrier” includes a pharmaceutically acceptable salt, pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting a compound(s) (e.g. SVV and / or a checkpoint inhibitor) of the present invention within or to the subject such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each salt or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; diluent; granulating agent; lubricant; binder; disintegrating agent; wetting agent; emulsifier; coloring agent; release agent; coating agent;sweetening agent; flavoring agent; perfuming agent; preservative; antioxidant; plasticizer; gelling agent; thickener; hardener; setting agent; suspending agent; surfactant; humectant; carrier; stabilizer; and other non-toxic compatible substances employed in pharmaceutical formulations, or any combination thereof. As used herein, ‘'pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.

[0043] As used herein, the term ‘'effective amount” or “therapeutically effective amount” means the amount of the virus genome, virus particle, or infectious units generated from a vector, which is required to prevent the particular disease condition, or which reduces the severity of and / or ameliorates the disease condition or at least one symptom thereof or condition associated therewith.

[0044] A “subj ecf ’ or “patient,” as used herein, may 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.

[0045] As used herein, the terms “cold tumor” refers to a tumor that is resistant to the antitumor immune response of the subject having the tumor (“cold tumor”). As used herein, the terms “hot tumor” refers to a tumor that is susceptible to the antitumor immune response of the subject having the tumor (“hot tumor’). For example, cold tumors refer to cancers that contain a low number of infiltrating T cells and are not recognized and do not provoke a strong response by the immune system, making them difficult to treat with current immunotherapies. Cancers that are classically immunologically cold include but are not limited to glioblastomas as well as ovarian, prostate, pancreatic, and most breast cancers. In contrast, immunologically hot tumors contain high levels of infiltrating T cells and more antigens, making them more recognizable by the immune system and more likely to trigger a strong immune response. Non limiting examples of cancers considered to be immunologically hot are bladder, head and neck, kidney, melanoma, and non-small cell lung cancers.

[0046] 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 an inflexible limitation onthe scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, inclusive of each of the range endpoints, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.SVV Structure

[0047] The structure of wild type SVV has been described in a number of studies, including by Venkataraman et a!., Structure. 2008; 16(10): 1555-1561, and Wen et al., Virology Journal (2022) 19:65. The SVV virion is known as an icosahedral structure with no capsule. The SVV virion has a diameter of 27 nm. The genome for SVV contains 7280 nucleotides and includes a single open reading frame (ORF) of 6543 nt, which encodes a single polypeptide. The polypeptide is cleaved into four structural proteins and eight nonstructural proteins. The Pl polypeptide is cleaved by 3C protease into VP0, VP3, and VP1 subunits, which constitute the viral nucleocapsid, and the mature VP0 is cleaved to form VP2 and VP4 subunits. VP1, VP2, and VP3 proteins are distributed on the outer surface of the capsid, while VP4 protein is on the inner surface of the capsid. The VP1. VP2. and VP3 proteins can induce the production of neutralizing antibodies and their antigenicity is relatively strong and conservative (Wen et al.. Virology Journal (2022) 19:65).

[0048] The wild ty pe SVV genome sequence is presented as SEQ ID NOs: 1 and 2 in Table 4. A nucleic acid sequence for an exemplary plasmid comprising a nucleic acid molecule that encodes a wild type SVV and a nucleic acid molecule that encodes for the luciferase enzyme NanoLuc® (Promega Corporation, Madison, WI) is presented as SEQ ID NO: 3 in Table 4. The nucleic acid sequences for the nucleic acid molecules encoding wild type VP1, VP2, and VP3 are presented in Table 5. Amino acid sequences for the wild type VP1, VP2, and VP3 are presented in Table 6.

[0049] Disclosed are three approaches that allow for repeat administrations of SVV to the same patient, where the SVV evades neutralizing antibodies (NAbs):1) SVV mutants through serotype switching that evade previously developed neutralizing antibody (NAb) epitopes or identifying those that were not neutralized by human or murine serum containing SVV neutralizing antibodies;2) Tolerization to SVV capsid proteins to avoid developing NAbs - this includes bioassay of nanoparticles with any one or more of VP1, VP2, VP3, and VP4 plus rapamycin (VP1 through VP4 recombinant proteins), or any one or more of mutant VP1, mutant VP2, mutant VP3, and mutant VP4 plus rapamycin (VP1 through VP4 recombinant mutant proteins), and IV administration of the nanoparticles to test for tolerization against SVV and3) Cyclophosphamide (CYP), low dose, multiple administration to decrease B cells and NAbs to SVV capsid proteins.SVV Mutants

[0050] Provided herein are nucleic acid molecules encoding mutant SVV that evade neutralizing antibodies with a binding specificity to SVV. Also provided are the mutant SVV and methods of producing and using the mutant SVV for treating cancer.

