Oncolytic viruses for delivery of therapeutic polypeptides

Abstract

Provided are oncolytic viruses that encode a death domain (DD) polypeptide fragment of human CD95 or a serine protease, and related compositions and methods for treating cancers.

Description

[0001] Attorney Docket No. OPNI-003 / 04WO 332575-2098

[0002] ONCOLYTIC VIRUSES FOR DELIVERY OF THERAPEUTIC POLYPEPTIDES

[0003] Cross-Reference to Related Applications

[0004] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 682,495, filed August 13, 2024, which is incorporated by reference in its entirety.

[0005] Statement Regarding Sequence Listing

[0006] The Sequence Listing XML associated with this application is provided in XML file format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing XML is OPNI_003_04WO_ST26.xml. The XML file is about 38,304 bytes, was created on August 11, 2025, and is being submitted electronically via USPTO Patent Center.

[0007] BACKGROUND

[0008] Technical Field

[0009] The present disclosure relates to oncolytic viruses that encode a death domain (DD) polypeptide fragment of human CD95 or a serine protease, and related compositions and methods for treating cancers.

[0010] Description of the Related Art

[0011] Precision medicine, which is designed to optimize efficiency or therapeutic benefit for particular groups of patients by using genetic or molecular profiling, has gained tremendous traction for treating cancer. Identifying the specific genomic abnormalities that (i) confer risk of developing cancer, (ii) influence tumor growth, and (iii) regulate metastasis have defined how cancer is diagnosed, determined how targeted therapies are developed and implemented, and shaped cancer prevention strategics.

[0012] The need for precision medicine in cancer is largely based on the failure to identify targetable properties in cancerous tumor cells that distinguish them from healthy, non-cancerous cells. Indeed, although radiation and / or chemotherapies have the capacity to effectively kill many if not most cancer cells, their efficacy is severely limited by cytotoxic effects on non-cancer cells. These findings demonstrate that rapid cell division, a property' targeted by radiation therapy and chemotherapy, is not unique enough to cancer cells to achieve the specificity required to limit extensive side effects.

[0013] It has been shown that certain serine protease enzymes and their proteolytic substrates, for example, CD95. are selectively toxic to cancer cells but relatively non-toxic to normal or otherwise healthy cells. However, there is a need in the art to identify optimal enzymes that are capable of such selective cancer cell-toxicity, and refine the clinical utility and delivery of such enzymes. Attorney Docket No. OPNI-003 / 04WO 332575-2098

[0014] BRIEF SUMMARY

[0015] Embodiments of the present disclosure include an oncolytic virus, comprising a recombinant polynucleotide that encodes:

[0016] (a) a death domain (DD) polypeptide, wherein the DD polypeptide is a C-terminal fragment of human CD95 (SEQ ID NO: 1) that induces apoptosis of cancer cells; or

[0017] (b) a serine protease selected from porcine pancreatic elastase (PPE) polypeptide and human neutrophil elastase (ELANE) polypeptide.

[0018] In some embodiments, the DD polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95. 96, 97, 98, 99. or 100% identical to a selected from SEQ ID NOs: 2-5 and induces apoptosis of cancer cells. In some embodiments, the DD polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95. 96, 97, 98, 99, or 100% identical to SEQ ID NO: 2 and induces apoptosis of cancer cells. In some embodiments, the DD polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 3 and induces apoptosis of cancer cells. In some embodiments, the DD polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85. 90. 95, 96, 97. 98. 99, or 100% identical to SEQ ID NO: 4 and induces apoptosis of cancer cells. In some embodiments, the DD polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90. 95, 96, 97, 98, 99. or 100% identical to SEQ ID NO: 5 and induces apoptosis of cancer cells.

[0019] In certain embodiments, the PPE polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90. 95, 96, 97, 98. 99, or 100% identical to SEQ ID NO: 6 (WT pro-PPE), or the ELANE polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 7 (WT pro-ELANE).

[0020] In some embodiments, the PPE polypeptide or ELANE polypeptide comprises, in an N- tenninal to C-terminal orientation, a signal peptide, a modified activation peptide, and a peptidase domain, wherein the modified activation peptide comprises a heterologous protease cleavage site that is cleavable by a protease selected from a metalloprotease, an aspartyl protease, and a cysteine protease. In certain embodiments, the metalloprotease, aspartyl protease, or cysteine protease is selected from matrix metalloproteinase- 12 (MMP12), cathepsin D (CTSD), cathepsin C (CTSD), and cathepsin L (CTSL), and optionally wherein the heterologous protease cleavage site is selected from Table S3, including the MMP12 cleavage site of SEQ ID NOs: 8-10. the CTSD cleavage site of SEQ ID NOs: 11-12, the CTSC cleavage site of SEQ ID NO: 13, or the CTSL cleavage site of SEQ ID NOs: 14-16.

[0021] In some embodiments, the PPE polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95. 96, 97, 98, 99. or 100% identical to a sequence selected from SEQ ID NOs: 17-25 (Table S4), which retains the heterologous protease cleavage site. Attorney Docket No. OPNI-003 / 04WO 332575-2098

[0022] In some embodiments, the PPE polypeptide comprises at least one amino acid alteration in the peptidase domain (SEQ ID NO: 26), wherein the at least one alteration is at a residue selected from one or more of Q211, T55, D74, R75, S214, R237, and N241, the residue numbering being defined by SEQ ID NO: 6 (WT pro-PPE). In some embodiments, the at least one amino acid alteration is selected from one or more of Q211F, T55A, D74A, R75A, R75E, Q211A, S214A, R237A, N241A, and N241Y, the residue numbering being defined by SEQ ID NO: 6 (WT pro-PPE).

[0023] In specific embodiments, the PPE polypeptide comprises a peptidase domain selected from: an amino acid sequence that is at least 80. 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 27. and which retains the Q211F amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95. 96, 97, 98, 99. or 100% identical to SEQ ID NO: 28, and which retains the T55A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95. 96, 97, 98, 99. or 100% identical to SEQ ID NO: 29, and which retains the N241 A amino acid substitution; an amino acid sequence that is at least 80, 85. 90, 95, 96, 97. 98. 99, or 100% identical to SEQ ID NO: 30, and which retains the N241Y amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96. 97. 98, 99, or 100% identical to SEQ ID NO: 31, and which retains the R75A amino acid substitution; an amino acid sequence that is at least 80. 85, 90, 95, 96. 97, 98, 99, or 100% identical to SEQ ID NO: 32, and which retains the R75E amino acid substitution; an amino acid sequence that is at least 80. 85, 90, 95, 96, 97. 98, 99, or 100% identical to SEQ ID NO: 33, and which retains the Q211 A amino acid substitution; an amino acid sequence that is at least 80, 85. 90, 95, 96, 97. 98, 99, or 100% identical to SEQ ID NO: 34, and which retains the R237A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97. 98, 99, or 100% identical to SEQ ID NO: 35, and which retains the S214A amino acid substitution; and an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 36, and which retains the D74A amino acid substitution.

[0024] In certain embodiments, the oncolytic virus is selected from a pox virus, a herpes virus, an adenovirus, a retrovirus, a rhabdovirus, a paramyxovirus, and a reovirus. In some embodiments, the poxvirus is a myxoma virus or a vaccinia virus. In particular embodiments, the poxvirus is a myxoma virus, optionally wherein the myxoma virus comprises an attenuated or deleted Ml 1 protein, M062R protein, or Ml 53 protein, including combinations thereof.

