Artificial oncolytic viruses and related methods

Abstract

The disclosure provides artificial viral compositions for use in treating cancer or a hyperproliferative disorder in a subject to whom the compositions are administered, as well as to methods of making and using the compositions.

Description

[0001]

[0002] ARTIFICIAL ONCOLYTIC VIRUSES AND RELATED METHODS

[0003] RELATED APPLICATIONS

[0004] This application is related to U.S. Provisional Application No. 63 / 632,325, filed on April 10, 2024, and U.S. Provisional Application No. 63 / 688,101, filed on August 28, 2024. The entire teachings of the above application are incorporated herein by reference.

[0005] BACKGROUND OF THE INVENTION

[0006] In 2024, an estimated 2,001,140 new cases of cancer will be diagnosed in the United States and 611,720 people will die from the disease. The treatment of cancer has progressed as understanding of the underlying biological processes has increased. However most current treatment options, including surgery, radiation, chemotherapy, immunotherapy, and newer targeted therapies, continue to be deployed relatively late in cancer development and have undesirable side effects even if successful in addressing the cancer.

[0007] Solid tumors present particular therapeutic challenges; for example, cells of a solid tumor do not all present the same mix of antigens on their surfaces, complicating targeting strategies. Additionally, solid tumors can comprise masses of cells thousands of layers thick, making it difficult for therapeutic agents to infiltrate the tumor mass before being eradicated by the patient’s immune system. Some solid tumors also produce immune-suppressing agents such as the checkpoint molecule PD- Ll, which hamper strategies that enlist the host immune system in killing the tumor cells. Accordingly, treatments having increased effectiveness, precision, impact on quality of life and survivability are still needed. SUMMARY OF THE INVENTION

[0008] The inventions described herein relate to artificial oncolytic viruses for use in the treatment of cancer, as well as to methods of making these viruses and methods of treating cancer using them.

[0009] Disclosed herein is an artificial oncolytic virus. The oncolytic virus may comprise a glycoprotein engineered to selectively bind to a target cell via a binding partner identified as highly expressed on the target cell as compared with a non-target cell; and an aptazyme engineered to target a highly expressed protein, wherein the aptazyme controls replication of the oncolytic virus.

[0010] In some embodiments, the glycoprotein is engineered to comprise a nanobody, antibody or protein that targets a receptor. In some embodiments, the glycoprotein is engineered to comprise a nanobody targeting a cancer receptor. In some embodiments, the glycoprotein is engineered to comprise a nanobody that targets the glypican 3 (GPC3) receptor, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), transferrin receptor (TFRC), mucin 1 (MUC1), MET proto-oncogene receptor tyrosine kinase (MET), CD33, CD19, CD47, chemokine receptor 4 (CXCR4), fibroblast growth factor receptor (FGFR), death receptor 5 (DR5), TRAIL receptor 2 (TRAILR2), or trophoblast cell surface antigen 2 (TROP2). In some embodiments, the glycoprotein is engineered to comprise a nanobody that targets the GPC3 receptor or EpCAM. The glycoprotein may be engineered to remove its natural infectivity.

[0011] In some embodiments, the aptazyme is engineered to target a cancer- specific protein. The aptazyme may be engineered to target beta catenin (CTNNB1), alpha fetoprotein (AFP), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), telomerase reverse transcriptase (TERT), matrix metalloproteinase-9 (MMP9), oncostatin M protein (OSM), secreted phosphoprotein 1 (SPP1), p53, phosphorylated ERK1, phosphorylated ERK2, or estrogen receptor (ER). In one aspect, the aptazyme comprises a Hammer head ribozyme (HH Rbz). In other aspects, the aptazyme comprises a Hepatitis D virus ribozyme, a Twister ribozyme, or a Pistol ribozyme. In some embodiments, the aptazyme comprises a HH Rbz and is engineered to target CTNNB 1. In some embodiments, the aptazyme comprises a HH Rbz and is engineered to target AFP. In one embodiment, the aptazyme comprises a HH Rbz and is engineered to target CTNNB 1 and wherein the glycoprotein is engineered to comprise a nanobody that targets the GPC3 receptor. In one embodiment, the aptazyme comprises a HH Rbz and is engineered to target CTNNB 1 and wherein the glycoprotein is engineered to comprise a nanobody that targets EpCAM. In one embodiment, the aptazyme comprises a HH Rbz and is engineered to target AFP and wherein the glycoprotein is engineered to comprise a nanobody that targets the GPC3 receptor. In one embodiment, the aptazyme comprises a HH Rbz and is engineered to target AFP and wherein the glycoprotein is engineered to comprise a nanobody that targets EpCAM.

[0012] In some embodiments, the target cell is a cancer cell. In some embodiments, the target cell is a cell associated with a hyperproliferative disorder. In some embodiments, the target cell is a tumor cell.

[0013] In some embodiments, the virus is non-pathogenic in a subject to whom it is intended to be administered. In some embodiments, the virus is one to which a subject to whom it is intended to be administered is not immune. In some embodiments, the subject is Homo sapiens.

[0014] In some embodiments, the virus comprises at least one domain which is identical to a reference naturally-occurring virus. The reference naturally-occurring virus may be a member of the Rhabdoviridae family. For example, the reference naturally-occurring virus is from the genus Vesiculovirus. In one aspect, the reference naturally-occurring virus comprises Vesicular stomatitis virus (VSV).

[0015] In some embodiments, the engineered glycoprotein is derived from the Togaviridae family. For example, the engineered glycoprotein is derived from the genus Alphavirus. In some embodiments, the engineered glycoprotein is derived from Sindbis virus (SINV). In some embodiments, the aptazyme regulates production of the nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), or large polymerase subunit (L).

[0016] In some embodiments, when administered to a subject said virus causes cell death via apoptosis, necrosis, and / or cytopathic effect (CPE) of one or more target cells in said subject. Cell death may occur within 3 months, preferably within 1 month, preferably within 28 days, preferably within 21 days, more preferably within 7 days, more preferably within 2-4 days. In some embodiments, the target cell is selected from the group consisting of cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, and uterus. In some embodiments, the target cell is a cancer cell and wherein the cancer is selected from the group consisting of melanoma, non- small cell lung cancer, smallcell lung cancer, lung cancer, hepatocarcinoma, retinoblastoma, astrocytoma, glioblastoma, gum cancer, tongue cancer, leukemia, neuroblastoma, head cancer, neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, lymphoma, brain cancer, colon cancer, and bladder cancer.

[0017] In some embodiments, said virus additionally comprises one or more heterologous functional domains wherein said functional domain is selected from the group consisting of a therapeutic agent, a kill switch for said target cell, an agent which facilitates the ability of the virus to evade the recipient immune system, a watermark, a barcode, an agent which degrades the extracellular matrix of a solid tumor, and a diagnostic agent. For example, the one or more heterologous functional domains are inserted in frame.

[0018] Also disclosed herein are pharmaceutical compositions comprising the artificial oncolytic virus as described herein. Further disclosed herein are methods of treating a hyperproliferative disorder in a subject comprising administering to the subject the pharmaceutical composition described herein.

[0019] In some embodiments, the hyperproliferative disorder is cancer. For example, the cancer is selected from the group consisting of melanoma, non- small cell lung cancer, small-cell lung cancer, lung cancer, hepatocarcinoma, retinoblastoma, astrocytoma, glioblastoma, gum cancer, tongue cancer, leukemia, neuroblastoma, head cancer, neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, lymphoma, brain cancer, colon cancer, and bladder cancer. In some embodiments, the subject is Homo sapiens.

[0020] Also disclosed herein are methods of slowing, inhibiting or reducing the growth or size of a tumor comprising administering to the subject the pharmaceutical composition described herein. In some embodiments, the tumor is selected from the group consisting of non- small cell lung cancer, small-cell lung cancer, lung cancer, hepatocarcinoma, retinoblastoma, astrocytoma, glioblastoma, neuroblastoma, head cancer, neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, brain cancer, colon cancer, and bladder cancer. The subject may be Homo sapiens.

[0021] In some embodiments, the methods further include administering at least one interferon (IFN) inhibitor, such as a Type I IFN inhibitor and / or blocker. In some embodiments, the IFN inhibitor is co-administered with the artificial oncolytic virus. In some embodiments, the IFN inhibitor is sequentially administered with the artificial oncolytic virus (e.g., before or after administration of the artificial oncolytic virus or pharmaceutical compositions comprising the artificial oncolytic virus).

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0024] FIG. 1 shows immunocytochemistry (ICC) staining of HepG2, HepaRG, and NIH / 3T3 cells with wildtype VSV and HGI627 virus. Wildtype VSV is infectious in HepG2, but induced lysis and cell lifting in normal cells. HGI627 was shown to readily infect target liver cancer cells (HepG2).

[0025] FIG. 2 shows HGI267 demonstrated safety in normal, healthy mouse epithelial (NIH / 3T3) and human hepatic (HEPARG) cells.

[0026] FIG. 3 provides a schematic of an experimental design for assessing the efficacy of an oncolytic virus described herein. At the start mice are inoculated with 5xl06Hep3B cells. An oncolytic virus may be administered doses of IxlO6TCID50 (293T) by intraperitoneal (IP) injection on Day 1 and 17, followed by a dose of 3xl06TCID50 (293T) intratumoral (IT) injection on Day 24.

[0027] FIGS. 4A-4B demonstrate that intratumoral (IT) administration of an exemplary oncolytic virus shows significant therapeutic efficacy with durable survival benefit (FIG. 4A) and robust anti-tumor effects (FIG. 4B) in Hep3B xenograft model of childhood HCC.

[0028] FIG. 5 shows staining (H&E, Masson’s Trichrome, Human Mitochondria, and TUNEL) of lesions removed from mice treated with an exemplary oncolytic virus (HGI627) and a vehicle.

[0029] FIG. 6 shows stains of lesions removed from mice treated with an exemplary oncolytic virus (HGI627) and a vehicle for the presence of neutrophils and macrophages.