[0051] The nucleic acid molecules encoding the mutant SVV comprise modified nucleic acid sequence of SEQ ID NOs: 1 or 2 encoding SVV-001 (Table 4), modified nucleic acid sequences encoding NTX-010, and modified nucleic acid sequences encoding the SVV strain having ATCC Patent Deposit Number PTA-5343. The modified nucleic acid sequence may comprise one or more nucleotide substitutions relative to the nucleic acid sequence of SEQ ID NOs: 1 or 2 encoding SVV-001 (Table 4), the nucleic acid sequences encoding NTX-010, and the nucleic acid sequences encoding the SVV strain having ATCC Patent Deposit Number PTA-5343. The one or more nucleotide substitutions may comprise nucleotide substitutions encoding any one of the mutations listed in Tables 1, 2, and 3.

[0052] The nucleic acid molecules encoding mutant SVV encode one or more mutations in one or more SVV epitopes that allow the mutant SVV to evade neutralizing antibodies with a binding specificity to SVV. The one or more mutations of the mutant SVV may be in one or more SVV epitopes in VP1, VP2, and / or VP3 subunits of SVV. The one or more SVV epitopes may be selected from the epitopes listed in Table 1.

[0053] The mutant SVV may comprise one or more point mutations listed in any one of Tables 1, 2, and / or 3. The mutant SVV may comprise one or more mutations selectedfrorn VP1 E22A, VP1 D155A, VP1 H158M, VP1 H211M, VP1 D213S, VP1 E226A, VP1 K228A, VP2 R13A, VP2 K73A. VP2 K144V, VP2 D146A, VP2 K148S. VP2 E157A, VP2 Ml 73V, VP2 T227V, VP3 R8V, VP3 E9S, VP3 E57A, VP3 R58L. VP3 E61S, VP3 P62S, VP3 D158A, VP3 N162D, VP3 I183V, VP3 Y188I, and VP3 D191A. For example, the mutant SVV can comprise mutations selected from VP1 E22A, VP1 D155A, VP1 H158M, VP1 E226A, VP2 K148S, VP2 T227V, VP3 E57A, VP3 P62S, and VP3 I183V. The mutant SVV can comprise any combination of the mutations listed herein and presented in Tables 1, 2, and 3. Exemplary combinations of mutations in mutant SVV are presented in Tables 2 and 3 and Figures 3 and 4.

[0054] In some embodiments, the mutant SVV may comprise one or more mutations selected from E22A, D155A, H158M, and E226A mutations in VP1 subunit, E157A, K148S and T227V mutations in VP2 subunit, and E57A. P62S, and I183V mutations in the VP3 subunit. For example, the mutant SVV can comprise a double mutant comprising VP2 E157A and VP3 1183V mutations, VP2 El 57 A and VP 1 E226A mutations, VP2 E157A and VP3 P62S mutations, and / or VP2 E157A and VP1 D155A mutations. The mutant SVV may comprise a triple mutant comprising VP2 E157A and VP3 I183V mutations and any one of the following mutations: VP1 E22A, VP1 D155A, VP1 H158M, VP1 H21 IM, VP1 D213S, VP1 E226A, VP1 K228A, VP2 R13A, VP2 K73A, VP2 K144V, VP2 D146A, VP2 K148S, VP2 M173V, VP2 T227V, VP3 R8V, VP3 E9S, VP3 E57A, VP3 R58L, VP3 E61S, VP3 P62S, VP3 D158A, VP3 N162D, VP3 Y188I, and VP3 D191A. The mutant SVV may comprise a triple mutant comprising VP2 E157A and VP1 E226A mutations and any one of the following mutations: VP1 E22A, VP1 D155A, VP1 H158M, VP1 H211M, VP1 D213S, VP1 K228A, VP2 R13A, VP2 K73A, VP2 K144V, VP2 D146A, VP2 K148S, VP2 M173V, VP2 T227V, VP3 R8V, VP3 E9S, VP3 E57A, VP3 R58L, VP3 E61S, VP3 P62S, VP3 D158A, VP3 N162D, VP3 I183V. VP3 Y188I, and VP3 D191A. The mutant SVV may comprise a triple mutant comprising VP2 E157A and VP3 P62S mutations and any one of the following mutations: VP1 E22A, VP1 D155A, VP1 H158M, VP1 H211M, VP1 D213S, VP1 E226A, VP1 K228A, VP2 R13A, VP2 K73A, VP2 K144V, VP2 D146A, VP2 K148S, VP2 M173V, VP2 T227V, VP3 R8V, VP3 E9S, VP3 E57A, VP3 R58L. VP3 E61S, VP3 D158A, VP3 N162D. VP3 I183V, VP3 Y188I, and VP3 D191A. The mutant SVV may comprise a triple mutant comprising VP2 E157A and VP1 D155A mutations and any one of the following mutations: VP1 E22A, VP1 H158M, VP1 H211M, VP1 D213S, VP1 E226A, VP1K228A, VP2 R13A, VP2 K73A, VP2 K144V, VP2 D146A, VP2 K148S, VP2 M173V, VP2 T227V, VP3 R8V, VP3 E9S, VP3 E57A, VP3 R58L, VP3 E61S, VP3 P62S, VP3 D158A, VP3 N162D. VP3 I183V, VP3 Y188I. and VP3 D191A. In some embodiments, the double and triple mutant SVV comprise the double or triple mutations listed in Tables 2 and 3, respectively.

