Attenuated oncolytic virus and uses thereof

Abstract

The present invention relates to an attenuated oncolytic virus including a recombinant polynucleotide, where the recombinant polynucleotide includes a 5'UTR, an internal ribosomal entry site (IRES), an encoding region, at least one protease cleavage site, and a 3' UTR, where the encoding region encodes an immune modulator polypeptide and uses of the attenuated oncolytic virus.

Description

Jecho Labs ref no: 240419 McAndrews ref no: 68466WO01ATTENUATED ONCOLYTIC VIRUS AND USES THEREOFREFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0001] The instant application contains a Sequence Listing, which has been submitted electronically in .xml format. The contents of the electronic sequence listing (Attenuated Oncolytic Virus.xml; Size; 32,182 bytes; and Date of Creation: September 22, 2024) are herein incorporated by reference in its entirety.BACKGROUND

[0002] Poliovirus is a non-enveloped single- stranded RNA virus that belongs to the Picomaviridae family. It is a simple virus about 25 nm in size consisting of an RNA genome and a protein capsid. Poliovirus infects human cells through a cell surface receptor CD155 (cluster of differentiation 155). After entering the host cell, the polio genome is translated by the host cell machinery into a long polypeptide chain, in a manner strictly dependent on its long 5 ’ end sequence also known as the internal ribosomal entry site (IRES). This long polypeptide is self-processed by internal proteases into various viral components for RNA genome replication, peptide cleavage, and virus packaging. Once the virus particles are assembled, they exit the host cell by lysis.

[0003] The poliovirus mainly propagates in the gastrointestinal tract. The poliovirus can replicate in other cells such as adipocyte and myocyte cells, as well as many tumor cells. The poliovirus may also attack motor neurons that causes poliomyelitis with symptoms ranging from permanent paralysis to death.

[0004] There are three serotypes of poliovirus, PV-1, PV-2, and PV-3. Vaccines have been developed against all three serotypes to prevent poliomyelitis. The vaccine, Sabin 1, which is effective against PV-1, is an attenuated PV-1 with 57 mutations in the genome. Sabin 1 can replicate efficiently in the GI tract but poorly in neuron cells.

[0005] The genomic composition of poliovirus, like all RNA viruses, is highly susceptible to mutations during replication. The genetic stability of Sabin 1 poliovirus was assessed previously. A limited number of loci with high mutation rates were identified. However, even high passage virus stocks with 6x higher mutations had acceptable monkey neurovirulence, suggesting the stability of the attenuation phenotype.Jecho Labs ref no: 240419McAndrews ref no: 68466WO01SUMMARY OF THE INVENTION

[0006] In one aspect, the disclosure provides an attenuated oncolytic virus including a recombinant polynucleotide, where the recombinant polynucleotide includes a 5’UTR, an internal ribosomal entry site (IRES), an encoding region, at least one protease cleavage site, and a 3’ UTR, where the encoding region encodes an immune modulator polypeptide. In some embodiments, the recombinant polynucleotide includes a mutation at one or more nucleic acid positions resulting in higher genetic stability without phenotypical impact relative to the attenuated oncolytic virus. In some embodiments, the mutation is selected from the group consisting of C2053T, A3896T, G3257A, T3974A, A5397G, A5154G, A610G, T2251C, T6580C, T4723A, G3960C, T5320A, G6694A, C1737T, G3032A, A3950T, C2053T, A3896T, C5755T, C642T, C4819T, T503C, G1351A, G3052A, T984C, G3617A, C3928T, and combinations thereof.

[0007] In some embodiments, the immune modulator polypeptide has at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 2 or 4. In some embodiments, the immune modulator polypeptide is a GM-CSF protein. In some embodiments,

[0008] In one aspect, the disclosure provides an attenuated oncolytic virus comprising a recombinant polynucleotide sequence having at least about 80% sequence identity to the nucleic acid sequence of SEQ ID NOs: 5, 6, or 7. In some embodiments, the recombinant polynucleotide encodes a polypeptide having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the polypeptide is a GM-CSF protein. In some embodiments, the attenuated oncolytic virus is derived from a poliovirus. In some embodiments, the attenuated oncolytic virus is capable of preferential replication in a tumor cell.

[0009] In one aspect, the disclosure provides a composition including an attenuated oncolytic virus described herein. In some embodiments, the composition is a therapeutic composition or a vaccine. In some embodiments, the composition is used in a cancer immunotherapy. In some embodiments, the composition further includes one or more one or more therapeutically acceptable carriers, therapeutically acceptable diluents, therapeutically acceptable excipients, or other therapeutic agents. In some embodiments, the therapeutically acceptable excipients are selected from the group consisting of salts, buffering agents, preservatives, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers, filmJecho Labs ref no: 240419McAndrews ref no: 68466WO01 formers, coatings, flavors, fragrances, glidants, lubricants, sorbents, suspending or dispersing agents, sweeteners, waters of hydration, and combinations thereof.

[0010] In one aspect, the disclosure provides a method of treating cancer, for inhibiting tumor cell growth, and / or for enhancing an immune response, the method comprising administering an effective amount of the attenuated oncolytic virus or a composition to a subject in need thereof. In some embodiments, the attenuated oncolytic virus targets CD 155 positive cancer cells. In some embodiments, the subject has elevated levels of CD155. In some embodiments, the subject has or is suspected to have a cancer selected from the group consisting of head and neck squamous cell carcinomas (HNSCC), melanoma, breast cancer, colorectal cancer, prostate cancer, glioblastoma, hepatocellular carcinomas, lung cancer, liver cancer, gastric cancer, brain cancer, colon cancer, cervical cancer, kidney cancer, ovarian cancer cells, pancreatic cancer cells, and esophageal cancer. In some embodiments, the attenuated oncolytic virus is administered to the subject intravenously, intratumorally, systemically, intraperitoneally, subcutaneously, or intramuscularly. In some embodiments, the method further includes administering one or more additional therapies to the subject in need thereof. In some embodiments, the one or more additional therapies are selected from the group consisting of surgery, radiation, chemotherapy, immunotherapy, hormone therapy, and a combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Various aspects of the present disclosure will now be described, by way of example only, with reference to the attached Figures, wherein:

[0012] FIG. 1 is a schematic illustrating the poliovirus polypeptide and its self-processing regions. The polyprotein is divided into a structural region and a non- structural region. VP0, VP1, VP2, VP3, and VP4 form procapsid and capsid during the assembly of new viral particles. 2AProand 3CPro. 3CDProare proteases that process the polyprotein, while 3B (VPg), 2BC, 2B, 2C (ATPase), and 3Dpol (RNA-dependent RNA polymerase) are required for viral genome replication. The viral protease 3Cproand 2Aprorecognize and cleave characteristic amino acid sequences of XXQTG and TYTGX, respectively.

[0013] FIG. 2 is a schematic illustrating exemplary attenuated poliovirus constructs.

[0014] FIGS. 3A-3C are images illustrating the transfection and virus production of various attenuated polioviruses. Vero cells were transfected using the pJL182, pJL183, or pJL184Jecho Labs ref no: 240419McAndrews ref no: 68466WO01 construct as a template to generate the recombinant polioviruses. FIG. 3A shows Vero cells using the pJL182 construct. FIG. 3B shows Vcro cells using the pJL183 construct. FIG. 3C shows Vcro cells using the pJL184 construct.

[0015] FIG. 4 is a line graph illustrating the attenuated virus, JL128B, replication in various cancer cell lines.

[0016] FIG. 5 is an image of a gel illustrating GM-CSF expression from JL182B infected cells. Lane 1: MW marker; 2: no virus infection control; 3: PVS-RIPO with 10% FBS; 4: JL182B with 10% FBS; 5: JL182B with 20% FBS; 6: Recombinant human GM-CSF.

[0017] FIG. 6 is a bar graph illustrating TF-1 cell proliferation induced by JL182B derived GM-CSF.

[0018] FIG. 7A and 7B are images illustrating PMBC differentiation induction. FIG. 7A shows PBMCs treated with 100 pg / mL recombinant GM-CSF. FIG. 7B shows PBMCs treated with infection supernatant containing 100 pg / mL expressed GM-CSF.

[0019] FIG. 8 is a bar graph illustrating PBMC proliferation induced by JL182B derived GM- CSF and recombinant IL-15 after 7 days.

[0020] FIG. 9 is a bar graph illustrating PBMC proliferation induced by JL182B derived GM- CSF and recombinant IL-15 after 10 days.

[0021] FIG. 10 is a bar graph illustrating the effect of JL182B derived GM-CSF on macrophage phagocytosis.DETAILED DESCRIPTION

[0022] I. Introduction

[0023] PVS-RIPO is a genetically modified Sabin 1 poliovirus. The entire Sabin 1 IRES was replaced by the IRES from another enterovirus human rhinovirus type 2. PVS-RIPO showed no neurocytopathic activity, while still retaining its replication ability in other tissues. PVS-RIPO is a nonpathogenic oncolytic virus with potential antineoplastic activity. Upon intratumoral administration of PVS-RIPO, the poliovirus is selectively taken up by and replicates in tumor cells expressing CD155 and eventually causes tumor cell lysis.

[0024] GM-CSF is a cytokine that stimulates hematopoietic stem cells to produce granulocytes and monocytes. The monocytes mature into macrophage and dendritic cells. GM-CSF is secreted by macrophages, T cells, NK, and a variety of other cells, usually following antigen stimulation orJecho Labs ref no: 240419McAndrews ref no: 68466WO01 activation by inflammatory cytokines. GM-CSF promotes macrophages proliferation, and this positive feedback can lead to a rapid expansion of macrophages. Macrophages clear pathogens such as cell debris, bacteria, tumor cells, and foreign particles through phagocytosis. Besides producing pro-inflammatory cytokine, macrophages also help initiate adaptive immune response by functioning as antigen presenting cells.

[0025] Mature GM-CSF is a polypeptide with 127 amino acid residues. The protein structure contains two pairs of disulfide bonds. GM-CSF is a heavily glycosylated protein with two N-linked and four O-linked sites.

[0026] Some tumor cells also produce GM-CSF that stimulates some tumor associated macrophages to be involved in invasion and immune suppression in tumor microenvironment.

[0027] Imlygic, a genetically engineered oncolytic herpes virus carrying the GM-CSF coding sequence, can produce GM-CSF in virus infected tumor cells. GM-CSF is released after cell lysis and its proinflammatory effect elevates immune response in the tumor region.