[0030] FIG. 7 provides schematics of a wildtype vesicular stomatitis virus (VSV) and an exemplary embodiment of an oncolytic virus described herein. An oncolytic virus may include various modifications including deletion of VSV-G glycoprotein, insertion of detargeted sindbis virus (SIN) glycoproteins, insertion of a synthetic anti- GPC3 nanobody for retargeting, insertion of a cancer protein dependent aptazyme as a genetic ON switch to control viral replication.

[0031] FIGS. 8A-8D provide a schematic of an experimental design for assessing an exemplary oncolytic virus described herein in vivo (FIG. 8A) and results of the assessment (FIGS. 8B-8D). Tumor volume was measured at 7, 14, 21 and 28 days post-treatment of mice administered an exemplary oncolytic virus (FIG. 8B) or a vehicle (FIG. 8C) and an average of the results was provided in FIG. 8D.

[0032] FIG. 9 provides a graph showing treatment with an exemplary oncolytic virus (HGI627) led to delayed and transient tumor regression, followed by eventual tumor regrowth.

[0033] FIG. 10 provides a graph showing the effect of administering an exemplary oncolytic virus (HGI627) with an interferon inhibitor. Administration of an interferon inhibitor in combination with an exemplary oncolytic virus at a lower MOI resulted in similar results of administering the exemplary oncolytic virus at a higher MOI.

[0034] FIG. 11 provides a graph showing that cytotoxicity increases when an interferon inhibitor in administered in combination with an exemplary oncolytic virus (HGI627).

[0035] DETAILED DESCRIPTION OF THE INVENTION Oncolytic viruses are a subset of lytic viruses that selectively infect and replicate in cancer cells with little or no effect on normal cells. Oncolytic viral therapies harness the basic biological principles of the virus; the virus replicates in cancer cells, and as the infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining cancer cells or tumor. Often these viruses replicate in dividing cells preferentially over non-dividing cells. Importantly, the viral replication cycle allows local amplification of the virus, and the oncolytic process continues as long as target cells exist. While the potential of oncolytic viral therapies has been recognized for some time, real world obstacles to broad utility of this therapeutic approach remain and are addressed by the inventions described herein. Although a degree of natural tumor-selectivity can be demonstrated for some virus species, new approaches are still needed to engineer and / or enhance tumor- selectivity for oncolytic viruses in order to maximize safety. This selectivity is particularly important when intravenous administration is used, and / or when potentially toxic therapeutic genes are added to the viruses to enhance antitumoral potency; gene expression must be tightly limited in normal tissues.

[0036] The inventions disclosed herein provide viral compositions for use in inducing the regression of a tumor or neoplasia, reducing the size of or eliminating a tumor or neoplasia, and treating or eliminating cancer (e.g., solid tumor cancers or blood cancers) or a hyperproliferative disorder in a subject to whom the compositions are administered. The viral compositions of the invention are artificial (i.e., non-naturally occurring and not identical to naturally occurring compositions), and in some embodiments fully synthetic (i.e., synthesized completely de novo as opposed to beginning with and modifying a naturally occurring virus). The inventions also provide methods of making these compositions and methods of using these compositions therapeutically, particularly methods of selectively killing cancer cells by contacting them with the viral compositions described herein.

[0037] Although numerous benefits can be derived from the inventions described herein, of particular note are the cancer cell targeting specificity (i.e., selective infection) and cancer cell replication specificity (i.e., selective replication) exhibited by the artificial viruses of the invention. Both specificities are generally conferred by engineering the artificial virus based on bioinformatic analysis of the cancer cell to be targeted as described further herein. The bioinformatic analysis can be conducted on one or more samples from a single individual (i.e., giving rise to a personalized therapeutic approach) or on groups of samples (stratified, for example, by type of cancer, origin of cancer, stage of cancer, time course of cancer progression, time course of cancer treatment, ethnicity, gender, age, etc.) to inform a pan-generic therapeutic approach. The bioinformatic analysis is conducted to identify genes that are differentially expressed between the cells of the sample(s) and normal or nontarget cells, and / or to determine expression levels of differentially expressed genes. As a result of this analysis, one or more artificial oncolytic viruses can be engineered to preferentially or specifically bind to (and thereby infect) a cancer cell by binding to a gene product identified as preferentially or specifically expressed on the cancer cell. In certain embodiments the gene product is highly expressed on the cancer cell in addition to being preferentially or specifically expressed. Additionally or alternatively, one or more artificial oncolytic viruses can be engineered such that transcription or replication of the viral genome is under the control of a regulatory region of a gene identified as preferentially or specifically expressed by the cancer cell, resulting in cell- or tumor- specific or -preferential expression or replication of the viral genome. In certain embodiments the gene product is highly expressed by the cancer cell in addition to being preferentially or specifically expressed. In this manner binding and / or expression of the artificial oncolytic viruses can be engineered to greatly reduce or eliminate binding to / infection of and / or replication in non-target cells. These and other advantages of the disclosed inventions will be apparent from the description.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art. The following references provide one of skill with a general definition of many of the terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

[0039] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, tissue culture and transformation, protein purification, etc. Enzymatic reactions and purification techniques may be performed according to the manufacturer's specifications or as commonly accomplished in the art or as described herein. The following procedures and techniques may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the specification. See, e.g., Sambrook et al., 2001, Molecular Cloning: A Laboratory Manuel, 3.sup.rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclature used in connection with, and the laboratory procedures and techniques of, analytic chemistry, organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques may be used for chemical synthesis, chemical analyses, pharmaceutical preparation, formulation, and delivery and treatment of patients.

[0040] Viral Compositions

[0041] Embodiments of the invention relate to artificial oncolytic viruses comprising a domain engineered to selectively bind to a target cell via a binding partner identified as highly expressed or preferentially expressed on the target cell as compared with a non-target cell. In some embodiments the binding partner is both highly expressed and preferentially expressed on the target cell as compared with a non-target cell. Embodiments of the invention also relate to artificial oncolytic viruses wherein expression of the viral genome is engineered to be under the control of a regulatory region of a gene identified as highly expressed or preferentially expressed by the target cell as compared with a non-target cell. In some embodiments the gene is both highly expressed and preferentially expressed by the target cell as compared with a non-target cell. In some embodiments either or both of the binding partner and the gene are specifically expressed by the target cell. In some embodiments the target cell is a cancer cell, such as a tumor cell. In certain embodiments the artificial oncolytic virus both comprises a domain engineered to selectively bind to a target cell via a binding partner identified as highly expressed or preferentially expressed on the target cell as compared with a non-target cell and wherein expression of the viral genome is engineered to be under the control of a regulatory region of a gene identified as highly expressed or preferentially expressed by the target cell as compared with a non-target cell.

[0042] Viral compositions of the present invention are preferably non-pathogenic in a subject to whom the virus is intended to be administered. Additionally, viral compositions of the invention are preferably those to which a subject to whom it is intended to be administered has reduced or no pre-existing immunity, as it is desirable to delay any immune response to the virus to enable the virus to infect and destroy target cells without interference from the subject’s immune system.

[0043] Viral compositions of the present invention, while in some embodiments similar to a reference naturally-occurring or wild type virus, are artificial and do not exist in nature. Viral compositions of the invention may be substantially identical to (i.e., derived from) a reference naturally-occurring virus. As used herein, an oncolytic virus which is derived from a reference virus comprises a nucleic acid sequence or amino acid sequence which is possessed by the reference virus. In some embodiments an oncolytic virus which is "derived from" a reference virus comprises one or more genes possessed by the reference virus. In some embodiments an oncolytic virus which is derived from a reference virus encodes one or more proteins encoded by the reference virus. In some embodiments, an oncolytic virus which is derived from a reference virus may comprise a nucleic acid sequence encoding one or more functional elements of the reference virus. A "functional element" may, e.g., be a transcriptional regulator, a regulator of post-transcriptional processing, a translational regulator, a regulator of post-transcriptional processing, a response element, a repeat sequence, or a viral protein (e.g., viral protease). In some embodiments, an oncolytic virus which is derived from a reference virus may comprise one or more genes of, or proteins encoded by, the reference virus.

[0044] In some embodiments, the virus comprises at least one domain which is identical to a domain of a reference naturally-occurring virus. In some embodiments the virus is identical to a reference naturally-occurring virus except for one or more engineered domains and / or the engineered regulatory domains. In some embodiments, the viral sequence of the oncolytic virus comprises portions from one or more viruses. For example, a viral sequence may be based on a reference naturally-occurring virus (e.g., VSV), but that sequence may be modified or engineered to include aspects from other viruses or to include one or more supplemental agents (e.g., modified proteins and / or addition of ON / OFF switches).

[0045] Reference naturally-occurring viruses can include, but are not limited to measles virus (MV), rabies virus (RABV), Gibbon Ape Leukemia Virus (GALV), Sendai Virus (SeV), Seneca valley virus (SVV), an adenovirus (Ad), vesicular stomatitis virus (VSV), autonomous parvovirus, herpes simplex virus (HSV), vaccinia virus (VV), myxoma virus (MYXV), Newcastle disease virus (NDV), reovirus, retrovirus, influenza virus, Sindbis virus (SIN), canine distemper virus (CDV), cowpea mosaic virus (CPMV), Nipah virus (NiV), Tupaia paramyxovirus (TPMV), or poxvirus, as examples. In some aspects, a reference virus is one or more of non- pathogenic, has low or no seroprevalence, is selective or cancer-specific, supports transgene insertion and expression, and is genetically stable. For example, in some embodiments the reference naturally-occurring virus can be a member of the Rhabdoviridae family, such as from the genus Vesiculovirus. In certain instances, the reference naturally-occurring virus can be Indiana vesiculovirus or New Jersey vesiculovirus. Exemplary substrains of Indiana vesiculovirus include, but are not limited to, San Juan, Mudd-Summers, Orsay, and Glasgow. In one embodiment, vesicular stomatitis virus (VSV) is an exemplary reference naturally-occurring virus because it exhibits low seroprevalence, robust oncolysis, is well characterized, and has high transgene carrying capacity. See Basu et al. “Rational Selection of an Ideal Oncolytic Virus to Address Current Limitations in Clinical Translation,” Inti. Rev. Cell Mol. Biol. 2023:379:241-261, incorporated herein by reference.