[0025] In some embodiments, the recombinant polynucleotide further encodes a cytokine, an activator of co-stimulatory receptor, or cell matrix protein. In some embodiments, the cytokine is selected from tumor necrosis factor alpha (TNFa). interleukin- 12 subunit alpha (IL-12a), interleukin- 12 subunit beta (IL-120), and interleukin- 15 (IL-15) optionally IL-15a, granulocyte-macrophage colony-stimulating factor (GM-CSF). interleukin-2 (IL-2), interleukin-21 (IL-21): and / or wherein the activator of co-stimulatory receptors is selected from one or more of 4-1BB, 0X40, Glucocorticoid- induced TNFR-related protein (GITR), and B7-1 (CD80); and / or wherein the cell matrix protein is a human decorin protein. Attorney Docket No. OPNI-003 / 04WO 332575-2098

[0026] Also included are pharmaceutical compositions, comprising an oncolytic virus described herein and a pharmaceutically -acceptable carrier.

[0027] Some embodiments include methods of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition or oncolytic virus described herein. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological malignancy.

[0028] In some embodiments, the cancer is selected from one or more of bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain / nervous system cancer (optionally glioblastoma), breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer (optionally small cell lung cancer (SCLC)), nasopharynx cancer, neck cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, prostate cancer, skin cancer (optionally melanoma), stomach cancer, testicular cancer, tongue cancer, uterine cancer, multiple myeloma, and embryonal rhabdomyosarcoma.

[0029] Certain embodiments comprise administering the pharmacal composition to the subject by systemic administration, intratumoral injection, or intranasal administration. Some embodiments comprise administering the pharmaceutical composition to the subject by parenteral administration, optionally intravenous or subcutaneous administration. Specific embodiments comprise coadministering to the subject an immune checkpoint modulatory agent, a chemotherapeutic agent, a hormonal therapeutic agent, and / or a kinase inhibitor.

[0030] In some embodiments, administering the oncolytic virus increases an anti-cancer immune response in the subject by about or at least about 5, 10, 15, 20, 25, 30. 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900. 1000. 2000% or more, relative to a control. In some embodiments, administering the oncolytic virus increases cancer cell-killing in the subject by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70. 80, 90, 100, 200, 300, 400. 500, 600, 700, 800, 900, 1000, 2000% or more, relative to a control.

[0031] Also included is the use of a pharmaceutical composition or oncolytic virus described herein in the preparation of a medicament for treating a cancer in a subject. Some embodiments include a pharmaceutical composition or oncolytic virus described herein for use in treating a cancer in a subject.

[0032] DETAILED DESCRIPTION

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs. Although any methods, materials, compositions, reagents, cells, similar or equivalent similar or equivalent to those described herein can be used in the practice or testing of the subject matter of the present disclosure, preferred methods and materials are described. All publications and references, including but not limited to patents and patent applications, cited in this specification are herein Attorney Docket No. OPNI-003 / 04WO 332575-2098 incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references.

[0034] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer’s specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures 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 present specification. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic 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 recombinant technology, molecular biological, microbiological, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0035] For the purposes of the present disclosure, the following terms are defined below.

[0036] The articles “a” and ‘‘an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” includes “one element”, “one or more elements” and / or “at least one element”.

[0037] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0038] An “antagonist” refers to a biological or chemical agent that interferes with or otherwise reduces the physiological action of another agent or molecule. In some instances, the antagonist specifically binds to die other agent or molecule. Included are full and partial antagonists.

[0039] An “agonist” refers to a biological or chemical agent that increases or enhances the physiological action of another agent or molecule. In some instances, the agonist specifically binds to the other agent or molecule. Included are full and partial agonists.

[0040] As used herein, the term “amino acid” is intended to mean both naturally occurring and non- naturally occurring amino acids as well as amino acid analogs and mimetics. Naturally -occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4-hydroxyproline. hydroxylysine, desmosine. isodesmosine. homocysteine, citrulline and ornithine, for example. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine. ethionine and the like, which are known to a person skilled in the art. Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and Attorney Docket No. OPNI-003 / 04WO 332575-2098 moieties on the amino acid or by derivatization of the amino acid. Amino acid mimetics include, for example, organic structures which exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics arginine (Arg or R) would have a positive charge moiety located in similar molecular space and having the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid or of the amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0041] As used herein, a subject “at risk” of developing a disease, or adverse reaction may or may not have detectable disease, or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to the treatment methods described herein. “At risk” denotes that a subject has one or more risk factors, which are measurable parameters that correlate with development of a disease, as described herein and known in the art. A subject having one or more of these risk factors has a higher probability of developing disease, or an adverse reaction than a subject without one or more of these risk factor(s).

[0042] “Biocompatible” refers to materials or compounds which are generally not injurious to biological functions of a cell or subject and which will not result in any degree of unacceptable toxicity, including allergenic and disease states.

[0043] The term “binding” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.

[0044] By “coding sequence” is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene. By contrast, the term “non-coding sequence” refers to any nucleic acid sequence that docs not directly contribute to the code for the polypeptide product of a gene.

[0045] Throughout this disclosure, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0046] By “consisting of is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements. Attorney Docket No. OPNI-003 / 04WG 332575-2098

[0047] The term “endotoxin free” or “substantially endotoxin free” relates generally to compositions, solvents, and / or vessels that contain at most trace amounts (e.g., amounts having no clinically adverse physiological effects to a subject) of endotoxin, and preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain micro-organisms, such as bacteria, typically gramnegative bacteria, although endotoxins may be found in gram-positive bacteria, such as Listeria monocytogenes. The most prevalent endotoxins are lipopolysaccharides (LPS) or lipo-oligo- saccharides (LOS) found in the outer membrane of various Gram-negative bacteria, and which represent a central pathogenic feature in the ability of these bacteria to cause disease. Small amounts of endotoxin in humans may produce fever, a lowering of the blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects.

[0048] Therefore, in pharmaceutical production, it is often desirable to remove most or all traces of endotoxin from drug products and / or drug containers, because even small amounts may cause adverse effects in humans. A depyrogenation oven may be used for this purpose, as temperatures in excess of 300°C are ty pically required to break down most endotoxins. For instance, based on primary packaging material such as syringes or vials, the combination of a glass temperature of 250°C and a holding time of 30 minutes is often sufficient to achieve a 3 log reduction in endotoxin levels. Other methods of removing endotoxins are contemplated, including, for example, chromatography and filtration methods, as described herein and known in the art.

[0049] Endotoxins can be detected using routine techniques known in the art. For example, the Limulus Amoebocyte Lysate assay, which utilizes blood from the horseshoe crab, is a very sensitive assay for detecting presence of endotoxin. In this test, very low levels of LPS can cause detectable coagulation of the limulus lysate due a powerful enzymatic cascade that amplifies this reaction. Endotoxins can also be quantitated by enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin free, endotoxin levels may be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU / mg of active compound. Typically, 1 ng lipopolysaccharide (LPS) corresponds to about 1-10 EU.