[0055] Also provided are expression vectors comprising the nucleic acid molecules encoding the mutant SVV. The expression vectors may be plasmids, viral vectors, or bacmids.

[0056] Also provided are pharmaceutical compositions comprising the mutant SVV. The pharmaceutical compositions may comprise the mutant SVV and a pharmaceutically acceptable carrier.

[0057] Also provided are methods of producing the mutant SVV. The methods of producing the mutant SVV may comprise expressing the nucleic acid molecules encoding the mutant SVV. The expression may include cell-free expression of the nucleic acid molecules encoding the mutant SVV. The expression may include transfection of eukaryotic cells with the nucleic acid molecules encoding the mutant SVV. The nucleic acid molecules encoding the mutant SVV may be in expression vectors, such as plasmids, viral vectors, or bacmids. The nucleic acid molecules may be under the control of a viral or a mammalian gene promoter in the expression vectors. For example, the nucleic acid molecules may be under the control of a T7 promoter. The methods of producing the mutant SVV may also comprise a method of detecting the number of viral genomes (VG). The detecting may use polymerase chain reaction to detect the viral genomes.Tolerization to SVV capsid proteins with nanoparticles

[0058] Polylactide nanoparticles that contain rapamycin and recombinant proteins VP1, VP2, VP3 and / or VP4 can tolerize immunity to SVV proteins after uptake by dendritic cells followed by increased numbers of SVV-specific T suppressor cells. Exemplary nanoparticles are shown in a diagram in Figure 5. The development of nanoparticles for tolerizing immunity to antigens has been described (Kishimoto TK (2020) Development of ImmTOR Tolerogenic Nanoparticles for the Mitigation of Anti-drug Antibodies. Front. Immunol. 11:969; pl-14).

[0059] The nanoparticles may comprise polymeric compounds. The nanoparticles may comprise polymeric compounds PLA (poly(D,L-lactide) and PLA-PEG [poly(D,L- lactide)-block-poly(ethylene-glycol)] polymers encapsulating rapamycin. The size of the nanoparticles may be between about 100 nm and about 700 nm. For example, the size of the nanoparticles may be between about 100 nm and about 700 nm, about 150 nm and about 650 nm, about 200 nm and about 600 nm, about 250 nm and about 550 nm, about 300 nm and about 500 nmsabout 350 nm and about 550 nm. or about 450 nm and about 550 nm. The size of the nanoparticles may be about 100 nm, about 150 nm. about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, or about 700 nm.

[0060] The nanoparticles may comprise rapamycin and SVV-001. The nanoparticles may comprise rapamycin and mutant SVV. The nanoparticles may comprise rapamycin and any one or more of VP1, VP2, VP3, and VP4. The nanoparticles may comprise rapamycin and any one or more of mutant VP1, mutant VP2, mutant VP3, and mutant VP4.

[0061] The nanoparticles may be administered to a subject intravenously. The nanoparticles may be administered to a subject intratumorally.Cyclophosphamide (CYP) for reduced NAb production

[0062] Also described are methods comprising administering to the subject cyclophosphamide and any one of SVV-001, mutant SVV, VP1, VP2, VP3, VP4, mutant VP1, mutant VP2, mutant VP3, and mutant VP4 for reduced anti-SVV NAb production. The cyclophosphamide may be administered at a dose between about 10 mg / kg and about 400 mg / kg. The cyclophosphamide may be administered at a dose between about 10 mg / kg and about 400 mg / kg, between about 20 mg / kg and about 350 mg / kg. between about 30 mg / kg and about 300 mg / kg , or between about 40 mg / kg and about 250 mg / kg. The cyclophosphamide may be administered at a dose of about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg. about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, about 100 mg / kg, about 150 mg / kg, about 200 mg / kg, about 250 mg / kg, about 300 mg / kg, about 350 mg / kg, or about 400 mg / kg,

[0063] The cyclophosphamide may be administered to a subject intravenously. The cyclophosphamide may be administered to a subject intratumorally.Multiple Administrations of SVV, mutant SVV, or Nanoparticles to the Same Subject