[0046] In some embodiments, the virus (e.g., a virus derived from a reference virus) comprises one or more untranslated regions (UTRs). In some embodiments, the virus comprises a 5' and / or 3' untranslated region (UTR). In one embodiment, the virus described herein does not comprise a 5' or 3' UTR. In another embodiment, the virus comprises either a 5' or 3' UTR. In another embodiment, the virus described herein comprises both a 5' and a 3' UTR. In some embodiments, the 5' UTR, the 3' UTR, or both comprise one or more modified nucleosides.

[0047] In some embodiments, the reference virus is modified or engineered to include one or more portions of the sequence from a second virus, and optionally from a third, fourth, fifth, etc. virus. In some embodiments, the oncolytic virus comprises one or more genes selected from the group consisting of nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and large polymerase subunit (L). In some embodiments, a sequence comprises one or more genes selected from the group consisting of nonstructural protein 1 (nsPl), nonstructural protein 2 (nsP2), nonstructural protein 3 (nsP3), nonstructural protein 4 (nsP4), capsid protein (C), spike glycoprotein El (El), spike glycoprotein E2 (E2), assembly protein E3 (E3), assembly protein (6K), fusion (F) glycoprotein, and hemagglutinin (H) glycoprotein. In some embodiments the artificial oncolytic virus additionally comprises one or more heterologous functional domains, wherein said functional domain is selected from the group consisting of a therapeutic agent, a kill switch for said target cell, an agent which facilitates replication, an agent which facilitates the ability of the virus to evade the recipient immune system, a watermark, a barcode, an agent which degrades the extracellular matrix of a solid tumor, and a diagnostic agent.

[0048] In some embodiments, an oncolytic virus comprises a modified glycoprotein. In some aspects, a glycoprotein is modified to remove its natural infectivity, e.g., is de-targeted. In some aspects, a glycoprotein is modified to include a nanobody, protein, or antibody for retargeting. In some embodiments, the glycoprotein is modified to target glypican 3 (GPC3) receptor, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), transferrin receptor (TFRC) also known as CD71, mucin 1 (MUC1), MET proto-oncogene receptor tyrosine kinase (MET), CD33, CD19, CD47, chemokine receptor 4 (CXCR4), fibroblast growth factor receptor (FGFR), death receptor 5 (DR5), TRAIL receptor 2 (TRAILR2), or trophoblast cell surface antigen 2 (TROP2). In one embodiment, the glycoprotein is modified to target GPC3 receptor. In one embodiment, the glycoprotein is modified to target EpCAM. In some aspects, the oncolytic virus may be based on VSV, but the sequence is modified to remove the VSV glycoprotein and replace it with a scaffold based on the Sindbis virus (E3-E2- 6K-E1). In some aspects, a VSV glycoprotein is replaced by a Sindbis virus glycoprotein scaffold that is modified to remove its natural infectivity. In one embodiment, a Sindbis virus glycoprotein scaffold is further modified or additionally modified to target a receptor. The modified glycoprotein of the oncolytic virus facilitates the selective infection of the virus.

[0049] In some embodiments, an oncolytic virus according to the present disclosure may possess one or more of the following functional properties: ability to replicate in, and / or cause cell killing of, cancer cells; reduced ability to replicate in and / or cause cell killing of, non-cancerous cells as compared to the ability to replicate in, and / or cause cell killing of, cancer cells; comparable or improved ability to cause cell killing of cancer cells as compared to the ability of one or more oncolytic viruses known in the art; ability to help replication of helper-dependent adenovirus (HD Ad); comparable or improved ability to replicate in cancer cells as compared to the ability of one or more oncolytic viruses known in the art.

[0050] As used herein "wild-type" refers to the naturally occurring sequence of a nucleic acid at a genetic locus in the genome of an organism, and sequences transcribed or translated from such a nucleic acid. Thus, the term "wild-type" also may refer to the amino acid sequence encoded by the nucleic acid. As a genetic locus may have more than one sequence or alleles in a population of individuals, the term "wild-type" encompasses all such naturally occurring alleles. As used herein the term "polymorphic" means that variation exists (i.e., two or more alleles exist) at a genetic locus in the individuals of a population. As used herein, "mutant" refers to a change in the sequence of a nucleic acid or its encoded protein, polypeptide, or peptide that is the result of recombinant DNA technology.

[0051] A nucleic acid may be made by any technique known to one of ordinary skill in the art. Non-limiting examples of a synthetic nucleic acid, particularly a synthetic oligonucleotide, include a nucleic acid made by in vitro chemical synthesis using phosphotriester, phosphite or phosphoramidite chemistry and solid phase techniques such as described in EP 266,032, or via deoxy nucleoside H-phosphonate intermediates as described by Froehler et al., 1986, and U.S. Pat. No. 5,705,629. A non-limiting example of enzymatically produced nucleic acid includes one produced by enzymes in amplification reactions such as PCR.TM. (see for example, U.S. Pat. Nos. 4,683,202 and 4,682,195), or the synthesis of oligonucleotides described in U.S. Pat. No. 5,645,897. A non-limiting example of a biologically produced nucleic acid includes recombinant nucleic acid production in living cells, such as recombinant DNA vector production in bacteria (see for example, Sambrook et al. 1989). In some embodiments, the oncolytic virus may be designed and engineered using methods described in WO 2022 / 133154, which is incorporated herein by reference.

[0052] The nucleic acid(s), regardless of the length of the sequence itself, may be combined with other nucleic acid sequences, including but not limited to, promoters, enhancers, polyadenylation signals, restriction enzyme sites, multiple cloning sites, coding segments, noncoding segments, untranslated regions, cleavage sites, genetic barcodes, primer sites, and the like, to create one or more nucleic acid construct(s). The overall length may vary considerably between nucleic acid constructs. Thus, a nucleic acid segment of almost any length may be employed, with the total length preferably being limited by the ease of preparation or use in the intended recombinant nucleic acid protocol.

[0053] In some embodiments, an oncolytic virus includes an agent to control replication of the virus. For example, the oncolytic virus may include one or more aptazymes to facilitate selective replication of the virus. Aptazymes are self-cleaving units composed of a ribozyme / riboswitch and an aptamer. The aptamer may be a single stranded sequence or oligonucleotide that may bind a specific target protein. If the aptamer is present, the riboswitch may either turn “ON” or “OFF” depending on the design. In some embodiments, the aptazyme acts as an “ON” switch in the presence of an endogenous protein. In some embodiments, the aptazyme acts as an “OFF” switch in the presence of an endogenous protein. In some aspects, the endogenous protein is highly expressed. In some embodiments, the aptazyme is a cancer protein dependent aptazyme. In some embodiments, an aptamer targets beta catenin (CTNNB1), alpha fetoprotein (AFP), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), telomerase reverse transcriptase (TERT), matrix metalloproteinase- 9 (MMP9), oncostatin M protein (OSM), secreted phosphoprotein 1 (SPP1), p53, phosphorylated ERK1, phosphorylated ERK2, or estrogen receptor (ER). In one embodiment, an aptamer targets AFP. In one embodiment, an aptamer targets CTNNB 1. In some embodiments, a riboswitch comprises a Hammer head ribozyme (HH Rbz). For example, the riboswitch may comprise satellite RNA of tobacco ringspot virus (sTRSV) sequence for the HHRbz. In one embodiment, an aptazyme comprises an aptamer targeting CTNNB1 and further comprises a HH Rbz riboswitch. In other embodiments, a riboswitch comprises a Hepatitis D virus (HDV) ribozyme, Twister ribozyme, or a Pistol ribozyme.

[0054] In some aspects, the aptazyme is located or placed behind the nucleocapsid (N) protein, the glycoprotein (G) (e.g., the retargeted glycoprotein), matrix (M) protein, phosphoprotein (P), and / or large polymerase subunit (L). In some aspects, the aptazyme regulates the production of the nucleocapsid (N) protein of the oncolytic virus. In some aspects, the aptazyme regulates the production of the retargeted glycoprotein (G) of the oncolytic virus. In some aspects, the aptazyme regulates VSV-P or VSV-L. In some aspects, an aptazyme is located in the 5’ UTR of the controlled gene. In other aspects, an aptazyme is located in the 3’ UTR of the controlled gene (e.g., behind a gene / coding sequence). In one aspect, a first aptazyme is located in the 5’ UTR and a second aptazyme is located in the 3’ UTR of the controlled gene (e.g., VSV-N, VSV-P, VSV-L, and / or a retargeted glycoprotein).

[0055] In some embodiments, the aptazyme is incorporated into a coding sequence. For example, the aptazyme may be incorporated into the phosphoprotein (P) coding sequence (e.g., VSV-P coding sequence). In one aspect, the aptazyme is incorporated into the VSV-P coding sequence at amino acid position 196. In some aspects, the aptazyme present in the coding sequence (e.g., the P coding sequence) may cleave the RNA sequence into multiple pieces in the absence of a target protein (e.g., a target cancer protein) thereby resulting in non-complete, non-functional protein (e.g., P protein) and limiting viral replication. In contrast, the presence of the target protein (e.g., the target cancer protein) results in the aptamer region of the aptazyme to bind the protein thereby causing a conformational shift in the RNA sequence resulting in no cleavage. Thus, the mRNA of the coding sequence (e.g., VSV-P) is maintained as a single sequence strand that may be translated into a complete, functional protein. In some embodiments, linker and / or spacer sequences are inserted immediately upstream and / or downstream of the aptazyme sequence. The insertion of linker and / or spacer sequences may increase flexibility and tolerance for the insertion of the aptazyme into the coding sequence.