[0050] The term “half maximal effective concentration” or “ECso” refers to the concentration of an agent (for example, an oncolytic virus encoding a serine protease or a CD95 death domain polypeptide) as described herein at which it induces a response halfway between the baseline and maximum after some specified exposure time; the EC50 of a graded dose response curve therefore represents the concentration of a compound at which 50% of its maximal effect is observed. EC50 also represents the plasma concentration required for obtaining 50% of a maximum effect in vivo. Similarly, the “EC90” refers to the concentration of an agent or composition at which 90% of its maximal effect is observed. The “EC90” can be calculated from the “EC50” and the Hill slope, or it can be determined from the data directly, using routine knowledge in the art. In some embodiments, the EC50 of an agent is less than about 0.01. 0.05. 0.1. 0.2. 0.3. 0.4. 0.5. 0.6, 0.7, 0.8, 0.9, 1, 2. 3. 4, 5, 6, 7, Attorney Docket No. OPNI-003 / 04WO 332575-2098

[0051] 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200 or 500 nM. In some embodiments, an agent will have an ECso value of about 1 nM or less.

[0052] The “half-life’’ of an agent can refer to the time it takes for the agent to lose half of its pharmacologic, physiologic, or other activity, relative to such activity at the time of administration into the serum or tissue of an organism, or relative to any other defined time-point. “Half-life” can also refer to the time it takes for the amount or concentration of an agent to be reduced by half of a starting amount administered into the serum or tissue of an organism, relative to such amount or concentration at the time of administration into the serum or tissue of an organism, or relative to any other defined time-point. The half-life can be measured in serum and / or any one or more selected tissues.

[0053] The term “heterologous” refers to a feature or element (e.g., protease cleavage site) in a polypeptide or encoding polynucleotide that is derived from a different source than the wild-type polypeptide or encoding polynucleotide, for example, a feature from a different species than the wildtype, or a non-natural, engineered feature.

[0054] The terms “modulating” and “altering” include “increasing.” “enhancing” or “stimulating,” as well as “decreasing” or “reducing.” typically in a statistically significant or a physiologically significant amount or degree relative to a control. An “increased,” “stimulated” or “enhanced” amount is ty pically a “statistically significant” amount, and may include an increase that is about or at least about 1.1, 1.2, 1.5, 2. 3, 4, 5, 6, 7. 8. 9, 10. 15, 20, 30, 40, 50. 60, 70, 80, 90. 100, 200, 300. 400, 500, or 1000-fold more than the amount produced by no composition (e.g.. the absence of agent) or a control composition. A “decreased” or “reduced” amount is typically a “statistically significant” amount, and may include a decrease that about or at least about 1.1. 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000-fold less than the amount produced by no composition (e.g., the absence of an agent) or a control composition. Examples of comparisons and “statistically significant” amounts arc described herein.

[0055] The terms “polypeptide,” “protein”, and “peptide” are used interchangeably and refer to a polymer of amino acids not limited to any particular length. The term “enzyme” includes polypeptide or protein catalysts. As used herein a “proprotein”, “proenzyme”, or “zy mogen" refers to an inactive (or substantially inactive) protein or enzyme, which typically is activated by protease cleavage of an activation peptide to generate an active protein or enzyme. The terms include modifications such as myristoylation, sulfation, glycosylation, phosphorylation and addition or deletion of signal sequences. The terms “polypeptide” or “protein” means one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or protein can comprise a plurality of chains non-covalently and / or covalently linked together by peptide bonds, having the sequence of native proteins, that is. proteins produced by naturally -occurring and specifically non-recombinant cells, or genetically -engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from. Attorney Docket No. OPNI-003 / 04WG 332575-2098 additions to, and / or substitutions of one or more amino acids of the native sequence. In certain embodiments, the polypeptide is a ‘’recombinant” polypeptide, produced by a recombinant virus or cell that comprises one or more “recombinant polynucleotides” (e.g., DNA, RNA), which are typically made of heterologous polynucleotide sequences or combinations of polynucleotide sequences that would not otherwise be found in the virus or cell.

[0056] The term “polynucleotide” and “nucleic acid” includes mRNA. RNA. cRNA, cDNA, and DNA. The term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA. The terms “isolated DNA” and “isolated polynucleotide” and “isolated nucleic acid” refer to a molecule that has been isolated free of total genomic DNA of a particular species. Therefore, an isolated DNA segment encoding a polypeptide refers to a DNA segment that contains one or more coding sequences yet is substantially isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Also included are non-coding polynucleotides (e.g., primers, probes, oligonucleotides), which do not encode a polypeptide. Also included are recombinant vectors, including, for example, expression vectors, viral vectors, plasmids, cosmids, phagemids, phage, viruses, and the like.

[0057] Additional coding or non-coding sequences may. but need not, be present within a polynucleotide described herein, and a polynucleotide may, but need not, be linked to other molecules and / or support materials. Hence, a polynucleotide or expressible polynucleotide, regardless of the length of the coding sequence itself, may be combined with other sequences, for example, expression control sequences.

[0058] “Expression control sequences” include regulatory sequences of nucleic acids, or the corresponding amino acids, such as promoters, leaders, enhancers, introns, recognition motifs for RNA, or DNA binding proteins, polyadenylation signals, terminators, internal ribosome entry’ sites (IRES), secretion signals, subccllular localization signals, and the like, which have the ability’ to affect the transcription or translation, or subcellular, or cellular location of a coding sequence in a host cell. Exemplary’ expression control sequences are described in Goeddel; Gene Expression Technology: Methods in Enzy mology 185, Academic Press, San Diego, Calif. (1990).

[0059] A “promoter” is a DNA regulatory region capable of binding RNA poly merase in a cell and initiating transcription of a downstream (3’ direction) coding sequence. As used herein, the promoter sequence is bounded at its 3’ terminus by the transcription initiation site and extends upstream (5’ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. A transcription initiation site (conveniently defined by mapping with nuclease SI) can be found within a promoter sequence, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters can often, but not always, contain “TATA” boxes and "CAT” boxes. Prokaryotic promoters contain Shine- Dalgamo sequences in addition to the -10 and -35 consensus sequences. Attorney Docket No. OPNI-003 / 04WO 332575-2098

[0060] A large number of promoters, including constitutive, inducible and repressible promoters, from a variety of different sources are well known in the art. Representative sources include for example, viral, mammalian, insect, plant, yeast, and bacterial cell types), and suitable promoters from these sources are readily available, or can be made synthetically, based on sequences publicly available on line or, for example, from depositories such as the ATCC as well as other commercial or individual sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in either a 3’ or 5’ direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, pBAD (araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter. Inducible promoters include the Tet system. (US Patents 5,464,758 and 5,814,618), the Ecdysone inducible system (No et al., Proc. Natl. Acad. Sci. (1996) 93 (8): 3346-3351; the T-RExTM system (Invitrogen Carlsbad, CA). LacSwitch® (Stratagene, (San Diego, CA) and the Cre-ERT tamoxifen inducible recombinase system (Indra et al. Nuc. Acid. Res. (1999) 27 (22): 4324-4327; Nuc. Acid. Res. (2000) 28 (23): e99; US Patent No. 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol. (2005) 308: 123-144) or any promoter known in the art suitable for expression in the desired cells.

[0061] An “expressible polynucleotide” includes a cDNA. RNA. mRNA or other polynucleotide that comprises at least one coding sequence and optionally at least one expression control sequence, for example, a transcriptional and / or translational regulatory element, and which can express an encoded polypeptide (for example, a serine protease or a CD95 death domain polypeptide) upon introduction into a cell, for example, a cell in a subject.