[0064] Also provided are methods of treating subjects with cancer by administering to the subjects the mutant SVV, the nanoparticles, cyclophosphamide and SVV, or cyclophosphamide and mutant SVV. The methods of treating the subjects may comprise administering the mutant SVV, the nanoparticles, cyclophosphamide and SVV, or cyclophosphamide and mutant SVV two, three, four, or more times to the same subject. The mutant SVV. the nanoparticles, cyclophosphamide and SVV, or cyclophosphamide and mutant SVV may be administered intratumorally to the subjects. The mutant SVV, the nanoparticles, cyclophosphamide and SVV, or cyclophosphamide and mutant SVV may be administered systemically, e.g., intravenously, to the subjects. The methods of treating subjects may comprise administering the mutant SVV, the nanoparticles, cyclophosphamide and SVV, or cyclophosphamide and mutant SVV two, three, four, or more times to the same subject during the course of treatment. The mutant SVV, the nanoparticles, cyclophosphamide and SVV, or cyclophosphamide and mutant SVV allow the administered SVV or mutant SVV to evade neutralizing antibodies with a binding specificity to SVV, which permit multiple administrations of the mutant SVV to the same subject.Combination Therapy with SVV and an Immune Checkpoint Inhibitor

[0065] An exemplary' method of combination therapy for treating cancer in a subject is a method of administering an effective amount of a treatment comprising a SVV mutant, the nanoparticles, cyclophosphamide and SVV, or cyclophosphamide and mutant SVV and an immune checkpoint inhibitor to the subject. The method may be for treating a cancer in a subject who has previously been injected with SVV or SVV mutant.

[0066] The method may comprise administering the treatment comprising an effective amount of a SVV mutant, the nanoparticles, cyclophosphamide and SVV, or cyclophosphamide and mutant SVV and an effective amount of an immune checkpoint inhibitor to the subject throughout the treatment period. The method may comprise administering an effective amount of an immune checkpoint inhibitor to the subject after the treatment period.

[0067] The method may comprise administering an effective amount of SVV mutant, the nanoparticles, cyclophosphamide and SVV, or cyclophosphamide and mutant SVV intratumorally and administering an effective amount of the immune checkpoint inhibitor intravenously. The method may comprise administering the immune checkpoint inhibitor intravenously after administering SVV mutant intratumorally.

[0068] The mutant SVV may be administered at a total dose between about 2xl03viral genomes and 5xlO10viral genomes per subject per day. For example, the mutant SVV may be administered intratumorally once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, or 24 times during the treatment period. The mutant SVV may be administered intratumorally at a total dose between about 2xl03viral genomes (VG) and about 5xl010VG per subject per day. For example, the mutant SVV may be administered at a total dose between about 2xl03VG and about 5xl010VG, between about 2xl03VG and about 4xlO10VG, between about 2xl03VG and about 3xl010VG, between about 2xl03VG and about 2xlO10VG, or between about 2xl05VG and about 2xlO10VG per subject per day. The mutant SVV may be administered at a total dose of about 2xl03VG, about 2xl04VG, about 2xl05VG, about 2xl06VG, about 2xl07VG, about 2xl08VG, about 2xl09VG, about 2xlO10VG, about 3xl03VG, about 3xl04VG, about 3xlO5VG, about 3xl06VG, about 3xl07VG, about 3xl08VG, about 3xl09VG, about 3xl010VG, about 4xl03VG, about 4xl04VG, about 4xl05VG, about 4xl06VG, about 4xl07VG. about 4xl08VG. about 4xl09VG. about 4x1010VG, about 5xl03VG, about 5xl04VG, about 5xl05VG, about 5xl06VG, about 5x107VG, about 5x108VG, about 5xl09VG, or about 5xl010VG, per subject per day.Pharmaceutical compositions

[0069] Also provided herein are pharmaceutical compositions for treating a cancer in a subject in need thereof. The pharmaceutical compositions can comprise an SVV mutant, the nanoparticles, cyclophosphamide and SVV, or cyclophosphamide and mutant SVV. The pharmaceutical compositions can comprise a SVV mutant and a pharmaceutically acceptable carrier.

[0070] The pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The various components of the pharmaceutical composition may bepresent in the form of a physiologically acceptable salt, such as in combination with a phy siologically acceptable cation or anion, as is well known in the art.

[0071] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it is understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, 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 an equine, an ovine, a bovine, a porcine, a canine, a feline and a murine. In one embodiment, the subject is a human.

[0072] In one embodiment, the compositions are formulated using one or more pharmaceutically acceptable excipients or carriers. In one aspect a pharmaceutical composition is disclosed for treating a cancer in a subject. Pharmaceutically acceptable carriers, which are useful, include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may 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, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0073] The disclosed composition may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the invention included but are not limited to those selected from the group consisting of benzy l alcohol, sorbic acid, parabens, imidurea and combinations thereof. A particularly preferred preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

[0074] The composition may include an antioxidant and a chelating agent which inhibit the 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% and more preferably BHT in the range of 0.03% to 0. 1% by weight by total weight of the composition. Preferably, the chelating agent is present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Particularly preferred chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the w eight range of about 0.01% to 0.20% and more preferably in the range of 0.02% to 0. 10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition which may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are the particularly preferred antioxidant and chelating agent respectively for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.