[0056] The aptazyme activity is designed to focus on maintaining (e.g., in the presence of a target protein, such as a cancer protein) or cleaving (e.g., in the absence of the target protein) a poly A tail. The loss of the poly A tail may lead to destabilization of RNA and, optionally, degradation. In some embodiments, degradation domains and / or stabilization domains may be added. In some aspects, the orientation comprises a viral gene / stabilization domain / aptazyme / degradation domain / motif. In some embodiments, the more downstream sequence takes preference (e.g., the degradation domain / motif). In some embodiments, the aptazyme is designed / selected such that cleavage does not naturally occur, but in the presence of the target protein (e.g., the target cancer protein) cleavage occurs. Thus, the degradation domain is cleaved in the presence of the target protein and the stabilization domain is dominant, thereby resulting in stable RNA that can be made into protein. In some aspects, the aptazyme is an OFF switch aptazyme acting as a genetic ON switch.

[0057] An oncolytic virus as described herein may comprise a vesicular stomatitis virus (VSV) as a platform that is modified to replace the native VSV glycoprotein with a detargeted sindbis virus (SIN) glycoprotein scaffold (E3-E2-6K-E1) and the insertion of a synthetic anti-GPC3 nanobody for retargeting. In addition, the VSV platform of the oncolytic virus is further modified to include the insertion of a cancer protein dependent aptazyme as a genetic ON switch to control viral replication. In some aspects, the aptazyme regulates the production of the N protein. In other aspects, the aptazyme regulates the production of the glycoprotein. In one embodiment, an oncolytic virus comprises a glycoprotein modified to target GPC3 and an aptamer targeting CTNNB 1. In one embodiment, an oncolytic virus comprises glycoprotein modified to target GPC3 and an aptamer targeting AFP. In one embodiment, an oncolytic virus comprises a glycoprotein modified to target EpCAM and an aptamer targeting CTNNB 1. In one embodiment, an oncolytic virus comprises a glycoprotein modified to target EpCAM and an aptamer targeting AFP.

[0058] By "expression construct" or "expression cassette" is meant a nucleic acid molecule that is capable of directing transcription. An expression construct includes, at a minimum, one or more transcriptional control elements that direct gene expression in one or more desired cell types, tissues or organs. Additional elements, such as a transcription termination signal, may also be included. A "vector" or "construct" (sometimes referred to as a gene delivery system or gene transfer "vehicle") refers to a macromolecule or complex of molecules comprising a polynucleotide to be delivered to a host cell, either in vitro or in vivo. A "plasmid," a common type of a vector, is an extra-chromosomal DNA molecule separate from the chromosomal DNA that is capable of replicating independently of the chromosomal DNA. In certain cases, it is circular and double- stranded. In other cases, it is linear.

[0059] By "operably linked" or co-expressed" with reference to nucleic acid molecules is meant that two or more nucleic acid molecules (e.g., a nucleic acid molecule to be transcribed and an enhancer element) are connected in such a way as to permit transcription of the nucleic acid molecule. "Operably linked" or "coexpressed" with reference to peptide and / or polypeptide molecules means that two or more peptide and / or polypeptide molecules are connected in such a way as to yield a single polypeptide chain, i.e., a fusion polypeptide, having at least one property of each peptide and / or polypeptide component of the fusion. The fusion polypeptide is preferably chimeric, i.e., composed of heterologous molecules.

[0060] Pharmaceutical Compositions and Methods of Treatment

[0061] Some embodiments of the present invention relate to methods of treatment for a hyperproliferative disease, such as cancer, by the delivery of a pharmaceutical composition comprising an effective amount of one or more artificial oncolytic viruses described herein. An effective amount of the pharmaceutical composition is an amount sufficient to induce oncolysis in a cell to which the composition is administered and / or the slowing, inhibition or reduction (including complete eradication) in the growth or size of a tumor and / or to treat a cancer in a subject to whom the composition is administered. The cytotoxic effects under in vitro or in vivo conditions can be detected by various means as known in the art, for example, by detecting tumor size using gadolinium enhanced MRI scanning, by radiolabeling of a tumor, and the like.

[0062] In some embodiments, the one or more artificial oncolytic viruses are administered in combination with one or more supplemental agents. In one embodiment, the one or more artificial oncolytic viruses are administered in combination with an interferon (IFN) inhibitor and / or blocker (e.g., an IFN pathway inhibitor, such as a Type I IFN inhibitor). In some embodiments, a pharmaceutical composition comprising one or more artificial oncolytic viruses further comprises one or more interferon inhibitors. In some embodiments, an IFN inhibitor comprises one or more inhibitors. In one embodiment, an IFN inhibitor comprises a Type I IFN antibody mixture, such as anifrolumab. In some aspects, an IFN inhibitor or blocker may include a Janus kinase (JAK) inhibitor. Non-limiting examples of JAK inhibitors include ruxolitinib, baricitinib, tofacitinib, Upadacitinib, abrocitinib, filgotinib, peficitinib, delgocitinib, pacritinib, fedratinib, and deucravacitinib. Administration of an interferon inhibitor in combination with an artificial oncolytic virus may result in enhanced oncolysis and / or acceleration of cell death, e.g., in vitro. Further, administration of an interferon inhibitor in combination with an artificial oncolytic virus may result in accelerated onset and / or progression of cytotoxicity. In some aspects, the IFN inhibitor is co-administered with the artificial oncolytic virus. In alternative embodiments, the IFN inhibitor is sequentially administered with the artificial oncolytic virus (e.g., administered before or after administration of the artificial oncolytic virus or compositions comprising the artificial oncolytic virus).

[0063] As used herein, the terms "treat" and "treating" refers to a treatment / therapy from which a subject receives a beneficial effect, such as the reduction, decrease, attenuation, diminishment, stabilization, remission, suppression, inhibition or arrest of the development or progression of cancer, or a symptom thereof. In certain embodiments, the treatment / therapy that a subject receives results in at least one or more of the following effects: (i) the reduction or amelioration of the severity of cancer and / or a symptom associated therewith; (ii) the reduction in the duration of a symptom associated with cancer; (iii) the prevention in the recurrence of a symptom associated with cancer; (iv) the regression of cancer and / or a symptom associated therewith; (v) the reduction in hospitalization of a subject; (vi) the reduction in hospitalization length; (vii) the increase in the survival of a subject; (viii) the inhibition of the progression of cancer and / or a symptom associated therewith; (ix) the enhancement of or improvement in the therapeutic effect of another therapy; (x) a reduction or elimination in the cancer cell population; (xi) a reduction in the growth of a tumor or neoplasm; (xii) a decrease in tumor size; (xiii) a reduction in the formation of a tumor; (xiv) eradication, removal, or control of primary, regional and / or metastatic cancer; (xv) a decrease in the number or size of metastases; (xvi) a reduction in mortality; (xvii) an increase in cancer- free survival rate of patients;

[0064] (xviii) an increase in relapse-free survival; (xix) an increase in the number of patients in remission; (xx) a decrease in hospitalization rate; (xxi) the size of the tumor is maintained and does not increase in size or increases the size of the tumor by less than 5% or 10% after administration of a therapy as measured by conventional methods available to one of skill in the art, such as MRI, X-ray, and CAT Scan; (xxii) the prevention of the development or onset of cancer and / or a symptom associated therewith; (xxiii) an increase in the length of remission in a subject to whom the therapy is administered; (xxiv) the reduction in the number of symptoms associated with cancer; (xxv) an increase in symptom-free survival of cancer patients; and / or (xxvi) limitation of or reduction in metastasis. In some embodiments, the treatment / therapy that a subject receives does not cure cancer, but prevents the progression or worsening of the disease. In certain embodiments, the treatment / therapy that a subject receives does not prevent the onset / development of cancer, but may prevent the onset of cancer symptoms.

[0065] As used herein, the terms "patient" or "subject" are used interchangeably and mean a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline. Preferably, the patient is a human (Homo sapiens) or a canine (e.g., Canis lupus familiaris). The subject may be of any gender. A subject may have been diagnosed with a cancer requiring treatment, may be suspected of having such a cancer, or may be at risk of developing such a cancer.

[0066] Examples of cancer contemplated for treatment in accordance with the invention include, but are not limited to, liver cancer, lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, leukemias, lymphomas, pre- neoplastic lesions in the lung, colon cancer, melanoma, and bladder cancer.

[0067] In some embodiments the composition is administered to a subject who has a tumor. The tumor can be, for example, a brain cancer tumor, a head & neck cancer tumor, an esophageal cancer tumor, a skin cancer tumor, a lung cancer tumor, a thymic cancer tumor, a stomach cancer tumor, a colon cancer tumor, a liver cancer tumor, an ovarian cancer tumor, a uterine cancer tumor, a bladder cancer tumor, a testicular cancer tumor, a rectal cancer tumor, a breast cancer tumor, or a pancreatic cancer tumor. The tumor can be a primary tumor or a metastatic tumor or a recurrent tumor.

[0068] Cancer cells that may be treated by methods and compositions of the invention include cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin’s disease; Hodgkin’s; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified nonHodgkin’s lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0069] The present invention contemplates methods for inhibiting or preventing local invasiveness and / or metastasis of any type of primary cancer. For example, the primary cancer may be melanoma, non-small cell lung, small-cell lung, lung, hepatocarcinoma, hepatocellular carcinoma, hepatoblastoma, retinoblastoma, astrocytoma, glioblastoma, gum, tongue, leukemia, neuroblastoma, head, neck, breast, lung, pancreatic, prostate, renal, bone, testicular, ovarian, mesothelioma, cervical, gastrointestinal, lymphoma, brain, colon, or bladder. Moreover, the present invention can be used to prevent cancer or to treat pre-cancers or premalignant cells, including metaplasias, dysplasias, and hyperplasias. It may also be used to inhibit undesirable but benign cells, such as squamous metaplasia, dysplasia, benign prostate hyperplasia cells, hyperplastic lesions, and the like. The progression to cancer or to a more severe form of cancer may be halted, disrupted, or delayed by methods of the invention as discussed herein.