[0028] An attenuated recombinant poliovirus carrying a therapeutic polypeptide payload was attenuated to be selectively delivered to the virus propagating site. PVS-RIPO, a chimeric poliovirus (Sabin 1) with HRV type 2 IRES, lost its ability to propagate in normal neutral origin cells due to the replacement of IRES. However, PVS-RIPO can infect and reproduce in many tumor cell lines with surface receptor CD155 expression. Infected tumor cells eventually disintegrate and release viral particles to the surrounding area.

[0029] Poliovirus’ ability of self-processing after the viral polypeptide is translated inside a host cell was advantageously used. GM-CSF coding sequence, flanked with protease cleavage sites (3C or 2A), was inserted in-frame into the poliovirus genome. For the N-terminal insertion, the coding sequence for GM-CSF without its signal peptide was inserted at glutamine 4 where a 3Cprocleavage site (GAQ G) was created at the N-terminus of GM-CSF and a second 3Cprocleavage site (QEQJ.G) at the C-terminus of GM-CSF. For the internal insertion, a 2Aprocleavage site (TYJ.GA) and a 3Cprocleavage site (EQ|GF) were designed to flank the GM-CSF coding sequence. For the C-terminal insertion, only one 3Cprocleavage site (SFQ|G) was inserted at the 5’ end of GM-CSF. GM-CSF polypeptide was expressed along with poliovirus polyprotein. Full length GM-CSF was separated from the polyprotein after protease processing and eventually released into the surrounding area after cell lysis. The expressed GM-CSF was intended to be released only after cell lysis; therefore, no secretion signal peptide was engineered in the construct.Jecho Labs ref no: 240419McAndrews ref no: 68466WO01

[0030] To facilitate an understanding of the present disclosure, a number of terms and phrases arc defined below.

[0031] IL Definitions

[0032] As used herein, the term "about" refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "comprising essentially of" can mean within one or more than one standard deviation per the practice in the art. "About" or "comprising essentially of" can mean a range of up to 10% (i.e., + / - 10%). Thus, "about" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1 %, 0.05%, 0.01 %, or 0.001 % greater or less than the stated value. For example, about 5 mg can include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the instant disclosure, unless otherwise stated, the meaning of "about" or "comprising essentially of" should be assumed to be within an acceptable error range for that particular value or composition.

[0033] It will be understood that disclosure of any range of numerical values includes the endpoints of said range and all intervening values between said endpoints, as well as all sub-ranges contained within said range. By way of illustration, reference to a range of between 1 and 5 or a range from 1 to 5 is a disclosure of a range of values which is at least 1 and at most 5, as well as a range of values of, for example, at least 1.01 and at most 5, at least 1.02 and at most 5, at least 1 and at most 4.99, and so on.

[0034] The term "administering" as used herein refers to the physical introduction of an agent to a subject, such as a modified T cell disclosed herein, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,Jecho Labs ref no: 240419McAndrews ref no: 68466WO01 subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parcntcral route, e.g., orally. Other non-parenteral routes include a topical, epidermal, or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually, or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0035] The terms, "activated" and "activation", refer to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. In one embodiment, activation may also be associated with induced cytokine production and detectable effector functions. The term "activated T cells" refers to, among other things, T cells that are proliferating. Signals generated through the TCR alone may be insufficient for full activation of the T cell and one or more secondary or costimulatory signals may also be required. Thus, T cell activation comprises a primary stimulation signal through the TCR / CD3 complex and one or more secondary costimulatory signals. Costimulation may be evidenced by proliferation and / or cytokine production by T cells that have received a primary activation signal, such as stimulation through the TCR / CD3 complex.

[0036] The term "antigen" refers to a compound, composition, or substance that may stimulate the production of antibodies or a T cell response in a human or animal, including compositions (such as one that includes a tumor- specific protein) that are injected or absorbed into a human or animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens. A "target antigen" or "target antigen of interest" is an antigen that is not substantially found on the surface of other normal (desired) cells and to which a binding domain of a TCR or CAR contemplated herein, is designed to bind. A person of skill in the art would readily understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. An antigen can be endogenously expressed, i.e. expressed by genomic DNA, or can be recombinantly expressed. An antigen can be specific to a certain tissue, such as a cancer cell, or it can be broadly expressed. In addition, fragments of larger molecules can act as antigens. In one embodiment, antigens are tumor antigens.

[0037] The term "cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth result inJecho Labs ref no: 240419McAndrews ref no: 68466WO01 the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. A "cancer" or "cancer tissue" can include a tumor. Examples of cancers that can be treated by the methods of the present disclosure include, but are not limited to, cancers expressing B7H6 on the cell surface of the cancer cells. In some embodiments, the methods of the present disclosure can be used to reduce the tumor size of a tumor derived from, for example, head and neck squamous cell carcinomas (HNSCC), melanoma, breast cancer, colorectal cancer, prostate cancer, glioblastoma, hepatocellular carcinomas, lung cancer, liver cancer, gastric cancer, brain cancer, colon cancer, cervical cancer, kidney cancer, ovarian cancer cells, pancreatic cancer cells, and esophageal cancer.

[0038] Chemokines" are a type of cytokine that mediates cell chemotaxis, or directional movement. Examples of chemokines include, but are not limited to, IL-8, IL-15, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1. alpha. (MIP-1. alpha., MIP-la), MIP-Lbeta. (MIP-lb), gamma-induced protein 10 (IP- 10), and thymus and activation regulated chemokine (TARC or CCL17).

[0039] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, praline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues within a viral polypeptide can be replaced with an amino acid residue with a similar side chain. In general, two sequences are generally considered to be "substantially similar" if they contain a conservative amino acid substitution in corresponding positions. For example, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids, and / or as having "polar" or "non-polar" side chains. Substitution of one amino acid for another of the same type may be considered a conservative substitution.

[0040] The "control elements" or "regulatory sequences" present in an expression vector are those non-translated regions of the vector-origin of replication, selection cassettes, promoters,Jecho Labs ref no: 240419McAndrews ref no: 68466WO01 enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence), introns, a polyadcnylation sequence, 5' and 3' untranslated regions - which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters may be used.

[0041] The term "dosing regimen" may be used to refer to a set of one or more unit doses that are administered individually to a subject. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, a dosing regimen comprises a plurality of doses and consecutive doses are separated from one another by time periods of equal length; in some embodiments, a dosing regimen comprises a plurality of doses and consecutive doses are separated from one another by time periods of at least two different lengths. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen is periodically adjusted to achieve a desired or beneficial outcome.

[0042] The term "identity" refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Methods for the calculation of a percent identity as between two provided polypeptide sequences are known. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, may be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences may be disregarded for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, optionally taking into account the number of gaps, and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. Comparison orJecho Labs ref no: 240419McAndrews ref no: 68466WO01 alignment of sequences and determination of percent identity between two sequences may be accomplished using a mathematical algorithm, such as BLAST (basic local alignment search tool). In some embodiments, polymeric molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95- 100%).

[0043] The terms "improve," "increase," "inhibit," and "reduce" indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may comprise a measurement in certain system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and / or after) an agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may comprise a measurement in a comparable system known or expected to respond in a comparable way, in the presence of the relevant agent or treatment.

[0044] An "immune response" refers to the action of a cell of the immune system (for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including Abs, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and / or elimination from a vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

[0045] The term "in vitro" refers to events occurring in an artificial environment, e.g., in a test tube, reaction vessel, cell culture, etc., rather than within a multi-cellular organism. The term "in vitro cell" refers to any cell which is cultured ex vivo. In particular, an in vitro cell can include a T cell. The term "in vivo" refers to events that occur within a multi-cellular organism, such as a human or a non-human animal.

[0046] The term "isolated" refers to a substance that (1) has been separated from at least some components with which it was associated at an earlier time or with which the substance would otherwise be associated, and / or (2) is present in a composition that comprises a limited or defined amount or concentration of one or more known or unknown contaminants. An isolated substance, in some embodiments, may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91 %, about 92%, about 93%,Jecho Labs ref no: 240419McAndrews ref no: 68466WO01 about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% (c.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) of other non-substancc components with which the substance was associated at an earlier time, e.g., other components or contaminants with which the substance was previously or otherwise would be associated. In certain instances, a substance is isolated if it is present in a composition that comprises a limited or reduced amount or concentration of molecules of a same or similar type. For instance, in certain instances, a nucleic acid, DNA, or RNA substance is isolated if it is present in a composition that comprises a limited or reduced amount or concentration of non-substance nucleic acid, DNA, or RNA molecules. For instance, in certain instances, a polypeptide substance is isolated if it is present in a composition that comprises a limited or reduced amount or concentration of non-substance polypeptide molecules. In certain embodiments, an amount may be, e.g., an amount measured relative to the amount of a desired substance present in a composition. In certain embodiments, a limited amount may be an amount that is no more than 100% of the amount of substance in a composition, e.g., no more than 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the amount of substance in a composition (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90- 100%, or 95-100% ). In certain instances, a composition is pure or substantially pure with respect to a selected substance. In some embodiments, an isolated substance is about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). A substance is "pure" if it is substantially free of other components or of contaminants. In some embodiments, a substance may still be considered "isolated" or even "pure," after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without comprising such carriers or excipients.

[0047] The term "lymphocyte" includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell toxic) lymphocyte that represent a component of the inherent immune system. NK cells reject tumors and cells infected by viruses. It works through the process of apoptosis or programmed cell death. They were termed "natural killers" because they do not require activation in order to kill cells. T cells play a role in cell-mediated-immunity (no antibody involvement). Its T cell receptors (TCR) differentiate themselves from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the T cell'sJecho Labs ref no: 240419McAndrews ref no: 68466WO01 maturation. There are six types of T cells, namely: Helper T cells (e.g., CD4+ cells), Cytotoxic T cells (also known as TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T cells or killer T cell), Memory T cells ((i) stem memory TSCM cells, like naive cells, are CD45Ro-' CCR7+, CD45RA+, CD62L+(L-selectin), CD27+, CD28+ and IL-7R alpha+, but they also express large amounts of CD95, IL-2RB, CXCR3, and LFA-1, and show numerous functional attributes distinctive of memory cells); (ii) central memory T.sub.CM cells express L-selectin and the CCR7, they secrete IL-2, but not IFN-gamma or IL-4, and (iii) effector memory TEM cells, however, do not express L-selectin or CCR7 but produce effector cytokines like IFN-gamma and IL-4), Regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells), Natural Killer T cells (NKT) and Gamma Delta T cells. B-cells, on the other hand, play a role in humoral immunity (with antibody involvement). It makes antibodies and antigens and performs the role of antigen-presenting cells (APCs) and turns into memory B-cells after activation by antigen interaction. In mammals, immature B-cells are formed in the bone marrow, where its name is derived from.