[0075] The pharmaceutical composition disclosed herein may be used in combination with an additional therapeutic agent such as an anti-tumor agent, including but not limited to a chemotherapeutic agent, an anti-cell proliferation agent or any combination thereof. For example, any conventional chemotherapeutic agents of the following nonlimiting exemplary classes are included in the invention: alkydating agents; nitrosoureas; antimetabolites: antitumor antibiotics; plant alkyloids; taxanes; hormonal agents; and miscellaneous agents. In another aspect, the pharmaceutical composition disclosed herein may be used in combination with a radiation therapy.Administration / Dosing

[0076] The SVV mutant is typically administered at a therapeutically effective dose. A therapeutically effective dose refers to that amount of the virus that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy ofviruses can be determined by standard procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population of animals or cells; for viruses, the dose is in units of vp / kg) and the ED50 (the dose, vp / kg. therapeutically effective in 50% of the population of animals or cells), or the TC50 (the therapeutic concentration or dose allowing inhibition of 50% of tumor cells and can be related to PFU) or the ECso (the effective concentration, vp / cell, in 50% of the population of animals or cells). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50 or ECso. The dosage of viruses lies preferably within a range of circulating concentrations that include the ED50 or EC50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed- and the route of administration utilized.

[0077] The infectivity of the SVV mutant can be manifested, such as by increased titer or half-life of the oncolytic virus when exposed to a bodily fluid, such as blood or serum. 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.

[0078] Administration of the compositions of the present invention to a patient subject, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat cancer in the subject. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, w eight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.Kits

[0079] In yet another aspect, also provided herein is a kit comprising an SVV mutant.

[0080] In further embodiments a kit is provided for treating or ameliorating a cancer, as described elsewhere herein wherein the kit comprises: a) a pharmaceutical composition as described herein; and b) an additional agent or therapy as described herein. The kit can further include instructions or a label for using the kit to treat or ameliorate the cancer.EXAMPLES

[0081] The examples detail three approaches that allow for repeat administrations of SVV to the same patient, where the SVV evades neutralizing antibodies (NAbs):1) Serotype switching that evades previously developed NAbs - this includes testing serotypes containing neutralizing antibody (NAb) epitopes or identifying those that were not neutralized by human or murine serum containing SVV neutralizing antibodies;2) Tolerization to SVV capsid proteins to avoid developing NAbs - this includes bioassay of nanoparticles with VP1 through VP4 plus rapamycin (VP1 through VP4 recombinant proteins) and IV administration of the nanoparticles to test for tolerization against SVV and3) Cyclophosphamide (CYP), low dose, multiple administration to decrease B cells and NAbs to SVV capsid proteins.Example 1. Single, double, and triple mutants of SVV evade neutralizing antibodies present in human sera.

[0082] Amino acid substitutions have been targeted (forced) or selected naturally using in vitro culture with neutralizing polyclonal antisera. Targeted (in silico high probability) or selected (in vitro immune selection) substitutions have been made in the capsid of SVV-001.

[0083] Figure 1 A shows a dominant neutralizing antibody epitope identified as QELNEE (SEQ ID NO: 20). lower bottom of the diagram. The outer middle ribbons looping to the right are the center of a pentamer, and a self-assembly of five clumps each containing VP1, VP2 and VP3. Each of the five clumps has contact with the SVV receptor TEM8. One contact amino acid is in the upper labeled loop in which the lysine (k) is shown as a white k on black background. Therefore, QELNEE is in close proximity to TEM8 contact points.

[0084] Several other amino acid sequences that are recognized by anti-SVV antibodies have been published others are identified here using polyclonal antisera. Materials and MethodsMaterials

[0085] Plasmids encoding mutant SVV were first transfected in PerC6 cells and tested for viability for visual cytopathic effect (CPE) after 1-5 days. Cell virus lysate was collected and 5 pL was used to infect a previously seeded 6-well plate with PerC6 cells to check the replication viability of the virus. Viruses (plasmids) that did not replicate or had a delay were discarded. Figure IB shows purposeful alterations within epitopes and substitutions detected by natural immune selection were made into plasmids that were tested for viral fitness by transfection of PerC6 cells. The parent SVV-001 also contained Nanoluc® luciferase for quantitation of luminescence. Viral fitness was tested simultaneously with escape from polyclonal neutralizing antibodies. The first step was to test single substitutions.

[0086] Plasmids encoding the mutant SVV were generated to include one or more mutations in the viral epitopes - epitopes in the viral proteins VP1, VP2, and / or VP3. A list of mutations in the viral epitopes that were used to generate mutant SVV is presented in Tables 1, 2, and 3.Table 1. List of SVV proteins containing single point mutations.