[0070] In accordance with methods of the invention, treatment comprises contacting one or more cancer cells or tumors with a composition (e.g., an oncolytic virus) according to the invention. The routes of administration will vary, naturally, with the location and nature of the lesion, and include, e.g., intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, regional (e.g., in the proximity of a tumor, particularly with the vasculature or adjacent vasculature of a tumor), percutaneous, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, and oral administration and formulation. Treatment regimens may vary as well, and often depend on tumor type, tumor location, disease progression, and health and age of the patient.

[0071] The term "intravascular" is understood to refer to delivery into the vasculature of a patient, meaning into, within, or in a vessel or vessels of the patient. In certain embodiments, the administration is into a vessel considered to be a vein (intravenous), while in others administration is into a vessel considered to be an artery. Veins include, but are not limited to, the internal jugular vein, a peripheral vein, a coronary vein, a hepatic vein, the portal vein, great saphenous vein, the pulmonary vein, superior vena cava, inferior vena cava, a gastric vein, a splenic vein, inferior mesenteric vein, superior mesenteric vein, cephalic vein, and / or femoral vein. Arteries include, but are not limited to, coronary artery, pulmonary artery, brachial artery, internal carotid artery, aortic arch, femoral artery, peripheral artery, and / or ciliary artery. It is contemplated that delivery may be through or to an arteriole or capillary.

[0072] In some embodiments, the oncolytic virus is administered via intravenous injection, intraperitoneal injection or intratumoral injection. Intravenous or intraperitoneal injection may be administered intermittently, e.g., every 1-20 days, every 5-15 days, every 10-20 days, every 5-10 days, every 1-5 days, every 1-3 days, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the oncolytic virus is administered once or multiple times over a 14-day period. In some embodiments, the oncolytic virus to be administered via intravenous or intraperitoneal injection is at a dose of TCID50 by weight. In one embodiment, the oncolytic virus is administered to a mouse subject at a dose of IxlO4to IxlO8TCID50 / kg. In one embodiment, the oncolytic virus is administered to a human subject a dose of IxlO8to IxlO11TCID50 / kg.

[0073] Intratumoral injection, or injection directly into the tumor vasculature, is specifically contemplated for discrete, solid, accessible tumors. Local, regional or systemic administration also may be appropriate. Intratumoral injection may be guided by ultrasound, CT or a combination of both. In some embodiments the volume to be administered can be, for example, up to 5 mL at a concentration of 109plaque-forming units (PFU) or fluorescent-forming units (FFU) per mL. The viral particles may advantageously be delivered by administering multiple injections to the tumor, spaced at approximately 1 cm intervals. Intratumoral injection may be administered intermittently, e.g., every 1-20 days, every 5-20 days, every 5-15 days, every 10-20 days, every 5-10 days, every 1-5 days, every 1-3 days or every 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. The oncolytic virus may be administered to supplement intraperitoneal or intravenous injection of the oncolytic virus. For example, the virus may be administered via intratumoral once per week as needed or per physician’s direction. In some embodiments, the oncolytic virus to be administered via intraperitoneal injection is at a dose of IxlO4to IxlO7TCID50 / kg for a human subject. The oncolytic virus may be diluted and injected with a volume ranging from 0.1 to 120 mL.

[0074] In some embodiments, a composition of the present invention (e.g., an oncolytic virus) is administered via one or more of intraperitoneal injection, intravenous injection and / or intratumoral injection. For example, an oncolytic virus may be administered via intravenous or intraperitoneal injection (e.g., one to three doses administered intermittently) and by intratumoral rejection as needed (e.g., one to three doses administered intermittently). Continuous administration also may be applied where appropriate, for example, by implanting a catheter into a tumor or into tumor vasculature. Such continuous perfusion may take place for a period from about 1-2 hours, to about 2-6 hours, to about 6-12 hours, or about 12-24 hours following the initiation of treatment. Generally, the dose of the therapeutic composition via continuous perfusion will be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the perfusion occurs. It is further contemplated that limb perfusion may be used to administer therapeutic compositions of the present invention, particularly in the treatment of melanomas and sarcomas.

[0075] In the case of surgical intervention, the present invention may be used preoperatively, to render an inoperable tumor suitable for resection or to address nonresected cells that may remain in the subject locally or metastatically. In certain embodiments, the tumor being treated may not, at least initially, be resectable. Treatments with therapeutic viral compositions may increase the resectability of the tumor due to shrinkage at the margins or by elimination of certain particularly invasive portions. Following treatments, resection may be possible. Additional treatments subsequent to resection will serve to eliminate microscopic residual disease at the tumor site.

[0076] The treatments may include various "unit doses" defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a specified period of time. Unit dose of the present invention may conveniently be described in terms of plaque forming units (pfu) or TCID50 for a viral construct.

[0077] The pharmaceutical compositions disclosed herein may be administered intratumor ally, parenterally, intravenously, intradermally, intramuscularly, transdermally or even intraperitoneally as described in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363, each of which is incorporated herein by reference.

[0078] Injection of nucleic acid constructs may be delivered by syringe or any other method used for injection of a solution, as long as the expression construct can pass through the particular gauge of needle required for injection and the dosage can be administered with the required level of precision.

[0079] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

[0080] The phrase "pharmaceutically-acceptable" or "pharmacologically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a subject (e.g., a canine or a human). As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the viral agent, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0081] The compositions and methods of the present invention may be used in the context of hyperproliferative diseases / conditions including cancer. In order to increase the effectiveness of a treatment with the compositions of the present invention, it may be desirable to combine these compositions with other agents effective in the treatment of those diseases and conditions. For example, the treatment of a cancer may be implemented with therapeutic compositions of the present invention in combination with other anti-cancer therapies, such as anti-cancer agents or surgery. As used herein, the term "in combination" in the context of the administration of (a) therapy(ies) to a subject, refers to the use of more than one therapy. The use of the term "in combination" does not restrict the order in which therapies are administered to a subject. A first therapy can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to a subject.

[0082] Administration of the viral compositions of the present invention to a patient in combination with a secondary therapy will follow general protocols for the administration of that particular secondary therapy, repeating treatment cycles as necessary. The secondary anti-cancer agent or therapy can be one or more therapies selected from the group consisting of, for example, chemotherapy, biological therapy, radiotherapy, immunotherapy, hormone therapy, anti-vascular therapy, cryotherapy, toxin therapy and surgery.

[0083] As used herein, an anti-cancer agent or therapy is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis, necrosis, and / or cytopathic effect (CPE) in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. Anti-cancer agents include biological agents (biotherapy), chemotherapy agents, and radiotherapy agents. More generally, these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with the viral composition and the secondary therapy or agent at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the viral expression construct and the other includes the second agent(s).

[0084] Alternatively, the viral therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agent and artificial virus are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and virus would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one may contact the cell with both modalities within about 12-24 h of each other and, more preferably, within about 6-12 h of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[0085] The inventions disclosed herein will be exemplified in a non-limiting manner by the following. The teachings of all references, patents and patent applications cited herein are incorporated fully by reference herein.

[0086] EXAMPLES

[0087] Example 1: Oncolytic Virus Therapy with Two-Factor Authentication Shows Robust Safety & Efficacy in Liver Cancer

[0088] INTRODUCTION

[0089] Hepatoblastoma (HB) and hepatocellular carcinoma (HCC) are common pediatric liver tumors with low survival rates [1-2]. Oncolytic virus therapy (OVT) is a promising modality, but efficacy has fallen short of expectations thus far. Vesicular stomatitis virus (VSV) is an ideal candidate for development (e.g., low seroprevalence, robust oncolysis, well-characterized, high transgene carrying capacity), but cancer specificity and safety need to be addressed [3].

[0090] GOALS

[0091] The inventors will engineer lytic VSV-based therapies for liver cancer with excellent safety through two-factor authentication (selective infection and selective replication). Nonclinical characterization of candidate oncolytic virus therapies and evaluation of safety and efficacy will be conducted.

[0092] METHODS

[0093] Viruses

[0094] HGI627 is a novel vesicular stomatitis indiana virus (VSIV) rationally designed for the treatment of liver cancer. Modifications include: deletion of VSV-G glycoprotein, insertion of detargeted sindbis virus (SIN) glycoproteins (E3-E2-6K- El), insertion of a synthetic anti-GPC3 nanobody for retargeting, insertion of a cancer protein dependent aptazyme as a genetic ON switch to control viral replication (FIG. 7).

[0095] In Vitro

[0096] TCID50 assays were conducted on HepG2, HepaRG, and NIH / 3T3 for virus quantification and calculated using Spearman-Karber. In addition, endpoint dilution assays were performed and immunocytochemistry (ICC) staining was carried out using anti-VSV (REA005; Imanis Life Sciences) to evaluate selectivity.

[0097] In Vivo

[0098] The efficacy of HGI627 was evaluated on Hep3B (human childhood hepatocellular carcinoma) cell line derived xenograft (CDX) in NSG mice (FIG. 3). HGI627 was administered doses of IxlO6TCID50 (293T) by intraperitoneal (IP) injection on Day 1 and 17, followed by a dose of 3xl06TCID50 (293T) intratumoral (IT) injection on Day 24 (FIG. 3). Histology was performed by HistoWiz Inc. (histowiz.com) using a Standard Operation Procedure and fully automated workflow. Samples were processed, embedded in paraffin, and sectioned at 4pm. Immunohistochemistry (IHC) was performed on a Bond Rx auto Stainer (Leica Biosystems) with enzyme treatment (1:1000) using standard protocols. Staining with anti-mitochondria (ab92824) was performed to differentiate human Hep3B xenograft from mouse cells. H&E, Masson’s Trichrome, and TUNEL (Promega) staining were performed by HistoWiz Inc. (histowiz.com). RESULTS

[0099] In Vitro

[0100] VSV wildtype is infectious in HepG2, but induced lysis and cell lifting in normal cells (FIG. 1). HGI627 readily infected target liver cancer cells (HepG2), but demonstrated safety in normal, heathy mouse epithelial (NIH / 3T3) and human hepatic cells (HepaRG) (FIGS. 1-2). Two-factor authentication in HGI627 showed significant increase in selectivity as seen by ON vs. OFF target titers by TCID50 assay (FIG. 2).