[0048] The term "nucleic acid" refers to any polymeric chain of nucleotides. A nucleic acid may be DNA, RNA, or a combination thereof. In some embodiments, a nucleic acid comprises one or more natural nucleic acid residues. In some embodiments, a nucleic acid comprises one or more nucleic acid analogs. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60,65, 70, 75,80, 85,90,95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long (e.g., 20 to 100, 20 to 500, 20 to 1000, 20 to 2000, or 20 to 5000 or more residues). In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide.

[0049] The term "operably linked" refers to a juxtaposition where the components described are in a relationship permitting them to function in their intended manner. For example, a control element "operably linked" to a functional element is associated in such a way that expressionJecho Labs ref no: 240419McAndrews ref no: 68466WO01 and / or activity of the functional element is achieved under conditions compatible with the control element.

[0050] 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 contains at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's 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.

[0051] In other embodiments, a vector for use in practicing the embodiments described herein including, but not limited to expression vectors and viral vectors, will include exogenous, endogenous, or heterologous sequences such as promoters and / or enhancers. An "endogenous" control sequence is one which is naturally linked with a given gene in the genome. An "exogenous" control sequence is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter. A "heterologous" sequence is an exogenous sequence that may be from a different protein of the same species or a different species than the protein or cell being genetically manipulated.

[0052] The term "promoter" as used herein refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds. An RNA polymerase initiates and transcribes polynucleotides operably linked to the promoter. In some embodiments, promoters operative in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and / or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide.

[0053] The term "enhancer" refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances may function independent of theirJecho Labs ref no: 240419McAndrews ref no: 68466WO01 orientation relative to another control sequence. An enhancer may function cooperatively or additively with promoters and / or other enhancer elements. The term "promotcr / cnhanccr" refers to a segment of DNA which contains sequences capable of providing both promoter and enhancer functions.

[0054] The term "pharmaceutically acceptable" or “therapeutically acceptable” refers to a molecule or composition that, when administered to a recipient, is not deleterious to the recipient thereof, or that any deleterious effect is outweighed by a benefit to the recipient thereof. With respect to a carrier, diluent, or excipient used to formulate a composition as disclosed herein, a pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof, or any deleterious effect must be outweighed by a benefit to the recipient. The term "pharmaceutically acceptable carrier" means a pharmaceutically- acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one portion of the body to another (e.g., from one organ to another). Each carrier present in a pharmaceutical composition must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient, or any deleterious effect must be outweighed by a benefit to the recipient. Some examples of materials which 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; pH buffered solutions; polyesters, polycarbonates and / or poly anhydrides; and other nontoxic compatible substances employed in pharmaceutical formulations.

[0055] The term "pharmaceutical composition" or “therapeutical composition” refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significantJecho Labs ref no: 240419McAndrews ref no: 68466WO01 probability of achieving a predetermined therapeutic effect when administered to a relevant subject or population. In some embodiments, a pharmaceutical composition may be formulated for administration in solid or liquid form, comprising, without limitation, a form adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosa surfaces.

[0056] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dosage" of a therapeutic agent, e.g., engineered CAR-T cells, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0057] The terms "transduction" and "transduced" refer to the process whereby foreign DNA is introduced into a cell via viral vector (see Jones et al., "Genetics: principles and analysis," Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, a RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adenovirus associated vector, a lentiviral vector, or any combination thereof.

[0058] Treatment" or "treating" of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down, or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease. In one embodiment, "treatment" or "treating"Jecho Labs ref no: 240419McAndrews ref no: 68466WO01 includes a partial remission. Tn another embodiment, "treatment" or "treating" includes a complete remission. In some embodiments, treatment may be of a subject who docs not exhibit signs of the relevant disease, disorder and / or condition and / or of a subject who exhibits only early signs of the disease, disorder, and / or condition. In some embodiments, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with an increased risk of development of the relevant disease, disorder, and / or condition.

[0059] The term "vector" or "lentiviral vector" refers to a recipient nucleic acid molecule modified to comprise or incorporate a provided nucleic acid sequence. One type of vector is a "plasmid," which refers to a circular double stranded DNA molecule into which additional DNA may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors comprise sequences that direct expression of inserted genes to which they are operatively linked. Such vectors may be referred to herein as "expression vectors." Standard techniques may be used for the engineering of vectors, e.g., as found in Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference.

[0060] The term "sequence" refers to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular, or branched, and can be either single-stranded or double stranded. The term "donor sequence" refers to a nucleotide sequence that is inserted into a genome. A donor sequence can be of any length, for example between 2 and 10,000 nucleotides in length (or any integer value therebetween or thereabove), preferably between about 100 and 1,000 nucleotides in length (or any integer therebetween), more preferably between about 200 and 500 nucleotides in length.Jecho Labs ref no: 240419McAndrews ref no: 68466WO01

[0061] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods arc described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps.

[0062] As a general matter, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.

[0063] III. Attenuated Virus

[0064] In one aspect, the present disclosure provides an attenuated virus carrying a polypeptide payload. In some cases, the attenuated virus is an oncolytic virus. In some cases, the attenuated virus is an attenuated virus from the Picomaviridae family. In some cases, the attenuated virus is a poliovirus. In some cases, the attenuated virus is poliovirus Sabin 1 or PVS-RIPO. In some cases, the attenuated virus is capable of preferential replication in a tumor cell. In some cases, the payload is an immune modulator polypeptide. In some cases, the payload is a GM-CSF protein.

[0065] In one aspect, the present disclosure provides an attenuated oncolytic virus including a recombinant polynucleotide that includes a 5’UTR, an internal ribosomal entry site (IRES), an encoding region, at least one protease cleavage site, and a 3’ UTR, where the encoding region encodes an immune modulator polypeptide.

[0066] A recombinant polynucleotide sequence encoding an attenuated virus carrying a therapeutic payload of the invention may be prepared by a variety of methods. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. A recombinant polynucleotide sequence encoding an attenuated virus carrying a therapeutic payload of the invention may be obtained using standard techniques, (e.g., gene synthesis). Alternatively, a recombinant polynucleotide sequence encoding an attenuated virus carrying a therapeutic payload may be altered to contain specific amino acid substitutions using standard techniques in the art (e.g, mutagenesis). Recombinant polynucleotide sequences can be synthesized using a nucleotide synthesizer or PCR techniques.

[0067] A recombinant polynucleotide sequence encoding an attenuated virus carrying a therapeutic payload of the invention may be inserted into a vector capable of replicating andJecho Labs ref no: 240419McAndrews ref no: 68466WO01 expressing the recombinant polynucleotide in prokaryotic or eukaryotic host cells. Many vectors arc available in the art and can be used for the purpose of the invention. Each vector may contain various components that may be adjusted and optimized for compatibility with the particular host cell. For example, the vector components may include, but are not limited to, an origin of replication, a selection marker gene, a promoter, a ribosome binding site, a signal sequence, the nucleic acid sequence encoding the protein of interest, and a transcription termination sequence. Vectors can be linearized or supercoiled exhibiting improved transfection efficiency.

[0068] Common methods of genetic engineering are well known to those of skill in the art, e.g., homologous recombination, site directed mutagenesis, zinc finger nucleases, shRNA, transposons, See e.g., Cytotechnology. 2007 Apr; 53(1-3): 65-73.

[0069] Examples of mammalian cell types which may be manipulated to be used as host cells include, but are not limited to, Vero cells, human embryonic kidney (HEK) (e.g., HEK293, HEK 293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NSO, VERY, BHK, MDCK W13K BT4g3, Hs5nT, HTB2, BT20, T47D), CRL7030, and HssnBst cells. In other embodiments, E. coli cells are used as host cells for the invention. Different host cells have characteristic and specific mechanisms for the posttranslational processing and modification of protein products. Appropriate cell lines or host systems may be chosen to ensure the correct polypeptide payload expressed. The above described expression vectors may be introduced into appropriate host cells using conventional techniques in the art, e.g., transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. Once the vectors are introduced into host cells for protein production, host cells are cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Methods for expression of the polypeptide payload are known in the art, see, for example, Paulina Baibas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2004 (July 20, 2004) and Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd ed. 2012 (June 28, 2012).

[0070] Host cells used to produce the polypeptide payload of the invention may be grown in media known in the art and suitable for culturing of the selected host cells. Examples of suitable media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco's ModifiedJecho Labs ref no: 240419McAndrews ref no: 68466WO01Eagle's Medium (DMEM), Expi293™ Expression Medium, DMEM with supplemented fetal bovine scrum (FBS), and RPMI-1640. Examples of suitable media for bacterial host cells include Luria broth (LB) plus necessary supplements, such as a selection agent, e.g., ampicillin. Host cells are cultured at suitable temperatures, such as from about 20 °C to about 39 °C, e.g., from 25 °C to about 37 °C, preferably 37 °C, and CO2 levels, such as 5 to 10% (preferably 8%). The pH of the medium is generally from about 6.8 to 7.4, e.g., 7.0, depending mainly on the host organism. If an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions suitable for the activation of the promoter. Conventional cell culture conditions for the production of polypeptide payload are known in the art, e.g., see Butler, Cell Culture and Upstream Processing, Taylor & Francis; 1st edition (May 25, 2007).

[0071] Protein recovery typically involves disrupting the host cell, generally by such means as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris may be removed by centrifugation or filtration. The proteins may be further purified. A polypeptide of the invention may be purified by any method known in the art of protein purification, for example, by protein A affinity, other chromatography (e.g., ion exchange, affinity, and size-exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins, (see Process Scale Purification of Antibodies, Uwe Gottschalk (ed.) John Wiley & Sons, Inc., 2009).