[0062] In certain instances, the expressible polynucleotides described herein are engineered for localization within a cell, potentially within a specific compartment such as the nucleus, or are engineered for secretion from the cell or translocation to the plasma membrane of the cell. In exemplary' embodiments, the expressible poly nucleotides are engineered for nuclear localization.

[0063] The term “isolated” polypeptide or protein referred to herein means that a subject protein (1) is free of at least some other proteins with which it would typically be found in nature. (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or non-covalent interaction) with portions of a protein with which the “isolated protein” is associated in nature, (6) is operably associated (by covalent or non-covalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated protein can be encoded by genomic DNA, cDNA, mRNA or other RNA, of may be of synthetic origin, or any combination thereof. In certain embodiments, the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise). Attorney Docket No. OPNI-003 / 04WO 332575-2098

[0064] In certain embodiments, the “purity ” of any given agent in a composition may be defined. For instance, certain compositions may comprise an agent such as a polypeptide agent that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure on a protein basis or a weight-weight basis, including all decimals and ranges in between, as measured, for example and by no means limiting, by high performance liquid chromatography (HPLC), a well-known form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds.

[0065] The term “reference sequence” refers generally to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as references sequences, including those described by name and those described in the Tables and the Sequence Listing.

[0066] Certain embodiments include biologically active “variants” and “fragments” of the proteins / polypeptides described herein, and the polynucleotides that encode the same. “Variants” contain one or more substitutions, additions, deletions, and / or insertions relative to a reference polypeptide or polynucleotide (see, e.g.. the Tables and the Sequence Listing). A variant polypeptide or polynucleotide comprises an amino acid or nucleotide sequence with at least about 50%. 55%, 60%, 65%, 70%. 75%, 80%, 85%, 90%. 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98% , 99% or more sequence identity or similarity or homology to a reference sequence, as described herein, and substantially retains the activity of that reference sequence. Also included are sequences that consist of or differ from a reference sequences by the addition, deletion, insertion, or substitution of 1, 2. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. 17, 18, 19, 20, 30, 40, 50, 60,70, 80, 90, 100, 110. 120, 130, 140, 150 or more amino acids or nucleotides and which substantially retain at least one activity of that reference sequence. In certain embodiments, the additions or deletions include C-terminal and / or N- terminal additions and / or deletions.

[0067] The terms “sequence identity ” or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by -nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g.. A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He. Phe, Tyr, Trp. Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FAST A, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e.. resulting in the Attorney Docket No. OPNI-003 / 04WO 332575-2098 highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., Nucl. Acids Res. 25:3389, 1997.

[0068] A “subject” or a “subject in need thereof’ or a “patient” or a “patient in need thereof ’ includes a mammalian subject such as a human subject.

[0069] “Substantially” or “essentially” means nearly totally or completely, for instance. 95%, 96%, 97%, 98%, 99% or greater of some given quantity.

[0070] By “statistically significant,” it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.

[0071] “Therapeutic response” refers to improvement of symptoms (whether or not sustained) based on administration of one or more therapeutic agents.

[0072] As used herein, the terms “therapeutically effective amount”, “therapeutic dose,” “prophylactically effective amount,” or “diagnostically effective amount” is the amount of an agent needed to elicit the desired biological response following administration.

[0073] As used herein, “treatment” of a subject (e.g., a mammal, such as a human) or a cell is any type of intervention used in an attempt to alter the natural course of the individual or cell. Treatment includes, but is not limited to, administration of a pharmaceutical composition, and may be performed either prophylactically or subsequent to the initiation of a pathologic event or contact with an etiologic agent. Also included are “prophylactic” treatments, which can be directed to reducing the rate of progression of the disease or condition being treated, delay ing the onset of that disease or condition, or reducing the severity' of its onset. “Treatment” or “prophy laxis” docs not necessarily indicate complete eradication, cure, or prevention of the disease or condition, or associated symptoms thereof.

[0074] The term “wild-type” refers to a gene or gene product (e.g., a polypeptide) that is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene.

[0075] Each embodiment in this specification is to be applied to every other embodiment unless expressly stated otherwise.

[0076] Oncolytic Viruses

[0077] Embodiments of the present disclosure include an oncolytic virus, comprising a recombinant polynucleotide that encodes: a death domain (DD) polypeptide, wherein the DD polypeptide is a C- terminal fragment of human CD95 (SEQ ID NO: 1) that induces apoptosis of cancer cells; or a serine Attorney Docket No. OPNI-003 / 04WG 332575-2098 protease such as porcine pancreatic elastase (PPE) polypeptide or human neutrophil elastase (ELANE) polypeptide.

[0078] The term “oncolytic virus” or “oncovirus” refers to a virus that preferentially infects and kills cancer cells relative to non-cancerous cells (see, for example, Kaufman et al., Nat Rev Drug Discov. 14(9):642-62, 2015). General examples of oncolytic viruses include certain pox viruses, herpes viruses, adenoviruses, retroviruses, rhabdoviruses. picomaviruses, paramyxoviruses, and reoviruses. Specific examples of oncolytic viruses include pox viruses such as myxoma viruses (see, for example, Rahman and McFadden, J Clin Med. 9(1): 171, 2020) and vaccinia viruses (see, for example. Deng et al., Oncol Rep. 41(l):686-692, 2019). Specific examples of oncolytic viruses include talimogene laherparepvec (T-VEC), coxsackievirus A21 (CAVATAK™), Oncorine (H101), pelareorep (REOLYSIN®). Seneca Valley virus (NTX-010), Senecavirus SVV-001. ColoAdl. SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1. MV-N1S, and DNX-2401.

[0079] In specific embodiments, the oncolytic virus is a myxoma virus. In some embodiments, the myxoma virus comprises a wild-type strain of myxoma virus or a genetically modified strain of myxoma virus. In some embodiments, the myxoma virus comprises a Lausanne strain. In some embodiments, the Lausanne strain comprises GenBank Accession Number AF170726.2, or a variant thereof. In some instances, the myxoma virus comprises a South American MYXV strain that circulates in Sylvilagus brasiliensis. In some instances, the myxoma virus comprises a Californian myxoma virus strain that circulates in Sylvilagus bachmani. In some instances, the myxoma virus comprises 6918, an attenuated Spanish field strain that comprises modifications in genes M009L. M036L, M135R. and M148R (see, for example, GenBank Accession number EU552530). In some instances, the myxoma virus comprises 6918VP60-T2 (see, for example, GenBank Accession Number EU552531). In some instances, the myxoma virus comprises a Standard laboratory Strain (SLS). In some embodiments, the myxoma virus comprises a nucleic acid construct or myxoma virus genome as described herein. In some instances, the myxoma virus comprises a polynucleotide sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a sequence disclosed in Cameron, et al. (Virology 264: 298-318, 1999).

[0080] In some embodiments, the myxoma virus is engineered to inactivate or attenuate an activity or expression level of a viral gene or protein. In some embodiments, the viral gene or protein is Ml 53. In some embodiments, the inactivated or attenuated activity or expression level of the viral gene or protein results in the myxoma virus exhibiting enhanced anti-cancer activity in relation to a wild-type myxoma virus, or in relation to a myxoma virus that lacks the inactivated or attenuated activity or expression level of the viral gene or protein, for example, a myxoma virus that comprises a wild type M153 gene and / or expresses a wild type M153 protein. In some embodiments, the myxoma virus is engineered to inactivate or attenuate an activity or expression level of more than one viral gene or protein. For instance, in specific embodiments, the myxoma virus comprises an attenuated or deleted Attorney Docket No. OPNI-003 / 04WG 332575-2098

[0081] Mil protein, M062R protein, or M153 protein, including combinations thereof (see, for example, U.S. Application No. 2021 / 0061864; and Rahman and McFadden, 2020, supra).