[0101] In Vivo

[0102] Intratumoral (IT) administration of HGI627 demonstrated significant therapeutic efficacy with durable survival benefit and robust anti-tumor effects in a Hep3B xenograft model of childhood HCC (FIG. 4). H&E highlighted differences in harvested tumor size and morphology (FIG. 5). Trichrome staining showed the widespread presence of collagen fibers in tumors treated with HGI627, indicating residual tumor volume measurements were primarily fibrotic tissue (FIG. 5). Mitochondria staining and quantitative analysis demonstrated that 3% of tumor volume is human cells (Hep3B), and the remainder are mouse cells and fibrosis (FIG. 5). TUNEE staining showed significant apoptosis is present in the remaining Hep3B cells (FIG. 5).

[0103] CONCEUSIONS

[0104] HGI627 demonstrated selective targeting and robust killing of liver cancer in vitro. Treatment with HGI627 showed potent anti-tumor activity and significantly improved survival of HCC in vivo. These initial results are encouraging and support further evaluation of HGI627 (additional in vivo studies are ongoing).

[0105] REFERENCES

[0106] [1] Czauderna P, Eopez-Terrada D, Hiyama E, Haberle B, Malogolowkin MH, Meyers RE. Hepatoblastoma state of the art: pathology, genetics, risk stratification, and chemotherapy. Curr Opin Pediatr. 2014 Feb ;26(1): 19-28. PMID: 24322718. [2] Czauderna P, Mackinlay G, Perilongo G, Brown J, Shafford E, Aronson D, Pritchard J, Chapchap P, Keeling J, Plaschkes J, Otte JB; Liver Tumors Study Group of the International Society of Pediatric Oncology. Hepatocellular carcinoma in children: results of the first prospective study of the International Society of Pediatric Oncology group. J Clin Oncol. 2002 Jun 15;20(12):2798-804. PMID: 12065556.

[0107] [3] Basu R, Moles CM. Rational selection of an ideal oncolytic virus to address current limitations in clinical translations. Int Rev Cell Mol Biol. 2023;379:241-261. Pub 2023 Apr 1. PMID: 27541726.

[0108] Example 2: Oncolytic Virus Therapy with Two-Factor Authentication Shows Robust Safety & Efficacy in Liver Cancer

[0109] Background: Multi-factored authenticated oncolytic virotherapy (OVT) is being pioneered using glycoprotein receptor matching and aptazyme technology to positively control replication in cytoplasm. Using VSV as a platform the OVTs show excellent safety and remain highly lytic. Sindbis virus glycoprotein was mutated to eliminate natural infectivity and retargeted to GPC3 with a codon optimized nanobody. An aptazyme was used to regulate viral replication.

[0110] Methods: 293T cells were used for virus rescue and titration by TCID50. Identity was confirmed by Nanopore sequencing. The inventors evaluated safety on off-target SK-Hep-1 and NCI-H28 cell lines, and efficacy on target HepG2 and patient derived HB17 cells. Cell health and GFP was measured at multiple timepoints up to 72 hours.

[0111] Results: Safety testing of HGI498 on SK-Hep-1 (GPC3 negative) and NCI- H28 (GPC3 negative, biomarker negative) showed no virus induced cell cytopathic effects in a range of 0.01 < MOI <1. HGI498’s efficacy testing on HepG2 and HB17 showed tumor cytotoxicity at MOI 0.01 (p<0.001). Endpoint dilution assay showed a 159-fold and 316-fold difference between off-target NCI-H28 and on-target HepG2 and HB17, respectively. The inventors incorporated an optimized aptazyme with the GPC3 targeted glycoprotein (HGI572), which was similarly rescued, titered, and evaluated for selectivity. HGI572 demonstrated approximately 1000-fold higher titer on target HepG2 and HB17 cells than off-target cells. Initial in vivo MTD study shows that both HGI498 and HGI572 are well tolerated, and are currently being investigated in vivo for efficacy on Hep3B in NSG mice. HGI498 has shown no seroprevalence (in vitro) and is stable at cryostorage for a minimum of 11 months.

[0112] Conclusion: The inventors have successfully engineered a two-factor authentication enabled OVT to address an unmet need for a therapy for liver cancer. This is the first report of an ON switch using a cancer protein to control viral replication.

[0113] Example 3 -

[0114] Introduction

[0115] An in vivo efficacy experiment was conducted to test the latest oncolytic viral therapy, HGI627, for its efficacy against liver cancer and specifically Hep3B cells.

[0116] Therapeutic Design and Assumptions

[0117] The oncolytic virus used in this experiment is based on platform work and uses the vesicular stomatitis virus (VSV), which is a negative sense RNA virus from the Rhabdoviridae family. To make the virus a safe oncolytic virus it is modified as follows: 1) To enable selective infection, the natural glycoprotein, which facilitates the infection of cells is removed and replaced by a scaffold based on the Sindbis virus. This scaffold is modified to remove its natural infectivity and accept a nanobody which targets the glypican 3 (GPC3) receptor. In this way the resulting virus can only infect cells that express the glypican 3 receptor. 2) The virus is further modified with an aptazyme, which acts as an active “ON " switch in the presence of another highly expressed protein.

[0118] The resulting virus, called HGI627, thus targets and replicates in cells that express the GPC3 receptor and another highly expressed protein. The replication eventually leads to cell lysis and cell death. It is known that liver cancer (HCC) and hepatoblastoma highly express the combination of the targeted proteins. The presence of each of these proteins by themselves in healthy cells is not common and the combination is rare. The hypothesis is that the virus should infect and kill Hepatocellular carcinoma or Hepatoblastoma cells and not infect or kill healthy cells, leading to maximum efficacy with low or no toxic side effects.

[0119] The safety of the approach using selective infection and selective replication has been established with earlier versions of the therapies with healthy NSG mice.

[0120] Experimental Design

[0121] The experiment was done with NSG mice, which are immunodeficient to accept xenografting the human Hep3B cells. At the start all mice were inoculated with 5xl06Hep3B cells. After 8 days the tumors started to become palpable at 4- 16mm3and the mice were randomized in groups of 5 animals. Group 1 was treated with placebo and Group 2 was treated with HGI627 (FIG. 3). The tumors were measured on Monday, Wednesday and Friday and the mice were given dietary supplements (DietGel) as needed to maintain weight.

[0122] Results

[0123] The Kaplan Meier graph of the experimental groups is provided in FIG. 4A. By day 31 post-treatment, all the vehicle / placebo group mice had to be euthanized due to excessive tumor burden (>2000mm3). All mice in the treated group made it until the endpoint of the study, which was chosen to be 8 weeks.

[0124] In the graph presented in FIG. 4A, the inventors illustrated the measured lesion volume over time, with measurements made on Monday, Wednesday, and Friday. Several observations can be made. The word lesion was used, not tumor, because especially for the treated group the inventors do not know how much of the volume is cancerous, how much is due to inflammation, collages or other cells.

[0125] The slight decrease observed in the control group's lesion volume at day 29 post-treatment can be attributed to the removal of mice bearing the largest lesions once they reach the maximum tumor burden, consequently lowering the average volume. The initial and subsequent intraperitoneal injections on days 1 and day 17 did not yield a large response. The inventors observed tumor inhibition of up to 32% before the intratumoral injection. Following the intratumoral injection administered on day 24, there is a rapid decrease in lesion volume within the treated group, continuing until a plateau is reached on day 37. The mice were then observed for an additional 19 days before the conclusion of the study. A significant unanswered question remained: was the final measured lesion volume indicative of a cancer, or could it have been something else resulting from the study, such as scar tissue (collagen), or fat? Are there remaining cancer cells?

[0126] After euthanizing the mice, the lesions were removed and prepared for histopathology. The inventors initially used four different stains, H&E, Trichrome, Human Mitochondria and TUNEL, which are depicted in FIG. 5. H&E is a basic stain to clearly show cells. Trichrome shows collagens and scarring. Human Mitochondria stains for human cells, which in the mouse would indicate what cells are specifically Hep3B and TUNEL stains for apoptosis or cell death. In the images in FIG. 5 the inventors have samples from representative lesions treated with vehicle or HGI627. Each at different levels of magnification to provide overview and zoomed in for specific details. In the first row, where the inventors study the cell morphology it is quite obvious that the cells have a different size and morphology between treated and untreated. Striation in extracellular structure and openings point towards fibrosis. In the second row, the trichrome staining confirms the presence of collagens (blue) and scarring. The tumors were large and oncolysis was fast enough that a “wound” was created, mouse cells traveled inward to close the gap and secrete large amounts of collagens and disorganized extracellular matrix (ECM). In the third row, the inventors show that in the treated tumor, most cells were not human, but primarily mouse cells. There were some small isolated and encapsulated Hep3B cells present in the HGI627 stain. To establish if they were alive the inventors stained them with TUNEL and the brown stain shows these cells as apoptotic or dying.

[0127] Next to the previous stains, the inventors also stained the lesions for the presence of neutrophils and macrophages (FIG. 6). In the presence of viruses, these “first responders” immune cells could be present. As can be seen in FIG. 6 this is definitely the case in the tumors of the treated animals. It can clearly be seen that the remaining lesion in the treated group was mostly made up of macrophages. They are there to help kill cancer cells, neutralize the virus and help with wound healing.