[0072] In some embodiments, the attenuated oncolytic virus includes a mutation at one or more nucleic acid positions. As used herein, the term “mutation” refers to a change introduced into the nucleic acid sequence of the recombinant polynucleotide, including but not limited to substitutions, insertions, deletions, point mutations, transpositions, inversions, frame shifts, nonsense mutations, truncations, or other forms of aberrations. A mutation may produce no discernible changes or result in a new property, function, or trait of the mutated attenuated oncolytic virus. In some embodiments, a mutation at one or more nucleic acid positions results in higher genetic stability without phenotypical impact relative to an attenuated oncolytic virus.

[0073] A mutation can result in one or more nucleotides being substituted for an alternative nucleotide including adenine, guanine, thymine, cytosine, and / or uracil. In some embodiments, the attenuated oncolytic virus includes one or more of the following mutations: C2053T, A3896T, G3257A, T3974A, A5397G, A5154G, A610G, T2251C, T6580C, T4723A, G3960C, T5320A, G6694A, C1737T, G3032A, A3950T, C2053T, A3896T, C5755T, C642T, C4819T, T503C,Jecho Labs ref no: 240419McAndrews ref no: 68466WO01G1351A, G3052A, T984C, G3617A, and C3928T. By way of example, in the aforementioned C2053T mutation, the cytosine at position 2053 is changed to a thymine.

[0074] In some embodiments, the attenuated oncolytic virus includes the mutation selected from the group consisting of C2053T, A3896T, G3257A, T3974A, A5397G, A5154G, A610G, T2251C, T6580C, T4723A, G3960C, T5320A, G6694A, C1737T, G3032A, A3950T, C2053T, A3896T, C5755T, C642T, C4819T, T503C, G1351A, G3052A, T984C, G3617A, C3928T, and combinations thereof.

[0075] In some embodiments, the immune modulator polypeptide has at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: XXX. In some embodiments, the recombinant polynucleotide sequence has at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NOs: XXX. In some embodiments, the recombinant polynucleotide encodes a polypeptide having at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: XXX.

[0076] IV. Methods of Treatment

[0077] In one aspect, the present disclosure provides a composition including the attenuated oncolytic virus. In some embodiments, the composition is a therapeutic composition or a vaccine. In some embodiments, the composition is used in a cancer immunotherapy.

[0078] In some embodiments, the composition includes one or more one or more therapeutically acceptable carriers, therapeutically acceptable diluents, therapeutically acceptable excipients, or other therapeutic agents. In some embodiments, the therapeutically acceptable excipients are selected from the group consisting of salts, buffering agents, preservatives, antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers, film formers, coatings, flavors, fragrances, glidants, lubricants, sorbents, suspending or dispersing agents, sweeteners, waters of hydration, and combinations thereof. In some cases, the composition can be about 0.1 % to about 85%, about 0.5% to about 75% by weight of the polypeptide or recombinant polynucleotide of the disclosure, with the remainder consisting essentially of suitable pharmaceutical excipients. In some embodiments, the amount of active component, e.g., one or more polypeptides or recombinant polynucleotides of the invention included in the pharmaceutical preparations is such that a suitable dose within the designated range is provided (e.g., a dose within the range of 0.01-500 mg / kg of body weight).Jecho Labs ref no: 240419McAndrews ref no: 68466WO01

[0079] Acceptable carriers and excipients in the therapeutic or pharmaceutical compositions arc nontoxic to recipients at the dosages and concentrations employed. Pharmaceutical or therapeutic compositions of the invention can be administered parenterally in the form of an injectable formulation. Pharmaceutical compositions for injection (i.e., intravenous injection) can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture media (e.g., Dulbecco's Modified Eagle Medium (DMEM), a-Modified Eagles Medium (a-MEM), F-12 medium). Formulation methods are known in the art, e.g., Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (2nd ed.) Taylor & Francis Group, CRC Press (2006).

[0080] In some cases, a pharmaceutical composition can be administered by a method selected from the group consisting of local, oral, sublingual, buccal, mucosal, nasal, intravenous, subcutaneous, enteral, intra-arterial, intramuscular, intraperitoneal, epidural, intrathecal, intracerebroventricular, intra-articular, intraosseous infusion, intracardiac, intravitreal, parenteral, vaginal, intracavemous, intravesical, rectal, topical, transdermal, inhalation, perivascular, ocular, ear canal, and any combination thereof.

[0081] In some embodiments, administering a composition herein can be performed at least about: 1 time per day, 2 times per day, 3 times per day, 4 times per day, or more than 4 times per day. In some cases, administering can be performed daily, weekly, monthly, or as needed. In some cases, administration or application of a composition herein can be performed for a treatment duration of at least about at least about 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, 26, 27, 28, 29, or 30 days. In some cases, a treatment duration can be from about: 1 to about 30 days, 1 to about 60 days, 1 to about 90 days, 30 days to about 90 days, 60 days to about 90 days, 30 days to about 180 days, from 90 days to about 180 days, or from 180 days to about 360 days. In some embodiments, administration of a composition disclosed herein can be performed for a treatment duration of at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 1 year or for life. Administration can be performed repeatedly over a lifetime of a subject, such as once a month or once a year for the lifetime of a subject.Jecho Labs ref no: 240419McAndrews ref no: 68466WO01

[0082] In some embodiments, the composition to be administered can contain a quantity of the selected compound in a pharmaceutically effective amount for therapeutic use in a biological system, including a patient or subject according to the present disclosure.

[0083] In some embodiments, methods of treating patients or subjects for a particular disease state or infection can comprise administering an effective amount of a pharmaceutical composition comprising a polypeptide or recombinant polynucleotide (e.g., attenuated virus) described herein and / or at least one additional bioactive (e.g., anticancer) agent according to the present disclosure. In some cases, the therapeutically effective amount of a composition herein ranges from about 0.000001 mg / kg to about 1000 mg / kg, wherein mg can be mg of the composition and kg can be kg of body weight of the subject. For example, a composition of about: 0.000001 mg / kg, 0.00001 mg / kg, 0.0001 mg / kg, 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg. 1.0 mg / kg, 10 mg / kg, 100 mg / kg or 1000 mg / kg can be administered to a subject in need thereof. In some cases, one or more additional treatments are administered to a subject in need thereof. Additional treatments may include one or more surgery, radiation, chemotherapy, immunotherapy, and hormone therapy.

[0084] Disclosed herein are methods of treatment using a composition or pharmaceutical composition disclosed herein. In some embodiments, the methods described herein can be used in the development of polypeptides or recombinant polynucleotides (e.g., attenuated virus) that can be used to produce medicaments. In some cases, cells can be used in manufacturing of biological products including vaccines, attenuated viruses, and polypeptides.

[0085] In some embodiments, the methods described herein can be used for polypeptide or attenuated virus delivery in vivo. In some cases, a method can be used herein to deliver apolypeptide or attenuated virus to a subject in need thereof, for example a subject in need of treatment or prevention of a disease (e.g., a cancer). In some embodiments, the subject in need thereof has elevated levels of CD155 positive cancer cells. In some embodiments, the polypeptide or attenuated virus targets CD 155 cancer cells. For example, cancers including head and neck squamous cell carcinomas (HNSCC), melanoma, breast cancer, colorectal cancer, prostate cancer, glioblastoma, hepatocellular' carcinomas, lung cancer, liver cancer, gastric cancer, brain cancer, colon cancer, cervical cancer, kidney cancer, ovarian cancer cells, pancreatic cancer cells, and esophageal cancer can be treated. In some embodiments, a vaccine comprising a composition or pharmaceutical composition disclosed herein can be administered to a subject to increaseJecho Labs ref no: 240419McAndrews ref no: 68466WO01 immunogenicity in the subject. In some embodiments, a composition or pharmaceutical composition can be used to prevent, ameliorate, and / or reduce the severity of a cancer.

[0086] In some embodiments, the methods, systems, and compositions can be used in animal models. For example, mice or any mammal can be produced by a method according to the disclosure specifically designed for the study of cancer.

[0087] In some embodiments, a method of treatment can comprise a therapeutically effective amount of a dose (e.g., a unit dose) of an attenuated virus or polypeptide. In some cases, a composition disclosed herein can be administered to a subject in need of treatment in order to effect treatment. In some cases, a treatment may be preventive and / or therapeutic, and it may be directed to any cancer (e.g., CD155 positive cancer). In some cases, a treatment scheme can be determined by a physician in each case depending on such factors as the cancer to be treated, age, and weight of the patient In some embodiments, the attenuated virus or polypeptide is administered to the patient through a route selected from the group consisting of local, sublingual, buccal, intravenous, subcutaneous, enteral, intra-arterial, intramuscular, intraperitoneal, epidural, intrathecal, intracerebroventricular, intra- articular, intraosseous infusion, intracardiac, intravitreal, parenteral, vaginal, intracavemous, intravesical, rectal, transdermal, and perivascular.

[0088] V. Sequences

[0089] Human granulocyte-macrophage colony-stimulating factor (GM-CSF) nucleic acid sequence (homo sapiens colony stimulating factor 2 (CSF2), mRNA NCBI Reference Sequence; NM_000758.4; SEQ ID NO: 1):AGTACACAGAGAGAAAGGCTAAAGTTCTCTGGAGGATGTGGCTGCAGAGCCTGCTG CTCTTGGGCACTGTGGCCTGCAGCATCTCTGCACCCGCCCGCTCGCCCAGCCCCAGC ACGCAGCCCTGGGAGCATGTGAATGCCATCCAGGAGGCCCGGCGTCTCCTGAACCTG AGTAGAGACACTGCTGCTGAGATGAATGAAACAGTAGAAGTCATCTCAGAAATGTTT GACCTCCAGGAGCCGACCTGCCTACAGACCCGCCTGGAGCTGTACAAGCAGGGCCT GCGGGGCAGCCTCACCAAGCTCAAGGGCCCCTTGACCATGATGGCCAGCCACTACAA GCAGCACTGCCCTCCAACCCCGGAAACTTCCTGTGCAACCCAGATTATCACCTTTGA AAGTTTCAAAGAGAACCTGAAGGACTTTCTGCTTGTCATCCCCTTTGACTGCTGGGA GCCAGTCCAGGAGTGAGACCGGCCAGATGAGGCTGGCCAAGCCGGGGAGCTGCTCT CTCATGAAACAAGAGCTAGAAACTCAGGATGGTCATCTTGGAGGGACCAAGGGGTG GGCCACAGCCATGGTGGGAGTGGCCTGGACCTGCCCTGGGCCACACTGACCCTGATAJecho Labs ref no: 240419McAndrews ref no: 68466WO01CAGGCATGGCAGAAGAATGGGAATATTTTATACTGACAGAAATCAGTAATATTTATATA TTTATATTTTTAAAATATTTATTTATTTATTTATTTAAGTTCATATTCCATATTTATTCAAGA TGTTTTACCGTAATAATTATTATTAAAAATATGCTTCTACTTG