[0082] Oncovirus therapy can have a variety of beneficial effects, including, for example, selectively infecting and killing cancer cells, and inducing anticancer activity through release of cancer antigens and immune stimulation (see, for example, Cook and Chauhan, hit J Mol Sci. 21(20):7505, 2020). As described herein, oncoviruses can also be used to selectively deliver therapeutic polypeptides to cancers or cancerous tumor sites (e.g., tumor microenvironment), where expression of the therapeutic polypeptides in target cancer cells enhances cancer cell-killing. In such instances, an oncolytic virus is engineered to comprise a recombinant polynucleotide that encodes the therapeutic polypeptide. Typically, the recombinant polynucleotide is an “expressible polynucleotide”, as described herein. General examples of therapeutic polypeptides include pro-apoptotic polypeptides, including propeptides or proenzymes thereof. As noted above, specific examples of therapeutic polypeptides including CD95 DD polypeptides and a serine proteases such as PPE or human ELANE.

[0083] Thus, in certain embodiments, an oncolytic virus comprises a recombinant polynucleotide that encodes a CD95 DD polypeptide that induces apoptosis of cancer cells, for example, a C-terminal fragment of human CD95, including active variants and fragments thereof that induce apoptosis of cancer cells. Exemplary CD95 and DD sequences are provided in Table DI.

[0084] Thus, in some embodiments, a recombinant polynucleotide encodes a DD polypeptide that comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a selected from Table DI (for example. SEQ ID NOs: 2-5) and which induces apoptosis of cancer cells. Induction of apoptosis in cancer cells can be measured according to Attorney Docket No. OPNI-003 / 04WG 332575-2098 routine techniques in the art (see, for example, WO 2020 / 132465; and WO 2018 / 232273, incorporated by reference).

[0085] In embodiments, an oncolytic virus comprises a recombinant polynucleotide that encodes a serine protease that (directly or indirectly) induces cancer cell-killing, for example, by inducing apoptosis. Generally, serine proteases in nature are produced as an inactive “proprotein’' (or “proenzyme”, “zymogen”) composed of a signal peptide, an activation peptide comprising a native or wild-type protease cleavage site, and an active peptidase domain. The proprotein is activated by protease cleavage of the activation peptide to release the enzymatically -active, peptidase domain. In some embodiments, the serine protease is PPE or human ELANE, including proproteins (e.g., pro- PPE, pro-ELANE) and fragments and variants thereof. Exemplary pro-PPE and pro-ELANE polypeptide sequences are provided in Table SI.

[0086] Therefore, in some embodiments, a recombinant polynucleotide encodes a PPE polypeptide that comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 6 (WT pro-PPE). In some instances, a recombinant polynucleotide encodes an ELANE polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99. or 100% identical to SEQ ID NO: 7 (WT pro-ELANE). Also included are variants and / or fragments thereof, some of which include “activatable” proproteins (i.e., activated by proteolytic cleavage at the activation peptide domain to release the proteolytically -active peptidase domain), and some of which include activated polypeptide variants and fragments thereof (e.g.. peptidase domain). In some embodiments, expression of the pro-PPE or pro-ELANE and protease cleavage of the activation peptide, for example, at a cancer or tumor site in vivo, generates an active PPE or ELANE peptidase domain, which has increased cancer cell-killing activity relative to the proprotein form. In certain embodiments, the cancer cell-killing activity of the active peptidase domain is increased by about or Attorney Docket No. OPNI-003 / 04WO 332575-2098 at least about 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold or more relative to that of the proprotein.

[0087] Certain examples of variants include PPE and ELANE polypeptides (e.g., proproteins of SEQ ID NOs: 6 or 7) that comprise a modified “activation peptide”, which allows them to be selectively activated by proteases associated with the tumor microenviromnent (see U.S. Provisional Application No. 63 / 067,059, incorporated by reference). That is, the activation peptide containing the native protease cleavage site is replaced or otherwise modified so that it is not cleavable (or not substantially cleavable) by its native protease under suitable conditions (e g., in vivo, in vitro, for example, using a colorimetric substrate activity assay), but is instead cleavable by a protease that is present at relatively higher levels in cancer tissues or at tumor sites.

[0088] For instance, in some embodiments, a variant or modified PPE or ELANE polypeptide comprises, in an N-terminal to C-terminal orientation, a signal peptide, a modified activation peptide, and a peptidase domain, wherein the modified activation peptide comprises a heterologous protease cleavage site that is cleavable by a protease selected from a metalloprotease, an aspartyl protease, and a cysteine protease. In particular embodiments, the heterologous protease cleavage site is cleavable by matrix metalloproteinase- 12 (MMP12), cathepsin D (CTSD). cathepsin C (CTSC). or cathepsin L

[0089] (CTSL). The amino acid sequences of exemplary protease cleavage sites are provided in Table S3. Attorney Docket No. OPNI-003 / 04WO 332575-2098

[0090] In certain embodiments, a modified activation peptide has a heterologous protease cleavage site that comprises, consists, or consists essentially of an amino acid sequence selected from Table 21 S3. For example, in some embodiments, the modified activation peptide comprises a protease cleavage site selected from SEQ ID NOs: 8-10, and is cleavable by MMP12; or the modified activation peptide comprises a protease cleavage site selected from SEQ ID NOs: 11-12. and is cleavable by CTSD; or the modified activation peptide comprises a protease cleavage site of SEQ ID NO: 13. and is cleavable by CTSC; or the modified activation peptide comprises a protease cleavage site selected from SEQ ID NOs: 14-16, and is cleavable by CTSL.

[0091] Examples of modified PPE proproteins having a heterologous protease cleavage site, as described herein, are provided in Table S4 below.

[0092] Thus, in certain embodiments, the PPE polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97. 98, 99, or 100% identical to a Attorney Docket No. OPNI-003 / 04WO 332575-2098 sequence selected from Table S4 (e.g., SEQ ID NOs: 17-25), which retains the heterologous protease cleavage site.

[0093] In some embodiments, the PPE polypeptide (e.g., pro-PPE) comprises the wild-type PPE peptidase domain (SEQ ID NO: 26), and in some embodiments, the PPE peptidase domain is a variant or fragment thereof, for example, which comprises an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 7, and which has serine protease activity and / or cancer cellkilling activity. Thus, additional examples of variants include PPE polypeptides (e.g., pro-PPE) that comprise at least one amino acid alteration in the peptidase domain (SEQ ID NO: 26). some of which have increased cancer cell-killing activity, and / or reduced binding to or interaction with a human alpha-1 antitrypsin (Al AT) protein, relative to that of the wild-type PPE peptidase domain. Particular examples of variants include a PPE peptidase domain that has at least one alteration at a residue selected from one or more of Q211, T55, D74, R75. S214. R237. and N241, the residue numbering being defined by SEQ ID NO: 6 (WT pro-PPE). In specific embodiments, the at least one amino acid alteration is selected from one or more of Q21 IF, T55A, D74A, R75A, R75E, Q211A, S214A, R237A, N241 A, and N241 Y, the residue numbering being defined by SEQ ID NO: 6 (WT pro-PPE) (see U.S. Provisional Application No. 63 / 067,058, incorporated by reference).