[0128] Conclusions

[0129] Intraperitoneal (IP) administration did not yield a large response; tumor inhibition of up to 32% was observed. The first hypothesis is that the dosing was too low to be effective. With IP administration it is known that only a small amount of the therapy actually reaches subcutaneous tumors. Several studies have evaluated virus delivery in vivo and reported 0.001-0.01% of systemic injections reach subcutaneous tumors [1, 2, 3].

[0130] Another reason can be that the virus is not replicating fast enough. Basically, the cancer cell growth is fast enough to compensate for cell death caused by viral replication and oncolysis.

[0131] The intratumoral injection was very effective. After the injection, the lesion volume decreased for all 5 mice and eventually reached a stable volume of about 150 mm3. No adverse or negative clinical observations were made, so the mice appeared to be healthy and the therapy was well tolerated. Staining of the harvested lesions shows that only 3-5% of the cells in the lesion are human or Hep3B cancer cells. However, further staining with TUNEL assay shows that the majority of these are apoptotic or dead. This experiment also shows that large tumors can be treated without significant toxic side effects, proving that the viral therapy is very targeted.

[0132] The last stains of the treated lesions shows that the majority of the cells in the lesions are macrophages. That is a result of the presence of viruses, which were killing cells. The macrophages probably helped to kill cancer cells and assisted in the healing process.

[0133] Example 4 - In Vivo Efficacy of HGI627 on Hep3B CDX in NSG Mice

[0134] Introduction and Study Objective

[0135] The objective of the study is to test the efficacy of the client’s test article on Hep3B CDX hepatocellular carcinoma in NSG mice. Study Design

[0136] Model Study Groups and Dosing Regimen

[0137] *BID dosing IP at least 4 hours apart, keep virus solution at 4°C between dosing

[0138] Tumor Cell Preparation Cryogenic vials containing tumor cells will be thawed and cultured according to the manufacturer’s protocol. On the day of injection, cells will be washed in serum-free media, counted, and resuspended in PBS at a concentration of 5M viable cells / 100 pL. Cell suspensions will be mixed with an equal volume of Cultrex ECM resulting in a final concentration of 5M viable cells / 200 p L. The ECM-cell mixture will be kept on ice during transport to the vivarium. Cells will be prepared for injection by withdrawing the ECM-cell mixture into a chilled 1 ml slip-tip syringe. The filled syringes will be kept on ice to avoid solidification of the ECM.

[0139] Tumor Implantation

[0140] Animals will be shaved and ear tagged prior to injection. One mouse at a time will be immobilized and the site of injection will be disinfected with an alcohol swab. 200 pL of the PBS-cell mixture will be subcutaneously injected into the rear flank of the mouse. Up to 5 animals will be injected with 200 pL of PBS-cell mixture per syringe. Animals will be undisturbed for up to seven days before observing for tumor growth.

[0141] Tumor Measurement

[0142] Animals will be monitored weekly for palpable tumors, or any changes in appearance or behavior. Once tumors are palpable, tumors will be measured at least once a week using calipers. Tumor volume will be calculated using the following equation: (longest diameter * shortest diameter2) / 2. Once tumors are of appropriate size to begin the study, tumors and body weights will be measured at least 3 times per week for the duration of the study. One individual will be responsible for tumor measurements for the duration of the study, except in unavoidable circumstances.

[0143] Randomization and Dose Selection

[0144] When tumors are 80- 100mm3, mice will be randomly assigned to the respective treatment groups and dosed within 24 hours of randomization.

[0145] Reference Day: Day 1 is defined by dosing start date.

[0146] Study Termination The study will be terminated seven weeks after the first dose. During the study, changes to the length of treatment and / or observation period may be considered.

[0147] Body Weight Measurement and Guidelines

[0148] Body weight will be measured at least 3 times a week following randomization and initiation of treatment. Hydrogel / DietGel and / or dosing holidays may be given to animals due to body weight loss; body weight loss will be calculated based on the BW of the mouse on the first day of treatment.

[0149] Animals will be euthanized if more than body weight loss of >20% is observed. Dosing holiday and / or nutritional supplements may be provided to the animal based on assessment of animal health. If there are no signs of recovery, the animal will be sacrificed for humane reasons as per IACUC protocol regulations.

[0150] Clinical Observations

[0151] Clinical observations will be performed at the time of tumor and body weight measurements. During routine monitoring, the animals will be checked for any adverse effects of tumor growth and treatments on behavior such as mobility, food and water consumption, eye / hair matting and any other abnormalities. Mortality and observed abnormal clinical signs will be recorded for individual animals in detail. Daily monitoring will take place for mice showing severe signs of pain or distress and any moribund animals will be euthanized the same day that they are recognized as moribund.

[0152] Tissue Collection

[0153] Tumors will be collected at EOS and moribundity.

[0154] **Specific conditions: mean tumor volume either 1) decreases >15% on a measurement day, or 2) decreases in two consecutive measurement days.

[0155] Example 1 : In the current study on HepG2, Group 4 mean tumor volume decreases from 1408.9 mm3on measurement day 29 to 1015.62 mm3on measurement day 31. This represents a 27.91% decrease in mean tumor volume, which would satisfy the first condition (>15% decrease). Example 2: In the previous study on Hep3B, Group 4 mean tumor volume decreases from 1129.8 mm3on measurement day 26 to 1020.7 mm3on measurement day 29. This represents a 9.66% decrease in mean tumor volume, which would not satisfy the first condition (>15% decrease). However, mean tumor volume decreases again on the subsequent measurement (Day 31), which would satisfy the second condition (decreases in two consecutive measurement days).

[0156] Formulation Instructions

[0157] Thaw virus and vehicle on ice. The titer for HGI627 is 1.5xl09TCID50 / mL on Hep3B.

[0158] For Group 1 dosing, no formulation is necessary before administration.

[0159] For Group 2 dosing, combine 67 pF virus with 33 pF vehicle to produce a 100 pF dose of IxlO8TCID50 (Hep3B). To formulate with overage: 938 ul virus plus 462 ul vehicle = 1.4mE (enough for 14 mice). For Group 3 dosing, no formulation is necessary before administration.

[0160] Bring the TA and vehicle to room temperature before dosing.

[0161] Example 5

[0162] Introduction

[0163] In an in vivo efficacy study using a Hep3B subcutaneous xenograft model in NSG mice, treatment with HGI627 resulted in complete tumor regression in 100% of animals (n=9). In contrast, in the HepG2 subcutaneous model, HGI627 treatment led to delayed and transient tumor regression, followed by eventual tumor regrowth, as shown in FIG. 9.

[0164] It is hypothesized that the differential response between Hep3B and HepG2 is due to intact interferon (IFN) signaling in HepG2, which mounts a robust antiviral response that limits viral replication and oncolysis. Hep3B cells, by comparison, are deficient in IFN signaling and thus more permissive to viral infection and killing.

[0165] To test this hypothesis, the inventors initiated in vitro studies assessing the combination of HGI627 with IFN pathway inhibitors in HepG2 cells. Preliminary results using a research-grade type I IFN-blocking antibody cocktail demonstrated a marked improvement in oncolytic efficacy. Ongoing efforts are focused on evaluating clinically approved IFN-modulating agents in combination with HGI627. An in vivo efficacy study using a HepG2 CDX subcutaneous model is currently in preparation to determine whether this strategy translates to improved outcomes in a more clinically relevant setting.

[0166] Methods

[0167] HepG2 cells were seeded in 96-well plates at a density of 3xl04cells per well and incubated overnight at 37°C in a humidified atmosphere with 5% CO2. After 24 hours, viral stocks were thawed at room temperature and kept on ice during assay setup. Cells were infected with serial dilutions of virus corresponding to MOIs ranging from 1 down to IO4, based on titers determined by TCIDso assays in HepG2 cells and validated via immunocytochemistry (ICC) using an anti-VSV antibody.

[0168] To assess the effect of interferon blockade, Human Type I IFN Neutralizing Antibody Mixture (PBL Assay Science; Catalog No. 39000) was added to the culture media 0-4 hours prior to infection (defined as t = 0 hours). Control conditions included uninfected cells (to establish a baseline for cell health and proliferation) as well as cells treated with IFN blocker alone (to evaluate any antibody-dependent effects on cell viability).

[0169] Real-time imaging and quantification of cell health and proliferation were performed using the Incucyte Live-Cell Analysis System (Sartorius), measuring confluence over time. Cytotoxicity kinetics were evaluated using the CellTox Green Cytotoxicity Assay (Promega), following the manufacturer's protocol. Phase-contrast and GFP fluorescence images (lOx magnification) were acquired every 3 hours for the duration of the experiment, which was concluded up to 168 hours post- infection. Images were assessed for cytopathic effect (CPE), and key metrics — including confluence percentage, GFP mean intensity, and integrated fluorescence — were analyzed on a per-image basis, averaged per well, and exported to Google Sheets and GraphPad Prism 9 (GraphPad Software, San Diego, CA) for further analysis of technical and experimental replicates.

[0170] Results Co-treatment with an interferon (IFN) inhibitor alongside HGI627 significantly enhances oncolysis and accelerates cell death in vitro. Cell confluence was monitored over time to assess the impact of treatment with and without the IFN blocker. HGI627 monotherapy at an MOI of 1x103led to a 25% reduction in confluence by approximately 84 hours post-treatment, while the same level of reduction was not reached until -168 hours with a lower MOI of I x 104. Remarkably, co-administration of an anti-IFN agent at MOI 1x104restored oncolytic potency, with a comparable loss in confluence occurring around 96 hours, indicating enhanced therapeutic efficacy through IFN signaling blockade. (FIG. 10)

[0171] In addition to confluence and cell area measurements, cytotoxicity was assessed using Promega’s CellTox Assay. The data indicate that the addition of the IFN inhibitor mix accelerates both the onset and progression of cytotoxicity, as reflected by the steeper increase in fluorescence beginning around 48 hours (blue squares). Furthermore, the combination therapy group exhibits a stronger overall response, evidenced by the higher fluorescence signal on the y-axis. This heightened response may be attributed to a more rapid induction of cytotoxic effects under the combination treatment. (FIG. 11)

[0172] Next Steps

[0173] Building on the promising in vitro data using the Type I IFN inhibitor from PBL Assay Science, the inventors are evaluating additional IFN type 1 inhibitors and blockers with greater translational potential and implementation in the clinic. More specifically, the inventors are / have looked to evaluate HGI627 with already approved FDA drugs and therapies, as these have already demonstrated safety in human trials. Using these, as opposed to current research products will enable a faster path to clinic.