[0090] GM-CSF amino acid sequence (SEQ ID NO: 2):MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVI SEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFE SFKENLKDFLLVIPFDCWEPVQE

[0091] DNA sequence encoding the polypeptide payload (SEQ ID NO: 3): gcacccgcccgctcgcccagccccagcacgcagccctgggagcatgtgaatgccatccaggaggcccggcgtctcctgaacctgagta gagacactgctgctgagatgaatgaaacagtagaagtcatctcagaaatgtttgacctccaggagccgacctgcctacagacccgcctgga gctgtacaagcagggcctgcggggcagcctcaccaagctcaagggccccttgaccatgatggccagccactacaagcagcactgccctc caaccccggaaacttcctgtgcaacccagattatcacctttgaaagtttcaaagagaacctgaaggactttctgcttgtcatcccctttgactgc tgggagccagtccaggag

[0092] Polypeptide payload amino acid sequence (SEQ ID NO: 4):APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLE LYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQnTFESFKENLKDFLLVIPFDC WEPVQE

[0093] GM-CSF and flanking region sequence in pJL182 (SEQ ID NO: 5):CACGTGGCGGCTAGTACTCCGGTATTGCGGTACCCTTGTACGCCTGTTTTATACTCCC TTCCCGTAACTTAGGAATTCAACTTAGAAGTTTTTCACAAAGACCAATAGCCGGTAA TCAGCCAGATTACTGAAGGTCAAGCACTTCTGTTTCCCCGGTCAATGTTGATATGCT CCAACAGGGCAAAAACAACTGCGATCGTTAACCGCAAAGCGCCTACGCAAAGCTTA GTAGCATCTTTGAAATCGTTTGGCTGGTCGATCCGCCATTTCCCCTGGTAGACCTGGCAGATGAGGCTAGAAATACCCCACTGGCGACAGTGTTCTAGCCTGCGTGGCTGCCTGC ACACCCTATGGGTGTGAAGCCAAACAATGGACAAGGTGTGAAGAGCCCCGTGTGCT CGCTTTGAGTCCTCCGGCCCCTGAATGTGGCTAACCTTAACCCTGCAGCTAGAGCAC GTAACCCAATGTGTATCTAGTCGTAATGAGCAATTGCGGGATGGGACCAACTACTTTJecho Labs ref no: 240419McAndrews ref no: 68466WO01GGGTGTCCGTGTTTCACTTTCTCCTTTATATTTGCTTATGGTGACAATATATACAATA TATATATTGGCACCATGGGAGCTCAGGGCgcacccgcccgctcgcccagccccagcacgcagccctgggag catgtgaatgccatccaggaggcccggcgtctcctgaacctgagtagagacactgctgctgagatgaatgaaacagtagaagtcatctcag aaatgtttgacctccaggagccgacctgcctacagacccgcctggagctgtacaagcagggcctgcggggcagcctcaccaagctcaag ggccccttgaccatgatggccagccactacaagcagcactgccctccaaccccggaaacttcctgtgcaacccagattatcacctttgaaag tttcaaagagaacctgaaggactttctgcttgtcatcccctttgactgctgggagccagtccaggagCAAGGAGCTCAGGTTT CATCACAGAAAGTGGGCGCACATGAAAACTCAAATAGAGCGTATGGTGGTTCTACC ATTAATTACACCACCATTAATTATTATAGAGATTCAGCTAGTAACGCGGCTTCGAAA CAGGACTTCTCTCAAGACCCTTCCAAGTTCACCGAGCCCATCAAGGATGTCCTGATA AAAACATCCCCAATGCTAAACTCGCCAAACATAGAGGCTTGCGGGTATAGCGATAG AGTACTGCAATTAACACTGGGAAACTCCACTATAACCACACAGGAGGCGGCTAATT CAGTAGTCGCTTATGGGCGTTGGCCTGAATATCTGAGGGACAGCGAAGCCAATCCA GTGGACCAGCCGACAGAACCAGACGTCGCTGCATGCAGGTTTTATACGCTAGACAC CGTGTCTTGGACGAAAGAGTCGCGAGGGTGGTGGTGGAAGTTGCCTGATGCACTGC GGGACATGGGACTCTTTGGCCAAAATATGTACTACCACTACCTAGGTAGGTCCGGGT ACACCGTGCATGTACAGTGTAACGCCTCCAAATTCCACCAGGGGGCACTAGGGGTATTCGCCGTACCAGAGATGTGT

[0094] GM-CSF and flanking region sequence in pJL183 (SEQ ID NO: 6):CTATTTACAGTGTGGAAGATCACTTATAAAGATACTGTCCAGTTACGGAGGAAATTGG AGTTCTTCACCTATTCTAGATTTGATATGGAATTTACCTTTGTGGTTACTGCAAATTTCA CTGAGACTAACAATGGGCATGCCTTAAATCAAGTGTACCAAATTATGTACGTACCACC AGGCGCTCCAGTGCCCGAGAAATGGGACGACTACACATGGCAAACCTCATCAAATCC ATCAATCTTTTACACCTACGGAACAGCTCCAGCCCGGATCTCGGTACCGTATGTTGGT ATTTCGAACGCCTATTCACACTTTTACGACGGTTTTTCCAAAGTACCACTGAAGGACC AGTCGGCAGCACTAGGTGACTCCCTCTATGGTGCAGCATCTCTAAATGACTTCGGTAT TTTGGCTGTTAGAGTAGTCAATGATCACAACCCGACCAAGGTCACCTCCAAAATCAG AGTGTATCTAAAACCCAAACACATCAGAGTCTGGTGCCCGCGTCCACCGAGGGCAGT GGCGTACTACGGCCCTGGAGTGGATTACAAGGATGGTACGCTTACACCCCTCTCCACC AAGGATCTGACCACATATGGCgcacccgcccgctcgcccagccccagcacgcagccctgggagcatgtgaatgccat ccaggaggcccggcgtctcctgaacctgagtagagacactgctgctgagatgaatgaaacagtagaagtcatctcagaaatgtttgacctc caggagccgacctgcctacagacccgcctggagctgtacaagcagggcctgcggggcagcctcaccaagctcaagggccccttgacca tgatggccagccactacaagcagcactgccctccaaccccggaaacttcctgtgcaacccagattatcacctttgaaagtttcaaagagaac ctgaaggactttctgcttgtcatcccctttgactgctgggagccagtccaggagCAAGGATTCGGACACCAAAACAAA GCGGTGTACACTGCAGGTTACAAAATTTGCAACTACCATTTGGCCACTCAGGAAGATTJecho Labs ref no: 240419McAndrews ref no: 68466WO01TGCAAAACGCAGTGAACGTCATGTGGAATAGAGACCTCTTAGTCACAGAATCAAGAG CCCAGGGCACCGATTCAATCGCAAGGTGCAATTGCAACGCAGGGGTGTACTACTGCG AGTCTAGAAGGAAATACTACCCAGTATCCTTCGTTGGCCCAACGTTCCAGTACATGGA GGCTAATAACTATTACCCAGCTAGGTACCAGTCCCATATGCTCATTGGCCATGGATTCGCATCTCCAGGGGATTGTGGTGGCATACTCAGATGTCACCACGGGGTGATAGGGATCAT TACTGCTGGTGGAGAAGGGTTGGTTGCATTTACAGACATTAGAGACTTGTATGCCTACGAAGAAGAAGCCATGGAACAAGGCATCACCAATTACATAGAGTCACTTGGGGCCGCA TTTGGAAGTGGATTTACTCAGCAGATTGGAGACAAAATAACAGAGTTGACTAATATGG TGACCAGTACCATCACTGAAAAGCTACTTAAGAACTTGATCAAGATCATATCCTCA

[0095] GM-CSF and flanking region sequence in pJL184 (SEQ ID NO: 7):TACATCGACTACCTAAACCACTCACACCACCTGTACAAGAATAAAACATACTGTGTCAAGGGCGGTATGCCATCTGGTTGCTCAGGCACTTCAATTTTTAACTCAATGATTAACAACTTGATTATCAGGACACTCTTACTGAAAACCTACAAGGGCATAGATTTAGACCACCTAAAAATGATTGCCTATGGTGATGATGTAATTGCTTCCTACCCCCATGAAGTTGACGCTAGTCTCCTAGCCCAATCAGGAAAAGACTATGGACTAACTATGACTCCAGCTGACAAATCAGCTATATTTGAAACAGTCACATGGGAGAATGTAACATTCTTGAAGAGATTCTTCAGGGCAGACGAGAAATACCCATTTCTTATTCATCCAGTAATGCCAATGAAGGAAATTCATGAATCAATTAGATGGACAAAAGATCCTAGGAACACTCAGGATCACGTTCGCTCTCTGTGCCTATTAGCTTGGCACAATGGCGAAGAAGAATATAACAAATTCCTAGC TAAAATCAGGAGTGTGCCAATTGGAAGAGCTTTATTGCTCCCAGAGTACTCAACATTGTACCGCCGTTGGCTTGACTCATTTCAAGGAgcacccgcccgctcgcccagccccagcacgcagccctggg agcatgtgaatgccatccaggaggcccggcgtctcctgaacctgagtagagacactgctgctgagatgaatgaaacagtagaagtcatctc agaaatgtttgacctccaggagccgacctgcctacagacccgcctggagctgtacaagcagggcctgcggggcagcctcaccaagctca agggccccttgaccatgatggccagccactacaagcagcactgccctccaaccccggaaacttcctgtgcaacccagattatcacctttgaa agtttcaaagagaacctgaaggactttctgcttgtcatcccctttgactgctgggagccagtccaggagTAGTAACCCTACCTCAGTCGAATTGGATTGGGTCATACTGCTGTAGGGGTAAATTTTTCTTTAATTCGGAGAAAAAAAAAAAACCCGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAAT GAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTJecho Labs ref no: 240419McAndrews ref no: 68466WO01CAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATG TGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTT TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAG GTGGCGAAACCCGACAGGA

[0096] For SEQ ID NO: 5, 6, 7, flanking region sequences (600bps for both upstream and downstream) are displayed in capital letters, and the GM-CSF sequence is displayed in lowercase letters.