[0087] The single mutants were designed to maximize the effect of the change in the epitope by mutating polar ammo acids to non-polar or neutral amino acids.Table 2. List of SVV proteins containing double point mutations.Table 3. List of SVV proteins containing triple point mutations.Neutralizing Assay with human or murine serum

[0088] The cell virus lysate previously obtained (1.5 pl) was used to infect previously seeded PerC6 cells in a 96 well plate as observed in the plate layout (Figure 1C).

[0089] Blank corresponds to no cells seeded. As a positive control, SVV401 (SVV- NanoLuc®) was used, since all plasmids have the Nanoluc® luciferase gene inserted. The last three samples correspond to 3 different concentrations 1E5 viral particles (VP,) 1E4 VP and 1E3 VP in each well.

[0090] The NanoLuc® luciferase activity was measured 24 hrs post-infection by pulling a small sample from each well and mixing it with the reagent in a new plate and reading it on the plate reader for luminescence.For the single mutants, treatments were the following:1. No serum2. Human serum 008 (New) dilution 1:20,0003. Murine serum 4A4 dilution 1 :200For the doubles and triple mutants, treatments are the following:1. No serum2. Human serum 008 (New) dilution 1:20,0003. Human serum 009 dilution 1:2,0004. Human serum 005 dilution 1:20,0005. Murine serum 4A4 dilution 1 :2006. Murine serum 8G11 dilution 1:200

[0091] Five JJ.1 of each diluted serum was added to each well in the appropriate plate. PBS was added in the no serum treatment.

[0092] Dilutions chosen for each serum were determined in a previous neutralization assay with SVV001 with serial dilutions of each serum. All sera are polyclonal neutralizing tested elsewhere before. Murine sera are hybridomas. Human sera was previously obtained in Neotropix clinical trial (Clinical trial number: N05-10564; Clinical Trials gov ID: NCT00314925).Calculations

[0093] To determine the % of reduction of luminescence (% LMU reduction) as a sign of neutralization of the virus by the presence of neutralizing antibodies, the following calculations were made.

[0094] First the background luminescence from the cells was subtracted from each well of each sample for all treatments:LMU = RawLMUsample- AverageLMUCells

[0095] For the treatments that w ere in the presence of serum, the percentage of reduction was calculated as follows:Results

[0096] The results from the neutralizing assay are presented in FIGs. 2-4. The results showed that for the single mutants, some of the mutants were not able to replicate and therefore were discarded. Figure 2 shows the percentage of luminescence reduction as a reflection of neutralization of the virus by the polyclonal human or murine neutralizing serum. Only the mutant SVV that had less than 60% reduction were considered for combining with other mutants to make a mutant SVV with two or more mutations in VP1, VP2 and / or VP3. Figures 3 and 4 show the percentage of luminescence reduction as a reflection of neutralization of the virus by the polyclonal human (Figure 4) or murine neutralizing serum (Figure 3). Figure 3 shows that the mutant SVV were able to replicate even better than without any serum in the presence of one of the two sera, but the other serumis still neutralizing the mutant SVV. Figure 4 shows that the mutants in one serum replicate better than without any serum. However, in the presence of the other sera there are still neutralizing antibodies that neutralize some of the mutant SVV. Mutant SVV that had an overall lower percentage of reduction in the presence of all polyclonal sera (human or murine) were considered of interest: M270 (VP2 E157A VP1 E226A VP3 I183V), M275 (VP2 E157A VP1 E226A VP1 H158M), M282 (VP2 E157A VP3 P62S VP3 E57A), M288 (VP2 E157A VP1 D155A VP1 E22A) and M292 (VP2 E157A VP1 D155A VP1 H158M).Example 2. Tolerization to SVV immunogen

[0097] Poly lactide nanoparticles that contain recombinant proteins VP1, VP2, VP3 and VP4 and rapamycin would tolerize immunity to SVV proteins after uptake by dendritic cells followed by increased numbers of SVV-specific T suppressor cells. Exemplary nanoparticles are shown in a diagram in Figure 5.

[0098] A cell-based indirect potency assay for biological effect of rapamycin has shown that rapamycin within 500nm nanoparticles is active on dendritic cells, measured bycell adherence (Jhunjhunwala et al.. Journal of Controlled Release 133 (2009) 191-197). Capsid native conformations are not expected to be required for generation of tolerance.

[0099] Development of the nanoparticles to achieve multiple administrations of SVV-001 was as follows: PLA nanoparticles 200-500nm in diameter have been constructed that contain recombinant capsid proteins as well as rapamycin. Presence of recombinant capsid proteins in the particles was verified by SDS-PAGE.Example 3. Pharmacologic inhibition of NAbs: cyclophosphamide.