[0174] Anifrolumab is a fully human, effector- null, IgGl monoclonal antibody that was approved by the FDA in July, 2021 for the treatment of moderate to severe systemic lupus erythematosus; the primary mechanism of action is binding to IFNAR1 and blocks type I IFN signalling.

[0175] An alternative to blocking IFN ARI (receptor), Janus kinase (JAK) is a known, downstream signaling molecule for IFN Type 1 that is a promising target. The FDA has approved multiple JAK inhibitors (11 total), including ruxolitinib and baricitinib, which are JAK1 / 2 inhibitors. These have previously demonstrated synergistic effects and improvements in efficacy for VSV-based therapy for IFN-responsive, resistant ovarian cancers (Geoffrey et al.).

Claims

CLAIMSWhat is claimed is:

1. An artificial oncolytic virus comprising: a glycoprotein engineered to selectively bind to a target cell via a binding partner identified as highly expressed on the target cell as compared with a non-target cell; and an aptazyme engineered to target a highly expressed protein, wherein the aptazyme controls replication of the oncolytic virus.

2. The artificial oncolytic virus according to claim 1, wherein the glycoprotein is engineered to comprise a nanobody, antibody or protein that targets a receptor.

3. The artificial oncolytic virus according to claim 1 or claim 2, wherein the glycoprotein is engineered to comprise a nanobody targeting a cancer receptor.

4. The artificial oncolytic virus according to any one of claims 1-3, wherein the glycoprotein is engineered to comprise a nanobody that targets the glypican 3 (GPC3) receptor, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), transferrin receptor (TFRC), mucin 1 (MUC1), MET protooncogene receptor tyrosine kinase (MET), CD33, CD19, CD47, chemokine receptor 4 (CXCR4), fibroblast growth factor receptor (FGFR), death receptor 5 (DR5), TRAIL receptor 2 (TRAILR2), or trophoblast cell surface antigen 2 (TR0P2).

5. The artificial oncolytic virus according to any one of claim 1-4, wherein the glycoprotein is engineered to remove its natural infectivity.

6. The artificial oncolytic virus according to any one of claims 1-5, wherein the aptazyme is engineered to target a cancer- specific protein.

7. The artificial oncolytic virus according to any one of claims 1-6, wherein the aptazyme is engineered to target beta catenin (CTNNB1), alpha fetoprotein(AFP), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), telomerase reverse transcriptase (TERT), matrix metalloproteinase-9 (MMP9), oncostatin M protein (OSM), secreted phosphoprotein 1 (SPP1), p53, phosphorylated ERK1, phosphorylated ERK2, or estrogen receptor (ER).

8. The artificial oncolytic virus according to any one of claims 1-7, wherein the aptazyme comprises a Hammer head ribozyme (HH Rbz).

9. The artificial oncolytic virus according to any one of claims 1-8, wherein the aptazyme comprises a HH Rbz and is engineered to target CTNNB 1.

10. The artificial oncolytic virus according to any one of claims 1-9, wherein the aptazyme comprises a HH Rbz and is engineered to target CTNNB 1 and wherein the glycoprotein is engineered to comprise a nanobody that targets the GPC3 receptor.

11. The artificial oncolytic virus according to any one of claims 1-9, wherein the aptazyme comprises a HH Rbz and is engineered to target CTNNB 1 and wherein the glycoprotein is engineered to comprise a nanobody that targets EpCAM.

12. The artificial oncolytic virus according to any one of claims 1-8, wherein the aptazyme comprises a HH Rbz and is engineered to target AFP.

13. The artificial oncolytic virus of claim 12, wherein the aptazyme comprises a HH Rbz and is engineered to target AFP and wherein the glycoprotein is engineered to comprise a nanobody that targets EpCAM.

14. The artificial oncolytic virus of claim 12, wherein the aptazyme comprises a HH Rbz and is engineered to target AFP and wherein the glycoprotein is engineered to comprise a nanobody that targets the GPC3 receptor.

15. The artificial oncolytic virus according to any of claims 1-14, wherein the target cell is a cancer cell.

16. The artificial oncolytic virus according to any of the claims 1-14, wherein the target cell is a cell associated with a hyperproliferative disorder.

17. The artificial oncolytic virus according to any of claims 1-14, wherein the target cell is a tumor cell.

18. The artificial oncolytic virus according to any of claims 1-17, wherein the virus is non-pathogenic in a subject to whom it is intended to be administered.

19. The artificial oncolytic virus according to any of claims 1-18, wherein the virus is one to which a subject to whom it is intended to be administered is not immune.

20. The artificial oncolytic virus according to claim 18 or claim 19, wherein the subject is Homo sapiens.

21. The artificial oncolytic virus according to any of claims 1-20, wherein the virus comprises at least one domain which is identical to a reference naturally- occurring virus.

22. The artificial oncolytic virus according to claim 21, wherein the reference naturally-occurring virus is a member of the Rhabdoviridae family.

23. The artificial oncolytic virus according to claim 21 or claim 22, wherein the reference naturally-occurring virus is from the genus Vesiculovirus.

24. The artificial oncolytic virus according to any one of claims 21-23, wherein the reference naturally-occurring virus comprises Vesicular stomatitis virus (ySN).

25. The artificial oncolytic virus according to any one of claims 1-24, wherein the engineered glycoprotein is derived from the Togaviridae family.

26. The artificial oncolytic virus according to any one of claims 1-25, wherein the engineered glycoprotein is derived from the genus Alphavirus.

27. The artificial oncolytic virus according to any one of claims 1-26, wherein the engineered glycoprotein is derived from Sindbis virus (SINV).

28. The artificial oncolytic virus according to any one of claims 1-27, wherein the aptazyme regulates production of the nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), or large polymerase subunit (L).

29. The artificial oncolytic virus according to any of claims 1-28, wherein when administered to a subject said virus causes cell death via apoptosis, necrosis, and / or cytopathic effect (CPE) of one or more target cells in said subject.

30. The artificial oncolytic virus according to claim 29, wherein cell death occurs within 3 months, preferably within 1 month, preferably within 28 days, preferably within 21 days, more preferably within 7 days, more preferably within 2-4 days.

31. The artificial oncolytic virus according to any of claims 1-30, wherein the target cell is selected from the group consisting of cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, and uterus.

32. The artificial oncolytic virus according to any of claims 1-31, wherein the target cell is a cancer cell and wherein the cancer is selected from the group consisting of melanoma, non- small cell lung cancer, small-cell lung cancer, lung cancer, hepatocarcinoma, retinoblastoma, astrocytoma, glioblastoma, gum cancer, tongue cancer, leukemia, neuroblastoma, head cancer, neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, lymphoma, brain cancer, colon cancer, and bladder cancer.

33. The artificial oncolytic virus according to any of claims 1-32, wherein said virus additionally comprises one or more heterologous functional domains wherein said functional domain is selected from the group consisting of a therapeutic agent, a kill switch for said target cell, an agent which facilitates the ability of the virus to evade the recipient immune system, a watermark, abarcode, an agent which degrades the extracellular matrix of a solid tumor, and a diagnostic agent.

34. The artificial oncolytic virus according to claim 33, wherein the one or more heterologous functional domains are inserted in frame.

35. A pharmaceutical composition comprising the artificial oncolytic virus according to any one of claims 1-34.

36. A method of treating a hyperproliferative disorder in a subject comprising administering to the subject the pharmaceutical composition of claim 35.

37. The method according to claim 36, wherein the hyperproliferative disorder is cancer.

38. The method according to claim 37, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, small-cell lung cancer, lung cancer, hepatocarcinoma, retinoblastoma, astrocytoma, glioblastoma, gum cancer, tongue cancer, leukemia, neuroblastoma, head cancer, neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, lymphoma, brain cancer, colon cancer, and bladder cancer.

39. The method according to any of claims 36-38, wherein the subject is Homo sapiens.

40. The method of any one of claims 36-39, further comprising administering at least one interferon (IFN) inhibitor.

41. The method of claim 40, wherein the IFN inhibitor is a Type I IFN inhibitor.

42. The method of claim 40 or claim 41, wherein the IFN inhibitor is coadministered with the pharmaceutical composition.

43. The method of claim 40 or claim 41, wherein the IFN inhibitor is administered after administration of the pharmaceutical composition.

44. The method of claim 40 or claim 41, wherein the IFN inhibitor is administered prior to administering the pharmaceutical composition.

45. A method of slowing, inhibiting or reducing the growth or size of a tumor comprising administering to the subject the pharmaceutical composition of claim 35.

46. The method according to claim 45, wherein the tumor is selected from the group consisting of non- small cell lung cancer, small-cell lung cancer, lung cancer, hepatocarcinoma, retinoblastoma, astrocytoma, glioblastoma, neuroblastoma, head cancer, neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, brain cancer, colon cancer, and bladder cancer.

47. The method according to claim 45 or claim 46, wherein the subject is Homo sapiens.

48. The method of any one of claims 45-47, further comprising administering at least one interferon (IFN) inhibitor.

49. The method of claim 48, wherein the IFN inhibitor is a Type I IFN inhibitor.

50. The method of claim 48 or claim 49, wherein the IFN inhibitor is coadministered with the pharmaceutical composition.

51. The method of claim 48 or claim 49, wherein the IFN inhibitor is administered after administration of the pharmaceutical composition.

52. The method of claim 48 or claim 49, wherein the IFN inhibitor is administered prior to administering the pharmaceutical composition.

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