[0097] pJL182 Attenuated Viral Construct (SEQ ID NO: 20): ttaaaacagctctggggttgtacccaccccagaggcccacgtggcggctagtactccggtattgcggtacccttgtacgcctgttttatactcc cttcccgtaacttaggaattcaacttagaagtttttcacaaagaccaatagccggtaatcagccagattactgaaggtcaagcacttctgtttcc ccggtcaatgttgatatgctccaacagggcaaaaacaactgcgatcgttaaccgcaaagcgcctacgcaaagcttagtagcatctttgaaat cgtttggctggtcgatccgccatttcccctggtagacctggcagatgaggctagaaataccccactggcgacagtgttctagcctgcgtggct gcctgcacaccctatgggtgtgaagccaaacaatggacaaggtgtgaagagccccgtgtgctcgctttgagtcctccggcccctgaatgtg gctaaccttaaccctgcagctagagcacgtaacccaatgtgtatctagtcgtaatgagcaattgcgggatgggaccaactactttgggtgtcc gtgtttcactttctcctttatatttgcttatggtgacaatatatacaatatatatattggcaccatgggagctcagggcgcacccgcccgctcgccc agccccagcacgcagccctgggagcatgtgaatgccatccaggaggcccggcgtctcctgaacctgagtagagacactgctgctgagat gaatgaaacagtagaagtcatctcagaaatgtttgacctccaggagccgacctgcctacagacccgcctggagctgtacaagcagggcct gcggggcagcctcaccaagctcaagggccccttgaccatgatggccagccactacaagcagcactgccctccaaccccggaaacttcct gtgcaacccagattatcacctttgaaagtttcaaagagaacctgaaggactttctgcttgtcatcccctttgactgctgggagccagtccagga gCAAggagctcaggtttcatcacagaaagtgggcgcacatgaaaactcaaatagagcgtatggtggttctaccattaattacaccaccatt aattattatagagattcagctagtaacgcggcttcgaaacaggacttctctcaagacccttccaagttcaccgagcccatcaaggatgtcctga taaaaacatccccaatgctaaactcgccaaacatagaggcttgcgggtatagcgatagagtactgcaattaacactgggaaactccactata accacacaggaggcggctaattcagtagtcgcttatgggcgttggcctgaatatctgagggacagcgaagccaatccagtggaccagccg acagaaccagacgtcgctgcatgcaggttttatacgctagacaccgtgtcttggacgaaagagtcgcgagggtggtggtggaagttgcctg atgcactgcgggacatgggactctttggccaaaatatgtactaccactacctaggtaggtccgggtacaccgtgcatgtacagtgtaacgcct ccaaattccaccagggggcactaggggtattcgccgtaccagagatgtgtctggccggggatagcaacaccactaccatgcacaccagct atcaaaatgccaatcctggcgagaaaggaggcactttcacgggtacgttcactcctgacgacaaccagacatcacctgcccgtaggttctg cccggtggattacctctttggaaatggcacgttattggggaatgcctttgtgttcccgcaccagataataaacctacggaccaacaactgtgct acactggtactcccttacgtgaactccctctcgatagatagtatggtaaagcacaataattggggaattgcaatattaccattggccccattaaa ttttgctagtgagtcctccccagagattccaatcaccttgaccatagcccctatgtgctgtgagttcaatggattaagaaacattaccctgccacJecho Labs ref no: 240419McAndrews ref no: 68466WO01 gcttacagggcctgccggtcatgaacacccctggtagcaatcaatatcttactgcagacaacttccagtcaccgtgtgcgctgcctgaatttg atgtgaccccacctattgacatacccggtgaagttaagaacatgatggaattggcagaaatcgacaccatgattccctttgacttaagtgcaaa aaaaaagaacaccatggaaatgtatagggttcggttaagtgacaaaccacatacagacgatcccatactctgcctgtcactctctccagcttc agatcctaggttgtcacatactatgcttggagaaatcctaaattactacacacactgggcaggatccctgaagttcacgtttctgttctgtggatc catgatggcaactggcaaactgttggtgtcatacgcgcctcctggagccgacccaccaaagaagcgtaaggaggcgatgttgggaacaca tgtgatctgggacataggactgcagtcctcatgtactatggtagtgccatggattagcaacaccacgtatcggcaaaccatagatgatagtttc accgaaggcggatacatcagcgtcttctaccaaaccagaatagtcgtccctctttcgacacccagagagatggacatccttggttttgtgtca gcgtgtaatgacttcagcgtgcgcttgatgcgagataccacacatatagagcaaaaagcgctagcacaggggttaggtcagatgcttgaaa gcatgattgacaacacagtccgtgaaacggtgggggcggcaacgtctagagacgctctcccaaacactgaagccagtggaccagcacac tccaaggaaattccggcactcaccgcagtggaaactggggccacaaatccactagtcccttctgatacagtgcaaaccagacatgttgtaca acataggtcaaggtcagagtctagcatagagtctttcttcgcgcggggtgcatgcgtggccattataaccgtggataactcagcttccaccaa gaataaggataagctatttacagtgtggaagatcacttataaagatactgtccagttacggaggaaattggagttcttcacctattctagatttga tatggaatttacctttgtggttactgcaaatttcactgagactaacaatgggcatgccttaaatcaagtgtaccaaattatgtacgtaccaccagg cgctccagtgcccgagaaatgggacgactacacatggcaaacctcatcaaatccatcaatcttttacacctacggaacagctccagcccgg atctcggtaccgtatgttggtatttcgaacgcctattcacacttttacgacggtttttccaaagtaccactgaaggaccagtcggcagcactagg tgactccctctatggtgcagcatctctaaatgacttcggtattttggctgttagagtagtcaatgatcacaacccgaccaaggtcacctccaaaa tcagagtgtatctaaaacccaaacacatcagagtctggtgcccgcgtccaccgagggcagtggcgtactacggccctggagtggattacaa ggatggtacgcttacacccctctccaccaaggatctgaccacatatggattcggacaccaaaacaaagcggtgtacactgcaggttacaaa atttgcaactaccatttggccactcaggaagatttgcaaaacgcagtgaacgtcatgtggaatagagacctcttagtcacagaatcaagagcc cagggcaccgattcaatcgcaaggtgcaattgcaacgcaggggtgtactactgcgagtctagaaggaaatactacccagtatccttcgttgg cccaacgttccagtacatggaggctaataactattacccagctaggtaccagtcccatatgctcattggccatggattcgcatctccagggga ttgtggtggcatactcagatgtcaccacggggtgatagggatcattactgctggtggagaagggttggttgcatttacagacattagagacttg tatgcctacgaagaagaagccatggaacaaggcatcaccaattacatagagtcacttggggccgcatttggaagtggatttactcagcagat tggagacaaaataacagagttgactaatatggtgaccagtaccatcactgaaaagctacttaagaacttgatcaagatcatatcctcactagtt attataactaggaattatgaagacaccacaacagtgctcgctaccctggcccttcttgggtgtgatgcttcaccatggcagtggcttagaaaga aagcatgcgatgttctggagataccttatgtcaccaagcaaggtgacagttggttgaagaagtttactgaagcatgcaacgcagctaaggga ctggagtgggtgtcaaacaaaatctcaaaattcattgattggctcaaggagaaaattatcccacaagctagagataagttggaatttgtaacaa aacttagacaactagaaatgctggaaaaccaaatctcaactatacaccaatcatgccctagtcaggaacaccaggaaattctattcaataatgt cagatggttatccatccagtctaagaggtttgcccctctttacgcagtggaagccaaaagaatacagaaactagagcataccattaacaacta catacagttcaagagcaaacaccgtattgaaccagtatgtttgctagtacatggcagccccggaacaggtaaatctgtagcaaccaacctga ttgctagagccatagctgaaagagaaaacacgtccacgtactcgctacccccggatccatcacacttcgacggatacaaacaacagggagtJecho Labs ref no: 240419McAndrews ref no: 68466WO01 ggtgattatggacgacctgaatcaaaacccagatggtgcggacatgaagctgttctgtcagatggtatcaacagtggagtttataccacccat ggcatccctggaggagaaaggaatcctgtttacttcaaattacgttctagcatccacgaactcaagcagaatttccccccccactgtggcaca cagtgatgcattagccaggcgctttgcgttcgacatggacattcaggtcatgaatgagtattctagagatgggaaattgaacatggccatggc tactgaaatgtgtaagaactgtcaccaaccagcaaactttaagagatgctgtcctttagtgtgtggtaaggcaattcaattaatggataaatcttc cagagttagatacagtattgaccagatcactacaatgattatcaatgagagaaacagaagatccaacattggcaattgtatggaggctttgttc caaggaccactccagtataaagacttgaagattgacatcaagacgagtccccctcctgaatgtatcaatgacttgctccaagcagttgactcc caggaggtgagagattactgtgagaagaagggttggatagtcaacatcaccagccaggttcaaacagaaaggaacatcaacagggcaat gacaattctacaagcggtgacaaccttcgccgcagtggctggagttgtctatgtcatgtataaactgtttgctggacaccagggagcatacac tggtttaccaaacaaaaaacccaacgtgcccaccattaggacagcaaaggtacaagggccagggttcgattacgcagtggctatggctaaa agaaacattgttacagcaactactagcaagggagagttcactatgttaggagtccacgacaacgtggctattttaccaacccacgcttcacct ggtgaaagcattgtgatcgatggcaaagaagtggagatcttggatgccaaagcgctcgaagatcaagcaggaaccaatcttgaaatcacta taatcactctaaagagaaatgaaaagttcagagacattagaccacatatacctactcaaatcactgagacaaatgatggagtcttgatcgtgaa cactagcaagtaccccaatatgtatgttcctgtcggtgctgtgactgaacagggatatctaaatctcggtgggcgccaaactgctcgtactcta atgtacaactttccaaccagagcaggacagtgtggtggagtcatcacatgtactgggaaagtcatcgggatgcatgttggtgggaacggttc acacgggtttgcagcggccctgaagcgatcatacttcactcagagtcaaggtgaaatccagtggatgagaccttcgaaggaagtgggatat ccaatcataaatgccccgtccaaaaccaagcttgaacccagtgctttccactatgtgtttgaaggggtgaaggaaccagcagtcctcactaaa aacgatcccaggcttaagacaaactttgaggaggcaattttctccaagtacgtgggtaacaaaattactgaagtggatgagcacatgaaaga ggcagtagaccactatgctggccagctcatgtcactagacatcaacacagaacaaatgtgcttggaggatgccatgtatggcactgatggtc tagaagcacttgatttgtccaccagtgctggctacccttatgtagcaatgggaaagaagaagagagatatcttgaacaaacaaaccagagac actaaggaaatgcaaaaactgctcgacacatatggaatcaacctcccactggtgacttatgtaaaggatgaacttagatccaaaacaaaggtt gagcaggggaaatccagattaattgaagcttctagtttgaatgactcagtggcaatgagaatggcttttgggaacctatatgctgcttttcacaa aaacccaggagtgataacaggttcagcagtagggtgcgatccagatttgttttggagcaaaattccggtattgatggaagagaagctgtttgc ctttgactacacagggtatgatgcatctctcagccctgcttggttcgaggcactaaagatggtgcttgagaaaatcggattcggagacagagt tgactacatcgactacctaaaccactcacaccacctgtacaagaataaaacatactgtgtcaagggcggtatgccatctggttgctcaggcac ttcaatttttaactcaatgattaacaacttgattatcaggacactcttactgaaaacctacaagggcatagatttagaccacctaaaaatgattgcc tatggtgatgatgtaattgcttcctacccccatgaagttgacgctagtctcctagcccaatcaggaaaagactatggactaactatgactccag ctgacaaatcagctatatttgaaacagtcacatgggagaatgtaacattcttgaagagattcttcagggcagacgagaaatacccatttcttatt catccagtaatgccaatgaaggaaattcatgaatcaattagatggacaaaagatcctaggaacactcaggatcacgttcgctctctgtgcctat tagcttggcacaatggcgaagaagaatataacaaattcctagctaaaatcaggagtgtgccaattggaagagctttattgctcccagagtact caacattgtaccgccgttggcttgactcattttagtaaccctacctcagtcgaattggattgggtcatactgctgtaggggtaaatttttctttaatt cggagaaaaaaaaaaaacccJecho Labs ref no: 240419McAndrews ref no: 68466WO01