[0094] The wild-type PPE peptidase domain and exemplary PPE peptidase variants are provided in

[0095] Table S5. Attorney Docket No. OPNI-003 / 04WO 332575-2098

[0096] Thus, in certain embodiments, a modified PPE polypeptide (e.g., pro-PPE) comprises a peptidase domain selected from: an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99. or 100% identical to SEQ ID NO: 27, and which retains the Q211F amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 28, and which retains the T55A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99. or 100% identical to SEQ ID NO: 29, and which retains the N241 A amino acid substitution; an amino acid sequence that is at least 80, 85. 90, 95, 96, 97. 98, 99, or 100% identical to SEQ ID NO: 30. and which retains the N241Y amino acid substitution; an amino acid sequence that is at least 80, 85. 90. 95, 96, 97, 98. 99, or 100% identical to SEQ ID NO: 31. and which retains the R75A amino acid substitution; an amino acid sequence that is at least 80. 85, 90, 95, 96. 97, 98, 99, or 100% identical to SEQ ID NO: 32. and which retains the R75E amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96. 97, 98, 99, or 100% identical to SEQ ID NO: 33, and which retains the Q211A amino acid substitution; an amino acid sequence that is at least 80, 85, 90. 95, 96. 97. 98, 99, or 100% identical to SEQ ID NO: 34, and which retains the R237A amino acid substitution; an amino acid sequence that is at least 80. 85, 90, 95, 96. 97, 98, 99, or 100% identical to SEQ ID NO: 35, and which retains the S214A amino acid substitution; and an amino acid sequence that is at least 80, 85. 90, 95, 96, 97. 98, 99, or 100% identical to SEQ ID NO: 36, and which retains the D74A amino acid substitution. In certain embodiments, prior to cleavage, the modified PPE polypeptide (e.g., pro-PPE) does not substantially bind to a serine protease inhibitor (Serpin) in vitro Attorney Docket No. OPNI-003 / 04WO 332575-2098 or in vivo, for example, wherein the Serpin is alpha-1 antitry psin (Al AT). In some embodiments, prior to cleavage, the modified PPE proprotein is substantially inactive as a serine protease in its proprote in form.

[0097] In some embodiments, expression and protease cleavage of the modified activation peptide, for example, at a cancer or tumor site in vivo, generates an active PPE peptidase domain (also referred to as an active PPE protein), which has increased serine protease activity relative to the modified PPE proprotein. In some embodiments, the serine protease activity of the active PPE protein is increased by about or at least about 2-fold, 5-fold. 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold or more relative to that of the modified PPE proprotein. In some embodiments, the active PPE protein has the same or substantially the same serine protease activity as a wild-type active PPE protein (e.g., SEQ ID NO: 26). In some embodiments, the active PPE protein is a variant that has about or at least about 50, 60, 70, 80, 90. 100, 200, 300. 400, 500. 600, 700, 800. 900, or 1000% or more of the serine protease activity of a wild-type active PPE protein (SEQ ID NO: 26).

[0098] In some embodiments, expression of the pro-PPE and protease cleavage of the modified activation peptide, for example, at a cancer or tumor site in vivo, generates an active PPE peptidase domain (or active PPE protein), which has increased cancer cell-killing activity relative to the PPE proprotein. In certain embodiments, the cancer cell-killing activity of the active PPE protein is increased by about or at least about 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold or more relative to that of the PPE proprotein. In some embodiments, the active PPE protein has the same or substantially the same cancer cell-killing activity as a wild-type active PPE protein (e.g., SEQ ID NO: 26). In some embodiments, the active PPE protein is a variant that has about or at least about 50, 60, 70. 80, 90, 100. 200, 300, 400, 500, 600, 700, 800, 900, or 1000% or more of the cancer cellkilling activity of a wild-type active PPE protein (SEQ ID NO: 26).

[0099] Serine protease activity and cancer cell-killing activity can be measured according to routine techniques in the art. For example, serine protease activity can be monitored using a colorimetric substrate activity' assay (N-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide), and cancer cell-killing activity can be measured in vitro or in vivo.

[0100] In some embodiments, the oncolytic virus, for example, a myxoma virus, comprises a recombinant polynucleotide that encodes one or more additional proteins, for example, a cytokine, an activator of co-stimulatory receptor, or cell matrix protein (see. for example, U.S. Application No. 2021 / 0061864, incorporated by reference). In some embodiments, the cytokine is selected from tumor necrosis factor alpha (TNFa), interleukin- 12 subunit alpha (IL-12a), interleukin- 12 subunit beta (IL- 120), and interleukin- 15 (IL-15) optionally IL-15a, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and interleukin-21 (IL-21). In some instances, the activator of co-stimulatory receptors is selected from one or more of 4- IBB, 0X40. Glucocorticoid-induced TNFR-related protein (GITR), and B7-1 (CD80). In specific instances, the cell matrix protein is a human decorin protein. Attorney Docket No. OPNI-003 / 04WO 332575-2098

[0101] Methods of Use and Pharmaceutical Compositions

[0102] Certain embodiments include methods of treating, ameliorating the symptoms of, and / or reducing the progression of, a disease or condition in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an oncolytic virus, as described herein. In particular embodiments, the disease is a cancer, that is, the subject in need thereof has, is suspected of having, or is at risk for having, a cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological malignancy. In particular embodiments, the cancer is a primary cancer or a metastatic cancer.

[0103] In specific embodiments, the cancer is selected from one or more of bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain / nervous system cancer (optionally glioblastoma), breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer (optionally small cell lung cancer (SCLC)), nasopharynx cancer, neck cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, prostate cancer, skin cancer (optionally melanoma), stomach cancer, testicular cancer, tongue cancer, uterine cancer, multiple myeloma, and embryonal rhabdomyosarcoma.

[0104] The methods for treating cancers can be combined with other therapeutic modalities. For example, a combination therapy described herein can be administered to a subject before, during, or after other therapeutic interventions, including symptomatic care, radiotherapy, surgery, transplantation, hormone therapy, photodynamic therapy, antibiotic therapy, or any combination thereof. Symptomatic care includes administration of corticosteroids, to reduce cerebral edema, headaches, cognitive dysfunction, and emesis, and administration of anti-convulsants, to reduce seizures. Radiotherapy includes whole-brain irradiation, fractionated radiotherapy, and radiosurgery, such as stereotactic radiosurgery, which can be further combined with traditional surgery.

[0105] Certain embodiments thus include combination therapies for treating cancers, including methods of treating ameliorating the symptoms of, or inhibiting the progression of, a cancer in a subject in need thereof, comprising administering to the subject an oncolytic virus described herein in combination with at least one additional agent, for example, an immunotherapy agent, a chemotherapeutic agent, a hormonal therapeutic agent, and / or a kinase inhibitor. In some embodiments, administering the oncolytic virus enhances the susceptibility of the cancer to tire additional agent (for example, immunotherapy agent such as an immune checkpoint modulatory agent, chemotherapeutic agent, hormonal therapeutic agent, and or kinase inhibitor) by about or at least about 5, 10, 15. 20, 25, 30, 35, 40, 45, 50, 60, 70. 80, 90, 100, 200, 300, 400. 500, 600, 700. 800, 900, 1000, 2000% or more relative to the additional agent alone.