[0100] In a small in vivo study in a syngeneic murine tumor model, administration of cyclophosphamide (CYP, 250mg / kg) to tumor-bearing mice prior to administration of SVV resulted in very low tumor growth (Figure 6A, bottom line) compared to SVV alone or CYP alone (Figure 6A, middle line). Serum levels of neutralizing antibodies w ere very low or non-existent (Figure 6B). Figure 6A displays tumor growth over 30 days after SVV administration, CYP+SVV recipients are shown by the bottom line which illustrates no increase in tumor volume. To distinguish that the mechanism included blockade of development of neutralizing antibodies, levels of serum SVV-neutralizing antibodies are shown in Figure 6B.

[0101] CYP will be utilized in humans to do multiple IV administrations with SVV and works in animal models with SVV. CYP also reduces T-regs which inhibit anti-tumor immune response. CYP does not destroy an anti-tumor immune response.Table 4. Nucleic acid sequence for SVV-001.Table 5. Nucleic acid sequences for the nucleic acid molecules encoding wild type VP1.VP2. and VP3 subunits of SVV.Table 6. Amino acid sequences of wild type VP1, VP2 and VP3 proteins.EMBODIMENTSEmbodiment 1. A nucleic acid molecule encoding a mutant Seneca Valley Virus (SVV) comprising one or more mutations in one or more SVV epitopes.Embodiment 2. The nucleic acid molecule of embodiment 1, wherein the one or more SVV epitopes comprise epitopes in VP1, VP2, and / or VP3.Embodiment 3. The nucleic acid molecule of embodiment 1 or 2, wherein the one or more SVV epitopes are selected from the epitopes listed in Table 1.Embodiment 4. The nucleic acid molecule of any one of embodiments 1-3, wherein the one or more SVV epitopes comprise one or more point mutations listed in any one of Tables 1, 2, and / or 3.Embodiment 5. The nucleic acid molecule of any one of embodiments 1-4, wherein the one or more mutations comprise mutations selected from E22A, D155A, H158M, and E226A mutations in VP1 subunit (SEQ ID NO: 9), K148S and T227V mutations inVP2 subunit (SEQ ID NO: 7), and E57A, P62S, and 1183V mutations in the VP3 subunit (SEQ ID NO: 8).Embodiment 6. An expression vector comprising the nucleic acid molecule of any one of embodiments 1-5.Embodiment 7. The expression vector of embodiment 6, wherein the expression vector is a plasmid, a viral vector, or a bacmid.Embodiment 8. The expression vector of embodiment 6 or 7, wherein the nucleic acid molecule is under the control of a viral or a mammalian gene promoter.Embodiment 9. The expression vector of any one of embodiments 6-8. wherein the nucleic acid molecule is under the control of a T7 promoter.Embodiment 10. A mutant Seneca Valley Virus (SVV) comprising the nucleic acid molecule of any one of embodiments 1-5.Embodiment 11. A pharmaceutical composition comprising the mutant SVV of embodiment 10.Embodiment 12. The pharmaceutical composition of embodiment 1 1, further comprising a pharmaceutically acceptable carrier.Embodiment 13. A method of producing a mutant Seneca Valley Virus (SVV) comprising expressing the nucleic acid molecule of any one of embodiments 1-5.Embodiment 14. The method of embodiment 13 comprising transfecting a mammalian cell with the nucleic acid molecule of any one of embodiments 1 -5 or with the expression vector of any one of embodiments 6-9.Embodiment 15. The method of embodiment 13 or 14, further comprising detecting the number of viral genomes (VG).Embodiment 16. The method of embodiment 15, wherein the detecting uses polymerase chain reaction.Embodiment 17. A method of treating cancer in a subject comprising administering to the subject a mutant Seneca Valley Virus (SVV) comprising the nucleic acid molecule of any one of embodiments 1-6.Embodiment 18. The method of embodiment 17, wherein the step of administering is repeated two, three, four, or more times in the same subject.Embodiment 19. The method of embodiment 17 or 18, wherein the step of administering is repeated two, three, four, or more times over the course of treating the same subj ect.Embodiment 20. The method of any one of embodiments 17-19, wherein the step of administering is repeated between about three and about six times over six months of treating the same subject.Embodiment 21. The method of any one of embodiments 17-20, wherein the mutant SVV is administered intratumorally.Embodiment 22. The method of any one of embodiments 17-21, wherein the mutant SVV evades neutralizing antibodies with a binding specificity to SVV.Embodiment 23. A nanoparticle comprising: a) a nucleic acid molecule comprising a nucleic acid sequence of SEQ ID NO: 1, or b) a nucleic acid molecule of any one of embodiments 1-5, and c) rapamycin.Embodiment 24. A method of treating cancer in a subject comprising administering to the subject the nanoparticle of embodiment 23.Embodiment 25. The method of embodiment 24. wherein the step of administering is repeated two. three, four, or more times in the same subject.Embodiment 26. The method of embodiment 24 or 25, wherein the step of administering is repeated two, three, four, or more times over the course of treating the same subj ect.Embodiment 27. The method of any one of embodiments 24-26, wherein the step of administering is repeated between about three and about six times over six months of treating the same subject.Embodiment 28. A method of treating cancer in a subject comprising administering to the subject a Seneca Valley Virus (SVV), or a mutant SVV comprising the nucleic acid molecule of any one of embodiments 1-5, and cyclophosphamide.Embodiment 29. The method of embodiment 28, wherein the step of administering is repeated two. three, four, or more times in the same subject.Embodiment 30. The method of embodiment 28 or 29, wherein the step of administering is repeated two, three, four, or more times over the course of treating the same subj ect.Embodiment 31. The method of any one of embodiments 28-30, wherein the step of administering is repeated between about three and about six times over six months of treating the same subject.