[0098] The following examples further illustrate the invention but should not be construed in any way as limiting its scope.EXAMPLES

[0099] Example 1

[0100] Vector construction. Poliovirus’ ability of self-processing after the viral polypeptide is translated inside a host cell was advantageously used. FIG. 1 shows a poliovirus polypeptide and its self-processing steps. Vector constructs, shown in FIG. 2, were engineered from the human poliovirus 1 strain Sabin 1 (GenBank AY184219.1) and human rhinovirus IRES replacement (Pubmed ID: 8637880). The DNA sequence was cloned into a pUC19 vector. The resulting plasmid was named pIL 15003. The GM-CSF coding sequence, flanked with protease cleavage sites (3C or 2A), was inserted in-frame into the poliovirus genome. For the N-terminal insertion, the coding sequence for GM-CSF without its signal peptide was inserted at glutamine 4 where a 3Cprocleavage site (GAQJ.G) was created at the N-terminus of GM-CSF and a second 3Cprocleavage site (QEQJ.G) at the C-terminus of GM-CSF. For the internal insertion, a 2Aprocleavage site (TYJ.GA) and a 3Cprocleavage site (EQJ.GF) were designed to flank the GM-CSF coding sequence. For the C-terminal insertion, only one 3Cprocleavage site (SFQ J.G) was inserted at the 5’ end of GM-CSF.

[0101] Poliovirus / GM-CSF chimeras were constructed using the HiFi DNA assembly method (NEBuilder HiFi DNA Assembly Master Mix, NEB E2621L) in which both the insert (GM-CSF) and the vector (pJL15003) were amplified by PCR with additional overlapping sequences of 18- 22 bp at ends. The overlapping sequences would direct homologous recombination to insert the GM-CSF sequence into the viral genome seamlessly. The assembled PCR products were then transformed into NEB Stable Competent E. coli (NEB C3040H). The plasmids corresponding to the N-terminal insertion, the internal insertion, and the C-terminal insertion were designated as pJL182, pJL183, and pJL184. Sequencing the coding regions confirmed that GM-CSF was correctly inserted in pJL182, pJL183, and pJL184. PCR primer sequences are shown in Table 1.Table 1Construct Primer Primer location Primer sequence (5'- 3')Jecho Labs ref no: 240419 McAndrews ref no: 68466WO01IRES and Pl ctgagctcccatggtgccaatatatatattg (SEQ ID NO: 8) pJL 182 W 114 junctionIRES and GM-CSF ggcaccatgggagctcagggcgcacccgcccgctcgcccagc (SEQ IDW115 junction NO: 9)GM-CSF and Pl gtgatgaaacctgagctccTTGctcctggactggctcccag (SEQ IDW116 junction NO: 10)W117 5' Pl ggagctcaggtttcatcacagaaag (SEQ ID NO: 11) pJL183 W120 3’ Pl atatgtggtcagatccttggtg (SEQ ID NO: 12)Pl and GM-CSF caaggatctgaccacatatggcgcacccgcccgctcgcccag (SEQ IDW121 junction NO: 13)GM-CSF and P2 gttttggtgtccgaatccttgctcctggactggctcccagc (SEQ ID NO:W122 junction 14)W123 5' P2 ggattcggacaccaaaacaaagc (SEQ ID NO: 15) pJL184 W106 3' P3 aaatgagtcaagccaacggcgg (SEQ ID NO: 16)P3 and GM-CSF ccgccgttggcttgactcatttCAAGGAgcacccgcccgctcgcccagccW107 junction c (SEQ ID NO: 17) cgactgaggtagggttactactcctggactggctcccagc (SEQ ID NO:W108 3' GM-CSF 18)W109 5’ poly A tagtaaccctacctcagtcg (SEQ ID NO: 19)

[0102] GM-CSF polypeptide was expressed along with poliovirus polyprotein. Full length GM-CSF was separated from the polyprotein after protease processing and eventually released into the surrounding area after cell lysis. The expressed GM-CSF was intended to be released only after cell lysis; therefore, no secretion signal peptide was engineered in the construct.

[0103] Example 2

[0104] Transfection and virus production. The recombinant poliovirus genome was prepared by in vitro transcription using pJL182, pJL183, or pJL184 as a template. The templates were linearized with restriction enzyme Sall and purified before transcription. The resulting RNA was purified and transfected into Vero cells via electroporation (10 pg RNA, voltage: 110 V, duration: 20 ms, single shock). The transfected Vero cells were placed into T-25 flasks and incubated at 37 °C with 5% CO2. Cytopathic effect (CPE) was checked daily until >95% CPE was observed. The viral particles were harvested (P0) and tested for infectivity (CCID50) and genome sequencing. Construct JL182 (FIG. 3A) yielded a significantly higher titer than JL183 (FIG. 3B), while JL184 (FIG. 3C) failed to produce any viable virus, as indicated by CPE and CCID50 results shown in Table 2. Construct JL182 was selected for further development of a genomically stable virus strain.Jecho Labs ref no: 240419 McAndrews ref no: 68466WO01Table 2

[0105] Example 3

[0106] Virus cloning and plaque purification. Vero cells were diluted to 2.5-3.0 E+04 / cm2and seeded into 6-well plates. The plates were incubated at 37 °C, 5% CO2 overnight. The cells were washed twice with DPBS. Serial-diluted P0 vims seeds were added to the cells, and the plates were incubated at 36 °C, 5% CO2 lor 4 hours (gently shaken a few times every 20 min). After incubation, the liquid was carefully discarded, and 2 ml of DMEM / F12 medium with 10% FBS and 1% of agarose was added to the cells. The plates were placed at room temperature until the agarose was solidified. The plates were moved to a 36 °C, 5% CO2 incubator until plaques were observed. 0.5 ml of sterile 0.066% neutral red was added into each well to stain the plaques for at least 2 hours in a dark place. Plaques with round shape and sharp edge were carefully picked, mixed in 500 pl of DMEM complete medium (90% DMEM+10% FBS), and treated with 3 cycles of freeze / thaw. The liquid was added to fresh Vero cells for vims amplification. At CPE >95%, the supernatant was harvested and defined as viral clones from this round of plaque purification. Two rounds of cloning were performed for each virus clone (plaque) mentioned below.

[0107] Example 4

[0108] Genetic stability. Virus particles harvested from JL182 transfection were diluted and spread on Vero cell plates to form isolated plaques. 10 plaques were picked and serially passaged for genetic stability testing. Specifically, at passage 1 and 4, (Pl and P4), the RNA genome was extracted from the viruses and sequenced. There were noticeable differences in stability among individual plaques. Plaque 3-6-2 appeared to be stable after 4 passages, therefore, it was furtherJecho Labs ref no: 240419McAndrews ref no: 68466WO01 passaged up to PIO, and viruses at P5, P6, P7, and PIO were sequenced. As indicated in Table 3, the two most prominent mutation sites were at position 3257 (G to A) and 5154 (A to G) in the JL182 sequence.Table 3Jecho Labs ref no: 240419McAndrews ref no: 68466WO01

[0109] G3257A led to an amino acid change from Glu to Lys in capsid protein VP1 whileA5154G caused an Asn to Ser shift in ATPase 2C.

[0110] To test the potential impact of the two mutations on virus genetic stability as well as phenotype, two DNA templates were synthesized - JL182A with A5154G point mutation and JL182B with both G3257A and A5154G mutations. Vero cells were transfected with RNAs transcribed from those DNA templates and the resulting viruses were prepared from plaques. JL182A and JL182B were serially passaged up to 7 passages and the genetic stability was monitored by genome sequencing.Table 4Jecho Labs ref no: 240419McAndrews ref no: 68466WO01

[0111] Results indicated that virus bearing both G3257A and A5154G (JL182B) were more stable than the parental JL182.

[0112] Example 5

[0113] JL182B replication in cancer cell lines. Cancer cells (MCF7, DU 145, U138MG) were infected with JL182B and incubated at 37 °C. Samples were collected at various time points (Oh, 4h, 8h, 20h, 32h, 48h) after infection and the JL182B RNA copy numbers were determined by RT- PCR. JL182B was replication competent in cancer cells as shown in FIG. 4.