[0106] In some embodiments, the methods and compositions described herein increase an anti-cancer immune response in the subject by about or at least about 5, 10, 15. 20. 25, 30, 35, 40. 45, 50, 60, 70. 80, 90, 100. 200, 300, 400. 500, 600, 700. 800, 900. 1000, 2000% or more, relative to a control. In some embodiments, the methods and compositions described herein increase cancer cell-killing in the Attorney Docket No. OPNI-003 / 04WO 332575-2098 subject by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more, relative to a control. In some embodiments, the methods and compositions described herein increase median survival time of a subject by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, or longer. In certain embodiments, the methods and compositions described herein increase median survival time of a subject by 1 year, 2 years, 3 years, or longer. In some embodiments, the methods and pharmaceutical compositions increase progression-free survival by 2 weeks, 3 weeks. 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or longer. In certain embodiments, the methods and pharmaceutical compositions described herein increase progression-free survival by 1 year, 2 years, 3 years, or longer.

[0107] In certain embodiments, the methods and compositions described herein are sufficient to result in tumor regression, for example, as indicated by a statistically significant decrease in the amount of viable tumor, for example, at least a 10%. 20%. 30%, 40%, 50% or greater decrease in tumor mass, or by altered (e.g., decreased with statistical significance) scan dimensions. In certain embodiments, the methods and compositions described herein are sufficient to result in stable disease. In certain embodiments, the methods and compositions described herein are sufficient to result in clinically relevant reduction in symptoms of a particular disease indication known to the skilled clinician.

[0108] For in vivo use, as noted above, for the treatment of human or non-human mammalian disease or testing, the oncolytic viruses described herein are generally incorporated into one or more therapeutic or pharmaceutical compositions prior to administration, including veterinary' therapeutic compositions.

[0109] Thus, certain embodiments relate to pharmaceutical or therapeutic compositions that comprise an oncolytic virus, as described herein. In some instances, a pharmaceutical or therapeutic composition comprises one or more of the oncolytic viruses described herein in combination with a pharmaceutically- or physiologically-acceptable carrier or excipient. Certain pharmaceutical or therapeutic compositions further comprise at least one additional agent, for example, an immunotherapy agent, a chemotherapeutic agent, a hormonal therapeutic agent, and / or a kinase inhibitor as described herein.

[0110] To prepare a therapeutic or pharmaceutical composition, an effective or desired amount of one or more oncolytic viruses is mixed with any pharmaceutical carrier(s) or excipient known to those skilled in the art to be suitable for the particular agent and / or mode of administration. A pharmaceutical carrier may be liquid, semi-liquid or solid. Solutions or suspensions may include, for example, a sterile diluent (such as water), saline solution (e.g., phosphate buffered saline; PBS), fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvent; antimicrobial agents (such as benzyl alcohol and methyl parabens); antioxidants (such as ascorbic acid and sodium bisulfite) and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (such as Attorney Docket No. OPNI-003 / 04WO 332575-2098 acetates, citrates and phosphates). If administered parenterally (e.g., by IV infusion), suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol and mixtures thereof.

[0111] Carriers can include, for example, pharmaceutically - or physiologically -acceptable carriers, excipients, or stabilizers that are non-toxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically-acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as polysorbate 20 (TWEEN™) polyethylene glycol (PEG), and poloxamers (PLURONICS™), and the like.

[0112] Administration of an oncolytic virus described herein, in pure form or in an appropriate therapeutic or pharmaceutical composition, can be carried out via any of the accepted modes of administration of agents for serving similar utilities. Examples include administering the pharmaceutical composition to the subject by systemic administration, intratumoral injection, or intranasal administration, for example, parenteral administration, including intravenous and subcutaneous administration. Preferred modes of administration depend upon the nature of the condition to be treated or prevented. Particular embodiments include administration by IV infusion.

[0113] The precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating therefrom. Controlled clinical trials may also be performed. Dosages may also vary with the severity of the condition to be alleviated. A pharmaceutical composition is generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects. The composition may be administered one time, or may be divided into a number of smaller doses to be administered at intervals of time. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need.

[0114] The therapeutic or pharmaceutical compositions may be administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex. and diet of the subject; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy. Attorney Docket No. OPNI-003 / 04WO 332575-2098

[0115] The combination therapies described herein may include administration of a single pharmaceutical dosage formulation, which contains a modified serine protease proprotein and an additional therapeutic agent (e.g., immunotherapy agent, chemotherapeutic agent, hormonal therapeutic agent, kinase inhibitor), as well as administration of compositions comprising a modified serine protease proprotein and an additional therapeutic agent in its own separate pharmaceutical dosage formulation. For example, a modified serine protease proprotein and an additional therapeutic agent can be administered to the subject together in a single parenteral dosage composition such as in a saline solution or other physiologically acceptable solution, or each agent administered in separate parenteral dosage formulations. Where separate dosage formulations are used, the compositions can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially and in any order; combination therapy is understood to include all these regimens.

[0116] Also included are patient care kits, comprising (a) at least one oncolytic virus, as described herein; and optionally (b) at least one additional therapeutic agent (e.g.. immunotherapy agent, chemotherapeutic agent, hormonal therapeutic agent, kinase inhibitor). In certain kits, (a) and (b) are in separate therapeutic compositions. In some kits, (a) and (b) are in the same therapeutic composition.

[0117] The kits herein may also include a one or more additional therapeutic agents or other components suitable or desired for the indication being treated, or for the desired diagnostic application. The kits herein can also include one or more syringes or other components necessary' or desired to facilitate an intended mode of delivery (e.g., stents, implantable depots, etc.).

[0118] In some embodiments, a patient care kit contains separate containers, dividers, or compartments for the composition(s) and informational material(s). For example, the composition(s) can be contained in a bottle, vial, or syringe, and the informational material(s) can be contained in association with the container. In some embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of a modified serine protease proprotein and optionally at least one additional therapeutic agent. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of an oncolytic virus and optionally at least one additional therapeutic agent. The containers of the kits can be air tight, waterproof (e.g., impenneable to changes in moisture or evaporation), and / or light-tight.

[0119] The patient care kit optionally includes a device suitable for administration of the composition, e.g., a syringe, inhalant, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device. In some embodiments, the device is an implantable device that dispenses metered doses of the agent(s). Also included are methods of providing a kit, e.g.. by combining the components described herein. Attorney Docket No. OPNI-003 / 04WO 332575-2098

[0120] Although the foregoing embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary' skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

Attorney Docket No. OPNI-003 / 04WO 332575-2098CLAIMS1. An oncolytic virus, comprising a recombinant polynucleotide that encodes:(a) a death domain (DD) polypeptide, wherein the DD polypeptide is a C -terminal fragment of human CD95 (SEQ ID NO: 1) that induces apoptosis of cancer cells; or(b) a serine protease selected from porcine pancreatic elastase (PPE) polypeptide and human neutrophil elastase (ELANE) polypeptide.

2. The oncolytic virus of claim 1, wherein the DD polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95. 96, 97, 98, 99. or 100% identical to a selected from SEQ ID NOs: 5 and 2-4 and induces apoptosis of cancer cells.

3. The oncolytic virus of claim 2, wherein the DD polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95.

96. 97, 98, 99. or 100% identical to SEQ ID NO: 5 and induces apoptosis of cancer cells.