Claims

What is claimed:

1. A nucleic acid molecule encoding a mutant Seneca Valley Virus (SVV) comprising one or more mutations in one or more SVV epitopes.

2. The nucleic acid molecule of claim 1, wherein the one or more SVV epitopes comprise epitopes in VP1, VP2, and / or VP3.

3. The nucleic acid molecule of claim 1 or 2, wherein the one or more SVV epitopes are selected from the epitopes listed in Table 1.

4. The nucleic acid molecule of any one of claims 1-3, wherein the one or more SVV epitopes comprise one or more point mutations listed in any one of Tables 1, 2. and / or 3.

5. The nucleic acid molecule of any one of claims 1-4, wherein the one or more mutations comprise mutations selected from E22A, D155A, H158M, and E226A mutations in VP1 subunit (SEQ ID NO: 9), K148S and T227V mutations in VP2 subunit (SEQ ID NO: 7), and E57A, P62S, and II 83V mutations in the VP3 subunit (SEQ ID NO: 8).

6. An expression vector comprising the nucleic acid molecule of any one of claims 1-5.

7. The expression vector of claim 6, wherein the expression vector is a plasmid, a viral vector, or a bacmid.

8. The expression vector of claim 6 or 7, wherein the nucleic acid molecule is under the control of a viral or a mammalian gene promoter.

9. The expression vector of any one of claims 6-8, wherein the nucleic acid molecule is under the control of a T7 promoter.

10. A mutant Seneca Valley Virus (SVV) comprising the nucleic acid molecule of any one of claims 1-5.

11. A pharmaceutical composition comprising the mutant SVV of claim 10.

12. The pharmaceutical composition of claim 11, further comprising a pharmaceutically acceptable carrier.

13. A method of producing a mutant Seneca Valley Virus (SVV) comprising expressing the nucleic acid molecule of any one of claims 1-5.

14. The method of claim 13 comprising transfecting a mammalian cell with the nucleic acid molecule of any one of claims 1-5 or with the expression vector of any one of claims 6-9.

15. The method of claim 13 or 14. further comprising detecting the number of viral genomes (VG).1 . The method of claim 15, wherein the detecting uses polymerase chain reaction.

17. A method of treating cancer in a subject comprising administering to the subject a mutant Seneca Valley Virus (SVV) comprising the nucleic acid molecule of any one of claims 1-6.

18. The method of claim 17, wherein the step of administering is repeated two, three, four, or more times in the same subject.

19. The method of claim 17 or 18, wherein the step of administering is repeated two, three, four, or more times over the course of treating the same subject.

20. The method of any one of claims 17-19, wherein the step of administering is repeated between about three and about six times over six months of treating the same subject.

21. The method of any one of claims 17-20, wherein the mutant SVV is administered intratumorally.

22. The method of any one of claims 17-21, wherein the mutant SVV evades neutralizing antibodies with a binding specificity to SVV.

23. A nanoparticle comprising: a nucleic acid molecule comprising a nucleic acid sequence of SEQ ID NO: 1, or a nucleic acid molecule of any one of claims 1-5, and rapamycin.

24. A method of treating cancer in a subject comprising administering to the subject the nanoparticle of claim 23.

25. The method of claim 24. wherein the step of administering is repeated two. three, four, or more times in the same subject.

26. The method of claim 24 or 25, wherein the step of administering is repeated two, three, four, or more times over the course of treating the same subject.

27. The method of any one of claims 24-26, wherein the step of administering is repeated between about three and about six times over six months of treating the same subject.

28. A method of treating cancer in a subject comprising administering to the subject a Seneca Valley Virus (SVV), or a mutant SVV comprising the nucleic acid molecule of any one of claims 1-5, and cyclophosphamide.

29. The method of claim 28, wherein the step of administering is repeated two, three, four, or more times in the same subject.

30. The method of claim 28 or 29, wherein the step of administering is repeated two, three, four, or more times over the course of treating the same subject.

31. The method of any one of claims 28-30, wherein the step of administering is repeated between about three and about six times over six months of treating the same subject.

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