[0114] Example 6

[0115] In vitro cell killing by JL182B. Various CD155+ cancer cells and CD155+ normal cells (HUVEC) were seeded in a 96-well plate and infected with gradient diluted JL182B (initial MOI=100, 3-fold gradient dilution). Both cell and virus were co-incubated for 72 hours. After coincubation MTS was added and OD490 was determined using a microplate reader. JL182B showed an excellent killing effect on various cancer cell lines. In comparison, the CD155+ normal cells were approximately 20- to 100-fold more resistant to JL182B. The modified virus retained the parental vims’s selectivity of cytopathic effect.Table 5Jecho Labs ref no: 240419McAndrews ref no: 68466WO01

[0116] Example 7

[0117] GM-CSF expression from JL182B infected cells. A total of 1.0 E+07 Huh7 cells were seeded in a T-75 flask. The cells were infected with JL182B or PVS-R1PO and incubated at 37 °C for 1 day. Then, the cells were digested and collected by centrifugation. The cell debris was re-suspended and analyzed by Western blot to detect the presence of GM-CSF. As shown in FIG. 5, the JL182B infected cells produced free GM-CSF. Signals at higher molecular weight were also observed, and appeared to be the un-processed GM-CSF containing viral polyproteins.

[0118] Cancer cells (DU 145, MCF7, U138MG, Huh7, HepG2) were infected with JL182B at MOI 1.0 and incubated at 30 °C for 72 hours. The cells were lysed by 3 freeze / thaw cycles and supernatants were collected, and GM-CSF concentrations were determined by ELISA. PBS was used as a negative control (background).Table 6

[0119] GM-CSF expression levels in U138MG and Huh7 were further studied at various MOIs. It was found that at 24 hours post infection, the GM-CSF in Huh7 culture infected with JL182B could reach as high as 1.6 ng / mL.GM-CSF expression in Huh7 and U138MG at 24 hrs post infectionJecho Labs ref no: 240419McAndrews ref no: 68466WO01Table 7

[0120] Example 8

[0121] TF-1 cell proliferation induced by JL182B derived GM-CSF. Vero cells were infected with JL182B or PVS-RIPO. When CPE reached >95%, the supernatants were collected. Erythroblast cell TF-1 was treated with JL182B infection supernatant to probe the proliferation stimulation by GM-CSF. PVS-RIPO supernatant was used as a control. Briefly, TF-1 cells were adjusted to the concentration of 2.0 E+05 / ml and starved at 37 °C, 5% CO2 for 4-5 hours to deplete cytokine. The starved TF-1 cells were seeded into a 96- well plate at 100 pl per well. Then, serial- diluted JL182B or PVS-RIPO was added into cells and incubated at 37 °C, 5% CO2 for 72 hours. After incubation, MTS was added into each well and incubated at 37 °C, 5% CO2 for another 4-6 hours. Then, the plate was taken out and the OD490-OD650 was determined using a microplate reader. Results in FIG. 6 showed that the JL182B supernatant, compared to that of PVS-RIPO, was able to induce higher levels of proliferation of TF- 1 cells, and that the effect was overall dose- related.

[0122] Example 9

[0123] PBMC differentiation induced by JL182B supernatant. PBMCs were incubated with inactivated JL182B / U138MG infection supernatant to probe the expressed GM-CSF’s effect on differentiation and proliferation. Recombinant GM-CSF was used as a positive control. After 7 days of incubation, both GM-CSF and the infection supernatant induced similar morphology changes in PBMCs. Newly formed round and spindle shaped cells were observed under a microscope, as shown in FIG. 7A: PBMCs treated with 100 pg / mL recombinant GM-CSF and FIG. 7B: PBMCs treated with infection supernatant containing 100 pg / mL expressed GM-CSF).

[0124] Example 10Jecho Labs ref no: 240419McAndrews ref no: 68466WO01

[0125] PBMC proliferation induced by JL182B derived GM-CSF and recombinant IL- 15. To further elucidate the cell proliferation stimulation activity of JL182B derived GM-CSF, PBMCs were incubated with inactivated JL182B infection supernatant and IL- 15. JL182B infection was carried out in Huh7, U138MG, and DU145 cells. Recombinant GM-CSF and PVS- RIPO were used as controls. PBMCs were incubated with JL182B supernatant or controls for 5 days, then exposed to 10 ng / mL IL- 15 and incubated for another 10 days. Samples were taken on day 7 (FIG. 8) and day 10 (FIG. 9) post IL-15 addition. Total cell numbers were counted by the MTS method. Results showed that JL182B infection supernatants had a comparable stimulation effect that was significantly higher than IL- 15 alone (medium control) or PVS-RIPO infected supernatants. We attribute this effect to the elevated number of antigens presenting cells in GM- CSF or JL182B infected supernatant groups.

[0126] Example 11

[0127] Effect of JL182B derived GM-CSF on macrophage phagocytosis. THP- 1 cells were co-incubated with different concentrations of recombinant GM-CSF and JL182 / Huh7 infection supernatant (400 pg / ml GM-CSF by ELISA) at 37 °C, 5% CO2 for 48 hours. Prepared Huh7 cells were seeded into a 96-well plate with 2.0 E+3 per well and incubated at 37 °C, 5% CO2 for 4-5 hours for complete adherence. Then, treated THP-1 cells were added into Huh7 cells with a ratio of 10:1, and incubated at 37 °C, 5% CO2 for another 48 hours. Then, suspending THP-1 cells were carefully removed and total cell numbers were counted by the MTS method. The results showed (FIG. 10) that JL182B infection supernatant could significantly promote phagocytosis of THP-1 on Huh7 (the number of remaining Huh7 cells decreased by 24% compared to the control group), and the effect was similar with recombinant GM-CSF.

[0128] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of theJecho Labs ref no: 240419McAndrews ref no: 68466WO01 above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0129] All publications, patents, and patent applications cited herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

Jecho Labs ref no: 240419 McAndrews ref no: 68466WO01Claims:

1. An attenuated oncolytic virus comprising a recombinant polynucleotide, wherein the recombinant polynucleotide comprises a 5’UTR, an internal ribosomal entry site (IRES), an encoding region, at least one protease cleavage site, and a 3’ UTR, wherein the encoding region encodes an immune modulator polypeptide.

2. The attenuated oncolytic virus of claim 1, wherein the recombinant polynucleotide comprises a mutation at one or more nucleic acid positions resulting in higher genetic stability without phenotypical impact relative to the attenuated oncolytic virus.

3. The attenuated oncolytic virus of claim 2, wherein the mutation is selected from the group consisting of C2053T, A3896T, G3257A, T3974A, A5397G, A5154G, A610G, T2251C, T6580C, T4723A, G3960C, T5320A, G6694A, C1737T, G3032A, A3950T, C2053T, A3896T, C5755T, C642T, C4819T, T503C, G1351A, G3052A, T984C, G3617A, C3928T, and combinations thereof.

4. The attenuated oncolytic virus of any one of claims 1 to 3, wherein the immune modulator polypeptide has at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 2 or 4.

5. The attenuated oncolytic virus of any one of claims 1 to 4, wherein the immune modulator polypeptide is a GM-CSF protein.

6. An attenuated oncolytic virus comprising a recombinant polynucleotide sequence having at least about 80% sequence identity to the nucleic acid sequence of SEQ ID NOs: 5, 6, or7.Jecho Labs ref no: 240419 McAndrews ref no: 68466WO017. The attenuated oncolytic virus of claim 6, wherein the recombinant polynucleotide encodes a polypeptide having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 4.

8. The attenuated oncolytic virus of claim 7, wherein the polypeptide is a GM-CSF protein.

9. The attenuated oncolytic virus of any one of claims 1 to 8, wherein the attenuated oncolytic virus is derived from a poliovirus.

10. The attenuated oncolytic virus of any one of claims 1 to 9, wherein the attenuated oncolytic virus is capable of preferential replication in a tumor cell.

11. A composition comprising the attenuated oncolytic virus of any one of claims 1 to 10.

12. The composition of claim 11, wherein the composition is a therapeutic composition or a vaccine.

13. The composition of claim 11 or claim 12, wherein the composition is used in a cancer immunotherapy.

14. The composition of any one of claims 11 to 13, further comprising one or more one or more therapeutically acceptable carriers, therapeutically acceptable diluents, therapeutically acceptable excipients, or other therapeutic agents.Jecho Labs ref no: 240419McAndrews ref no: 68466WO0115. The composition of claim 14, wherein the therapeutically acceptable excipients are selected from the group consisting of salts, buffering agents, preservatives, anti- adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers, film formers, coatings, flavors, fragrances, glidants, lubricants, sorbents, suspending or dispersing agents, sweeteners, waters of hydration, and combinations thereof.

16. A method of treating cancer, for inhibiting tumor cell growth, and / or for enhancing an immune response, the method comprising administering an effective amount of the attenuated oncolytic virus of any one of claims 1 to 10 or a composition of any one of claims 11 to 15 to a subject in need thereof.

17. The method of claim 16, wherein the attenuated oncolytic virus targets CD155 positive cancer cells.

18. The method of claim 16 or claim 17, wherein the subject has elevated levels of CD155.

19. The method of any one of claims 16 to 18, wherein the subject has or is suspected to have a cancer selected from the group consisting of head and neck squamous cell carcinomas (HNSCC), melanoma, breast cancer, colorectal cancer, prostate cancer, glioblastoma, hepatocellular carcinomas, lung cancer, liver cancer, gastric cancer, brain cancer, colon cancer, cervical cancer, kidney cancer, ovarian cancer cells, pancreatic cancer cells, and esophageal cancer.

20. The method of any one of claims 16 to 19, wherein the attenuated oncolytic virus is administered to the subject intravenously, intratumorally, systemically, intraperitoneally, subcutaneously, or intramuscularly.Jecho Labs ref no: 240419 McAndrews ref no: 68466WO0121. The method of any one of claims 16 to 20, further comprising administering one or more additional therapies to the subject in need thereof.

22. The method of claim 21, wherein the one or more additional therapies are selected from the group consisting of surgery, radiation, chemotherapy, immunotherapy, hormone therapy, and a combination thereof.

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