4. The oncolytic virus of claim 2, wherein the DD polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85.

90. 95, 96, 97, 98. 99, or 100% identical to SEQ ID NO: 2 and induces apoptosis of cancer cells.

5. The oncolytic virus of claim 2, wherein the DD polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90. 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 3 and induces apoptosis of cancer cells.

6. The oncolytic virus of claim 2, wherein the DD polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 4 and induces apoptosis of cancer cells.

7. The oncolytic virus of claim 1, wherein the PPE polypeptide comprises, consists, or consists essentially of an ammo acid sequence that is at least 80, 85, 90, 95, 96, 97, 98. 99, or 100% identical to SEQ ID NO: 6 (WT pro-PPE), or the ELANE polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80. 85, 90, 95, 96, 97. 98, 99, or 100% identical to SEQ ID NO: 7 (WT pro- ELANE).

8. The oncolytic virus of claim 1 or 7, wherein the PPE polypeptide or ELANE polypeptide comprises, in an N-terminal to C-terminal orientation, a signal peptide, a modified activation peptide, and a peptidase domain, wherein the modified activation peptide comprises aAttorney Docket No. OPNI-003 / 04WO 332575-2098 heterologous protease cleavage site that is cleavable by a protease selected from a metalloprotease, an aspartyl protease, and a cysteine protease.

9. The oncolytic virus of claim 8, wherein the metalloprotease, aspartyl protease, or cysteine protease is selected from matrix metalloproteinase- 12 (MMP12). cathepsin D (CTSD), cathepsin C (CTSD), and cathepsin L (CTSL), and optionally wherein the heterologous protease cleavage site is selected from Table S3, including the MMP12 cleavage site of SEQ ID NOs: 8-10, the CTSD cleavage site of SEQ ID NOs: 11-12, the CTSC cleavage site of SEQ ID NO: 13, or the CTSL cleavage site of SEQ ID NOs: 14-16.

10. The oncolytic virus of claim 9, wherein the PPE polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95. 96, 97, 98, 99. or 100% identical to a sequence selected from SEQ ID NOs: 17-25 (Table S4), which retains the heterologous protease cleavage site.

11. The oncolytic virus of any one of claims 7-10, wherein the PPE polypeptide comprises at least one amino acid alteration in the peptidase domain (SEQ ID NO: 26), wherein the at least one alteration is at a residue selected from one or more of Q211, T55, D74. R75, S214, R237, and N241. the residue numbering being defined by SEQ ID NO: 6 (WT pro-PPE).

12. The oncolytic virus of claim 11, wherein the at least one amino acid alteration is selected from one or more of Q211F, T55A. D74A, R75A, R75E, Q211A, S214A, R237A, N241A, and N241Y, the residue numbering being defined by SEQ ID NO: 6 (WT pro-PPE).

13. The oncolytic virus of claim 12, wherein the PPE polypeptide comprises a peptidase domain selected from: an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:

27. and which retains the Q211F amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:

28. and which retains the T55A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96. 97, 98, 99, or 100% identical to SEQ ID NO:

29. and which retains the N241A amino acid substitution; an amino acid sequence that is at least 80. 85, 90, 95, 96. 97, 98, 99, or 100% identical to SEQ ID NO:

30. and which retains the N241Y amino acid substitution; an amino acid sequence that is at least 80. 85, 90, 95, 96. 97, 98, 99, or 100% identical to SEQ ID NO:

31. and which retains the R75A amino acid substitution;Attorney Docket No. OPNI-003 / 04WO 332575-2098 an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 32, and which retains the R75E amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:

33. and which retains the Q211A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:

34. and which retains the R237A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96. 97, 98, 99, or 100% identical to SEQ ID NO:

35. and which retains the S214A amino acid substitution; and an amino acid sequence that is at least 80. 85, 90, 95, 96. 97, 98, 99, or 100% identical to SEQ ID NO:

36. and which retains the D74A amino acid substitution.

14. The oncolytic virus of any one of claims 1-13. wherein the oncolytic virus is selected from a pox virus, a herpes virus, an adenovirus, a retrovirus, a rhabdovirus. a paramyxovirus, and a reovirus.

15. The oncolytic virus of claim 14, wherein the poxvirus is a myxoma vims or a vaccinia vims.

16. The oncolytic virus of claim 15, wherein the poxvims is a myxoma vims, optionally wherein the myxoma vims comprises an attenuated or deleted Mil protein, M062R protein, or M153 protein, including combinations thereof.

17. The oncolytic vims of any one of claims 1-16, wherein the recombinant polynucleotide further encodes a cytokine, an activator of co-stimulatorv receptor, or cell matrix protein.

18. The oncolytic virus of claim 17, wherein the cytokine is selected from tumor necrosis factor alpha (TNFa), interleukin-12 subunit alpha (IL-12a), interleukin- 12 subunit beta (IL-120), and interleukin- 15 (IL-15) optionally IL-15a, granulocyte-macrophage colony-stimulating factor (GM- CSF), interleukin-2 (IL-2), interleukin-21 (IL-21); and / or wherein the activator of co-stimulatory receptors is selected from one or more of 4- IBB. 0X40, Glucocorticoid-induced TNFR-related protein (GITR), and B7-1 (CD80); and / or wherein the cell matrix protein is a human decorin protein.

19. A pharmaceutical composition, comprising the oncolytic virus of any one of claims 1- 18 and a pharmaceutically-acceptable carrier.Attorney Docket No. OPNI-003 / 04WO 332575-209820. A method of treating cancer in a subject in need thereof, comprising administering to die subject the pharmaceutical composition of claim 19.

21. The method of claim 20, wherein the cancer is a solid tumor.

22. The method of claim 20, wherein the cancer is a hematological malignancy.

23. The method of any one of claims 20-21. wherein the cancer is selected from one or more of bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain / nervous system cancer(optionally glioblastoma), breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer (optionally small cell lung cancer (SCLC)). nasopharynx cancer, neck cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, prostate cancer, skin cancer (optionally melanoma), stomach cancer, testicular cancer, tongue cancer, uterine cancer, multiple myeloma, and embryonal rhabdomyosarcoma.

24. The method of any one of claims 20-23, comprising administering the pharmaceutical composition to the subject by systemic administration, intratumoral injection, or intranasal administration.

25. The method of claim 24, comprising administering tire pharmaceutical composition to the subject by parenteral administration, optionally intravenous or subcutaneous administration.

26. The method of any one of claims 20-25, further comprising administering to the subject an immune checkpoint modulatory' agent, a chemotherapeutic agent, a hormonal therapeutic agent, and / or a kinase inhibitor.

27. The method of any one of claims 20-26, wherein administering the oncolytic virus increases an anti-cancer immune response in the subject by about or at least about 5. 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80. 90, 100, 200, 300, 400, 500, 600. 700, 800, 900, 1000, 2000% or more, relative to a control.

28. The method of any one of claims 20-27. wherein administering the oncolytic virus increases cancer cell-killing in the subject by about or at least about 5, 10, 15. 20, 25, 30, 35. 40, 45, 50, 60, 70, 80. 90, 100, 200, 300. 400, 500, 600. 700, 800, 900. 1000. 2000% or more, relative to a control.Attorney Docket No. OPNI-003 / 04WO 332575-209829. Use of a pharmaceutical composition of claim 19 in the preparation of a medicament for treating a cancer in a subject.

30. A pharmaceutical composition of claim 19 for use in treating a cancer in a subject.

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